Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1
PLANTS PRODUCING MODIFIED LEVELS OF MEDIUM CHAIN FATTY
ACIDS
FIELD OF THE INVENTION
The present invention relates to methods of producing industrial products from
plant lipids, particularly from vegetative parts of plants. In particular, the
present
invention provides oil products such as biofuel, and processes for producing
these
products, as well as plants having an increased level medium chain fatty acids
such as
lauric acid and myristic acid. In one particular embodiment, the present
invention
relates to combinations of modifications in a fatty acid thioesterase and one
or more
acyltransferases. In an embodiment, the present invention relates to a process
for
extracting lipids. In another embodiment, the lipid is converted to one or
more
hydrocarbon products in harvested plant vegetative parts to produce alkyl
esters of the
fatty acids which are suitable for use as a renewable biofuel.
BACKGROUND OF THE INVENTION
Over recent years the global production of vegetable oils has experienced
constant growth, with over 179 million metric tons (MMT) being produced in
2015
(OECD/FAO, 2015), with the four major oil production crops being oil palm,
soybean,
canola and sunflower. An important component of global oil consumption is
medium-
chain fatty acids (MCFA), here defined as fatty acids in the range of 6-14
carbons in
length. As well as their application within the food industry MCFAs are an
ideal
source for biodiesel and also for a wide range of oleochemical feedstocks
including
pharmaceuticals, personal care products, lubricants and detergents (Arkcoll,
1988;
Basiron and Weng, 2004). Currently, the predominant crop sources of MCFA-
enriched
oils are coconut palm and oil palm (both palm oil and palm kernel oil)
(Arkcoll, 1988).
The production of these crops is limited to tropical and subtropical climates.
The
development of new crops that can produce MCFA-enriched oils in temperate
climates
has been proposed (Dehesh, 2001; Eccleston et al., 1996; Reynolds et al.,
2015;
Tjellstrom et al., 2013; Voelker et al., 1992; Wiberg et at., 2000) as a way
to meet the
growing global demand for MCFA in oleochemical production, pharmaceutical
applications, and personal care products.
Many studies have investigated the modification of seed oils to contain
increased MCFA content, predominantly focused on the engineering of lauric
acid
(C12:0) (Eccleston and Ohlrogge, 1998; Knutzon et al., 1999; Voelker et al.,
1992). In
oilseeds the engineered pathway begins with the overexpression of a
specialised
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thioesterase (FATB) that prematurely truncates the standard fatty acid
elongation cycle
within the plastid allowing export into the cytoplasm. The MCFA in the
cytoplasm is
available for incorporation into triacylglycerols (TAG) via the endogenous
oilseed
pathways which can occur via the acyl-CoA dependent reactions of the Kennedy
pathway (glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid
acyltransferase (LPAAT) and diacylglycerol acyltransferase (DGAT). Previous
studies
have investigated the incorporation of MCFA into seed oils following the
coordinated
over-expression of FATB and LPAAT, achieving up to 67% of laurate (C12:0) in
seed
oil (Knutzon et al., 1999). More recently, transcriptomic analyses have
enabled the
identification of new FATB and LPAAT genes from many Cuphea species, which
have
been used to both modify the fatty acid profiles and improve the incorporation
of
MCFA into the TAG, respectively, of transgenic C amelina sativa seeds (Kim et
al.,
2015a; Kim et al., 2015b).
Evidence, although, has found that endogenous TAG synthesis pathways in
developing oilseeds are not ideal for incorporating MCFA into TAG (Wiberg et
al.,
1997; Wiberg et al., 2000), and that newly-synthesised MCFA becomes
incorporated
into membrane bound lipids, impeding lipid flux, agronomic performance and can
even
result in cell death through chlorosis (Bates et al., 2014; Voelker et al.,
1996).
Therefore it would seem that although MCFA can be produced in plant cells
there is a
poor pathway for incorporation into seed TAG. It has also been recognised that
the
accumulation of unusual fatty acids in PC appears to be a bottleneck for their
enriched
incorporation into TAG (Bates and Browse, 2011; Reynolds et al., 2015). In the
example of engineering ricinoleic acid into oilseeds it has been demonstrated
that the
endogenous pathways need to be removed in conjunction with the ectopic
expression of
the specialised pathway counterpart (Adhikari et al., 2016; Bates and Browse,
2011;
Burgal et al., 2008; Chen et al., 2016; van Erp et al., 2011; van Erp et al.,
2015).
Recent work has demonstrated that engineering high oil levels in plant biomass
is a realistic proposition (Vanhercke et al., 2014a; Vanhercke et al., 2013;
Vanhercke et
al.. 2014b) with the accumulation of levels of TAG in Nicotiana tabacum leaves
of up
to 15% being attained by the coordinated transgenic expression of genes
normally
involved in oil production in seeds (Vanhercke et al., 2014a). Such approaches
have
uncovered a synergism involving an increase in the production of fatty acids
in the
plastid (WRINKLED] (WRI1)), improving the assembly of fatty acids into leaf
oils
(DGAT) and slowing the catabolism of these oils (OLEOSIN, OLEI (Winichayakul
et
al., 2013)); and sugar-dependent-1. SDP1 (Fan et al., 2014; Kelly et al.,
2013a and b;
Kim et al., 2014b; Vanhercke, 2014a). Although the production of TAG in
biomass
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=
offers a new source of common vegetable oils, these new expression platforms
could
also be adapted to produce high levels of novel fatty acids, such as MCFA
(Reynolds et
al., 2015; Wood, 2014).
The inventors first steps in this direction involved the overexpression of
thioesterases from Umbellularia californica, Cinnamomum camphora and Cocos
nucifera which resulted in the production of MCFA in leaf tissues (Reynolds et
al.,
2015). However, these metabolic pathways also resulted in high levels of MCFA
in PC
resulting in severe chlorosis and cell death (Bates et al., 2014; Wiberg et
al., 2000),
similar to conclusions drawn from oilseed engineering. The incorporation of
MCFA
into the membrane lipids of vegetative tissues is therefore particularly
problematic.
The inventors have improved the MCFA metabolic pathway by combining a
series of gene ensembles with three different DGAT1 genes isolated from Elaeis
guineensis (African oil palm). A functional GPAT9 from C. nucifera was
identified
that was included in the metabolic pathway for improving the incorporation of
MCFA
into seed oils. An improvement in MCFA utilisation was demonstrated in
vegetative
plant cells such as leaf cells, which resulted in more efficient sequestering
of MCFA in
TAG while also effectively limiting the accumulation of MCFA in membrane
lipids.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a process for producing
extracted
plant lipid, comprising the steps of:
a) obtaining one or more plant parts comprising lipid, preferably
vegetative
plant parts, the lipid comprising a total fatty acid content which comprises
fatty acids in
an esterified form, the fatty acids comprising a level of total, or new,
medium chain
fatty acids (MCFA) that is at least 25% of the total fatty acid content on a
weight basis,
and
b) extracting lipid from the plant part(s),
thereby producing the extracted plant lipid.
In an embodiment, the plant part comprises one or more exogenous
polynucleotides which encode polypeptides having fatty acid thioesterase (TE)
activity,
and either glycerol-3-phosphate acyltransferase (GPAT) activity, preferably
GPAT9
activity, or diacylglycerol acyltransferase (DGAT) activity, preferably DGAT1
activity,
or both GPAT and DGAT,
wherein the exogenous polynucleotide is operably linked to a promoter which is
capable of directing expression of the polynucleotide in a cell of the plant
part.
In a further embodiment, the plant part further comprises one or more or all
of:
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i. an exogenous polynucleotide which encodes a second polypeptide having
glycerol-3-phosphate acyltransferase (GPAT) activity, preferably GPAT9
activity, or diacylglycerol acyltransferase (DGAT) activity, preferably DGAT1
activity;
ii. an exogenous polynucleotide which encodes a third polypeptide having 1-
acyl-
glycerol-3-phosphate acyltransferase (LPAAT) activity;
iii. an exogenous polynucleotide which encodes a transcription factor
polypeptide
that increases the expression of one or more glycolytic and/or fatty acid
biosynthetic genes in a cell of the plant part compared to a corresponding
cell
lacking the exogenous polynucleotide;
iv. an exogenous polynucleotide which encodes a polypeptide which increases
the
export of fatty acids out of plastids of a cell in the plant part when
compared to
a corresponding cell lacking the exogenous polynucleotide; and
v. an exogenous polynucleotide which encodes an oil body coating (OBC)
polypeptide,
wherein each exogenous polynucleotide is operably linked to a promoter which
is capable of directing expression of the polynucleotide in a cell of the
plant part.
In an embodiment, the OBC polypeptide is an oleosin, such as a polyoleosin or
a
caleosin, or a lipid droplet associated protein (LDAP).
In an embodiment, the transcription factor polypeptide is selected from the
group consisting of Wrinkled 1 (WRI1), Leafy Cotyledon 1 (LEC1), LEC1-like,
Leafy
Cotyledon 2 (LEC2), BABY BOOM (BBM), FIJS3, AB13, ABI4, AB15, Dof4 and
Dofl 1, preferable WRI1, or the group consisting of MYB73, bZIP53, AGL15,
MYB115, MYB118, TANMEI, WUS, GFR2a1, GFR2a2 and PHR1.
In an embodiment, the polypeptide which increases the export of fatty acids
out
of plastids of the cell is a fatty acid thioesterase such as a FA Lk
polypeptide or a
FATB polypeptide, a fatty acid transporter such as an ABCA9 polypeptide or a
long-
chain acyl-CoA synthetase (LACS), preferably a FATB polypeptide.
In an embodiment, the fatty acid thioesterase is capable of hydrolysing a
substrate which is an acyl carrier protein (ACP) esterified to a medium chain
fatty acid
and/or a C16:0, preferably wherein the MCFA is a C10, C12 and/or C14.
In an embodiment, the plant part further comprises one or more or all of:
i. a genetic modification which down-regulates endogenous production and/or
activity of a polypeptide involved in the catabolism of triacylglycerols (TAG)
in
a cell of the plant part when compared to a corresponding cell lacking the
genetic modification;
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ii. a genetic modification which down-regulates endogenous production and/or
activity of a polypeptide involved in importing fatty acids into plastids of a
cell
in the plant part when compared to a corresponding cell lacking the genetic
modification; and
iii. a genetic modification which down-regulates endogenous production and/or
activity of a polypeptide involved in diacylglycerol (DAG) production in the
plastid when compared to a corresponding cell in the plant part lacking the
genetic modification.
In an embodiment, the polypeptide involved in the catabolism of
triacylglycerols
(TAG) in the plant, or part thereof, is an SDP1 lipase, a Cgi58 polypeptide,
an acyl-
CoA oxidase such as ACX1 or ACX2, or a polypeptide involved in 13-oxidation of
fatty
acids in the plant or part thereof such as a PXA1 peroxisomal ATP-binding
cassette
transporter, preferably an SDP1 lipase.
In an embodiment, the polypeptide involved in importing fatty acids into
plastids of the cell is a fatty acid transporter, or subunit thereof,
preferably a TGD
polypeptide.
In an embodiment, the polypeptide involved in diacylglycerol (DAG)
production in the plastid is a plastidial GPAT, a plastidial LPAAT or a
plastidial PAP.
In another embodiment, the plant part further comprises one or both of:
i. an exogenous polynucleotide which encodes a second transcription factor
polypeptide that increases the expression of one or more glycolytic and/or
fatty
acid biosynthetic genes in the cell; and
ii. a second genetic modification which down-regulates endogenous production
and/or activity of a polypeptide involved in importing fatty acids into
plastids of
the cell when compared to a corresponding cell lacking the second genetic
modification.
In a preferred embodiment, the presence of a) a genetic modification which
down-regulates endogenous production and/or activity of a polypeptide involved
in the
catabolism of triacylglycerols (TAG) in a cell of the plant part when compared
to a
corresponding cell lacking the genetic modification, b) an exogenous poly-
nucleotide
which encodes a polypeptide which increases the export of fatty acids out of
plastids of
a cell in the plant part when compared to a corresponding cell lacking the
exogenous
polynucleotide, or c) an exogenous polynucleotide which encodes a second
transcription factor polypeptide that increases the expression of one or more
glycolytic
and/or fatty acid biosynthetic genes in the cell, together with an exogenous
polynucleotide which encodes a WRI1 polypeptide and an exogenous
polynucleotide
=
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which encodes a polypeptide having DGAT1 activity, increases the total non-
polar
lipid content of the plant part, preferably a vegetative plant part such as a
leaf or stem,
relative to a corresponding plant part comprising the exogenous
polynucleotides
encoding the WRI1 and DGAT1 polypeptides but lacking each of the other
exogenous
polynucleotide and genetic modifications. Most preferably, at least the
promoter that
directs expression of the exogenous polynucleotide which encodes the
transcription
factor is a promoter other than a constitutive promoter. Alternatively for
Sorghum or
Zea mays, the promoter is preferably a constitutive promoter such as, for
example a
ubiquitin gene promoter.
In an embodiment, the addition of one or more of the exogenous polynucleotides
or genetic modifications, preferably the exogenous polynucleotide encoding an
OBC or
a fatty acyl thioesterase or the genetic modification which down-regulates
endogenous
production and/or activity of a polypeptide involved in the catabolism of
triacylglycerols (TAG) in the plant or part thereof, more preferably the
exogenous
polynucleotide which encodes a FATA thioesterase or an LDAP or which decreases
expression of an endogenous TAG lipase such as a SDP1 TAG lipase in the plant
or
part thereof, results in a synergistic increase in the total non-polar lipid
content of the
plant or part thereof when added to the pair of transgenes WRI1 and DGAT,
particularly before the plant flowers and even more particularly in the stems
and/or
roots of the plant.
In a preferred embodiment, the increase in the TAG content of a stem or root
is
at least 2-fold, more preferably at least 3-fold, relative to a corresponding
plant part
transformed with genes encoding WRI1 and DGAT1 but lacking the FATA
thioesterase, LDAP and the genetic modification which down-regulates
endogenous
production and/or activity of a polypeptide involved in the catabolism of
triacylglycerols (TAG) in the plant part. Most preferably, at least the
promoter that
directs expression of the exogenous polynucleotide which encodes the
transcription
factor is a promoter other than a constitutive promoter. Alternatively for
Sorghum or
Zea mays, the promoter is preferably a constitutive promoter such as, for
example a
ubiquitin gene promoter.
In an embodiment, each genetic modification is, independently, a mutation of
an
endogenous gene which partially or completely inactivates the gene, such as a
point
mutation, an insertion, or a deletion, or an exogenous polynucleotide encoding
an RNA
molecule which inhibits expression of the endogenous gene, wherein the
exogenous
polynucleotide is operably linked to a promoter which is capable of directing
expression of the polynucleotide in the plant, or part thereof. The point
mutation may
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be a premature stop codon, a splice-site mutation, a frame-shift mutation or
an amino
acid substitution mutation that reduces activity of the gene or the encoded
polypeptide.
The deletion may be of one or more nucleotides within a transcribed exon or
promoter
of the gene, or extend across or into more than one exon, or extend to
deletion of the
entire gene. Preferably the deletion is introduced by use of ZF, TALEN or
CRISPR
technologies. In an alternate embodiment, one or more or all of the genetic
modifications is an exogenous polynucleotide encoding an RNA molecule which
inhibits expression of the endogenous gene, wherein the exogenous
polynucleotide is
operably linked to a promoter which is capable of directing expression of the
polynucleotide in the plant, or part thereof. Examples of exogenous
polynucleotide
which reduces expression of an endogenous gene are selected from the group
consisting of an antisense polynucleotide, a sense polynucleotide, a microRNA,
a
polynucleotide which encodes a polypeptide which binds the endogenous enzyme,
a
double stranded RNA molecule and a processed RNA molecule derived therefrom.
In
an embodiment, the plant or part thereof comprises genetic modifications which
are an
introduced mutation in an endogenous gene and an exogenous polynucleotide
encoding
an RNA molecule which reduces expression of another endogenous gene. In an
alternate embodiment, all of the genetic modifications that provide for the
increased
TTQ and or TAG levels are mutations of endogenous genes.
In an embodiment, the activity of PDCT or CPT in a cell in the plant part is
increased relative to a wild-type plant part. Alternatively, the activity of
PDCT or CPT
is decreased, for example by mutation in the endogenous gene encoding the
enzyme or
by downregulation of the gene through an RNA molecule which reduces its
expression..
In an embodiment, when present, the two transcription factors are WRI1 and
LEC2, or WRI1 and LEC1.
In the above embodiments, the plant part preferably comprises an exogenous
polynucleotide which encodes a DGAT and a genetic modification which down-
regulates production of an endogenous SDP1 lipase. More preferably, the plant
part
does not comprise an exogenous polynucleotide encoding a PDAT, and/or is a
plant
part other than a Nicotiana benthamiana or part thereof, and/or the WRI1 is a
WRI1
other than Arabidopsis thaliana WRI1 and/or is a plant part other than a
Brassica
napus or part thereof. In an embodiment, at least one of the exogenous
polynucleotides
in the plant part is expressed from a promoter which is not a constitutive
promoter such
as, for example, a promoter which is expressed preferentially in green tissues
or stems
of the plant or that is up-regulated after commencement of flowering or during
senescence.
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In an embodiment, the plant part comprises an increased level or activity of
polypeptides which are:
i. a GPAT, a LPAAT, and a WRII polypeptide;
ii. a GPAT, a LPAAT, a DGAT and a WRI1 polypeptide;
iii. a GPAT9, a LPAAT, and a WRI1 polypeptide;
iv. a GPAT9, a LPAAT, a DGAT, and a WRI1 polypeptide;
v. a GPAT, a LPAAT, a DGAT1, and a WRI1 polypeptide;
vi. a GPAT9, a LPAAT, a DGAT1, and a WRI1 polypeptide;
vii. a GPAT, a LPAAT, a WRI1 polypeptide, and a fatty acid thioesterase,
preferably a FATB polypeptide;
viii. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
ix. a GPAT9, a LPAAT, a WRI1 polypeptide, and a fatty acid thioesterase,
preferably a FATB polypeptide;
x. a GPAT9, a LPAAT, a DGAT, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
xi. a GPAT, a LPAAT, a DGAT1, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide:
xii. a GPAT9, a LPAAT, a DGAT1, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
xiii. a GPAT, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and an OBC polypeptide such as an
oleosin, preferably a caleosin or a LDAP;
xiv. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xv. a GPAT9, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and an OBC polypeptide such as an
oleosin, preferably a caleosin or a LDAP;
xvi. a GPAT9, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xvii. a GPAT, a LPAAT, a DGAT1. a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
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xviii. a GPAT9, a LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xix. a GPAT, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and a silencing RNA which reduces the
expression of an endogenous gene which encodes a SDP1 lipase;
xx. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous Rene which encodes a SDP1
lipase;
xxi. a GPAT9, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and a silencing RNA which reduces the
expression of an endogenous gene which encodes a SDP I lipase;
xxii. a GPAT9, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxiii. a GPAT, a LPAAT, a DGATI, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP I
lipase;
xxiv. a GPAT9, a LPAAT, a DGATI, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxv. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
an oleosin, preferably a caleosin or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxvi. a GPAT9, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide. an OBC polypeptide such as an oleosin,
preferably a caleosin or a LDAP, and a silencing RNA which reduces the
expression of an endogenous gene which encodes a SDP I lipase;
xxvii. a GPAT9, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
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an oleosin, preferably a caleosin or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxviii. a GPAT, a LPAAT, a
DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide. an OBC polypeptide such as
an oleosin, preferably a caleosin, or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase; or a GPAT9, a LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
an oleosin, preferably a caleosin, or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase.
In an embodiment, the one or more or all of the polypeptides are encoded by
one
or more exogenous polynucleotides in the plant parts.
In a second aspect, the present invention provides a process for producing
extracted plant lipid, comprising the steps of:
a) obtaining
one or more plant parts comprising lipid, preferably vegetative
plant parts, the lipid comprising a total fatty acid content which comprises
fatty acids in
an esterified form, the fatty acids comprising an increased level of medium
chain fatty
acids (MCFA) relative to a corresponding wild-type plant part, wherein the
plant part
comprises an increased level or activity of polypeptides which are:
i. a GPAT, a LPAAT, and a WRI1 polypeptide;
a GPAT, a LPAAT, a DGAT and a WRI1 polypeptide;
a GPAT9, a LPAAT, and a WRI1 polypeptide;
iv. a GPAT9, a LPAAT, a DGAT, and a WRI1 polypeptide;
v. a GPAT, a LPAAT, a DGAT1, and a WRI1 polypeptide;
vi. a GPAT9, a LPAAT, a DGAT1, and a WRI1 polypeptide;
vii. a GPAT, a LPAAT, a WRI1 polypeptide, and a fatty acid thioesterase,
preferably a FATB polypeptide;
viii. a GPAT, a LPAAT, a
DGAT, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
ix. a GPAT9, a LPAAT, a WRI1 polypeptide, and a fatty acid thioesterase,
preferably a FATB polypeptide;
x. a GPAT9, a LPAAT. a DGAT, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
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xi. a GPAT, a LPAAT, a DGAT1, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
xii. a GPAT9, a LPAAT, a DGAT1, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
xiii. a GPAT, a LPAAT, a
WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and an OBC polypeptide such as an
oleosin, preferably a caleosin or a LDAP;
xiv. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xv. a GPAT9, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and an OBC polypeptide such as an
oleosin, preferably a caleosin or a LDAP;
xvi. a GPAT9, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xvii. a GPAT, a LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xviii. a GPAT9, a LPAAT, a
DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xix. a GPAT, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and a silencing RNA which reduces the
expression of an endogenous gene which encodes a SDP1 lipase;
xx. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxi. a GPAT9, a LPAAT, a
WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and a silencing RNA which reduces the
expression of an endogenous gene which encodes a SDP1 lipase;
xxii. a GPAT9, a LPAAT, a
DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
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xxiii. a GPAT, a
LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxiv. a GPAT9, a LPAAT,
a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxv. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
an oleosin, preferably a caleosin or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxvi. a GPAT9, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, an OBC polypeptide such as an oleosin,
preferably a caleosin or a LDAP, and a silencing RNA which reduces the
expression of an endogenous gene which encodes a SDP1 lipase;
xxvii. a GPAT9, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
an oleosin, preferably a caleosin or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxviii. a GPAT, a LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
an oleosin, preferably a caleosin, or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase; or
xxix. a GPAT9, a LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
an oleosin, preferably a caleosin, or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase,
and
b) extracting lipid from the plant part(s),
thereby producing the extracted plant lipid.
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13
In an embodiment, the one or more or all of the polypeptides are encoded by
one
or more exogenous polynucleotides in the plant parts.
In an embodiment, the level of total, or new, MCFA is increased relative to a
corresponding wild-type plant part, preferably the level is at least 25% of
the total fatty
acid content on a weight basis.
In an embodiment of the first and second aspects, the one or more or all of
the
encoded GPAT, LF'AAT and DGAT have a preference for utilising medium chain
fatty
acid substrates. GPAT, LPAAT and DGAT each use an acyl-CoA substrate, with a
second substrate that is G3P, LPA or DAG, respectively.
In an embodiment of the first and second aspects, the extracted lipid has one
or
more or all of the following features:
i. the level of medium chain fatty acids in the total fatty acid content of
the
extracted lipid, and/or in the total fatty acid content of the TAG of the
extracted
lipid, is at least about 30%, at least about 35%, at least about 40%, at least
about
50%, at least about 55%, or between about 25% and about 55%, between about
25% and about 50%, between about 30% and about 50%, between about 35%
and about 50%, between about 25% and about 40%, or between about 30% and
about 40%;
ii. the level of lauric acid (C12:0) in the total fatty acid content of the
extracted
lipid, and/or in the total fatty acid content of the TAG of the extracted
lipid, is,
or is increased by, at least about 15%, at least about 20%, at least about
25%, at
least about 30%, at least about 35%, at least about 40%, at least about 50%,
at
least or about 55%, or between about 15% and about 55%, between about 20%
and about 50%, between about 30% and about 50%, between about 35% and
about 50%, between about 15% and about 25%, or between about 20% and
about 30%;
iii. the level of myristic acid (C14:0) in the total fatty acid content of
the extracted
lipid, and/or in the total fatty acid content of the TAG of the extracted
lipid, is,
or is increased by, at least about 25%, at least about 30%, at least about
35%, at
least about 40%, at least about 45%, or between about 25% and about 45%,
between about 20% and about 50%, between about 30% and about 50%,
between about 35% and about 50%, between about 30% and about 40%,
between about 15% and about 25%, or between about 20% and about 30%;
iv. the level of palmitic acid (C16:0) in the total fatty acid content of
the extracted
lipid, and/or in the total fatty acid content of the TAG of the extracted
lipid, is,
or is increased by, between about 2% and about 18%, or between about 2% and
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about 16%, or between about 2% and about 15%, or between about 15% and
about 50%;
v. the level of lauric acid (C12:0) in the total fatty acid content of the
extracted
lipid, and/or in the total fatty acid content of the TAG of the extracted
lipid is,
or is increased by, at least about 25%, at least about 30%, at least about
40%, at
least about 45%, or at least about 50%, and the level of myristic acid (C14:0)
in
the total fatty acid content of the extracted lipid and/or in the total fatty
acid
content of the TAG of the extracted lipid is, or is increased by, at least
about
1%, at least about 2%, at least about 5%, or at least about 10%, or between
about 1% and about 10%, or between about 2% and 10%, or between about 2%
and about 6%, or less than about 10%, or less than about 8% or less than about
5%, or less than about 2%;
vi. the level of myristic acid (C14:0) in the total fatty acid content of
the extracted
lipid, and/or in the total fatty acid content of the TAG of the extracted
lipid is,
or is increased by, at least about 20%, at least about 25%, at least about
30%, or
at least about 40%, and the level of lauric acid (C12:0) in the total fatty
acid
content of the extracted lipid and/or in the total fatty acid content of the
TAG of
the extracted lipid is, or is increased by, at least about 1%, at least about
2%, at
least about 5%, or at least about 10%, or between about 1% and about 10%, or
between about 2% and about 10%, or between about 2% and about 6%, or less
than about 10%, or less than about 8% or less than about 5%, or less than
about
2%;
vii. the level of myristic acid (C14:0) in the total fatty acid content of
the extracted
lipid, and/or in the total fatty acid content of the TAG of the extracted
lipid is,
or is increased by, at least about 20%, at least about 25%, at least about
30%,
and the level of palmitic acid (C16:0) in the total fatty acid content of the
extracted lipid and/or in the total fatty acid content of the TAG of the
extracted
lipid is, or is increased by, at least about 2%, at least about 3%, at least
about
4%, or at least about 5%.
viii. the ratio of lauric acid (C12:0):myristic acid (C14:0) in the fatty
acid content of
the extracted lipid, and/or in the total fatty acid content of the TAG of the
extracted lipid, is increased, or is about 1:4, about 1:5, about 1:10, about
1:15,
about 1:20, about 1:25, or about 4:1. about 5:1, about 10:1, about 15:1, about
20:1, about 30:1, about 40:1, or about 45:1;
ix. the ratio of lauric acid (C12:0):palmitic acid (C16:0) in the fatty
acid content of
the extracted lipid, and/or in the total fatty acid content of the TAG of the
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extracted lipid, is increased, or is about 1:2, about 1:3, about 1:4, about
1:5,
about 1:10, about 1:15, about 10:1, about 20:1, about 30:1, about 40:1, or
about
45:1;
x. the ratio of myristic acid (C14:0):palmitic acid (C16:0) in the fatty
acid content
of the extracted lipid, and/or in the total fatty acid content of the TAG of
the
extracted lipid, is increased, or is about 1:2, about 1:3. about 1:4, about
1:5,
about 1:10, about 1:15, about 10:1, about 20:1, about 30:1, or about 40:1 ;
xi. the level of oleic acid in the total fatty acid content of the
extracted lipid, and/or
in the total fatty acid content of the TAG of the extracted lipid, is
decreased, or
is less than about 10%, less than about 8%, less than about 6%, less than
about
5%, less than about 4%, less than about 3%, less than about 2%, less than
about
1%;
xii. the level of linoleic acid (LA) in the total fatty acid content of the
extracted
lipid, and/or in the total fatty acid content of the TAG of the extracted
lipid, is
increased or decreased, or is less than about 20%, less than about 15%, less
than
about 10%, less than about 5%, less than about 4%, less than about 3%, less
than about 2%, or less than about 1%;
xiii. the level of a-linolenic acid (ALA) in the total fatty acid content
of the
extracted lipid, or in the total fatty acid content of the TAG of the
extracted
lipid, is decreased or is less than about 50%, less than about 40%, less than
about 30%, less than about 20%, less than about 10%, less than about 8%, less
than about 6%, less than about 5%, less than about 2%, or less than about 1%;
xiv. the level of total unsaturated fatty acids in the total fatty acid
content of the
extracted lipid, and/or in the total fatty acid content of the TAG of the
extracted
lipid, is decreased, or is less than about 50%, less than about 40%, less than
about 30%, less than about 20%, less than about 15%, less than about 10%, less
than about 8%, less than about 6%, less than about 5%, less than about 2%, or
less than about 1%;
xv. the level of total monounsaturated fatty acids in the total fatty acid
content of
the extracted lipid, and/or in the total fatty acid content of the TAG of the
extracted lipid, is decreased, or is less than about 10%, less than about 5%,
less
than about 4%, less than about 3%, less than about 2%, or less than about 1%;
xvi. the level of total polyunsaturated fatty acids in the total fatty acid
content of the
extracted lipid, and/or in the total fatty acid content of the TAG of the
extracted
lipid, is less than about 50%, less than about 40%, less than about 30%, less
than about 20%, less than about 15%, less than about 10%, less than about 8%,
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less than about 6%, less than about 5%, less than about 2%, or less than about
1%;
xvii. the triacylglycerol (TAG) content of the extracted lipid is at least
about 80%, at
least about 85%, at least about 90%, or least about 95%, and about 98%, or
between about 95% and about 98%, by weight of the extracted lipid;
xviii. the TAG content of the extracted lipid comprises, or is increased in
a level of,
one or more or all of the TAG species 36:0, 38:0, 40:0 and 42:0;
xix. the extracted lipid comprises tri-laurin (tri-C12:0) and/or tri-
myristin (tri-
C14:0); and
xx. the phosphocholine (PC) content of the extracted lipid comprises one or
both of
the PC species 28:0 and 30:0,
wherein any 'increase or decrease is relative to a corresponding wild-type
plant part.
In an embodiment, the plant part comprises one or more of the features defined
with respect to the first aspect.
In an embodiment of the first and second aspects, the plant part has one or
more
or all of the following features:
a) an increased soluble protein content relative to a corresponding wild-type
plant part,
b) an increased nitrogen content in plant part relative to a corresponding
wild-
type plant part,
a decreased carbon:nitrogen ratio relative to a corresponding wild-type plant
part,
g) increased photosynthetic gene expression relative to a corresponding wild-
type plant part,
h) increased photosynthetic capacity relative to a corresponding wild-type
plant
part,
i) decreased total dietary fibre (TDF) content relative to a corresponding
wild-
type plant part,
j) increased carbon content relative to a corresponding wild-type plant part,
and
k) increased energy content relative to a corresponding wild-type plant part,
wherein each exogenous polynucleotide is operably linked to a promoter which
is
capable of directing expression of the polynucleotide in the cell.
In an embodiment, the plant part, preferably a Sorghum sp. or Zea mays plant
part, further comprises:
1) an increased TTQ relative to a corresponding wild-type plant part,
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wherein each exogenous polynucleotide is operably linked to a promoter which
is
capable of directing expression of the polynucleotide in the plant, or part
thereof.
In an embodiment, the plant part is derived from an ancestor plant, for
example,
as described herein.
In an embodiment, the plant part has one or more or all of:
i) the plant part has an increased soluble protein relative to the
corresponding
wild-type plant part of at least about 10%, at least about 25%, at least about
50%, at
least about 75%, at least about 100%, between about 10% and about 200%,
between
about 50% and about 150%, or between about 50% and about 125%,
ii) the plant part has an increased nitrogen content relative to the
corresponding
wild-type plant part of at least about 10%, at least about 25%, at least about
50%, at
least about 75%, at least about 100%, between about 10% and about 200%,
between
about 50% and about 150% or between about 50% and about 125%,
iii) the plant part is a leaf which has an increased soluble protein content
relative
to a corresponding wild-type leaf of at least about 10%, at least about 25%,
at least
about 50%, at least about 75%, at least about 100%, between about 10% and
about
200%, between about 50% and about 150%, or between about 50% and about 125%,
iv) the plant part is a leaf which has an increased nitrogen content relative
to a
corresponding wild-type leaf of at least about 10%, at least about 25%, at
least about
50%, at least about 75%, at least about 100%, between about 10% and about
200%,
between about 50% and about 150%, or between about 50% and about 125%,
v) the plant part has a decreased carbon:nitrogen content relative to the
corresponding wild-type plant or part thereof of at least about 10%, at least
about 25%,
at least about 40%, between about 10% and about 50%, or between about 25% and
about 50%,
vi) expression of one or more genes involved in photosynthesis is increased in
the plant part relative to the corresponding wild-type plant part,
vii) the plant part has an increased carbon content relative to the
corresponding
wild-type plant part of at least about 10%, at least about 25%, at least about
50%, at
least about 75%, at least about 100%, at least about 125%, at least about
150%,
between about 10% and about 300%, between about 50% and about 250%, or between
about 100% and about 200%,
viii) the plant part has an increased energy content in the plant part
relative to
the corresponding wild-type plant part of at least about 10%, at least about
25%, at least
about 50%, at least about 75%, at least about 100%, at least about 125%, at
least about
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150%, at least about 200%, at least about 250%, between about 10% and about
400%,
between about 50% and about 300%, or between about 200% and about 300%,
ix) the plant part has a decreased starch content relative to the
corresponding
wild-type plant part of at least about 2 fold, at least about 5 fold, at least
about 10 fold,
at least about 15 fold, at least about 20 fold, at least about 25 fold,
between about 5 fold
and about 35 fold, between about 10 fold and about 30 fold, or between about
20 fold
and about 30 fold,
x) the plant part has a decreased TDF content relative to the corresponding
wild-
type plant part of at least about 10%, at least about 30%, at least about 50%,
between
about 10% and about 70%, or between about 30% and about 65%, and
xi) the plant part has a soluble sugar content relative to the corresponding
wild-
type plant part which is about 0.5 fold to 2 fold.
In an embodiment of the first and second aspects, plant part has one or more
or
all of;
i) the plant part comprises a total non-polar lipid content of at least about
8%, at
least about 10%, at least about 11%, at least about 12%, at least about 15%,
at least
about 20%, at least about 25%, at least about 30%, at least about 35%, at
least about
40%, at least about 45%, at least about 50%, at least about 55%, at least
about 60%, at
least about 65%, at least about 70%, between 8% and 75%, between 10% and 75%,
between 11% and 75%, between about 15% and 75%, between about 20% and 75%,
between about 30% and 75%, between about 40% and 75%, between about 50% and
75%, between about 60% and 75%, or between about 25% and 50% (w/w dry weight),
preferably before flowering,
ii) the plant part is a vegetative part that comprises a TAG content of at
least
about 8%, at least about 10%, at least about 11%, at least about 12%, at least
about
15%, at least about 20%, at least about 25%, at least about 30%, at least
about 35%, at
least about 40%, at least about 45%, at least about 50%, at least about 55%,
at least
about 60%, at least about 65%, at least about 70%, between 8% and 75%, between
10%
and 75%, between 11% and 75%, between about 15% and 75%, between about 20%
and 75%, between about 30% and 75%, between about 40% and 75%, between about
50% and 75%, between about 60% and 75%, or between about 25% and 50% (w/w dry
weight), preferably before flowering,
iii) one or more or all of the promoters are selected from a tissue-specific
promoter such as a leaf and/or stem specific promoter, a developmentally
regulated
promoter such as a senescence-specific promoter such as a SAG12 promoter, an
inducible promoter, or a circadian-rhythm regulated promoter,
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iv) the plant part is one member of a population or collection of at least
about
1,500, at least about 3,000 or at least about 5,000 such plant parts,
preferably vegetative
plant parts.
In a further embodiment, the plant part is:
i) a 16:3 plant part, and which comprises one or more or all of the following:
a) an exogenous polynucleotide which encodes a polypeptide which
increases the export of fatty acids out of plastids of the plant when compared
to a
corresponding plant lacking the exogenous polynucleotide,
b) a first genetic modification which down-regulates endogenous
production and/or activity of a polypeptide involved in importing fatty acids
into
plastids of the plant when compared to a corresponding plant lacking the first
genetic
modification, and
c) a second genetic modification which down-regulates endogenous
production and/or activity of a polypeptide involved in diacylglycerol (DAG)
production in the plastid when compared to a corresponding plant lacking the
second
genetic modification,
wherein the exogenous polynucleotide is operably linked to a promoter which is
capable of directing expression of the polynucleotide in the plant part, or
ii) a 18:3 plant part.
In an embodiment, the plant part has one or more or all of:
i) the plant part, preferably a vegetative plant part which has an increased
synthesis of total fatty acids relative to a corresponding plant part lacking
the
exogenous polynucleotide(s) and/or genetic modification(s),
ii) the plant part, preferably a vegetative plant part which has an increased
expression and/or activity of a fatty acyl acyltransferase which catalyses the
synthesis
of TAG, DAG or MAG, preferably TAG, relative to a corresponding plant part
lacking
the exogenous polynucleotide(s) and/or genetic modification(s),
iii) the plant part, preferably a vegetative plant part which has a decreased
production of lysophosphatidic acid (LPA) from acyl-ACP and G3P in its
plastids
relative to a corresponding plant part lacking the exogenous polynucleotide(s)
and/or
genetic modification(s),
iv) the plant part, preferably a vegetative plant part which has an altered
ratio of
C16:3 to C18:3 fatty acids in its total fatty acid content and/or its
galactolipid content
relative to a corresponding part lacking the exogenous polynucleotide(s)
and/or genetic
modification(s), preferably a decreased ratio,
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v) one or more or all of the promoters are selected from promoter other than a
constitutive promoter, preferably a tissue-specific promoter such as a leaf
and/or stem
specific promoter, a developmentally regulated promoter such as a senescense-
specific
promoter such as a SAG12 promoter, an inducible promoter, or a circadian-
rhythm
regulated promoter, preferably wherein at least one of the promoters operably
linked to
an exogenous polynucleotide which encodes a transcription factor polypeptide
is a
promoter other than a constitutive promoter,
vi) the plant part, preferably a vegetative plant part, comprises a total
fatty acid
content whose oleic acid level and/or palmitic acid level is increased by at
least 2%
relative to a corresponding plant, or part thereof, lacking the exogenous
polynucleotide(s) and/or genetic modification(s), and/or whose a-linolenic
acid (ALA)
level and /or linoleic acid level is decreased by at least 2% relative to a
corresponding
plant part lacking the exogenous polynucleotide(s) and/or genetic
modification(s),
vii) non-polar lipid in the plant part, preferably a vegetative plant part,
comprises a modified level of total sterols, preferably free (non-esterified)
sterols,
steroyl esters, steroyl glycosides, relative to the non-polar lipid in a
corresponding
plant, or part thereof, lacking the exogenous polynucleotide(s) and/or genetic
modification(s),
viii) non-polar lipid in the plant part comprises waxes and/or wax esters,
ix) the plant part comprises an exogenous polynucleotide encoding a silencing
suppressor, wherein the exogenous polynucleotide is operably linked to a
promoter
which is capable of directing expression of the polynucleotide in the plant,
x) the level of one or more non-polar lipid(s) and/or the total non-polar
lipid
content of the plant or part thereof, preferably a vegetative plant part, is
at least 2%
greater on a weight basis than in a corresponding plant or part, respectively,
which
comprises exogenous polynucleotides encoding an Arabidposis thaliana WRI1 and
an
Arahidopsis thaliana DGAT1 (SEQ ID NO:1),
xi) a total polyunsaturated fatty acid (PUFA) content which is decreased
relative
to the total PUFA content of a corresponding plant lacking the exogenous
polynucleotide(s) and/or genetic modification(s),
xii) if the plant part is a seed, the seed germinates at a rate substantially
the same
as for a corresponding wild-type seed or when sown in soil produces a plant
whose
seed germinate at a rate substantially the same as for corresponding wild-type
seed, and
xiii) the plant is an algal plant such as from diatoms (bacillariophytes),
green
algae (chlorophytes), blue-green algae (cyanophytes), golden-brown algae
(chrysophytes), haptophytes, brown algae or heterokont algae.
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In an embodiment, the plant part comprises a first exogenous polynucleotide
encoding a WRI1, a second exogenous polynucleotide encoding a DGAT or a PDAT,
preferably a DGAT1, a third exogenous polynucleotide encoding an RNA which
reduces expression of a gene encoding an SDP1 polypeptide, and a fourth
exogenous
polynucleotide encoding an oleosin. In preferred embodiments, the plant part
has one
or more or all of the following features:
i) a total lipid content of at least 8%, at least 10%, at least 12%, at least
14%, or
at least 15.5% (% weight),
ii) at least a 3 fold, at least a 5 fold, at least a 7 fold, at least an 8
fold, or least a
10 fold, at higher total lipid content in the plant part relative to a
corresponding the
plant part lacking the exogenous polynucleotides and/or genetic modifications,
iii) a total TAG content of at least 5%, at least 6%, at least 6.5% or at
least 7%
(% weight of dry weight or seed weight),
iv) at least a 40 fold, at least a 50 fold, at least a 60 fold, or at least 70
fold, at
least 100 fold, or at least a 120-fold higher total TAG content relative to a
corresponding the plant part lacking the exogenous polynucleotides and/or
genetic
modifications,
v) palmitic acid comprises at least 20%, at least 25%, at least 30% or at
least
33% (% weight) of the fatty acids in TAG,
vi) at least a 1.5 fold higher level of palmitic acid in TAG relative to a
corresponding the plant part lacking the exogenous polynucleotides and/or
genetic
modifications,
vii) linoleic acid comprises at least 22%, at least 25%, at least 30% or at
least
34% (% weight) of the fatty acids in TAG, and
viii) a-linolenic acid comprises less than 20%, less than 15%, less than 11%
or
less than 8% (% weight) of the fatty acids in TAG.
ix) at least a 5 fold, or at least an 8 fold, lower level of a-linolenic acid
in TAG
relative to a corresponding the plant or part thereof lacking the exogenous
polynucleotides and/or genetic modifications, In the above
embodiments, a preferred plant part is a leaf piece having a surface area of
at least 1cm2
or a stem piece having a length of at least lcm.
In an embodiment of the above aspects, the plant part has been treated so it
is no
longer able to be propagated or give rise to a living plant, i.e. it is dead
(for example a
brown leaf or stem). For example, the plant part has been dried and/or ground.
In
another embodiment, the plant part is alive (for example, a green leaf or
stem).
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In an embodiment, the part is a seed, fruit, or a vegetative part such as an
aerial
plant part or a green part such as a leaf or stem.
In the above embodiments, it is preferred that the plant part is a vegetative
plant
part which is growing in soil or which was grown in soil and the plant part
was
subsequently harvested, and wherein the vegetative part comprises at least 8%
TAG on
a weight basis (% dry weight) such as for example between 8% and 75% or
between
8% and 30%. More preferably, the TAG content is at least 10%, such as for
example
between 10% and 75% or between 10% and 30%. Preferably, these TAG levels are
present in the vegetative parts prior to or at flowering of the plant or prior
to seed
setting stage of plant development. In these embodiments, it is preferred that
the ratio
of the TAG content in the leaves to the TAG content in the stems of the plant
is
between 1:1 and 10:1, and/or the ratio is increased relative to a
corresponding
vegetative part comprising the first and second exogenous polynucleotides and
lacking
the first genetic modification. Preferably, the vegetative plant part has an
increased
soluble protein content relative to the corresponding wild-type vegetative
plant part of
at least about 100%, or between about 50% and about 125%. Preferably, the
vegetative
plant part has an increased nitorgen content relative to the corresponding
wild-type
vegetative part of at least about 100%, or between about 50% and about 125%.
Preferably, the vegetative plant part has an decreased carbon:nitrogen content
relative
to the corresponding wild-type vegetative part of at least about 40%, or
between about
25% and about 50%. Preferably, the vegetative plant part has a decreased TDF
content
in the part or at least a part of the transgenic plant relative to the
corresponding wild-
type vegetative plant part of at least about 30%, or between about 30% and
about 65%.
In an embodiment, the plant part, preferably a leaf, a grain, a stem, a root
or an
endosperm is from a monocotyledonous plant, which has a total fatty acid
content or
TAG content which is increased at least 5-fold on a weight basis when compared
to a
corresponding non-transgenic monocotyledonous plant. Alternatively, the
transgenic
monocotyledonous plant has endosperm comprising a TAG content which is at
least
2.0%, preferably at least 3%, more preferably at least 4% or at least 5%, on a
weight
basis. In an embodiment, the endosperm has a TAG content of at least 2% which
is
increased at least 5-fold relative to a corresponding non-transgenic
endosperm.
Preferably, the plant is fully male and female fertile, its pollen is
essentially 100%
viable, and its grain has a germination rate which is between 70% and 100%
relative to
corresponding wild-type grain. In an embodiment, the transgenic plant is a
progeny
plant at least two generations derived from an initial transgenic wheat plant,
and is
preferably homozygous for the transgenes. In embodiments, the monocotyledonous
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plant, or part thereof, preferably a leaf, stem, grain or endosperm, is
further
characterised by one or more features as defined in the context of a plant or
part thereof
of the invention. In embodiments, the monocotyledonous plant, or part thereof,
preferably a leaf, a grain, stem or an endosperm of the invention preferably
has an
increased level of monounsaturated fatty acids (MUFA) and/or a lower level of
polyunsaturated fatty acids (PUFA) in both the total fatty acid content and in
the TAG
fraction of the total fatty acid content, such as for example an increased
level of oleic
acid and a reduced level of LA (18:2), when compared to a corresponding plant
or part
thereof lacking the genetic modifications and/or exogenous polynucleotide(s).
Preferably, the linoleic acid (LA. 18:2) level in the total fatty acid content
of the grain
or endosperm of the monocotyledonous plant is reduced by at least 5% and/or
the level
of oleic acid in the total fatty acid content is increased by at least 5%
relative to a
corresponding wild-type plant or part thereof, preferably at least 10% or more
preferably at least 15%, when compared to a corresponding plant or part
thereof
lacking the genetic modifications and/or exogenous polynucleotide(s).
In an embodiment of the first and second aspects, the extracted lipid is in
the
form of an oil, wherein at least about 90%, or least about 95%, at least about
98%, or
between about 95% and about 98%, by weight of the oil is the lipid.
In an embodiment of the first and second aspects, the plant part is a
vegetative
plant part such as a plant leaf or stem, or the plant part is a seed or a
fruit.
In an embodiment of the first and second aspects the plant part is from a
species
selected from a group consisting of a Acrocomia aculeata (macauba palm),
Arabidopsis
thaliana, Aracinis hypogaea (peanut), Astrocaryum murumuru (murumuru),
Astrocaryum vulgare (tucuma), Attalea geraensis (Indaia-rateiro), Attalea
humilis
(American oil palm), Attalea oleifera (andaid), Attalea phalerata (uricuri),
Attalea
speciosa (babassu), Avena sativa (oats), Beta vulgaris (sugar beet), Brassica
sp. such as
Brassica carinata, Brassica juncea, Brassica napobrassica, Brassica napus
(canola),
Camelina sativa (false flax), Cannabis sativa (hemp), Carthamus tinctorius
(safflower), Caiyocar brasiliense (pequi), Cocos nucifera (Coconut), Crambe
abyssinica (Abyssinian kale), Cucumis melo (melon), Elaeis guineensis (African
palm),
Glycine max (soybean), Gossypium hirsutum (cotton), Helianthus sp. such as
Helianthus annuus (sunflower), Hordeum vulgare (barley), Jatropha curcas
(physic
nut), Joannesia princeps (arara nut-tree), Lemna sp. (duckweed) such as Lonna
aequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba (swollen
duckweed), Lemna japonica, Lemna minor, Lemna minuta, Lemna obscura, Lemna
paucicostata, Lemna perpusilla, Lemna tenera, Lemna trisulca, Lemna
turionifera,
CA 2998211 2018-03-16
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Lemna valdiviana, Lemna yungensis, Licania rigida (oiticica), Linum
usitatissimum
(flax), Lupinus angustifolius (lupin), Mauritia flexuosa (buriti palm),
Maximiliana
maripa (inaja palm), Miscanthus sp. such as Miscanthus x giganteus and
Miscanthus
sinensis, Nicotiana sp. (tabacco) such as Nicotiana tabacum or Nicotiana
benthamiana,
Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus bataua (pataud), Oenocarpus
distichus (bacaba-de-leque), Oryza sp. (rice) such as Oryza saliva and Oryza
glaberrima, Panicum virgatum (switchgrass), Paraqueiba paraensis (man), Persea
amencana (avocado), Pongamia pinnata (Indian beech), Populus trichocarpa,
Ricinus
communis (castor), Saccharum sp. (sugarcane), Sesamum indicum (sesame),
Solanum
tuberosum (potato), Sorghum sp. such as Sorghum bicolor, Sorghum vulgare,
Theobroma grandifor um (cupuassu), Trifblium sp., Trithrinax brasiliensis
(Brazilian
needle palm), Triticum sp. (wheat) such as Triticum aestivum and Zea mays
(corn). For
example, the plant part is from a monocotyledonous plant, preferably a plant
from the
family Poaceae, more preferably a Sorghum sp., a Zea mays, Miscanthus sp. such
as
Miscanthus x giganteus and Miscanthus sinensis, and/or a Panicum virgatum
(switchgrass) plant.
In an embodiment of the first and second aspects, the one or more or all of
the
promoters are expressed at a higher level in a vegetative plant part relative
to seed of a
plant.
In another aspect, the present invention provides extracted plant lipid
produced
by the process of both the first and second aspects, preferably comprising
plant leaf
lipid.
In another aspect, the present invention provides extracted plant lipid,
comprising fatty acids in an esterified form, wherein the level of medium
chain fatty
acids in the total fatty acid content of the lipid in the vegetative plant
part is at least
about 25%. In an embodiment, the lipid has one or more of the features defined
above
in relation to the first or second aspects.
In another aspect, the present invention provides a cell comprising an
increased
level or activity of polypeptides which are:
i. a GPAT, a LPAAT, and a WR11 polypeptide;
a GPAT, a LPAAT, a DGAT and a WRI1 polypeptide;
a GPAT9, a LPAAT, and a WRI1 polypeptide;
iv. a GPAT9, a LPAAT, a DGAT, and a WRI1 polypeptide;
v. a GPAT, a LPAAT, a DGAT1, and a WRI1 polypeptide;
vi. a GPAT9, a LPAAT, a DGAT1, and a WRI1 polypeptide;
CA 2998211 2018-03-16
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vii. a GPAT, a LPAAT, a WRI1 polypeptide, and a fatty acid thioesterase,
preferably a FATB polypeptide;
viii. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
ix. a GPAT9, a LPAAT, a WRI1 polypeptide, and a fatty acid thioesterase,
preferably a FATB polypeptide;
x. a GPAT9, a LPAAT, a DGAT. a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
xi. a GPAT, a LPAAT, a DGAT1, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
xii. a GPAT9, a LPAAT, a DGAT1, a WRI1 polypeptide, and a fatty acid
thioesterase, preferably a FATB polypeptide;
xiii. a GPAT. a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and an OBC polypeptide such as an
oleosin, preferably a caleosin or a LDAP;
xiv. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xv. a GPAT9, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and an OBC polypeptide such as an
oleosin, preferably a caleosin or a LDAP;
xvi. a GPAT9, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xvii. a GPAT, a LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xviii. a GPAT9, a LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and an OBC polypeptide such
as an oleosin, preferably a caleosin or a LDAP;
xix. a GPAT, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and a silencing RNA which reduces the
expression of an endogenous gene which encodes a SDP1 lipase;
xx. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
CA 2998211 2018-03-16
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reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxi. a GPAT9, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, and a silencing RNA which reduces the
expression of an endogenous gene which encodes a SDP I lipase;
xxii. a GPAT9, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxiii. a GPAT, a LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxiv. a GPAT9, a LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxv. a GPAT, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
an oleosin, preferably a caleosin or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP l
lipase;
xxvi. a GPAT9, a LPAAT, a WRI1 polypeptide, a fatty acid thioesterase,
preferably a FATB polypeptide, an OBC polypeptide such as an oleosin,
preferably a caleosin or a LDAP, and a silencing RNA which reduces the
expression of an endogenous gene which encodes a SDP1 lipase;
xxvii. a GPAT9, a LPAAT, a DGAT, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
an oleosin, preferably a caleosin or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase;
xxviii. a GPAT, a LPAAT, a DGAT1, a WR11 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
an oleosin, preferably a caleosin, or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase; or
CA 2998211 2018-03-16
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xxix. a GPAT9,
a LPAAT, a DGAT1, a WRI1 polypeptide, a fatty acid
thioesterase, preferably a FATB polypeptide, an OBC polypeptide such as
an oleosin, preferably a caleosin, or a LDAP, and a silencing RNA which
reduces the expression of an endogenous gene which encodes a SDP1
lipase.
In an embodiment, the one or more or all of the polypeptides are encoded by
one
or more exogenous polynucleotides in the plant parts.
In an embodiment, the level of total, or new, MCFA is increased relative to a
corresponding wild-type plant part, preferably at least 25% of the total fatty
acid
content on a weight basis.
In an embodiment, the one or more or all of the encoded GPAT, LPAAT and/or
DGAT have a preference for utilising medium chain fatty acid substrates.
In an embodiment, the cell further comprises one or more or all of:
i. a genetic modification which down-regulates endogenous production and/or
activity of a polypeptide involved in the catabolism of triacylglycerols (TAG)
in
the cell when compared to a corresponding cell lacking the genetic
modification;
ii. a genetic modification which down-regulates endogenous production and/or
activity of a polypeptide involved in importing fatty acids into plastids of
the
cell when compared to a corresponding cell lacking the genetic modification;
and
iii. a genetic modification which down-regulates endogenous production and/or
activity of a polypeptide involved in diacylglycerol (DAG) production in the
plastid when compared to a corresponding cell lacking the genetic
modification.
In an embodiment, the genetic modification is a mutation of an endogenous gene
which partially or completely inactivates the gene, such as a point mutation,
an
insertion, or a deletion, or the genetic modification is an exogenous
polynucleotide
encoding an RNA molecule which inhibits expression of the endogenous gene,
wherein
the exogenous polynucleotide is operably linked to a promoter which is capable
of
directing expression of the polynucleotide in the cell.
In an embodiment, the one or more or all of the promoters are expressed at a
higher level in a vegetative plant part relative to seed of a plant.
In an embodiment, the cell has one or more or all of the following features:
i. the level
of medium chain fatty acids in the total fatty acid content of the cell,
and/or in the total fatty acid content of the TAG of the cell, is at least
about
30%, at least about 35%, at least about 40%, at least about 50%, at least
about
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55%, or between about 25% and about 55%, between about 25% and about
50%, between about 30% and about 50%, between about 35% and about 50%,
between about 25% and about 40%, or between about 30% and about 40%;
ii. the level of lauric acid (C12:0) in the total fatty acid content of the
cell, and/or
in the total fatty acid content of the TAG of the cell, is, or is increased
by, at
least about 15%, at least about 20%, at least about 25%, at least about 30%,
at
least about 35%, at least about 40%, at least about 50%, at least or about
55%,
or between about 15% and about 55%, between about 20% and about 50%,
between about 30% and about 50%, between about 35% and about 50%,
between about 15% and about 25%, or between about 20% and about 30%;
iii. the level of myristic acid (C14:0) in the total fatty acid content of
the cell,
and/or in the total fatty acid content of the TAG of the cell, is, or is
increased
by, at least about 25%, at least about 30%, at least about 35%, at least about
40%, at least about 45%, or between about 25% and about 45%, between about
20% and about 50%, between about 30% and about 50%, between about 35%
and about 50%, between about 30% and about 40%, between about 15% and
about 25%, or between about 20% and about 30%;
iv. the level of palmitic acid (C16:0) in the total fatty acid content of
the cell,
and/or in the total fatty acid content of the TAG of the cell, is, or is
increased
by, between about 2% and about 18%, or between about 2% and about 16%, or
between about 2% and about 15%, or between about 15% and about 50%;
v. the level of lauric acid (C12:0) in the total fatty acid content of the
cell, and/or
in the total fatty acid content of the TAG of the cell is, or is increased by,
at
least about 25%, at least about 30%, at least about 40%, at least about 45%,
or
at least about 50%, and the level of myristic acid (C14:0) in the total fatty
acid
content of the cell and/or in the total fatty acid content of the TAG of the
cell is,
or is increased by, at least about 1%, at least about 2%, at least about 5%,
or at
least about 10%, or between about 1% and about 10%, or between about 2%
and 10%, or between about 2% and about 6%, or less than about 10%, or less
than about 8% or less than about 5%, or less than about 2%;
vi. the level of myristic acid (C14:0) in the total fatty acid content of
the cell,
and/or in the total fatty acid content of the TAG of the cell is, or is
increased by,
at least about 20%, at least about 25%, at least about 30%, or at least about
40%, and the level of lauric acid (C12:0) in the total fatty acid content of
the
cell and/or in the total fatty acid content of the TAG of the cell is, or is
increased by, at least about 1%, at least about 2%, at least about 5%, or at
least
CA 2998211 2018-03-16
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about 10%, or between about 1% and about 10%, or between about 2% and
about 10%, or between about 2% and about 6%, or less than about 10%, or less
than about 8% or less than about 5%, or less than about 2%;
vii. the level of myristic acid (C14:0) in the total fatty acid content of
the cell,
and/or in the total fatty acid content of the TAG of the cell is, or is
increased by,
at least about 20%, at least about 25%, at least about 30%, and the level of
palmitic acid (C16:0) in the total fatty acid content of the cell and/or in
the total
fatty acid content of the TAG of the cell is, or is increased by, at least
about 2%,
at least about 3%, at least about 4%, or at least about 5%.
viii. the ratio of lauric acid (C12:0):myristic acid (C14:0) in the fatty
acid content of
the cell, and/or in the total fatty acid content of the TAG of the cell, is
increased, or is about 1:4, about 1:5, about 1:10, about 1:15, about 1:20,
about
1:25, or about 4:1, about 5:1, about 10:1, about 15:1, about 20:1, about 30:1,
about 40:1, or about 45:1;
ix. the ratio of lauric acid (C12:0):palmitic acid (C16:0) in the fatty
acid content of
the cell, and/or in the total fatty acid content of the TAG of the cell, is
increased, or is about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about
1:15, about 10:1. about 20:1, about 30:1, about 40:1, or about 45:1;
x. the ratio of myristic acid (C14:0):palmitic acid (C16:0) in the fatty
acid content
of the cell, and/or in the total fatty acid content of the TAG of the cell, is
increased, or is about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about
1:15, about 10:1, about 20:1, about 30:1, or about 40:1;
xi. the level of oleic acid in the total fatty acid content of the cell,
and/or in the total
fatty acid content of the TAG of the cell, is decreased, or is less than about
10%,
less than about 8%, less than about 6%, less than about 5%, less than about
4%,
less than about 3%. less than about 2%, less than about 1%;
xii. the level of linoleic acid (LA) in the total fatty acid content of the
cell, and/or in
the total fatty acid content of the TAG of the cell, is increased or
decreased, or
is less than about 20%, less than about 15%, less than about 10%, less than
about 5%, less than about 4%, less than about 3%, less than about 2%, or less
than about 1%;
xiii. the level of a-linolenic acid (ALA) in the total fatty acid content
of the cell, or
in the total fatty acid content of the TAG of the cell, is decreased or is
less than
about 50%, less than about 40%, less than about 30%, less than about 20%, less
than about 10%, less than about 8%, less than about 6%, less than about 5%,
less than about 2%, or less than about 1%;
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xiv. the level of total unsaturated fatty acids in the total fatty acid
content of the cell,
and/or in the total fatty acid content of the TAG of the cell, is decreased,
or is
less than about 50%, less than about 40%, less than about 30%, less than about
20%, less than about 15%, less than about 10%, less than about 8%, less than
about 6%, less than about 5%, less than about 2%, or less than about 1%;
xv. the level of total monounsaturated fatty acids in the total fatty acid
content of
the cell, and/or in the total fatty acid content of the TAG of the cell, is
decreased, or is less than about 10%, less than about 5%, less than about 4%,
less than about 3%, less than about 2%, or less than about 1%;
xvi. the level of total polyunsaturated fatty acids in the total fatty acid
content of the
cell, and/or in the total fatty acid content of the TAG of the cell, is less
than
about 50%, less than about 40%, less than about 30%, less than about 20%, less
than about 15%, less than about 10%, less than about 8%, less than about 6%,
less than about 5%, less than about 2%, or less than about 1%;
xvii. the triacylglycerol (TAG) content of the cell is at least about 80%,
at least about
85%, at least about 90%, or least about 95%, and about 98%, or between about
95% and about 98%, by weight of the cell;
xviii. the TAG content of the cell comprises, or is increased in a level
of, one or more
or all of the TAG species 36:0, 38:0, 40:0 and 42:0;
xix. the cell comprises tri-laurin (tri-C12:0) and/or tri-myristin (tri-
C14:0);
xx. the phosphocholine (PC) content of the cell comprises one or both of
the PC
species 28:0 and 30:0,
xxi. the cell has a reduced level of medium chain fatty acids, preferably
C14:0, in
membrane lipids relative to a corresponding cell;
xxii. the cell has less chlorosis relative to a corresponding cell which
comprises the
exogenous polynucleotide encoding the thioesterase but lacks the exogenous
polynucleotide encoding the DGAT; and
xxiii. the cell is in a vegetative plant part and the part has an
alleviated chlorosis
phenotype relative to a corresponding vegetative plant part,
wherein any increase or decrease is relative to a corresponding wild-type
cell.
In another aspect, the present invention provides a plant or a part thereof
comprising the cell of the invention, or which is transgenic for one or more
exogenous
polynucleotides defined above.
In an embodiment, before the plant flowers, a vegetative part of the plant
comprises a total non-polar lipid content of at least about 8%, at least about
10%, about
11%, between 8% and 15%, or between 9% and 12% (w/w dry weight).
CA 2998211 2018-03-16
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In an embodiment, the plant is a monocotyledonous plant, or part thereof
preferably a leaf, a grain, a stem, a root or an endosperm, which has a total
fatty acid
content or TAG content which is increased at least 5-fold on a weight basis
when
compared to a correspouding non-transgenic monocotyledonous plant, or part
thereof.
Alternatively, the transgcnic monocotyledonous plant has endosperm comprising
a
TAG content which is at least 2.0%, preferably at least 3%, more preferably at
least 4%
or at least 5%, on a weight basis, or part of the plant, preferably a leaf, a
stem, a root, a
grain or an endosperm. In an embodiment, the endosperm has a TAG content of at
least 2% which is increased at least 5-fold relative to a corresponding non-
transgenic
endosperm. Preferably, the plant is fully male and female fertile, its pollen
is
essentially 100% viable, and its grain has a germination rate which is between
70% and
100% relative to corresponding wild-type grain. In an embodiment, the
transgenic plant
is a progeny plant at least two generations derived from an initial transgenic
wheat
plant, and is preferably homozygous for the transgenes. In embodiments, the
monocotyledonous plant, or part thereof, preferably a leaf, stem, grain or
endosperm, is
further characterised by one or more features as defined in the context of a
plant or part
thereof of the invention. In embodiments, the monocotyledonous plant, or part
thereof
preferably a leaf, a grain, stem or an endosperm of the invention preferably
has an
increased level of monounsaturated fatty acids (MUFA) and/or a lower level of
polyunsaturated fatty acids (PUFA) in both the total fatty acid content and in
the TAG
fraction of the total fatty acid content, such as for example an increased
level of oleic
acid and a reduced level of LA (18:2), when compared to a corresponding plant
or part
thereof lacking the genetic modifications and/or exogenous polynucleotide(s).
Preferably, the linoleic acid (LA, 18:2) level in the total fatty acid content
of the grain
or endosperm of the the monocotyledonous plant is reduced by at least 5%
and/or the
level of oleic acid in the total fatty acid content is increased by at least
5% relative to a
corresponding wild-type plant or part thereof, preferably at least 10% or more
preferably at least 15%, when compared to a corresponding plant or part
thereof
lacking the genetic modifications and/or exogenous polynucleotide(s).
In an embodiment, the plant, or part thereof, is a member of a population or
collection of at least about 1,500, at least about 3,000 or at least about
5,000 such plants
or parts.
In an embodiment, the TFA content, the the TAG content, the total non-polar
lipid content, or the one or more non-polar lipids, and/or the level of the
oleic acid or a
PUFA in the plant or part thereof is determinable by analysis by using gas
CA 2998211 2018-03-16
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chromatography of fatty acid methyl esters obtained from the plant or
vegetative part
thereof.
In a further embodiment, wherein the plant part is a leaf and the total non-
polar
lipid content of the leaf is determinable by analysis using Nuclear Magnetic
Resonance
(NMR).
In each of the above embodiments, it is preferred that the plant is a
transgenic
progeny plant at least two generations derived from an initial transgenic
plant, and is
preferably homozygous for the transgenes.
In an embodiment, the plant or the part thereof is phenotypically normal, in
that
it is not significantly reduced in its ability to grow and reproduce when
compared to an
unmodified plant or part thereof. In an embodiment, the biomass, growth rate,
germination rate, storage organ size, seed size and/or the number of viable
seeds
produced is not less than 70%, not less than 80% or not less than 90% of that
of a
corresponding wild-type plant when grown under identical conditions. In an
embodiment, the plant is male and female fertile to the same extent as a
corresponding
wild-type plant and its pollen (if produced) is as viable as the pollen of the
corresponding wild-type plant, preferably at least about 75%, or at least
about 90%, or
close to 100% viable. In an embodiment, the plant produces seed which has a
germination rate of at least about 75% or at least about 90% relative to the
germination
rate of corresponding seed of a wild-type plant, where the plant species
produces seed.
In an embodiment, the plant of the invention has a plant height which is at
least about
75%, or at least about 90% relative to the height of the corresponding wild-
type plant
grown under the same conditions. A combination of each of these features is
envisaged. In an alternative embodiment, the plant of the invention has a
plant height
which is between 60% and 90% relative to the height of the corresponding wild-
type
plant grown under the same conditions. In an embodiment, the plant or part
thereof of
the invention, preferably a plant leaf, does not exhibit increased necrosis,
i.e. the extent
of necrosis, if present, is the same as that exhibited by a corresponding wild-
type plant
or part thereof grown under the same conditions and at the same stage of plant
development. This feature applies in particular to the plant or part thereof
comprising
an exogenous polynucleotide which encodes a fatty acid thioesterase such as a
FATB
thioesterase.
In another aspect, the present invention provides a population of at least
about
1,500, at least about 3.000 or at least about 5,000 plants, each being a plant
of the
invention, growing in a field.
CA 2998211 2018-03-16
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In an embodiment, the exogenous polynucleotides are inserted at the same
chromosomal location in the genome of each of the plants, preferably in the
nuclear
genome of each of the plants.
In another aspect, the present invention provides a population of at least
about
1000 plants , each being a plant according to the invention, growing in a
field, or a
collection of at least about 1000 plant parts, each being a plant part
according to the
invention, wherein the plant parts have been harvested from plants growing in
a field.
Also provided is a storage bin comprising a collection of plants or plant
parts of
the invention.
In another aspect, the present invention provides an extract of a plant or a
part
thereof of the invention. The extract preferably has a different fatty acid
composition
relative to a corresponding wild-type extract.
In an embodiment, the extract is lacking at least 50% or at least 90% of the
chlorophyll and/or soluble sugars of the plant or part thereof.
In a further aspect, the present invention provides a process for selecting a
plant
or a part thereof with a desired phenotype, the process comprising
i) obtaining a plurality of candidate plants, or parts thereof, which each
comprise
a) a first exogenous polynucleotide which encodes a transcription factor
polypeptide that increases the expression of one or more glycolytic and/or
fatty acid
biosynthetic genes in a plant or part thereof, and
b) a second exogenous polynucleotide which encodes a polypeptide
involved in the biosynthesis of one or more non-polar lipids,
wherein each exogenous polynucleotide is operably linked to a promoter which
is
capable of directing expression of the polynucleotide in the plant, or part
thereof,
ii) analysing lipid in the plurality of parts, or at least a part of each
plant in the
plurality of candidate plants, from step i),
iii) analysing the plurality of parts, or at least a part of each plant in the
plurality
of candidate plants, from step i) for one or more or all of;
a) soluble protein content,
b) nitrogen content,
c) carbon:nitrogen ratio,
d) photosynthetic gene expression,
e) photosynthetic capacity,
0 total dietary fibre (TDF) content,
g) carbon content, and
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h) energy content, and
iv) selecting a plant or part thereof which comprises an increased
triacylglycerol
(TAG) content in the part or at least a part of the plant relative to a
corresponding wild-
type plant or part thereof and a desired phenotype selected from one or more
or all of
the following:
A) an increased soluble protein content in the part or at least a part of the
plant relative to a corresponding wild-type plant or part thereof,
B) an increased nitrogen content in the part or at least a part of the plant
relative to a corresponding wild-type plant or part thereof,
C) decreased carbon:nitrogen ratio in the part or at least a part of the plant
relative to a corresponding wild-type plant or part thereof,
D) increased photosynthetic gene expression in the part or at least a part of
the plant relative to a corresponding wild-type plant or part thereof,
E) increased photosynthetic capacity in the part or at least a part of the
plant relative to a corresponding wild-type plant or part thereof,
F) decreased total dietary fibre (TDF) content in the part or at least a part
of the plant relative to a corresponding wild-type plant or part thereof,
G) increased carbon content in the part or at least a part of the plant
relative to a corresponding wild-type plant or part thereof, and
H) increased energy content in the part or at least a part of the plant
relative to a corresponding wild-type plant or part thereof.
In an embodiment, the process further comprises a step of obtaining seed or a
progeny plant from the transgenic plant, wherein the seed or progeny plant
comprises
the exogenous polynucleotides.
In an embodiment, the increased triacylglycerol (TAG) content is determined by
analysing one or more of the total fatty acid content, TAG content, fatty acid
composition, by any means, which might or might not involve first extracting
the lipid.
In yet another embodiment, the selected plant or part thereof has one or more
of
the features as defined herein.
In another aspect, the present invention provides seed of, or obtained from, a
plant according to the invention.
In another aspect, the present invention provides a process for obtaining a
cell
according to the invention, the process comprising the steps of:
i) introducing into a cell at least one exogenous polynucleotide and/or at
least
one genetic modification as defined above to produce a cell as defined above,
CA 2998211 2018-03-16
35
ii) expressing the exogenous polynucleotide(s) in the cell or a progeny cell
thereof,
iii) analysing the lipid content of the cell or progeny cell, and
iv) selecting a cell according to the invention.
In another aspect, the present invention provides a method of producing a
plant
which has integrated into its genome a set of exogenous polynucleotides and/or
genetic
modifications as defined above, the method comprising the steps of:
i) crossing two parental plants, wherein one plant comprises at least one of
the
exogenous polynucleotides and/or at least one genetic modifications as defined
in any
one of claims 24 to 31; and the other plant comprises at least one of the
exogenous
polynucleotides and/or at least one genetic modifications as defined in any
one of
claims 24 to 31, and wherein between them the two parental plants comprise a
set of
exogenous polynucleotides and/or genetic modifications as defined in any one
of
claims 24 to 31,
ii) screening one or more progeny plants from the cross for the presence or
absence of the set of exogenous polynucleotides and/or genetic modifications
as
defined in any one of claims 24 to 31, and
iii) selecting a progeny plant which comprise the set of exogenous
polynucleotides and/or genetic modifications as defined in any one of claims
24 to 31,
thereby producing the plant.
In another aspect, the present invention provides a process for producing an
industrial product, the process comprising the steps of:
I) obtaining a cell of the invention, a plant or a part thereof of the
invention, or
seed the invention, and
ii) either
a) converting at least some of the lipid in the cell, plant or part thereof,
or seed, of step i) to the industrial product by applying heat, chemical, or
enzymatic means, or any combination thereof, to the lipid in situ in the cell,
or
plant or vegetative part thereof, or seed, or
b) physically processing the cell, plant or part thereof, or seed, of step
i), and subsequently or simultaneously converting at least some of the lipid
in the
processed cell, plant or part thereof, or seed, to the industrial product by
applying
heat, chemical, or enzymatic means, or any combination thereof, to the lipid
in the
processed cell, plant or part thereof, or seed, and
iii) recovering the industrial product,
thereby producing the industrial product.
CA 2998211 2018-03-16
36
In an embodiment, the step of physically processing the the cell, plant or
part
thereof, or seed, of step i), comprises one or more of rolling, pressing,
crushing or
grinding the plant or part thereof, or seed.
In an embodiment, the invention further comprises steps of:
(a) extracting at least some of the non-polar lipid content of the cell, or
plant or
part thereof, or seed, as non-polar lipid, and
(b) recovering the extracted non-polar lipid,
wherein steps (a) and (b) are performed prior to the step of converting at
least some of
the lipid in the cell, plant or part thereof, or seed, to the industrial
product.
In another aspect, the present invention provides a process for producing
extracted lipid, the process comprising the steps of:
i) obtaining a plant cell of the invention, or a plant or a part thereof of
the
invention, or seed of the invention,
ii) extracting lipid from the cell, or plant or part thereof, or seed, and
iii) recovering the extracted lipid,
thereby producing the extracted lipid.
In an embodiment, a process of extraction comprises one or more of drying,
rolling, pressing crushing or grinding the plant or part thereof, or seed,
and/or purifying
the extracted lipid or seedoil.
In an embodiment, the process uses an organic solvent in the extraction
process
to extract the oil.
In an embodiment, the process comprises recovering the extracted lipid by
collecting it in a container and/or one or more of degumming, deodorising,
decolourising, drying, fractionating the extracted lipid, removing wax esters
from the
extracted lipid, or analysing the fatty acid composition of the extracted
lipid.
In an embodiment, the volume of the extracted lipid or oil is at least 1
litre.
In a further embodiment, one or more or all of the following features apply:
(i) the extracted lipid or oil comprises triacylglycerols, wherein the
triacylglycerols comprise at least 90%, preferably at least 95% or at least
96%, of the
extracted lipid or oil,
(ii) the extracted lipid or oil comprises free sterols, steroyl esters,
steroyl
glycosides, waxes or wax esters, or any combination thereof, and
(iii) the total sterol content and/or composition in the extracted lipid or
oil is
significantly different to the sterol content and/or composition in the
extracted lipid or
oil produced from a corresponding plant or part thereof, or seed.
CA 2998211 2018-03-16
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In an embodiment, the process further comprises converting the extracted lipid
to an industrial product.
In an embodiment, the industrial product is a hydrocarbon product such as
fatty
acid esters, preferably fatty acid methyl esters and/or a fatty acid ethyl
esters, an alkane
such as methane, ethane or a longer-chain alkane, a mixture of longer chain
alkanes, an
alkene, a biofuel, carbon monoxide and/or hydrogen gas, a bioalcohol such as
ethanol,
propanol, or butanol, biochar, or a combination of carbon monoxide, hydrogen
and
biochar.
In a further embodiment, the plant part is an aerial plant part or a green
plant
part, preferably a vegetative plant part such as a plant leaf or stem.
In yet a further embodiment, the process further comprises a step of
harvesting
the plant or part thereof such as a vegetative plant part, tuber or beet, or
seed,
preferably with a mechanical harvester.
In another embodiment, the level of a lipid in the plant or part thereof, or
seed
and/or in the extracted lipid or oil is determinable by analysis by using gas
chromatography of fatty acid methyl esters prepared from the extracted lipid
or oil.
In yet another embodiment, the process further comprises harvesting the part
from a plant.
In an embodiment, the plant part is a vegetative plant part which comprises a
total non-polar lipid content of at least about 18%, at least about 20%, at
least about
25%, at least about 30%, at least about 35%, at least about 40%, at least
about 45%, at
least about 50%, at least about 55%, at least about 60%, at least about 65%,
at least
about 70%, between 18% and 75%, between about 20% and 75%, between about 30%
and 75%, between about 40% and 75%, between about 50% and 75%, between about
60% and 75%, or between about 25% and 50% (w/w dry weight).
In a further embodiment, the plant part is a vegetative plant part which
comprises a total TAG content of at least about 18%, at least about 20%, at
least about
25%, at least about 30%, at least about 35%, at least about 40%, at least
about 45%, at
least about 50%, at least about 55%, at least about 60%, at least about 65%,
at least
about 70%, between 18% and 75%, between about 20% and 75%, between about 30%
and 75%, between about 40% and 75%, between about 50% and 75%, between about
60% and 75%, or between about 25% and 50% (w/w dry weight).
In another embodiment, the plant part is a vegetative plant part which
comprises
a total non-polar lipid content of at least about 11%, at least about 12%, at
least about
15%, at least about 20%, at least about 25%, at least about 30%, at least
about 35%, at
least about 40%, at least about 45%, at least about 50%, at least about 55%,
at least
CA 2998211 2018-03-16
38
about 60%, at least about 65%, at least about 70%, between 8% and 75%, between
10%
and 75%, between 11% and 75%, between about 15% and 75%, between about 20%
and 75%, between about 30% and 75%, between about 40% and 75%, between about
50% and 75%, between about 60% and 75%, or between about 25% and 50% (w/w dry
weight), and wherein the vegetative plant part is from a 16:3 plant.
In yet another embodiment, the plant part is a vegetative plant part which
comprises a total TAG content of at least about 11%, at least about 12%, at
least about
15%, at least about 20%, at least about 25%, at least about 30%, at least
about 35%, at
least about 40%, at least about 45%, at least about 50%, at least about 55%,
at least
about 60%, at least about 65%, at least about 70%, between 8% and 75%, between
10%
and 75%, between 11% and 75%, between about 15% and 75%, between about 20%
and 75%, between about 30% and 75%, between about 40% and 75%, between about
50% and 75%, between about 60% and 75%, or between about 25% and 50% (w/w dry
weight), and wherein the vegetative plant part is from a 16:3 plant.
In an embodiment, the vegetative plant parts have a TAG/TFA Quotient (TTQ)
of between 0.01 and 0.6. In an embodiment, the vegetative plant parts have a
TTQ of
between 0.01 and 0.55, or between 0.01 and 0.5, or about 0.1, or about 0.2 or
about 0.3,
or about 0.4 or about 0.5. Preferably, the TTQ is between 0.60 and 0.84, which
corresponds to a TAG:TFA ratio of between 1.5:1 and 5:1, or between 0.84 and
0.95
which corresponds to a TAG:TFA ratio of between 5:1 and 20:1.
In an embodiment, the vegetative plant parts comprise an average TFA content
of about 6%, or about 8%, or about 9% or about 10% (w/w dry weight).
In an embodiment, the TFA content of the vegetative plant parts comprises a
palmitic acid content which is increased by at least 2% or at least 3%
relative to the
palmitic acid content of a corresponding wild-type vegetative plant part.
In an embodiment, the TFA content of the vegetative plant parts comprises a a-
linoleie acid (ALA) content which is decreased by at least 2% or at least 3%
relative to
the ALA content of a corresponding wild-type vegetative plant part.
In an embodiment, one or more or all of the following features apply:
(i) the vegetative plant parts are leaves and/or stems or parts thereof which
comprise one or more of an increased carbon content, an increased energy
content, an
increased soluble protein content, a reduced starch content, a reduced total
dietary fibre
(TDF) content and an increased nitrogen content, each on a weight basis
relative to a
corresponding wild-type leaf or stem or parts thereof from a wild-type Sorghum
sp. or
Zea mays plant at the same stage of growth.
CA 2998211 2018-03-16
39
(ii) the TFA content of the vegetative plant parts is at least about 6%, at
least
about 7%, at least about 8%, at least about 9%, at least about 10%, at least
about 11%,
at least about 12%. at least about 15%, at least about 20%, at least about
25%, at least
about 30%, at least about 35%, at least about 40%, at least about 45%, at
least about
50%, at least about 55%, at least about 60%, at least about 65%, at least
about 70%,
between about 6% and about 20%. between 8% and 75%, between 10% and 75%,
between 11% and 75%, between about 15% and 75%, between about 20% and 75%,
between about 30% and 75%, between about 40% and 75%, between about 50% and
75%, between about 60% and 75%, or between about 25% and 50% (w/w dry weight)
TFA,
(iii) the fatty acids esterified in the form of TAG in the vegetative plant
parts is
at least about 1%, at least about 2%, at least about 3%, at least about 4%, at
least about
5%, at least about 6%, at least about 7%, at least about 8%, at least about
9%, at least
about 10%, at least about 11%, at least about 12%, at least about 15%, at
least about
20%, at least about 25%, at least about 30%, at least about 35%, at least
about 40%, at
least about 45%, at least about 50%, at least about 55%, at least about 60%,
at least
about 65%, at least about 70%, between about 6% and about 20%, between 8% and
75%, between 10% and 75%, between 11% and 75%, between about 15% and 75%,
between about 20% and 75%, between about 30% and 75%, between about 40% and
75%, between about 50% and 75%, between about 60% and 75%, or between about
25% and 50% (w/w dry weight),
(iv) the vegetative plant parts comprise an increased content of a WRI1
polypeptide, an increased content of a DGAT polypeptide, and a decreased
content of a
SDP1 polypeptide, each relative to a corresponding wild-type vegetative plant
part,
(v) the vegetative plant parts comprise an increased content of a WRI1
polypeptide, an increased content of a DGAT polypeptide, and an increased
content of
a LEC2 polypeptide, each relative to a corresponding wild-type vegetative
plant part,
(vi) the vegetative plant parts comprise an increased content of a PDAT or
DGAT polypeptide, a decreased content of a TGD polypeptide, and a decreased
content
of a SDP1 polypeptide, each relative to a corresponding wild-type vegetative
plant part,
and
(vii) the vegetative plant parts comprise a decreased content of a TAG lipase
such as a SDP1 TAG lipase, a decreased content of a TGD polypeptide such as a
TGD5
polypeptide, and optionally a decreased content of a TST polypeptide such as a
TST1
polypeptide, each decrease being relative to a corresponding wild-type
vegetative plant
part.
CA 2998211 2018-03-16
40
In an embodiment, one or more or all of the following features apply:
(i) the vegetative plant parts are harvested from the plant between the time
of
first flowering of the plant and first maturity of seed,
(ii) the Sorghum sp. plant is a Sorghum bicolor plant,
(iii) the vegetative plant parts include leaves and/or stems or parts thereof,
(iv) the vegetative plant parts comprise an average total fatty acid content
of
about 8% or about 10% (w/w dry weight),
In another aspect, the present invention provides a process for producing
seed,
the process comprising:
i) growing a plant according to the invention, and
ii) harvesting seed from the plant.
In an embodiment, the above process comprises growing a population of at least
about 1,500, at least about 3,000 or at least about 5,000 plants, each being a
plant of the
invention, and harvesting seed from the population of plants.
In another aspect, the present invention provides recovered or extracted lipid
obtainable from a cell according to the invention, a plant or a part thereof
of the
invention, seed of the invention, or obtainable by the process of the
invention.
In another aspect, the present invention provides an industrial product
produced
by the process according to the invention, which is a hydrocarbon product such
as fatty
acid esters, preferably fatty acid methyl esters and/or a fatty acid ethyl
esters, an alkane
such as methane, ethane or a longer-chain alkane, a mixture of longer chain
alkanes, an
alkene, a biofuel, carbon monoxide and/or hydrogen gas, a bioalcohol such as
ethanol,
propanol, or butanol, biochar, or a combination of carbon monoxide, hydrogen
and
biochar. In an embodiment the industrial product comprises MCFA, preferably an
increased level of MCFA relative to a corresponding industrial product
produced from
a wild-type plant or part thereof.
In a further aspect, the present invention provides for the use of a
transgenic
plant or part thereof of the invention, seed of the invention, extract of the
invention or
the recovered or extracted lipid or soluble protein of the invention for the
manufacture
of an industrial product.
Examples of industrial products of the invention include, but are not limited
to, a
hydrocarbon product such as fatty acid esters, preferably fatty acid methyl
esters and/or
a fatty acid ethyl esters, an alkane such as methane, ethane or a longer-chain
alkane, a
mixture of longer chain alkanes, an alkene, a biofuel, carbon monoxide and/or
hydrogen gas, a bioalcohol such as ethanol, propanol, or butanol, biochar, or
a
combination of carbon monoxide, hydrogen andbiochar.
CA 2998211 2018-03-16
41
In another aspect, the present invention provides use of a cell according to
the
invention, a plant or part thereof of the invention, seed of the invention, or
the lipid of
the invention, for the manufacture of an industrial product.
In another aspect, the present invention provides a process for producing
fuel,
the process comprising:
i) reacting the lipid of the invention with an alcohol, optionally, in the
presence
of a catalyst, to produce alkyl esters, and
ii) optionally, blending the alkyl esters with petroleum based fuel.
In another aspect, the present invention provides a process for producing a
synthetic diesel fuel, the process comprising:
i) converting the lipid in a cell of the invention, or a plant or a part
thereof of the
invention, or seed of the invention, to a bio-oil by a process comprising
pyrolysis or
hydrothermal processing or to a syngas by gasification, and
ii) converting the bio-oil to synthetic diesel fuel by a process comprising
fractionation, preferably selecting hydrocarbon compounds which condense
between
about 150 C to about 200 C or between about 200 C to about 300 C, or
converting the
syngas to a biofuel using a metal catalyst or a microbial catalyst.
In another aspect, the present invention provides a process for producing a
biofuel, the process comprising converting the lipid in a cell of the
invention, a plant or
a part thereof of the invention, or seed of the invention, to bio-oil by
pyrolysis, a
bioalcohol by fermentation, or a biogas by gasification or anaerobic
digestion.
In another aspect, the present invention provides a process for producing a
feedstuff, the process comprising admixing a plant cell of the invention, a
plant or a
part thereof of the invention, seed of the invention, or the lipid of any one
of claims the
invention, or an extract or portion thereof, with at least one other food
ingredient.
In another aspect, the present invention provides feedstuffs, cosmetics or
chemicals comprising a plant cell of the invention, a plant or a part thereof
of the
invention, seed of the invention, or the lipid of the invention, or an extract
or portion
thereof.
In an embodiment, the feedstuff is silage, pellets or hay.
In another aspect, the present invention provides a process for feeding an
animal, the process comprising providing to the animal a plant or a part
thereof of the
invention, seed of the invention, or the lipid of the invention.
In an embodiment, the animal ingests an increased amount of MCFA, nitrogen,
protein, carbon and/or energy potential relative to when the animal ingests
the same
CA 2998211 2018-03-16
42
amount on a dry weight basis of a corresponding wild-type plant or part
thereof, seed or
extract or feedstuff produced from the corresponding wild-type plant or part
thereof.
Any embodiment herein shall be taken to apply mutatis mutandis to any other
embodiment unless specifically stated otherwise.
The present invention is not to be limited in scope by the specific
embodiments
described herein, which are intended for the purpose of exemplification only.
Functionally-equivalent products, compositions and methods are clearly within
the
scope of the invention, as described herein.
Throughout this specification, unless specifically stated otherwise or the
context
requires otherwise, reference to a single step, composition of matter, group
of steps or
group of compositions of matter shall be taken to encompass one and a
plurality (i.e.
one or more) of those steps, compositions of matter, groups of steps or group
of
compositions of matter.
The invention is hereinafter described by way of the following non-limiting
Examples and with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1. A representation of lipid synthesis in eukaryotic cells, showing
export of
some of the fatty acids synthesized in the plastids to the Endoplasmic
Reticulum (ER)
via the Plastid Associated Membrane (PLAM), and import of some of the fatty
acids
into the plastid from the ER for eukaryotic galactolipid synthesis.
Abbreviations:
Acetyl-CoA and Malonyl-CoA: acetyl-coenzyme A and malonyl-coenzymeA;
ACCase: Acetyl-CoA carboxylase;
FAS: fatty acid synthase complex;
16:0-ACP, 18:0-ACP and 18:1-ACP: C16:0-acyl carrier protein (ACP), C18:0-
acyl carrier protein, C18:1-acyl carrier protein;
KAS II: ketoacyl-ACP synthase II (EC 2.3.1.41);
PLPAAT: plastidial LPAAT;
PGPAT: plastidial GPAT;
PAP: PA phosphorylase (EC 3.1.3.4);
G3P: glycerol-3-phosphate;
LPA: lysophosphatidic acid;
PA: phosphatidic acid;
DAG: diacylglycerol;
TAG: tri acyl glycerol;
Acyl-CoA and Acyl-PC: acyl-coenzyme A and acyl- phosphatidylcholine;
CA 2998211 2018-03-16
43
=
PC: phosphatidylcholine;
GPAT: glycerol-3-phosphate acyltransferase;
LPAAT: lysophosphatidic acid acyltransferase (EC 2.3.1.51);
LPCAT: acyl-CoA:lysophosphatidylcholine acyltransferase; or synonyms 1-
acylglycerophosphocholine 0-acyltransferase; acyl-CoA:1-acyl-sn-glycero-3-
phosphocholine O-acyltransferase (EC 2.3.1.23);
CPT: CDP-choline:diacylglycerol cholinephosphotransferase; or synonyms 1-
alky1-2-acetylglycerol cholinephosphotransferase; alkylacylglycerol
cholinephosphotransferase: cholinephosphotransferase; phosphorylcholine-
glyceride transferase (EC 2.7.8.2);
PDCT: phosphatidylcholine:diacylglycerol cholinephosphotransferase;
PLC: phospholipase C (EC 3.1.4.3);
PLD: Phospholipase D; choline phosphatase; lecithinase D;
lipophosphodiesterase II (EC 3.1.4.4);
PDAT: phospholipid:diacylglycerol acyltransferase; or
synonym
phospholipid:1,2-diacyl-sn-glycerol 0-acyltransferase (EC 2.3.1.158);
FAD2: fatty acid Al2-desaturase; FAD3, fatty acid A15-desaturase;
UDP-Gal: Uridine diphosphate galactose;
MGDS: monogalactosyldiacylglycerol synthase;
MGDG: monogalactosyldiacylglycerol; DGDG: digalactosyldiacylglycerol
FAD6, 7, 8: plastidial fatty acid Al2-desaturase, plastidial o3-desaturase,
plastidial (o3-desaturase induced at low temperature, respectively.
Figure 2. Schematic diagram of vector pOIL122. Abbreviations: TER Agrtu-Nos,
Agrobacterium tumefaciens nopaline synthase terminator; NPTII, neomycin
phosphotransferase protein coding region; PRO CaMV35S-Ex2, Cauliflower Mosaic
Virus 35S promoter with double enhancer region; Arath-DGAT1, Arabidopsis
thaliana
DGAT1 acyltransferase protein coding region; PRO Arath-Rubisco SSU, A.
thaliana
Rubisco small subunit promoter; Arath-FATA2, A. thaliana FATA2 thioesterase
protein coding region; Arath-WRI, A. thaliana WRI1 transcription factor
protein
coding region; TER Glyma-Lectin, Glycine max lectin terminator; enTCUP2
promoter,
Nicotiana tabacum cryptic constitutive promoter; attB1 and attB2, Gateway
recombination sites; NB SDP1 fragment, Nicotiana benthamiana SDP1 region
targeted
for hpRNAi silencing; OCS terminator, A. tumefaciens octopine synthase
terminator.
Backbone features outside the T-DNA region are derived from pORE04 (Coutu et
al.,
2007).
CA 2998211 2018-03-16
44
Figure 3. TAG levels (% leaf dry weight) in N. benthamiana leaf tissue,
infiltrated
with genes encoding different WRI1 polypeptides either with (right hand bars)
or
without (left hand bars) co-expression of DGAT1 (n=3). All samples were
infiltrated
with the P19 construct as well.
Figure 4. Phylogenetic tree of LDAP polypeptides (Example 6).
Figure 5. Schematic representation of the genetic construct pJP3506 including
the T-
DNA region between the left and right borders. TAG, triacylglycerol; FFA, free
fatty
acids; DAG, diacylglycerol; Sesin-Oleosin, Sesame indicum oleosin protein
coding
region.
Figure 6. Triacylglycerol accumulation upon the expression of AtCaleosin 3 and
SiOleosin L.
Figure 7. Total fatty acid methyl ester (FAME) profiles (weight %)
illustrating the
effect of WRI1+DGAT1-mediated high oil background on MCFA production in
Nicotiana benthamiana leaf (n=4). Highest MCFA production was observed after
the
addition of Arath-WRII.
Figure 8. TAG content in leaf samples of transformed tobacco plants at seed-
setting
stage of growth, transformed with the T-DNA from pOIL049, lines #23c and #32b.
The
controls (parent) samples were from plants transformed with the T-DNA from
pJP3502. The upper line shows 18:2 percentage in the TAG and the lower line
shows
the 18:3 (ALA) percentage in the fatty acid content.
Figure 9. Leaf total FAME profiles (weight %) elucidating the effect of WRIl
on
MCFA accumulation (n=4). Addition of Arath-WRI1 greatly increased the
production
of the relevant fatty acid (C12:0, C14:0 or C16:0) relative to the previous
addition of
Cocnu-LPAAT alone.
Figure 10. TFA levels (% weight), TAG levels, levels of MCFA (C16:0 and C14:0,
%
of total fatty acids) in TFA and MCFA in TAG (% of total fatty acid content in
TAG)
in plant cells after expression of combinations of three oil palm DGATs with
FATB,
LPAAT and WRIL Numbers 1-10 are as listed in the text (Example 10).
CA 2998211 2018-03-16
45
Figure 11. Phylogenetic relationship of glycerol-3-phosphate acyltransferase
(GPAT)
genes from various species including the Arabidopsis thaliana (AtGPAT9) and
Cocos
nucifera (CnGPAT9) genes used in this study. The plant GPAT9 cluster is shaded
in
grey. BrGPAT3 = Brassica rapa glycerol-3-phosphate acyltransferase 3-like
(Accession: XM_009105753); BnGPAT3 = Brassica napus glycerol-3-phosphate
acyltransferase 3-like (Accession: XM_013896062); CsGPAT3 = Camelina sativa
glycerol-3-phosphate acyltransferase 3 (Accession: XM_010458322); AtGPAT9 = A.
thaliana glycerol-3-phosphate acyltransferase 9 (Accession: NM_125455);
ThGPAT3
= Tarenaya hassleriana glycerol-3-phosphate acyltransferase 3-like (Accession:
XM_010549847); RcGPAT3 = Ricinus communis glycerol-3-phosphate acyltransferase
3 (Accession: NM_001323761); JeGPAT3 = Jatropha curcas glycerol-3-phosphate
acyltransferase 3 (Accession: NM_001308751); EgGPAT3 = Elaeis guineensis
glycerol-3-phosphate acyltransferase 3-like (Accession: XM_010913693); CnGPAT9
=
C. nucifera GPAT9 (Accession: KX235871); Mouse GPAT = Mus musculus 1-
acylglycerol-3-phosphate 0-acyltransferase 9 (Accession: NM_172715); LrGPAT =
Lilium regale GPAT (Accession: 1X524740); LpGPAT = Lilium pensylvanicum GPAT
(Accession: JX524741); L1GPAT = Lilium longiflorum GPAT (Accession: JX524738);
EgGPAT mRMA = E. guineensis mRNA for acylation enzyme (Accession:
AJ272082); ChGPAT = Corylus heterophylla GPAT (Accession: JF428134); JcGPAT
= J. curcas glycerol-3-phosphate acyltransferase, chloroplastic (Accession:
NM_001305998); BnGPAT = B. napus glycerol-3-phosphate acyltransferase gene
(Accession: KM243174); PsGPAT = Pisum sativum chloroplast mRNA for acyl-
ACP:sn-glycerol-3-phosphate-acyltransferase (Accession: X59041); S1GPAT =
Solanum lycopersicum glycerol-3-phosphate acyltransferase (Accession:
NM_001306067); AtGPAT3 = A. thaliana putative sn-glycerol-3-phosphate 2-0-
acyltransferase (Accession: NM_116426); AtGPAT2 = A. thaliana glycerol-3-
phosphate sn-2-acyltransferase 2 (Accession: NM_100120); AtGPAT1 = A. thaliana
sn-glycerol-3-phosphate 2-0-acyltransferase (Accession: NM_100531); AtGPAT7 =
A.
thaliana glycerol-3-phosphate acyltransferase 7 (Accession: NM_120691);
AtGPAT5 ¨
A. thaliana glycerol-3-phosphate acyltransferase 5 (Accession: NM_111976);
QsGPAT
= Quercus suber glycerol-3-phosphate acyltransferase (Accession: JN819185);
EgGPAT5 = E. guineensis glycerol-3-phosphate acyltransferase 5 (Accession:
XM 010923983); EgGPAT6 = E. guineensis glycerol-3-phosphate 2-0-
acyltransferase
6 (Accession: XMO10924793); AtGPAT6 = A. thaliana bifunctional sn-glycerol-3-
phosphate 2-0-acyltransferase/phosphatase (Accession: NM_129367); AtGPAT8 = A.
CA 2998211 2018-03-16
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thaliana bifunctional sn-glycerol-3-phosphate 2-0-acyltransferase/phosphatase
(Accession: NM_116264); AtGPAT4 = A. thaliana glycerol-3-phosphate sn-2-
acyltransferase (Accession: NM 100043); GhGPAT = Gossypium hirsutum probable
glycerol-3-phosphate acyltransferase 3 (Accession: XM_016838669); EgGPAT4 = E.
guineensis glycerol-3-phosphate 2-0-acyltransferase 4-like (Accession:
XM 010942191).
Figure 12. Testing the effect of GPAT9 genes from Arabidopsis thaliana
(AtGPAT9)
and from Cocos nucifera (CnGPAT9) expression on TAG content, determined by
transient Nicotiana benthamiana leaf expression (n=4).
Figure 13. Fatty acid composition analysis of triacylglycerol (TAG),
determined by
the analysis of fatty acid methyl esters (FAME) via gas chromatography-flame
ionisation detection (GC-F1D) (n=3). Each thioesterase was infiltrated in
combination
with CnGPAT9 alone, CnGPAT9 + CnLPAAT or CnGPAT9 + CnLPAAT +
EgDGAT1, with all treatments including expression of AtWRI1 (Arabidopsis
thaliana
WRINKLED1). CcTE = Cinnarnomum camphora thioesterase; CnTE2 = Cocos
nucifera thioesterase; UcTE = Urnbellularia californica thioesterase; CnGPAT9
= C.
nucifera glycerol-3-phosphate acyltransferase 9; CnLPAAT = C. nucifera
lysophosphatidic acid acyltransferase; EgDGAT1 = Elaeis guineensis
diacylglycerol
acyltransferase.
KEY TO THE SEQUENCE LISTING
SEQ ID NO:1 Arabidopsis thaliana DGAT1 polypeptide (CAB44774.1)
SEQ ID NO:2 YFP tripeptide ¨ conserved DGAT2 and/or MGAT1/2 sequence motif
SEQ ID NO:3 HPHG tetrapeptide ¨ conserved DGAT2 and/or MGAT1/2 sequence
motif
SEQ ID NO:4 EPHS tetrapeptide ¨ conserved plant DGAT2 sequence motif
SEQ ID NO:5 RXGFX(K/R)XAXXXGXXX(LN)VPXXXFG(E/Q) ¨ long conserved
sequence motif of DGAT2 which is part of the putative glycerol phospholipid
domain
SEQ ID NO:6 FLXLXXXN ¨ conserved sequence motif of mouse DGAT2 and
MGAT1/2 which is a putative neutral lipid binding domain
SEQ ID NO:7 Conserved GPAT amino acid sequence GDLVICPEGTTCREP
SEQ ID NO:8 Conserved GPAT/phosphatase amino acid sequence (Motif I)
SEQ ID NO:9 Conserved GPAT/phosphatase amino acid sequence (Motif III)
SEQ ID NO:10 Sorbi-WRL1
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SEQ ID NO:11 Lupan-WRL1
SEQ ID NO:12 Ricco-WRL1
SEQ ID NO:13 Lupin angustifolius WRI1 polypeptide
SEQ ID NO:14 WRI1 motif (R G V T/S RHRWTG R)
SEQ ID NO:15 WR11 motif (F/Y EAHLWD K)
SEQ ID NO:16 WRI1 motif (D LAALKYW G)
SEQ ID NO:17 WRI1 motif (S X G F S/A R G X)
SEQ ID NO:18 WRI1 motif (H H H/Q N G R/K WEARIG R/K V)
SEQ ID NO:19 WR11 motif (Q EEAAAXY D)
SEQ ID NO:20 pJP3502 TDNA (inserted into genome) sequence
SEQ ID NO:21 pJP3507 vector sequence
SEQ ID NO:22 Linker sequence
SEQ ID NO:23 Partial Nicotiana benthamiana CG1-58 sequence selected for hpRNAi
silencing (pTV46)
SEQ ID NO:24 Partial N. tabacum AGPase sequence selected for hpRNAi silencing
(pTV35)
SEQ ID NO:25 GXSXG lipase motif
SEQ ID NO:26 HX(4)D acyltransferase motif
SEQ ID NO:27 VX(3)HGF probable lipid binding motif
SEQ ID NO:28 Arabidopsis thaliana BBM polypeptide (NP_197245.2)
SEQ ID NO:29 Inducible Aspergilus niger alcA promoter
SEQ ID NO:30 AlcR inducer that activates the AlcA promotor in the presence of
ethanol
SEQ NO:31 Arabidopsis thaliana LEC1; (AAC39488)
SEQ ID NO:32 Zea mays LEC1 (AAK95562)
SEQ ID NO:33 Arabidopsis thaliana LEC1-like (AAN15924)
SEQ ID NO:34 Arabidopsis thaliana FUS3 (AAC35247)
SEQ ID NO:35 Brassica napus FUS3
SEQ ID NO:36 Medicago truncatula FUS3
SEQ ID NO:37 Arabidopsis thaliana SDP1 cDNA sequence, Accession No.
NM 120486, 3275nt
SEQ ID NO:38 Sorghum bicolor SDP1 cDNA XM_002458486; 2724nt
SEQ ID NO:39 Nicotiana benthamiana SDP1 cDNA, Nbv5tr6404201SEQ ID NO:40
Nicotiana benthamiana SDP1 cDNA region targeted for hpRNAi silencing
SEQ ID NO:41 Promoter of Arabidopsis thaliana SDP] gene, 1.5kb
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SEQ ID NO:42 Nucleotide sequence of the complement of the pSSU-Oleosin gene in
the T-DNA of pJP3502. In order (complementary sequences): Glycine max Lectin
terminator 348nt, 3' exon 255nt, UBQ10 intron 304nt, 5' exon 213nt, SSU
promoter
1751nt
SEQ ID NO:43 Arabidopsis thaliana FATA1
SEQ ID NO:44 Arabidopsis thaliana FATA2
SEQ ID NO:45 Arabidopsis thaliana FATB
SEQ ID NO:46 Arabidopsis thaliana WRI3
SEQ ID NO:47 Arabidopsis thaliana WRI4
SEQ ID NO:48 Avena sativa WRI1
SEQ ID NO:49 Sorghum bicolor WRI1
SEQ ID NO:50 Zea mays WRI1
SEQ ID NO:51 Triadica sebifera WRI1
SEQ ID NO:52 S. tuberosum Patatin B33 promoter sequence
SEQ ID NO:53 Z. mays SEE1 promoter region (1970nt from Accession number
AJ494982)
SEQ ID NO:54 A. littoral is AlSAP promoter sequence, Accession No DQ885219
SEQ ID NO:55 A. rhizogenes ArRolC promoter sequence, Accession No. DQ160187
SEQ ID NO:56 Elaeis guineensis (oil palm) DGAT1
SEQ ID NO:57 G. max MYB73, Accession No. ABH02868
SEQ ID NO:58 A. thaliana bZIP53, Accession No. AAM14360
SEQ ID NO:59 A. thaliana AGL15, Accession No NP 196883
SEQ ID NO:60 A. thaliana MYB118, Accession No. AAS58517
SEQ ID NO:61 A. thaliana MYB115, Accession No. AAS10103
SEQ ID NO:62 A. thaliana TANMEI, Accession No. BAE44475
SEQ ID NO:63 A. thaliana WUS, Accession No. NP 565429
SEQ ID NO:64 B. napus GER2al, Accession No. AFB74090
SEQ ID NO:65 B. napus GFR2a2, Accession No. AFB74089
SEQ ID NO:66 A. thaliana PHRI, Accession No. AAN72198
SEQ ID NO:67 Sapium sebiferum LDAP-1 nucleotide sequence
SEQ ID NO:68 Sapium sebiferum LDAP-1 amino acid sequence
SEQ ID NO:69 Sapium sebiferum LDAP-2 nucleotide sequence
SEQ ID NO:70 Sapium sebiferum LDAP-2 amino acid sequence
SEQ ID NO:71 Sapium sebiferum LDAP-3 nucleotide sequence
SEQ ID NO:72 Sapium sebiferum LDAP-3 amino acid sequence
SEQ ID NO:73 S. bicolor SDPI (accession number XM_002463620)
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=
SEQ ID NO:74 T. aestivum SDP1 nucleotide sequence (Accession number AK334547)
SEQ ID NO:75 S. bicolor SDP1 hpRNAi fragment.
SEQ ID NO's 76 to 81 Oligonucleotide primer sequence
SEQ ID NO:82 Saccharum hybrid DIRIGENT (DIR16) promoter sequence
SEQ ID NO:83 Saccharum hybrid 0-Methyl transferase (OMT) promoter sequence
SEQ ID NO:84 Sequence of the Al promoter allele of the Saccharum hybrid R1MYB1
gene
SEQ ID NO:85 Saccharum hybrid Loading Stem Gene 5 (LSG5) promoter sequence
SEQ ID NO:86 Amino acid sequence of Sesamum indicum oleosinL polypeptide
(Accession No. AF091840)
SEQ ID NO:87 Amino acid sequence of Cinnamomum camphora 14:0-ACP
thioesterase (Accession.No. Q39473.1)
SEQ ID NO:88 Amino acid sequence of Cocos nucifera acyl-ACP thioesterase FatB1
(Accession No. AEM72519.1)
SEQ ID NO:89 Amino acid sequence of Cocos nucifera acyl-ACP thioesterase FatB2
(Accession No. AEM72520.1)
SEQ ID NO:90 Amino acid sequence of Cocos nucifera acyl-ACP thioesterase FatB3
(Accession No. AEM72521.1)
SEQ ID NO:91 Amino acid sequence of Cuphea lanceolata acyl-(ACP) thioesterase
type B (Accession No. CAB60830.1)
SEQ ID NO:92 Amino acid sequence of Cuphea viscosissima FatB1 (Accession No.
AEM72522.1)
SEQ ID NO:93 Amino acid sequence of and Umbellularia californica 12:0-ACP
thioesterase (Accession No. Q41635.1)
SEQ ID NO:94 Amino acid sequence of C. nucifera LPAAT (Accession No.
Q42670.1)
SEQ ID NO:95 Amino acid sequence of A. thaliana plastidial LPAAT1 (Accession
No. AEE85783.1)
SEQ ID NO:96 Codon optimised nucleotide sequence of Elaeis guineensis DGAT1
SEQ ID NO:97 Amino acid sequence of Cocos nucifera GPAT9
SEQ ID NO:98 Amino acid sequence of Arabidopsis thaliana GPAT9
SEQ ID NO:99 Amino acid sequence of Elaeis guineensis GPAT9
SEQ ID NO:100 Amino acid sequence of Phoenix dactylifera GPAT9
SEQ ID NO:101 Amino acid sequence of Musa acuminata GPAT9
SEQ ID NO:102 Amino acid sequence of Ananas comosus GPAT9
SEQ ID NO:103 Amino acid sequence of Asparagus officinalis GPAT9
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SEQ ID NO:104 Amino acid sequence of Oryza brachyantha GPAT9
SEQ ID NO:105 Amino acid sequence of Oryza saliva GPAT9
SEQ ID NO:106 Amino acid sequence of Nelumbo nucifera GPAT9
SEQ ID NO:107 Amino acid sequence of Vitis vinifera GPAT9
SEQ ID NO:108 Amino acid sequence of Nicotiana tomentosiformis GPAT9
SEQ ID NO:109 Amino acid sequence of Jatropha curcas GPAT9
SEQ ID NO:110 Amino acid sequence of Glycine max GPAT9
SEQ ID NO:111 Amino acid sequence of Sesamum indicum GPAT9
SEQ ID NO:112 Amino acid sequence of Brachypodium distachyon GPAT9
SEQ ID NO:113 Amino acid sequence of Setaria italica GPAT9
SEQ ID NO:114 Amino acid sequence of Cicer arietinum GPAT9
SEQ ID NO:115 Amino acid sequence of Zea mays GPAT9
SEQ ID NO:116 Amino acid sequence of Gossypium hirsutum GPAT9
SEQ ID NO:117 Amino acid sequence of Eucalyptus grandis GPAT9
SEQ ID NO:118 Amino acid sequence of Cucumis sativus GPAT9
SEQ ID NO:119 Amino acid sequence of Gossypium arboreum GPAT9
SEQ ED NO:120 Nucleotide sequence of Cocos nucifera GPAT9
SEQ ID NO:121 Nucleotide sequence of Arabidopsis thaliana GPAT9
SEQ ID NO:122 Nucleotide sequence of Elaeis guineensis GPAT9
SEQ ID NO:123 Nucleotide sequence of Phoenix dactylifera GPAT9
SEQ ID NO:124 Nucleotide sequence of Musa acuminata GPAT9
SEQ ID NO:125 Nucleotide sequence of Ananas comosus GPAT9
SEQ ID NO:126 Nucleotide sequence of Asparagus officinalis GPAT9
SEQ ID NO:127 Nucleotide sequence of Oryza brachyantha GPAT9
SEQ ID NO:128 Nucleotide sequence of Oryza sativa GPAT9
SEQ ID NO:129 Nucleotide sequence of Nelumbo nuctfera GPAT9
SEQ ID NO:130 Nucleotide sequence of Vitis vinifera GPAT9
SEQ ID NO:131 Nucleotide sequence of Nicotiana tornentosiformis GPAT9
SEQ ID NO:132 Nucleotide sequence of Jatropha curcas GPAT9
SEQ ID NO:133 Nucleotide sequence of Glycine max GPAT9
SEQ ID NO:134 Nucleotide sequence of Sesamum indicum GPAT9
SEQ 113 NO:135 Nucleotide sequence of Brachypodium distachyon GPAT9
SEQ ID NO:136 Nucleotide sequence of Setaria italica GPAT9
SEQ ID NO:137 Nucleotide sequence of Cicer arietinum GPAT9
SEQ ID NO:138 Nucleotide sequence of Zea mays GPAT9
SEQ ID NO:139 Nucleotide sequence of Gossypium hirsutum GPAT9
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SEQ ID NO:140 Nucleotide sequence of Eucalyptus grandis GPAT9
SEQ ID NO:141 Nucleotide sequence of Cucumis sativus GPAT9
SEQ ID NO:142 Nucleotide sequence of Gossypium arboreum GPAT9
SEQ ID NO:143 Amino acid sequence of E. guineensis NF-YB1
SEQ ID NO:144 Amino acid sequence of E. guineensis ZFP I
SEQ ID NO:145 Amino acid sequence of A. thaliana NF-YB2
SEQ ID NO:146 Amino acid sequence of A. thaliana NF-YB3
SEQ ID NO:147 Amino acid sequence of A. thaliana ZFP2
SEQ ID NO:148 Amino acid sequence of E. guineensis ABI5
SEQ ID NO:149 Amino acid sequence of E. guineensis NF-YC2
SEQ ID NO:150 Amino acid sequence of E. guineensis NF-YA3
SEQ ID NO:151 Amino acid sequence of G. max DOF4
SEQ ID NO:] 52 Amino acid sequence of G. max ZF351
DETAILED DESCRIPTION OF THE INVENTION
General Techniques
Unless specifically defined otherwise, all technical and scientific terms used
herein shall be taken to have the same meaning as commonly understood by one
of
ordinary skill in the art (e.g., in cell culture, molecular genetics, plant
biology, cell
biology, protein chemistry, lipid and fatty acid chemistry, animal nutrition,
biofeul
production, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and
immunological techniques utilized in the present invention are standard
procedures,
well known to those skilled in the art. Such techniques are described and
explained
throughout the literature in sources such as, J. Perbal, A Practical Guide to
Molecular
Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown
(editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2,
IRL
Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical
Approach, Volumes 1-4, IRL Press (1995 and 1996), F.M. Ausubel et al.
(editors),
Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-
Interscience (1988, including all updates until present), Ed Harlow and David
Lane
(editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory,
(1988),
and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley
& Sons
(including all updates until present).
CA 2998211 2018-03-16
52
Selected Definitions
The term "exogenous" in the context of a polynucleotide or polypeptide refers
to
the polynucleotide or polypeptide when present in a cell or a plant or part
thereof which
does not naturally comprise the polynucleotide or polypeptide. Such a cell is
referred
to herein as a "recombinant cell" or a "transgenic cell" and a plant
comprising the cell
as a "transgenic plant". In an embodiment, the exogenous polynucleotide or
polypeptide is from a different genus to the cell of the plant or part thereof
comprising
the exogenous polynucleotide or polypeptide. In another embodiment, the
exogenous
polynucleotide or polypeptide is from a different species. In one embodiment,
the
exogenous polynucleotide or polypeptide expressed in the plant cell is from a
different
species or genus. The exogenous polynucleotide or polypeptide may be non-
naturally
occurring, such as for example, a synthetic DNA molecule which has been
produced by
recombinant DNA methods. The DNA molecule may, preferably, include a protein
coding region which has been codon-optimised for expression in the plant cell,
thereby
producing a polypeptide which has the same amino acid sequence as a naturally
occurring polypeptide, even though the nucleotide sequence of the protein
coding
region is non-naturally occurring. The exogenous polynucleotide may encode, or
the
exogenous polypeptide may be, for example: a diacylglycerol acyltransferase
(DGAT)
such as a DGAT1 or a DGAT2, a Wrinkled 1 (WRI1) transcription factor, on OBC
such as an Oleosin or preferably an LDAP, a fatty acid thioesterase such as a
FATA or
FATB polypeptide, or a silencing suppressor polypeptide. In an embodiment, a
cell of
the invention is a recombinant cell.
As used herein, the term "triacylglycerol (TAG) content" or variations thereof
refers to the amount of TAG in the cell, plant or part thereof. TAG content
can be
calculated using techniques known in the art such as the sum of glycerol and
fatty acyl
moieties using a relation: % TAG by weight = 100x ((41x total mol
FAME/3)+(total g
FAME- (15x total mol FAME)))/g, where 41 and 15 are molecular weights of
glycerol
moiety and methyl group, respectively (where FAME is fatty acid methyl esters)
(see
Examples such as Example 1).
As used herein, the term "total fatty acid (TFA) content" or variations
thereof
refers to the total amount of fatty acids in the cell, plant or part thereof
on a weight
basis, as a percentage of the weight of the cell, plant or part thereof.
Unless otherwise
specified, the weight of the cell, plant or part thereof is the dry weight of
the cell, plant
or part thereof. TFA content is measured as described in Example 1 herein. The
method
involves conversion of the fatty acids in the sample to FAME and measurement
of the
amount of FAME by GC, using addition of a known amount of a distinctive fatty
acid
CA 2998211 2018-03-16
53
standard such as C17:0 as a quantitation standard in the GC. TFA therefore
represents
the weight of just the fatty acids, not the weight of the fatty acids and
their linked
moieties in the plant lipid.
As used herein, the"TAG/TFA Quotient" or "TTQ" parameter is calculated as
the level of TAG (%) divided by the level of TFA (%), each as a percentage of
the dry
weight of the plant material. For example, a TAG level of 6% comprised in a
TFA
level of 10% yields a TTQ of 0.6. The TAG and TFA levels are measured as
described
herein. It is understood that, in this context, the TFA level refers to the
weight of the
total fatty acid content and the TAG level refers to the weight of TAG,
including the
glycerol moiety of TAG.
As used herein, the tenn "soluble protein content" or variations thereof
refers to
the amount of soluble protein in the plant or part thereof. Soluble protein
content can
be calculated using techniques known in the art. For instance, fresh tissue
can be
ground, chlorophyll and soluble sugars extracted by heating to 80 C in 50-80%
(v/v)
ethanol in 2.5 mM HEPES buffer at pH 7.5, centriguation, washing pellet in
distilled
water, resuspending the pellet 0.1 M NaOH and heating to 95 C for 30 min, and
then
the Bradford assay (Bradford, 1976) is used determined soluble protein
content.
Alternatively, fresh tissue can be ground in buffer containing 100 mM Tris-HCl
pH 8.0
and 10 mM MgCl2.
As used herein, the term "nitrogen content" or variations thereof refers to
the
amount of nitrogen in the plant or part thereof. Nitrogen content can be
calculated
using techniques known in the art. For example, freeze-dried tissue can be
analysed
using a Europa 20-20 isotope ratio mass spectrometer with an ANCA preparation
system, comprising a combustion and reduction tube operating at 1000 C and 600
C,
respectively, to determine nitrogen content.
As used herein, the term "carbon content" or variations thereof refers to the
amount of carbon in the plant or part thereof. Carbon content can be
calculated using
techniques known in the art. For example, organic carbon levels can be
deteremined
using the method described by Shaw (1959), or as described in Example 1 of WO
2016/004473.
As used herein, the term "carbon:nitrogen ratio" or variations thereof refers
to
the relative amount of carbon in the cell, plant or part thereof when compared
to the
amount of nitrogen in the cell, plant or part thereof. Carbon and nitrogen
contents can
be calculated as described above and representated as a ratio.
As used herein, the term "photosynthetic gene expression" or variations
thereof
refers to one or more genes expressing proteins involved in photosynthetic
pathways in
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54
the plant ot part thereof. Examples of photosynthetic genes which may be
upregulated
in plants or parts thereof of the invention include, but are not limited to,
one or more of
the genes listed in Table 10 of WO 2016/004473.
As used herein, the term "photosynthetic capacity" or variations thereof
refers to
the ability of the plant or part thereof to photosynthesize (convert light
energy to
chemical energy). Photosynthetic capacity (Amax) is a measure of the maximum
rate at
which leaves are able to fix carbon during photosynthesis. It is typically
measured as
the amount of carbon dioxide that is fixed per metre squared per second, for
example as
m2jimol sec-1.
Photosynthetic capacity can be calculated using techniques known in
the art.
As used herein, the term "total dietary fibre (TDF) content" or variations
thereof
refers to the amount of fiber (including soluble and insoluble fibre) in the
cell, plant or
part thereof. As the skilled person would understand, dietary fiber includes
non-starch
polysaccharides such as arabinoxylans, cellulose, and many other plant
components
such as resistant starch, resistant dextrins, inulin, lignin, chitins,
pectins, p-glucans, and
oligosaccharides. fDF can be calculated using techniques known in the art. For
example, using the Prosky method (Prosky et al. 1985), the McCleary method
(McCleary et al., 2007) or the rapid integrated total dietary fiber method
(McCleary et
al.. 2015).
As used herein, the term "energy content" or variations thereof refers to the
amount of food energy in the plant or part thereof. More specifically, the
amount of
chemical energy that animals (including humans) derive from their food. Energy
content can be calculated using techniques known in the art. For example,
energy
content can be deteremined based on heats of combustion in a bomb calorimeter
and
corrections that take into consideration the efficiency of digestion and
absorption and
the production of urea and other substances in the urine. As another example,
energy
content can be calculated as described in Example 1 of WO 2016/004473.
As used herein, the term "extracted lipid" refers to a composition extracted
from
a cell, plant or part thereof of the invention, such as a transgenic cell,
plant or part
thereof of the invention, which comprises at least 60% (w/w) lipid.
As used herein, the term "non-polar lipid" refers to fatty acids and
derivatives
thereof which are soluble in organic solvents but insoluble in water. The
fatty acids
may be free fatty acids and/or in an esterified form. Examples of esterified
forms of
non-polar lipid include, but are not limited to, triacylglycerol (TAG),
diacylyglycerol
(DAG), monoacylglycerol (MAO). Non-polar lipids also include sterols, sterol
esters
and wax esters. Non-polar lipids are also known as "neutral lipids". Non-polar
lipid is
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55
typically a liquid at room temperature. In an embodiment, at least 50%, more
preferably at least 70%, more preferably at least 80%, more preferably at
least 90%,
more preferably at least 91%, more preferably at least 92%, more preferably at
least
93%, more preferably at least 94%, more preferably at least 95%, more
preferably at
least 96%, more preferably at least 97%, more preferably at least 98%, more
preferably
at least 99% of the fatty acids in non-polar lipid of the invention are
present as TAG.
The non-polar lipid may be further purified or treated, for example by
hydrolysis with a
strong base to release the free fatty acid, or by fractionation, distillation,
or the like.
Non-polar lipid may be present in or obtained from plant parts such as seed,
leaves,
tubers, beets or fruit. Non-polar lipid of the invention may form part of
"seedoil" if it is
obtained from seed.
The free and esterified sterol (for example, sitosterol, campesterol,
stigmasterol,
brassicasterol, A5-avenasterol, sitostanol, campestanol, and cholesterol)
concentrations
in the extracted lipid may be as described in Phillips et al. (2002). Sterols
in plant oils
are present as free alcohols, esters with fatty acids (esterified sterols),
glycosides and
acylated glycosides of sterols. Sterol concentrations in naturally occurring
vegetable
oils (seedoils) ranges up to a maximum of about 1100mg/100g. Hydrogenated palm
oil
has one of the lowest concentrations of naturally occurring vegetable oils at
about
60mg/100g. The recovered or extracted seedoils of the invention preferably
have
between about 100 and about 1000mg total sterol/100g of oil. For use as food
or feed,
it is preferred that sterols are present primarily as free or esterified forms
rather than
glycosylated forms. In the seedoils of the present invention, preferably at
least 50% of
the sterols in the oils are present as esterified sterols, except for soybean
seedoil which
has about 25% of the sterols esterified. The canola seedoil and rapeseed oil
of the
invention preferably have between about 500 and about 800 mg total
sterol/100g, with
sitosterol the main sterol and campesterol the next most abundant. The corn
seedoil of
the invention preferably has between about 600 and about 800 mg total
steroU100g,
with sitosterol the main sterol. The soybean seedoil of the invention
preferably has
between about 150 and about 350 mg total sterol/100g, with sitosterol the main
sterol
and stigmasterol the next most abundant, and with more free sterol than
esterified
sterol. The cottonseed oil of the invention preferably has between about 200
and about
350 mg total sterol/100g, with sitosterol the main sterol. The coconut oil and
palm oil
of the invention preferably have between about 50 and about 100mg total
sterol/100g,
with sitosterol the main sterol. The safflower seedoil of the invention
preferably has
between about 150 and about 250mg total sterol/100g, with sitosterol the main
sterol.
The peanut seedoil of the invention preferably has between about 100 and about
200mg
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total sterol/100g, with sitosterol the main sterol. The sesame seedoil of the
invention
preferably has between about 400 and about 600mg total sterol/100g, with
sitosterol the
main sterol. The sunflower seedoil of the invention preferably has between
about 200
and 400mg total sterol/100g, with sitosterol the main sterol. Oils obtained
from
vegetative plant parts according to the invention preferably have less than
200mg total
sterol/100g, more preferably less than 100mg total sterol/100g, and most
preferably less
than 50mg total sterols/100g, with the majority of the sterols being free
sterols. In an
embodiment, the lipid or oil is from a vegetative plant part which comprises
one or
more or all of sitosterol, campesterol, stigmasterol and cholesterol. In an
embodiment,
the lipid or oil is from a vegetative plant part and has more
galactosylglycerides than
phosphoglycerides. In an embodiment, the lipid or oil is from a seed and has
more
phosphoglycerides than galactosylglyeerides. Further guidance regarding
sterols and
other lipids components of plant cells can be found in Gunstone et al. (2007)
The Lipid
Handbook, Third Edition, CRC Press.
As used herein, the term "vegetative oil" refers to a composition obtained
from
vegetative parts of a plant which comprises at least 60% (w/w) lipid, or
obtainable from
the vegetative parts if the oil is still present in the vegetative part. That
is, vegetative
oil of the invention includes oil which is present in the vegetative plant
part, as well as
oil which has been extracted from the vegetative part (extracted oil). The
vegetative oil
is preferably extracted vegetative oil. Vegetative oil is typically a liquid
at room
temperature. The fatty acids are typically in an esterified form such as for
example,
TAG, DAG, acyl-CoA, galactolipid or phospholipid. The fatty acids may be free
fatty
acids and/or in an esterified form. In an embodiment, at least 50%, more
preferably at
least 70%, more preferably at least 80%, more preferably at least 90%, more
preferably
at least 91%, more preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more preferably at
least 96%,
more preferably at least 97%, more preferably at least 98%, more preferably at
least
99% of the fatty acids in vegetative oil of the invention can be found as TAG.
In an
embodiment, vegetative oil of the invention is "substantially purified" or
"purified" oil
that has been separated from one or more other lipids, nucleic acids,
polypeptides, or
other contaminating molecules with which it is associated in the vegetative
plant part or
in a crude extract. It is preferred that the substantially purified vegetative
oil is at least
60% free, more preferably at least 75% free, and more preferably, at least 90%
free
from other components with which it is associated in the vegetative plant part
or
extract. Vegetative oil of the invention may further comprise non-fatty acid
molecules
such as, but not limited to, sterols. In an embodiment, the vegetative oil is
canola oil
CA 2998211 2018-03-16
57
(Brassica sp. such as Brassica carinata, Brassica juncea, Brassica
napobrassica,
Brassica napus) mustard oil (Brassica juncea), other Brassica oil (e.g.,
Brassica
napobrassica, Brassica camelina), sunflower oil (Helianthus sp. such as
Helianthus
annuus), linseed oil (Linum usitatissimum), soybean oil (Glycine max),
safflower oil
(Carthamus tinctorius), corn oil (Zea mays), tobacco oil (Nicotiana sp. such
as
Nicotiana tabacum or Nicotiana benthamiana), peanut oil (Arachis hypogaea),
palm oil
(Elaeis guineensis), cotton oil (Gossypium hirsutum), coconut oil (Cocos
nucifera),
avocado oil (Persea americana), olive oil (Olea europaea), cashew oil
(Anacardium
occidentale), macadamia oil (Macadamia intergrifolia), almond oil (Prunus
amygdalus), oat oil (Avena sativa), rice oil (Oryza sp. such as Oryza sativa
and Oryza
glaberrima), Arab idopsis oil (Arabidopsis thaliana), Aracinis hypogaea
(peanut), Beta
vulgaris oil (sugar beet), Camelina sativa oil (false flax), Crambe abyssinica
oil
(Abyssinian kale), Cucumis melo oil (melon), Hordeum vulgare oil (barley),
Jatropha
curcas oil (physic nut), Joannesia princeps oil (arara nut-tree), Licania
rigida oil
(oiticica), Lupinus angustifolius oil (lupin), Miscanthus sp. oil such as
Miscanthus x
giganteus oil and Miscanthus sinensis oil, Panicum virgatum (switchgrass) oil,
Pongamia pinnata oil (Indian beech), Populus trichocarpa oil, Ricinus communis
oil
(castor), Saccharum sp. oil (sugarcane), Sesamum indicum oil (sesame), Solanum
tuberosum oil (potato), Sorghum sp. oil such as Sorghum bicolor oil, Sorghum
vulgare
oil, Theobroma grandiforum oil (cupuassu), Trifolium ,sp. oil, and Triticum
sp. oil
(wheat) such as Triticum aestivum. oil Vegetative oil may be extracted from
vegetative
plant parts by any method known in the art, such as for extracting seedoils.
This
typically involves extraction with nonpolar solvents such as diethyl ether,
petroleum
ether, chloroform/methanol or butanol mixtures, generally associated with
first
crushing of the seeds. Lipids associated with the starch or other
polysaccharides may
be extracted with water-saturated butanol. The seedoil may be "de-gummed" by
methods known in the art to remove polar lipids such as phospholipids or
treated in
other ways to remove contaminants or improve purity, stability, or colour. The
TAGs
and other esters in the vegetative oil may be hydrolysed to release free fatty
acids, or
the oil hydrogenated, treated chemically, or enzymatically as known in the
art. As used
herein, the term "seedoil" has an analogous meaning except that it refers to a
lipid
composition obtained from seeds of plants of the invention.
As used herein, the term "fatty acid" refers to a carboxylic acid with an
aliphatic
tail of at least 6 carbon atoms in length, either saturated or unsaturated.
Preferred fatty
acids have a carbon-carbon bonded chain of at least 12 carbons in length, more
preferably fatty acids having have a carbon-carbon bonded chain of 12 and/or
14
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carbons in length. Most naturally occurring fatty acids have an even number of
carbon
atoms because their biosynthesis involves acetate which has two carbon atoms.
The
fatty acids may be in a free state (non-esterified) or in an esterified form
such as part of
a TAG, DAG, MAG, acyl-CoA (thio-ester) bound, acyl-ACP bound, or other
covalently bound form. When covalently bound in an esterified form, the fatty
acid is
referred to herein as an "acyl" group. The fatty acid may be esterified as a
phospholipid such as a phosphatidylcholine (PC), phosphatidylethanolamine
(PE),
phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI),
or
diphosphatidylglycerol. Saturated fatty acids do not contain any double bonds
or other
functional groups along the chain. The term "saturated" refers to hydrogen, in
that all
carbons (apart from the carboxylic acid [-0001-1] group) contain as many
hydrogens as
possible. In other words, the omega (co) end contains 3 hydrogens (CH3-) and
each
carbon within the chain contains 2 hydrogens (-CH2-). Unsaturated fatty acids
are of
similar form to saturated fatty acids, except that one or more alkene
functional groups
exist along the chain, with each alkene substituting a singly-bonded "-CH2-CH2-
" part
of the chain with a doubly-bonded "-CH=CH-" portion (that is, a carbon double
bonded
to another carbon). The two next carbon atoms in the chain that are bound to
either
side of the double bond can occur in a cis or trans configuration.
As used herein, a fatty acid with a "medium chain length", also referred to as
"MCFA", comprises an acyl chain of 6 to 14 carbons. The acyl chain may be
modified
(for example it may comprise one or more double bonds, a hydroxyl group, an
expoxy
group, etc) or preferably is a saturated MCFA. This terms at least includes
one or more
or all of caproic acid (C6:0), caprylic acid (C8:0), capric acid (C10:0).
lauric acid
(C12:0), and myristic acid (C14:0). In an embodiment, the medium chain length
fatty
acids are lauric acid and/or myristic acid, or capric, lauric and myristic.
As used herein, "new medium chain fatty acids" or "new medium chain fatty
acid content" or the like refers to the difference between the total MCFA
content of the
extracted lipid, oil, recombinant cell, plant or plant part, or seed, of the
invention as the
context determines, expressed as a percentage of the total fatty acid content,
and the
total MCFA content of a corresponding wild-type extracted lipid, oil,
recombinant cell,
plant or plant part, or seed, obtained from a wild-type plant. That is, the
new MCFA
refers to the increased MCFA of the product of the invention relative to the
corresponding wild-type product. These new medium chain fatty acids are the
fatty
acids that are produced in the cells, plants and plant parts, or seeds, of the
invention by
the expression of the genetic constructs (exogenous polynucleotides)
introduced into
the cells, and include (if present) lauric acid and/or myristic acid.
Exemplary total
CA 2998211 2018-03-16
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medium chain fatty acid contents and new medium chain fatty acid contents are
determined by conversion of fatty acids in a sample to FAME and analysis by
GC, as
described in Example 1.
As used herein, "new medium chain fatty acids in the total fatty acid content
of
the TAG of the extracted lipid" or the like refers to the difference of the
total MCFA
content esterified in the form of triacylglycerols in the extracted lipid,
oil, recombinant
cell, plant or plant part, or seed, as the context determines, expressed as a
percentage of
the total fatty acid content esterified in TAG, and the total MCFA content
esterified in
the form of triacylglycerols in a corresponding wild-type extracted lipid,
oil,
recombinant cell, plant or plant part, or seed, obtained from a wild-type
plant.
As used herein, the terms "monounsaturated fatty acid" or "MUFA" refer to a
fatty acid which comprises at least 12 carbon atoms in its carbon chain and
only one
alkene group (carbon-carbon double bond), which may be in an esterified or non-
esterified (free) form. As used herein, the terms "polyunsaturated fatty acid"
or "PUFA"
refer to a fatty acid which comprises at least 12 carbon atoms in its carbon
chain and at
least two alkene groups (carbon-carbon double bonds), which may be in an
esterified or
non-esterified form.
"Monoacylglyceride" or "MAG" is glyceride in which the glycerol is esterified
with one fatty acid. As used herein, MAG comprises a hydroxyl group at an sn-
1/3
(also referred to herein as sn-1 MAG or 1-MAG or 1/3-MAG) or sn-2 position
(also
referred to herein as 2-MAG), and therefore MAG does not include
phosphorylated
molecules such as PA or PC. MAG is thus a component of neutral lipids in a
plant or
part thereof.
"Diacylglyceride" or "DAG" is glyceride in which the glycerol is esterified
with
two fatty acids which may be the same or, preferably, different. As used
herein, DAG
comprises a hydroxyl group at a sn-1,3 or sn-2 position, and therefore DAG
does not
include phosphorylated molecules such as PA or PC. DAG is thus a component of
neutral lipids in a plant or part thereof. In the Kennedy pathway of DAG
synthesis
(Figure 1), the precursor sn-glycerol-3-phosphate (G3P) is esterified to two
acyl
groups, each coming from a fatty acid coenzyme A ester, in a first reaction
catalysed by
a glycerol-3-phosphate acyltransferase (GPAT) at position sn-1 to form LysoPA,
followed by a second acylation at position sn-2 catalysed by a
lysophosphatidic acid
acyltransferase (LPAAT) to form phosphatidic acid (PA). This intermediate is
then de-
phosphorylated by PAP to form DAG. DAG may also be formed from TAG by
removal of an acyl group by a lipase, or from PC essentially by removal of a
choline
headgroup by any of the enzymes PDCT, PLC or PLD (Figure 1).
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"Triacylglyceride" or "TAG" is a glyceride in which the glycerol is esterified
with three fatty acids which may be the same (e.g. as in tri-olein) or, more
commonly,
different. In the Kennedy pathway of TAG synthesis, DAG is formed as described
above, and then a third acyl group is esterified to the glycerol backbone by
the activity
of DGAT. Alternative pathways for formation of TAG include one catalysed by
the
enzyme PDAT (Figure 1) and the MGAT pathway described herein.
As used herein, the term "wild-type" or variations thereof refers to a cell,
plant
or part thereof such as a cell, vegetative plant part, seed, tuber or beet,
that has not been
genetically modified, such as cells, plants or parts thereof that do not
comprise the one
or more exogenous polynucleotides, according to this invention.
The term "corresponding" refers to a cell, plant or part thereof such as a
cell,
vegetative plant part, seed, tuber or beet, that has the same or similar
genetic
background as a cell, plant or part thereof such as a vegetative plant part,
seed, tuber or
beet of the invention but which has not been modified as described herein (for
example,
a vegetative plant part or seed which lacks the defined exogenous
polynucleotide(s)).
In a preferred embodiment, the corresponding plant or part thereof such as a
vegetative
plant part is at the same developmental stage as the plant or part thereof
such as a
vegetative plant part of the invention. For example, if the plant is a
flowering plant,
then preferably the corresponding plant is also flowering. A corresponding
cell, plant
or part thereof such as a vegetative plant part, can be used as a control to
compare
levels of nucleic acid or protein expression, or the extent and nature of
trait
modification, for example MCFA and/or TAG content, with the cell, plant or
part
thereof such as a vegetative plant part of the invention which is modified as
described
herein. A person skilled in the art is readily able to determine an
appropriate
"corresponding" cell, plant or part thereof such as a vegetative plant part
for such a
comparison.
As used herein, "compared with" or "relative to" refers to comparing levels
of,
for example, MCFA or triacylglycerol (TAG) content, one or more or all of
soluble
protein content, nitrogen content, carbon:nitrogen ratio, photosynthetic gene
expression, photosynthetic capacity, total dietary fibre ( I'DF) content,
carbon content,
and energy content, or non-polar lipid content or composition, total non-polar
lipid
content, total fatty acid content or other parameter of the cell, plant or
part thereof
comprising the one or more exogenous polynucleotides, genetic modifications or
exogenous polypeptides with a cell, plant or part thereof such as a vegetative
plant part
lacking the one or more exogenous polynucelotides, genetic modifications or
polypeptides.
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As used herein, "synergism", "synergistic", "acting synergistically" and
related
terms are each a comparative term that means that the effect of a combination
of
elements present in a plant or part thereof of the invention, for example a
combination
of elements A and B, is greater than the sum of the effects of the elements
separately in
corresponding plants or parts thereof, for example the sum of the effect of A
and the
effect of B. Where more than two elements are present in the plant or part
thereof, for
example elements A, B and C, it means that the effect of the combination of
all of the
elements is greater than the sum of the effects of the individual effects of
the elements.
In a preferred embodiment, it means that the effect of the combination of
elements A, B
and C is greater than the sum of the effect of elements A and B combined and
the effect
of element C. In such a case, it can be said that element C acts
synergistically with
elements A and B. As would be understood, the effects are measured in
corresponding
cells, plants or parts thereof, for example grown under the same conditions
and at the
same stage of biological development.
As used herein, "germinate at a rate substantially the same as for a
corresponding wild-type plant" or similar phrases refers to seed of a plant of
the
invention being relatively able to germinate when compared to seed of a wild-
type
plant lacking the defined exogenous polynueleotide(s) and genetic
modifications.
Germination may be measured in vitro on tissue culture medium or in soil as
occurs in
the field. In one embodiment, the number of seeds which germinate, for
instance when
grown under optimal greenhouse conditions for the plant species, is at least
75%, more
preferably at least 90%, when compared to corresponding wild-type seed. In
another
embodiment, the seeds which germinate, for instance when grown under optimal
glasshouse conditions for the plant species, produce seedlings which grow at a
rate
which, on average, is at least 75%, more preferably at least 90%, when
compared to
corresponding wild-type plants. This is referred to as "seedling vigour". In
an
embodiment, the rate of initial root growth and shoot growth of seedlings of
the
invention is essentially the same compared to a corresponding wild-type
seedling
grown under the same conditions. In an embodiment, the leaf biomass (dry
weight) of
the plants of the invention is at least 80%, preferably at least 90%, of the
leaf biomass
relative to a corresponding wild-type plant grown under the same conditions,
preferably
in the field. In an embodiment, the height of the plants of the invention is
at least 70%,
preferably at least 80%, more preferably at least 90%, of the plant height
relative to a
corresponding wild-type plant grown under the same conditions, preferably in
the field
and preferably at maturity.
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As used herein, the term "an exogenous polynucleotide which down-regulates
the production and/or activity of an endogenous polypeptide" or variations
thereof,
refers to a polynucleotide that encodes an RNA molecule, herein termed a
"silencing
RNA molecule" or variations thereof (for example, encoding an amiRNA or
hpRNAi),
that down-regulates the production and/or activity, or itself down-regulates
the
production and/or activity (for example, is an amiRNA or hpRNA which can be
delivered directly to, for example, the plant or part thereof) of an
endogenous
polypeptide. This includes where the initial RNA transcript produced by
expression of
the exogenous polynucleotide is processed in the cell to form the actual
silencing RNA
molecule. The endogenous polypeptides whose production or activity are
downregulated include, for example, SDP1 TAG lipase, plastidial GPAT,
plastidial
LPAAT, TGD polypeptide such as TGD5, TST such as TST1 or TST2, AGPase,
PDCT, CPT or Al2 fatty acid desturase (FAD2), or a combination of two or more
thereof. Typically, the RNA molecule decreases the expression of an endogenous
gene
encoding the polypeptide. The extent of down-regulation is typically less than
100%,
for example the production or activity is reduced by between 25% and 95%
relative to
the wild-type. The optimal level of remaining production or activity can be
routinely
determined.
As used herein, the term "on a weight basis" refers to the weight of a
substance
(for example, TAG, DAG, fatty acid, protein, nitrogen, carbon) as a percentage
of the
weight of the composition comprising the substance (for example, seed, leaf
dry
weight). For example, if a transgenic seed has 25 ug total fatty acid per 120
ug seed
weight; the percentage of total fatty acid on a weight basis is 20.8%.
As used herein, the term "on a relative basis" refers to a parameter such as
the
amount of a substance in a composition comprising the substance in comparison
with
the parameter for a corresponding composition, as a percentage. For example, a
reduction from 3 units to 2 units is a reduction of 33% on a relative basis.
As used herein, "plastids" are organelles in plants, including algae, which
are
the site of manufacture of carbon-based compounds from photosynthesis
including
sugars, starch and fatty acids. Plastids include chloroplasts which contain
chlorophyll
and carry out photosynthesis, etioplasts which are the predecessors of
chloroplasts, as
well as specialised plastids such as chromoplasts which are coloured plastids
for
synthesis and storage of pigments, gerontoplasts which control the dismantling
of the
photosynthetic apparatus during senescence, amyloplasts for starch synthesis
and
storage, elaioplasts for storage of lipids, and proteinoplasts for storing and
modifying
proteins.
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As used herein, the term "biofuel" refers to any type of fuel, typically as
used to
power machinery such as automobiles, planes, boats, trucks or petroleum
powered
motors, whose energy is derived from biological carbon fixation. Biofuels
include
fuels derived from biomass conversion, as well as solid biomass, liquid fuels
and
biogases. Examples of biofuels include bioalcohols, biodiesel, synthetic
diesel,
vegetable oil, bioethers, biogas, syngas, solid biofuels, algae-derived fuel,
biohydrogen,
biomethanol, 2,5-Dimethylfuran (DMF), biodimethyl ether (bioDME), Fischer-
Tropsch
diesel, biohydrogen diesel, mixed alcohols and wood diesel.
As used herein, the term "bioalcohol" refers to biologically produced
alcohols,
for example, ethanol, propanol and butanol. Bioalcohols are produced by the
action of
microorganisms and/or enzymes through the fermentation of sugars,
hemicellulose or
cellulose.
As used herein, the term "biodiesel" refers to a composition comprising fatty
acid methyl- or ethyl- esters derived from lipids by transesterification, the
lipids being
from living cells not fossil fuels.
As used herein, the term "synthetic diesel" refers to a form of diesel fuel
which
is derived from renewable feedstock rather than the fossil feedstock used in
most diesel
fuels.
As used herein, the term "vegetable oil" includes a pure plant oil (or
straight
vegetable oil) or a waste vegetable oil (by product of other industries),
including oil
produced in either a vegetative plant part or in seed. Vegetable oil includes
vegetative
oil and seedoil, as defined herein.
As used herein, the term "biogas" refers to methane or a flammable mixture of
methane and other gases produced by anaerobic digestion of organic material by
anaerobes.
As used herein, the term "syngas" refers to a gas mixture that contains
varying
amounts of carbon monoxide and hydrogen and possibly other hydrocarbons,
produced
by partial combustion of biomass. Syngas may be converted into methanol in the
presence of catalyst (usually copper-based), with subsequent methanol
dehydration in
the presence of a different catalyst (for example, silica-alumina).
As used herein, the term "biochar" refers to charcoal made from biomass, for
example, by pyrolysis of the biomass.
As used herein, the term "feedstock" refers to a material, for example,
biomass
or a conversion product thereof (for example, syngas) when used to produce a
product,
for example, a biofuel such as biodiesel or a synthetic diesel.
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As used herein, the term "industrial product" refers to a hydrocarbon product
which is predominantly made of carbon and hydrogen such as, for example, fatty
acid
methyl- and/or ethyl-esters or alkanes such as methane, mixtures of longer
chain
alkanes which are typically liquids at ambient temperatures, a biofuel, carbon
monoxide and/or hydrogen, or a bioalcohol such as ethanol, propanol, or
butanol, or
biochar. The term "industrial product" is intended to include intermediary
products that
can be converted to other industrial products, for example, syngas is itself
considered to
be an industrial product which can be used to synthesize a hydrocarbon product
which
is also considered to be an industrial product. The term industrial product as
used
herein includes both pure forms of the above compounds, or more commonly a
mixture
of various compounds and components, for example the hydrocarbon product may
contain a range of carbon chain lengths, as well understood in the art.
As used herein, "progeny" means the immediate and all subsequent generations
of offspring produced from a parent, for example a second, third or later
generation
offspring.
As used herein, the term "ancestor" refers to any earlier generation of the
plant
comprising the first and second exogenous polynucleotides. The ancestor may be
the
parent plant, grandparent plant, great grandparent plant and so on.
As used herein, the term "selecting a plant" means actively selecting the
plant
on the basis that it has the desired phenotype, such as increased MCFA when
compared
to the corresponding wild-type plant.
As used herein, phrases such as "comprise a TFA content of about 5% (w/w dry
weight)", or "comprise a total TAG content of about 6% (w/w dry weight)", or
similary
structured phrases, mean that more than the defined level may be present. For
instance,
the phrase "comprise a TFA content of about 5% (w/w dry weight)" can be used
interchangeably with "comprises at least about 5% TFA (w/w dry weight)".
Extending
this example further, a vegetative plant part which comprise a TFA content of
about 5%
(w/w dry weight) may have a 6%, or 7.5% or higher TFA content.
As used herein, unless the context indicates otherwise, the term "increased
content" when used in reference to a polypeptide, or similar pharses including
refrence
to specific polypeptide, refers to either an exogenous polypeptide or an
endogenous
polypeptide. For example, a vegetative plant part of the invention may
comprise an
increased content of a WRI1 polypeptide, am increased GPAT9 content, an
increased
LPAAT content, an increased content of a DGAT polypeptide, and a decreased
content
of a SDP1 polypeptide, each relative to a corresponding wild-type vegetative
plant part,
wherein each of the WR11 and DGAT polypeptides is independently either an
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exogenous polypeptide or an endogenous polypeptide. As another example, a
vegetative plant part of the invention may comprise an increased content of a
WRI1
polypeptide, an increased content of a DGAT polypeptide, and an increased
content of
a LEC2 polypeptide, each relative to a corresponding wild-type vegetative
plant part,
wherein each of the WRIL DGAT and LEC2 polypeptides is independently either an
exogenous polypeptide or an endogenous polypeptide. As a further example, a
vegetative plant part of the invention may comprise an increased content of a
PDAT or
DGAT polypeptide, a decreased content of a TGD polypeptide, and a decreased
content
of a SDP1 polypeptide, each relative to a corresponding wild-type vegetative
plant part
wherein the PDAT or DGAT is either an exogenous polypeptide or an endogenous
polypeptide, and so on. An exogenous polypepetide may be the result of
expression of
a transgene encoding the polypeptide in the cell or plant or part thereof of
the
invention. The endogenous polypeptide may be the result of increased
expression of an
endogenous gene, such as inducing overexpression and/or providing increased
levels of
a transcription factor(s) for the gene.
Throughout this specification the word "comprise", or variations such as
"comprises" or "comprising", will be understood to imply the inclusion of a
stated
element, integer or step, or group of elements, integers or steps, but not the
exclusion of
any other element, integer or step, or group of elements, integers or steps.
The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X
and
Y" or "X or Y" and shall be taken to provide explicit support for both
meanings or for
either meaning.
As used herein, the term about, unless stated to the contrary, refers to +/-
10%,
more preferably +/- 5%, more preferably +/- 2%, more preferably +/- 1%, even
more
preferably +/- 0.5%, of the designated value.
Production of Plants with Modified Traits
The present invention is based on the finding that plant traits, such as MFCA
content and TAG content, in plants or parts thereof can be increased by a
combination
of two or more modifications selected from those designated herein as: (A).
Push, (B).
Pull, (C). Protect, (D). Package, (E). Plastidial Export, (F). Plastidial
Import and (G).
Prokaryotic Pathway.
Plants or parts thereof such as a vegetative plant parts of the invention
therefore
have a number of combinations of exogenous polynucleotides and/or genetic
modifications each of which provide for one of the modifications. These
exogenous
polynucleotides and/or genetic modifications include:
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(A) an exogenous polynucleotide which encodes a transcription factor
polypeptide that increases the expression of one or more glycolytic and/or
fatty acid
biosynthetic genes in the plant or part thereof such as a vegetative plant
part, providing
the "Push" modification,
(B) an exogenous polynucleotide which encodes a polypeptide involved in the
biosynthesis of one or more non-polar lipids in the plant or part thereof such
as a
vegetative plant part, providing the "Pull" modification,
(C) a genetic modification which down-regulates endogenous production and/or
activity of a polypeptide involved in the catabolism of triacylglycerols (TAG)
in the
plant or part thereof such as a vegetative plant part when compared to a
corresponding
plant or part thereof such as a vegetative plant part lacking the genetic
modification,
providing the "Protect" modification,
(D) an exogenous polynucleotide which encodes an oil body coating (OBC)
polypeptide such as a lipid droplet associated polypeptide (LDAP), providing
the
"Package" modification,
(E) an exogenous polynucleotide which encodes a polypeptide which increases
the export of fatty acids out of plastids of the plant or part thereof such as
a vegetative
plant part, when compared to a corresponding plant or part thereof such as a
vegetative
plant part lacking the exogenous polynucleotide, providing the "Plastidial
Export"
modification,
(F) a genetic modification which down-regulates endogenous production and/or
activity of a polypeptide involved in importing fatty acids into plastids of
the plant or
part thereof such as a vegetative plant part when compared to a corresponding
plant or
part thereof such as a vegetative plant part lacking the genetic modification,
providing
the "Plastidial Import" modification, and
G) a genetic modification which down-regulates endogenous production and/or
activity of a polypeptide involved in diacylglycerol (DAG) production in the
plastid of
the plant or part thereof such as a vegetative plant part when compared to a
corresponding plant or part thereof such as a vegetative plant part lacking
the genetic
modification, providing the "prokaryotic Pathway" modification.
Preferred combinations (also referred to herein as sets) of exogenous
polynucleotides and/or genetic modifications of the invention are;
1) A, B and optionally one of C, D, E, F or G;
2) A, C and optionally one of D, E, F or G;
3) A, D and optionally one of E, F or G;
4) A, E and optionally F or G;
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5) A, F and optionally G;
6) A and G;
7) A, B, C and optionally one of D, E, F or G;
8) A, B, D and optionally one of E, F or G:
9) A, B, E and optionally F or G;
10) A, B, F and optionally G;
11) A, B, C, D and optionally one of E, F or G;
12) A, B, C, E and optionally F or G;
13) A, B, C, F and optionally G;
14) A, B, D, E and optionally F or G;
15) A, B, D, F and optionally G;
16) A, B, E, F and optionally G;
17) A, C. D and optionally one of E, F or G;
18) A, C, E and optionally F or G;
19) A, C, F and optionally G;
20) A, C. D, E and optionally F or G;
21) A, C. D, F and optionally G;
22) A, C, E, F and optionally a fifth modification G;
23) A, D, E and optionally F or G;
24) A, D, F and optionally G;
25) A, D, E, F and optionally G;
26) A, E, F and optionally G;
27) Six of A, B, C, D, E, F and G omitting one of A, B, C, D, E, F or G, and
28) Any one of 1-26 above where there are two or more exogenous
polynucleotides encoding two or more different transcription factor
polypeptides that
increases the expression of one or more glycolytic and/or fatty acid
biosynthetic genes
in the plant or part thereof, for example one exogenous polynucleotide
encoding WRI1
and another exogenous polynucleotide encoding LEC2.
In each of the above preferred combinations there may be at least two
different
exogenous polynucleotides which encode at least two different transcription
factor
polypeptides that increases the expression of one or more glycolytic and/or
fatty acid
biosynthetic genes in theplant or part thereof such as a vegetative plant
part.
These modifications are described more fully as follows:
A. The
"Push" modification is characterised by an increased synthesis of total
fatty acids in the plastids of the plant or part thereof. In an embodiment,
this occurs by
the increased expression and/or activity of a transcription factor which
regulates fatty
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acid synthesis in the plastids. In one embodiment, this can be achieved by
expressing
in a transgenic plant or part thereof an exogenous polynucleotide which
encodes a
transcription factor polypeptide that increases the expression of one or more
glycolytic
and/or fatty acid biosynthetic genes in the plant or part thereof. In an
embodiment, the
increased fatty acid synthesis is not caused by the provision to the plant or
part thereof
of an altered ACCase whose activity is less inhibited by fatty acids, relative
to the
endogenous ACCase in the plant or part thereof. In an embodiment, the plant or
part
thereof comprises an exogenous polynucleotide which encodes the transcription
factor,
preferably under the control of a promoter other than a constitutive promoter.
The
transcription factor may be selected from the group consisting of WRI1, LEC1,
LEC1-
like, LEC2, BBM, FUS3, ABI3, ABI4, ABI5, Dof4, Dofl 1 or the group consisting
of
MYB73, bZIP53, AGL15, MYB115, MYB118, TANMEI, WUS, GFR2a1, GFR2a2
and PITRL and is preferably WRI1, LEC1 or LEC2, or WRI1 alone. In a further
embodiment, the increased synthesis of total fatty acids is relative to a
corresponding
wild-type plant or part thereof. In an embodiment, there are two or more
exogenous
polynueleotides encoding two or more different transcription factor
polypeptides. The
"Push" modification may also be achieved by increased expression of
polypeptides
which modulate activity of WRI1, such as MED15 or 14-3-3 polypeptides.
B. The
"Pull" modification is characterised by increased expression and/or
activity in the plant or part thereof of a fatty acyl acyltransferase which
catalyses the
synthesis of TAG, DAG or MAG in the plant or part thereof, such as a DGAT,
PDAT,
LPAAT, GPAT or MGAT, preferably a DGAT or a PDAT. In one embodiment, this
can be achieved by expressing in a transgenic plant or part thereof an
exogenous
polynucleotide which encodes a polypeptide involved in the biosynthesis of one
or
more non-polar lipids. In an embodiment, the acyltransferase is a membrane-
bound
acyltransferase that uses an acyl-CoA substrate as the acyl donor in the case
of DGAT,
LPAAT, GPAT or MGAT, or an acyl group from PC as the acyl donor in the case of
PDAT. The Pull modification can be relative to a corresponding wild-type plant
or part
thereof or, preferably, relative to a corresponding plant or part thereof
which has the
Push modification. In an embodiment, the plant or part thereof comprises an
exogenous polynucleotide which encodes the fatty acyl acyltransferase. The
"Pull"
modification can also be achieved by increased expression of a PDCT, CPT or
phospholipase C or D polypeptide which increases the production of DAG from
PC.
In a preferred embodiment, the cell comprises an exogenous polynucleotide(s)
encoding one or more or all of a GPAT, LPAAT and/or DGAT which have a
preference for utilising medium chain fatty acid substrates, particularly for
lauric acid
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and/or myristic acid. Such GPAT, LPAAT and/or DGAT having a preference for
utilising medium chain fatty acid substrates include those described herein,
as well as
those which can be isolated from plants which naturally produce high levels of
medium
chain fatty acids, such as but not limited to, Elaeis guineensis, Cocus
nucifera, Attalea
dubia, Orbignya phalerata, Astrocaryum murumuru, Bactris gasipaes, Pycnanthus
angolensis, Cuphea wrightii, Altalea colenda, Laurus nohilis, Umbellularia
californica,
Qualea grandiflora and Actinodaphne hookeri. The skilled person would
appreciate
that the sequences provided herein which readily be used to screen sequence
databases
to identify orthologous genes and proteins from the above species.
C. The "Protect"
modification is characterised by a reduction in the
catabolism of triacylglycerols (TAG) in the plant or part thereof. In an
embodiment,
this can be achieved through a genetic modification in the plant or part
thereof which
down-regulates endogenous production and/or activity of a polypeptide involved
in the
catabolism of triacylglycerols (TAG) in the plant or part thereof when
compared to a
corresponding plant or part thereof lacking the genetic modification. In an
embodiment, the plant or part thereof has a reduced expression and/or activity
of an
endogenous TAG lipase in the plant or part thereof, preferably an SDP1 lipase,
a Cgi58
polypeptide, an acyl-CoA oxidase such as the ACX1 or ACX2, or a polypeptide
involved in 13-oxidation of fatty acids in the plant or part thereof such as a
PXA1
peroxisomal ATP-binding cassette transporter. This may occur by expression in
the
plant or part thereof of an exogenous polynucleotide which encodes an RNA
molecule
which reduces the expression of, for example, an endogenous gene encoding the
TAG
lipase such as the SDP1 lipase, acyl-CoA oxidase or the polypeptide involved
in [3-
oxidation of fatty acids in the plant or part thereof, or by a mutation in an
endogenous
gene encoding, for example, the TAG lipase, acyl-CoA oxidase or polypeptide
involved in 13-oxidation of fatty acids. In an embodiment, the reduced
expression
and/or activity is relative to a corresponding wild-type plant or part thereof
or relative
to a corresponding plant or part thereof which has the Push modification.
D. The "Package" modification is characterised by an increased expression
and/or accumulation of an oil body coating (OBC) polypeptide. In an
embodiment, this
can be achieved by expressing in a transgenic plant or part thereof an
exogenous
polynucleotide which encodes an oil body coating (OBC) polypeptide. The OBC
polypeptide may be an oleosin, such as for example a polyoleosin, a caoleosin
or a
steroleosin, or preferably an LDAP. In an embodiment, the level of oleosin
that is
accumulated in the plant or part thereof is at least 2-fold higher relative to
the
corresponding plant or part thereof comprising the oleosin gene from the T-DNA
of
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pJP3502. In an embodiment, the increased expression or accumulation of the OBC
polypeptide is not caused solely by the Push modification. ht an embodiment,
the
expression and/or accumulation is relative to a corresponding wild-type plant
or part
thereof or, preferably, relative to a corresponding plant or part thereof
which has the
Push modification.
E. The "Plastidial Export" modification is characterised by an increased
rate
of export of total fatty acids out of the plastids of the plant or part
thereof. In one
embodiment, this can be achieved by expressing in a plant or part thereof an
exogenous
polynucleotide which encodes a polypeptide which increases the export of fatty
acids
out of plastids of the plant or part thereof when compared to a corresponding
plant or
part thereof lacking the exogenous polynucleotide. In an embodiment, this
occurs by
the increased expression and/or activity of a fatty acid thioesterase (TE), a
fatty acid
transporter polypeptide such as an ABCA9 polypeptide, or a long-chain acyl-CoA
synthetase (LACS). In an embodiment, the plant or part thereof comprises an
exogenous polynucleotide which encodes the TE, fatty acid transporter
polypeptide or
LACS. The TE may be a FATB polypeptide or preferably a FATA polypeptide. In an
embodiment, the TE is preferably a TE which has a preference for hydrolysing
MCFA,
or MCFA and C16:0 substrates. In an embodiment, the Plastidial Export
modification
is relative to a corresponding wild-type plant or part thereof or, preferably,
relative to a
corresponding plant or part thereof which has the Push modification.
F. The "Plastidial Import" modification is characterised by a reduced rate
of
import of fatty acids into the plastids of the plant or part thereof from
outside of the
plastids. In an embodiment, this can be achieved through a genetic
modification in the
plant or part thereof which down-regulates endogenous production and/or
activity of a
polypeptide involved in importing fatty acids into plastids of the plant or
part thereof
when compared to a corresponding plant or part thereof lacking the genetic
modification. For example, this may occur by expression in the plant or part
thereof of
an exogenous polynucleotide which encodes an RNA molecule which reduces the
expression of an endogenous gene encoding an transporter polypeptide such as a
TGD
polypeptide, for example a TGD1, TGD2, TGD3, TGD4 or preferably a TGD5
polypeptide, or by a mutation in an endogenous gene encoding the TGD
polypeptide.
In an embodiment, the reduced rate of import is relative to a corresponding
wild-type
plant or part thereof or relative to a corresponding plant or part thereof
which has the
Push modification.
G. The "Prokaryotic Pathway" modification is characterised by a decreased
amount of DAG or rate of production of DAG in the plastids of the plant or
part
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thereof. In an embodiment, this can be achieved through a genetic modification
in the
plant or part thereof which down-regulates endogenous production and/or
activity of a
polypeptide involved in diacylglycerol (DAG) production in the plastid when
compared
to a corresponding plant or part thereof lacking the genetic modification. In
an
embodiment, the decreased amount or rate of production of DAG occurs by a
decreased
production of LPA from acyl-ACF. and G3P in the plastids. The decreased amount
or
rate of production of DAG may occur by expression in the plant or part thereof
of an
exogenous polynucleotide which encodes an RNA molecule which reduces the
expression of an endogenous gene encoding a plastidial GPAT, plastidial LPAAT
or a
plastidial PAP, preferably a plastidial GPAT, or by a mutation in an
endogenous gene
encoding the plastidial polypeptide. In an embodiment, the decreased amount or
rate of
production of DAG is relative to a corresponding wild-type plant or part
thereof or,
preferably, relative to a corresponding plant or part thereof which has the
Push
modification.
The Push modification is highly desirable in the invention, and the Pull
modification is preferred. The
Protect and Package modifications may be
complementary i.e. one of the two may be sufficient. The plant or part thereof
may
comprise one, two or all three of the Plastidial Export, Plastidial Import and
Prokaryotic Pathway modifications. In an embodiment, at least one of the
exogenous
polynucleotides in the plant or part thereof, preferably at least the
exogenous
polynucleotide encoding the transcription factor which regulates fatty acid
synthesis in
the plastids, is expressed under the control of (H) a promoter other than a
constitutive
promoter such as. for example, a developmentally related promoter, a promoter
that is
preferentially active in photosynthetic cells, a tissue-specific promoter, a
promoter
which has been modified by reducing its expression level relative to a
corresponding
native promoter, or is preferably a senesence-specific promoter. More
preferably, at
least the exogenous polynucleotide encoding the transcription factor which
regulates
fatty acid synthesis in the plastids is expressed under the control of a
promoter other
than a constitutive promoter and the exogenous polynucleotide which encodes an
RNA
molecule which down-regulates endogenous production and/or activity of a
polypeptide
involved in the catabolism of triacylglycerols is also expressed under the
control of a
promoter other than a constitutive promoter, which promoters may be the same
or
different. Alternatively in monocotyledonous plants, the exogenous
polynucleotide
encoding the transcription factor which regulates fatty acid synthesis in the
plastids is
expressed under the control of a constitutive promoter such as, for example, a
ubiquitin
gene promoter or an actin gene promoter.
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Plants produce some, but not all, of their membrane lipids such as MGDG in
plastids by the so-called prokaryotic pathway (Figure 1). In plants, there is
also a
eukaryotic pathway for synthesis of galactolipids and glycerolipids which
synthesizes
FA first of all in the plastid and then assembles the FA into glycerolipids in
the ER.
MGDG synthesised by the eukaryotic pathway contains C18:3 (ALA) fatty acid
esterified at the sn-2 position of MGDG. The DAG backbone including the ALA
for
the MGDG synthesis by this pathway is assembled in the ER and then imported
into the
plastid. In contrast, the MGDG synthesized by the prokaryotic pathway contains
C16:3
fatty acid esterified at the sn-2 position of MGDG. The ratio of the
contribution of the
prokaryotic pathway relative to the eukaryotic pathway in producing MGDG
(16:3) vs
MGDG (18:3) is a characteristic and distinctive feature of different plant
species
(Mongrand et al. 1998). This distinctive fatty acid composition of MGDG allows
all
higher plants (angiosperms) to be classified as either so-called 16:3 or 18:3
plants. 16:3
species, exemplified by Arabidopsis and Brassica napus, generally have both of
the
prokaryotic and eukaryotic pathways of MGDG synthesis operating, whereas the
18:3
species exemplified by Sorghum bicolor, Zea mays, Nicotiana tabacum, Pisum
sativum
and Glycine max generally have only (or almost entirely) the eukaryotic
pathway of
MGDG synthesis, providing little or no C16:3 fatty acid accumulation in the
vegetative
tissues.
As used herein, a "16:3 plant" or "16:3 species" is one which has more than 2%
C16:3 fatty acid in the total fatty acid content of its photosynthetic
tissues. As used
herein, a "18:3 plant" or "18:3 species" is one which has less than 2% C16:3
fatty acid
in the total fatty acid content of its photosynthetic tissues. As described
herein, a plant
can be converted from being a 16:3 plant to an 18:3 plant by suitable genetic
modifications. The proportion of flux between the prokaryote and eukaryote
pathways
is not conserved across different plant species or tissues. In 16:3 species up
to 40% of
flux in leaves occurs via the prokaryotic pathway (Browse et al., 1986), while
in 18:3
species, such as pea and soybean, about 90% of FAs which are synthesized in
the
plastid are exported out of the plastid to the ER to supply the source of FA
for the
eukaryotic pathway (Ohlrogge and Browse, 1995; Somerville et al., 2000).
Therefore different amounts of 18:3 and 16:3 fatty acids are found within the
glycolipids of different plant species. This is used to distinguish between
18:3 plants
whose fatty acids with 3 double bonds are almost entirely C18 fatty acids and
the 16:3
plants that contain both C16- and Cis-fatty acids having 3 double bonds. In
chloroplasts
of 18:3 plants, enzymic activities catalyzing the conversion of phosphatidate
to
diacylglycerol and of diacylglycerol to monogalactosyl diacylglycerol (MGD)
are
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significantly less active than in 16:3 chloroplasts. In leaves of 18:3 plants,
chloroplasts
synthesize stearoyl-ACP2 in the stroma, introduce the first double bond into
the
saturated hydrocarbon chain, and then hydrolyze the thioester by thioesterases
(Figure
1). Released oleate is exported across chloroplast envelopes into membranes of
the
cell, probably the endoplasmic reticulum, where it is incorporated into PC. PC-
linked
oleoyl groups are desaturated in these membranes and subsequently move back
into the
chloroplast. The MGD-linked acyl groups are substrates for the introduction of
the
third double bond to yield MGD with two linolenoyl residues. This galactolipid
is
characteristic of 18:3 plants such as Asteraceae and Fabaceae, for example. In
photosynthetically active cells of 16:3 plants which are represented, for
example, by
members of Apiaceae and Brassicaceae, two pathways operate in parallel to
provide
thylakoids with MGD.
In one embodiment, the plant or part thereof such as a vegetative plant part
of
the invention produces higher levels of non-polar lipids such as TAG, or MFCA
content, preferably both, than a corresponding plant or part thereof such as a
vegetative
plant part which lacks the genetic modifications or exogenous polynucleotides.
In one
example, plants of the invention produce seeds, leaves, or have leaf portions
of at least
1cm2 in surface area, stems and/or tubers having an increased non-polar lipid
content
such as TAG or MCFA content, preferably both, when compared to corresponding
seeds, leaves, leaf portions of at least 1cm2 in surface area, stems or
tubers.
Preferably, the plant or part thereof such as a vegetative plant part of the
invention is transformed with one or more exogenous polynucleotides such as
chimeric
DNAs. In the case of multiple chimeric DNAs, these are preferably covalently
linked
on one DNA molecule such as, for example, a single T-DNA molecule, and
preferably
integrated at a single locus in the host cell genome, preferably the host
nuclear genome.
Alternatively, the chimeric DNAs are on two or more DNA molecules which may be
unlinked in the host genome, or the DNA molecule(s) is not integrated into the
host
genome, such as occurs in transient expression experiments. The plant or part
thereof
such as a vegetative plant part is preferably homozygous for the one DNA
molecule
inserted into its genome.
Transcription Factors
Various transcription factors are involved in plant cells in the synthesis of
fatty
acids and lipids incorporating the fatty acids such as TAG, and therefore can
be
manipulated for the Push modification. A preferred transcription factor is
WRIL As
used herein, the term "Wrinkled 1" or "WRI1 " or "WRL1" refers to a
transcription
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74
factor of the AP2/ERWEBP class which regulates the expression of several
enzymes
involved in glycolysis and de novo fatty acid biosynthesis. WRI1 has two plant-
specific (AP2/EREB) DNA-binding domains. WRI1 in at least Arabidopsis also
regulates the breakdown of sucrose via glycolysis thereby regulating the
supply of
precursors for fatty acid biosynthesis. In other words, it controls the carbon
flow from
the photosynthate to storage lipids. wril mutants in at least Arabidopsis have
a
wrinkled seed phenotype, due to a defect in the incorporation of sucrose and
glucose
into TAGs.
Examples of genes which are transcribed by WR11 include, but are not limited
to, one or more, preferably all, of genes encoding pyruvate kinase (At5g52920,
At3g22960), pyruvate dehydrogenase (PDH) Elalpha subunit (Atl g01090), acetyl-
CoA carboxylase (ACCase), BCCP2 subunit (At5g15530), enoyl-ACP reductase
(At2g05990; EAR), phosphoglycerate mutase (Atl g22170), cytosolic
fructokinase, and
cytosolic phosphoglycerate mutase, sucrose synthase (SuSy) (see, for example,
Liu et
al., 2010; Baud et al., 2007; Ruuska et al., 2002).
WRI1 contains the conserved domain AP2 (cd00018). AP2 is a DNA-binding
domain found in transcription regulators in plants such as APETALA2 and EREBP
(ethylene responsive element binding protein). In EREBPs the domain
specifically
binds to the ii bp GCC box of the ethylene response element (ERE), a promotor
element essential for ethylene responsiveness. EREBPs and the C-repeat binding
factor
CBF1, which is involved in stress response, contain a single copy of the AP2
domain.
APETALA2-like proteins, which play a role in plant development contain two
copies.
Other sequence motifs which may be found in WRI1 and its functional
homologs include:
I. RGVT/SRHRWTGR(SEQIDNO:14).
2. F/Y EAHL WDK (SEQ ID NO:15).
3. DLAALK YWG (SEQ ID NO:16).
4. SXGF S/A R G X (SEQ ID NO:17).
5. HHH/QNGR/KWEARIGR/K V (SEQ IDNO:18).
6. QEEA A A XYD (SEQ ID NO:19).
As used herein, the term "Wrinkled 1" or "WRIl" also includes "Wrinkled 1-
like" or "WRI1-like" proteins. Examples of WRI1 proteins include Accession
Nos:
A8MS57 (Arabidopsis thaliana), Q6X5Y6, (Arabidopsis thaliana), XP 002876251.1
(Arabidopsis lyrata subsp. Lyrata), ABD16282.1 (Brassica napus), AD016346.1
(Brassica napus), XP_003530370.1 (Glycine max), AE022131.1 (Jatropha curcas),
XP_002525305.1 (Ricinus communis), XP_002316459.1 (Populus trichocarpa),
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CBI29147.3 (Vitis vinifera), XP_003578997.1 (Brachypodium distachyon),
BAJ86627.1 (Hordeum vulgare subsp. vulgare), EAY79792.1 (Oryza sativa),
XP_002450194.1 (Sorghum bicolor), ACG32367.1 (Zea mays), XP_003561189.1
(Brachypodium distachyon), ABL85061.1 (Brachypodium sylvaticum), BAD68417.1
(Oryza sativa), XP_002437819.1 (Sorghum bicolor), XP_002441444.1 (Sorghum
bicolor), XP_003530686.1 (Glycine max), XP_003553203.1 (Glycine max),
XP_002315794.1 (Populus trichocarpa), XP_002270149.1 (Vitis vinifera),
XP_003533548.1 (Glycine max), XP_003551723.1 (Glycine max), XP_003621117.1
(Medicago truncatula), XP_002323836.1 (Populus trichocarpa), XP_002517474.1
(Ricinus communis), CAN79925.1 (Vitis vinifera), XP_003572236.1 (Brachypodium
distachyon), BAD10030.1 (Oryza sativa), XP_002444429.1 (Sorghum bicolor),
NP 001170359.1 (Zea mays), XP_002889265.1 (Arabidopsis lyrata subsp. lyrata),
AAF68121.1 (Arabidopsis thaliana), NP 178088.2 (Arabidopsis thaliana),
XP 002890145.1 (Arabidopsis lyrata subsp. lyrata), BAJ33872.1 (Thellungiella
halophila). NP 563990.1 (Arabidopsis thaliana), XP_003530350.1 (Glycine max),
XP 003578142.1 (Brachypodium distachyon), EAZ09147.1 (Oryza sativa),
XP_002460236.1 (Sorghum bicolor), NP 001146338.1 (Zea mays), XP_003519167.1
(Glycine max), XP_003550676.1 (Glycine max), XP 003610261.1 (Medicago
truncatula). XP_003524030.1 (Glycine max), XP_003525949.1 (Glycine max),
XP 002325111.1 (Populus trichocarpa), CBI36586.3 ( Vitis
vinifera),
XP 002273046.2 (Vitis vinifera), XP_002303866.1 (Populus trichocarpa), and
CBI25261.3 (Vitis vinifera). Further examples include Sorbi-WRL1 (SEQ ID
NO:10),
Lupan-WRL1 (SEQ ID NO:11), Ricco-WRL1 (SEQ ID NO:12), and Lupin
angustifolius WRI1 (SEQ ID NO:13). A preferred WRI1 is a maize WRI1 or a
sorghum WRIL In an embodiment, an exogenous polynucleotide of the invention
which encodes a WRI1 which comprises one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
More recently, a subset of WRI1-like transcription factors have been re-
classified as WRI2, WRI3 or WRI4 transcription factors, which are
characterised by
preferential expression in stems and/or roots of plants rather than in
developing seeds
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(To et al., 2012). Despite their re-classification, these are included in the
definition of
"WRI1" herein. Preferred WRI1 -like transcription factors are those which can
complement the function of a wril mutation in a plant, particularly the
function in
developing seed of the plant such as in an A. thaliana wril mutant. The
function of a
WRI1-like polypeptide can also be assayed in the N. benthamiana transient
assays as
described herein.
The WRI1 transcription factor may be endogenous to the plant or cell, or
exogenous to the plant or cell, for example expressed from an exogenous
polynucleotide. The WRI1 transcription factor may be a naturally occurring
WRI1
polypeptide or a variant thereof, provided it retains transcription factor
activity. The
level or activity of an endogenous WRI1 polypeptide may also be increased by
increased expression of a MED15 polypeptide (Kim et al., 2016), for example
polypeptides whose amino acid sequences are provided in Accession No:
NM_101446.4 or NM 001321633.1, or of a 14-3-3 polypeptide (Ma et al., 2016),
for
example Accession Nos: AY079350, AY079350, XM_002445734.1,
XM 002445734.1, NM 001203346, NM 001203346, XM 002445734.1, or
XM_002445734.1. MED15 polypeptide is thought to assist in directing WRI1 to
its
target promoters and expression of WRI1 expression itself, while 14-3-3
polypeptides
are thought to interact with WRI1 polypeptide to increase the WRI1 effect.
As used herein, a "LEAFY COTYLEDON" or "LEC" polypeptide means a
transcription factor which is a LEC I, LEC1-like, LEC2, ABI3 or FUS3
transcription
factor which exhibits broad control on seed maturation and fatty acid
synthesis. LEC2,
FUS3 and ABI3 are related polypeptides that each contain a B3 DNA-binding
domain
of 120 amino acids (Yamasaki et al., 2004) that is only found in plant
proteins. They
can be distinguished by phylogenetic analysis to determine relatedness in
amino acid
sequence to the members of the A. thaliana polypeptides having the Accession
Nos as
follows: LEC2, Accession No. AAL12004.1; FUS3 (also known as FUSCA3),
Accession No. AAC35247. LEC1 belongs to a different class of polypeptides and
is
homologous to a HAP3 polypeptide of the CBF binding factor class (Lee et al.,
2003).
The LEC, LEC2 and FUS3 genes are required in early embryogenesis to maintain
embryonic cell fate and to specify cotyledon identity and in later in
initiation and
maintenance of embryo maturation (Santos-Mendoza et al., 2008). They also
induce
expression of genes encoding seed storage proteins by binding to RY motifs
present in
the promoters, and oleosin genes. They can also be distinguished by their
expression
patterns in seed development or by their ability to complement the
corresponding
mutation in A. thaliana.
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As used herein, the term "Leafy Cotyledon 1" or "LEC1" refers to a NF-YB-
type transcription factor which participates in zygotic development and in
somatic
embryogenesis. The endogenous gene is expressed specifically in seed in both
the
embryo and endosperm. LEC1 activates the gene encoding WRI1 as well as a large
class of fatty acid synthesis genes. Ectopic expression of LEC2 also causes
rapid
activation of auxin-responsive genes and may cause formation of somatic
embryos.
Examples of LEC1 polypeptides include proteins from Arabidopsis thaliana
(AAC39488, SEQ ID NO:31), Medicago truncatula (AFK49653) and Brassica napus
(ADF81045), A. lyrata (XP_002862657), R. communis (XP_002522740). G. max
(XP 006582823), A. hypogaea (ADC33213), Z. mays (AAK95562, SEQ ID NO:32).
In an embodiment, an exogenous polynucleotide of the invention which encodes a
LEC1 which comprises one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
LEC1-like (L1I,) is closely related to LEC1 but has a different pattern of
gene
expression, being expressed earlier during embryogenesis (Kwong et al., 2003).
Examples of LEC1-like polypeptides include proteins from Arabidopsis thaliana
(AAN15924, SEQ ID NO:33), Brassica napus (AHI94922), and Phaseolus coccineus
LEC1 -like (AAN01148).
As used herein, the term "Leafy Cotyledon 2" or "LEC2" refers to a B3 domain
transcription factor which participates in zygotic development and in somatic
embryogenesis and which activates expression of a gene encoding WRIL Its
ectopic
expression facilitates the embryogenesis from vegetative plant tissues
(Alemanno et al.,
2008). Examples of LEC2 polypeptides include proteins from Arabidopsis
thaliana
(Accession No. NP 564304.1), Medicago truncatula (Accession No. CAA42938.1)
and Brassica napus (Accession No. AD016343.1). In an embodiment, an exogenous
polynucleotide of the invention which encodes a LEC2 which comprises one or
more of
the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
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thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
As used herein, the term "FUS3" refers to a B3 domain transcription factor
which participates in zygotic development and in somatic embryogenesis and is
detected mainly in the protodermal tissue of the embryo (Gazzarrini et al.,
2004).
Examples of FUS3 polypcptides include proteins from Arabidopsis thaliana
(AAC35247, SEQ ID NO:34), Brassica napus (XP 006293066.1, SEQ ID NO:35) and
Medicago truncatula (XP_003624470, SEQ ID NO:36). Over-expression of any of
LEC1, L1L, LEC2, FUS3 and ABI3 from an exogenous polynucleotide is preferably
controlled by a developmentally regulated promoter such as a senescence
specific
promoter, an inducible promoter, or a promoter which has been engineered for
providing a reduced level of expression relative to a native promoter,
particularly in
plants other than Arabidopsis thaliana and B. napus cv. Westar, in order to
avoid
developmental abnormalities in plant development that are commonly associated
with
over-expression of these transcription factors (Mu et al., 2008). In an
embodiment, an
exogenous polynucleotide of the invention which encodes a FUS3 which comprises
one
or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
As used herein, the term "BABY BOOM" or "BBM" refers an AP2/ERF
transcription factor that induces regeneration under culture conditions that
normally do
not support regeneration in wild-type plants. Ectopic expression of Brassica
napus
BBM (BnBBM) genes in B. napus and Arabidopsis induces spontaneous somatic
embryogenesis and organogenesis from seedlings grown on hormone-free basal
medium (Boutilier et al., 2002). In tobacco, ectopic BBM expression is
sufficient to
induce adventitious shoot and root regeneration on basal medium, but exogenous
cytokinin is required for somatic embryo (SE) formation (Srinivasan et al.,
2007).
Examples of BBM polypeptides include proteins from Arabidopsis thaliana
(Accession
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No. NP_197245.2, SEQ ID NO:28), maize (US 7579529), Sorghum bicolor (Accession
No. XP 002458927) and Medicago truncatula (Accession No. AAW82334.1). In an
embodiment, an exogenous polynucleotide of the invention which encodes a BBM
which comprises one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
An ABI3 polypeptide (A. thaliana Accession No. NP_189108) is related to the
maize VP1 protein, is expressed at low levels in vegetative tissues and
affects plastid
development. An ABI4 polypeptide (A. thaliana Accession NP_181551) belongs to
a
family of transcription factors that contain a plant-specific AP2 domain
(Finkelstein et
al., 1998) and acts downstream of ABI3. ABI5 (A. thaliana Accession No. NP
565840)
is a transcription factor of the bZ1P family which affects ABA sensitivity and
controls
the expression of some LEA genes in seeds. It binds to an ABA-responsive
element.
Each of the following transcription factors was selected on the basis that
they
functioned in embryogenesis in plants. Accession numbers are provided in Table
8.
Homologs of each can be readily identified in many other plant species and
tested as
described in Example 4.
MYB73 is a transcription factor that has been identified in soybean, involved
in
stress responses.
bZ1P53 is a transcription factor in the bZIP protein family, identified in
Arabidopsis.
AGL15 (Agamous-like 15) is a MADS box transcription factor which is natively
expressed during embryogenesis. AGL15 is also natively expressed in leaf
primordia,
shoot apical meristems and young floral buds, suggesting that AGL15 may also
have a
function during post-germinative development. AGL15 has a role in
embryogenesis
and gibberellic acid catabolism. It targets B3 domain transcription factors
that are key
regulators of embryogenesis.
MYl3115 and MYB118 are transcription factors in the MYB family from
Arabidopsis involved in embryogenesis.
TANMEI also known as EMB2757 encodes a WD repeat protein required for
embryo development in Arabidopsis.
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WUS, also known as Wuschel, is a homeobox gene that controls the stem cell
pool in embryos. It is expressed in the stem cell organizing center of
meristems and is
required to keep the stem cells in an undifferentiated state. The
transcription factor
binds to a TAAT element core motif.
GFR2a1 and GFR2a2 are transcription factors at least from soybean.
Fatty Acyl Acyltransferases
As used herein, the term "fatty acyl acyltransferase" refers to a protein
which is
capable of transferring an acyl group from acyl-CoA, PC or acyl-ACP,
preferably acyl-
CoA or PC, onto a substrate to form TAG, DAG or MAG. These acyltransferases
include DGAT, PDAT, MGAT, GPAT and LPAAT.
As used herein, the term "diacylglycerol acyltransferase" (DGAT) refers to a
protein which transfers a fatty acyl group from acyl-CoA to a DAG substrate to
produce TAG. Thus, the term "diacylglycerol acyltransferase activity" refers
to the
transfer of an acyl group from acyl-CoA to DAG to produce TAG. A DGAT may also
have MGAT function but predominantly functions as a DGAT, i.e., it has greater
catalytic activity as a DGAT than as a MGAT when the enzyme activity is
expressed in
units of nmoles product/min/mg protein (see for example. Yen et al., 2005).
The
activity of DGAT may be rate-limiting in TAG synthesis in seeds (Ichihara et
al.,
1988). DGAT uses an acyl-CoA substrate as the acyl donor and transfers it to
the sn-3
position of DAG to form TAG. The enzyme functions in its native state in the
endoplasmic reticulum (ER) of the cell.
There are three known types of DGAT, referred to as DGAT1, DGAT2 and
DGAT3, respectively. DGAT1 polypeptides are membrane proteins that typically
have
10 transmembrane domains, DGAT2 polypeptides are also membrane proteins but
typically have 2 transmembrane domains, whilst DGAT3 polypeptides typically
have
none and are thought to be soluble in the cytoplasm, not integrated into
membranes.
Plant DGAT1 polypeptides typically have about 510-550 amino acid residues
while
DGAT2 polypeptides typically have about 310-330 residues. DGAT1 is the main
enzyme responsible for producing TAG from DAG in most developing plant seeds,
whereas DGAT2s from plant species such as tung tree (Vernicia fordii) and
castor bean
(Ricinus communis) that produce high amounts of unusual fatty acids appear to
have
important roles in the accumulation of the unusual fatty acids in TAG. Over-
expression
of AtDGAT1 in tobacco leaves resulted in a 6-7 fold increased TAG content
(Bouvier-
Nave et al., 2000).
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Examples of DGAT1 polypeptides include DGAT1 proteins from Aspergillus
fumigatus (XP_755172.1), Arabidopsis thaliana (CAB44774.1; SEQ ID NO:!),
Ricinus communis (AAR11479.1), Vernicia fordii (ABC94472.1), Vernonia
galamensis
(ABV21945.1 and ABV21946.1), Euonymus alatus (AAV31083.1), Caenorhabditis
elegans (AAF82410.1), Rattus norvegicus (NP 445889.!), Homo sapiens
(NP_036211.2), as well as variants and/or mutants thereof. In an embodiment,
an
exogenous polynucleotide of the invention which encodes a DGAT1 which
comprises
one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
Examples of DGAT2 polypeptides include proteins encoded by DGAT2 genes
from Arabidopsis thaliana (NP 566952.1), Ricinus communis (AAY16324.1),
Vernicia
fordii (ABC94474.1), Mortierella ramanniana (AAK84 I 79.1), Homo sapiens
(Q96PD7.2) (Q58HT5.1), Bos taurus (Q7OVZ8.1), Mus muscu/us (AAK84175.1), as
well as variants and/or mutants thereof. DGAT1 and DGAT2 amino acid sequences
show little homology. Expression in leaves of an exogenous DGAT2 was twice as
effective as a DGAT1 in increasing oil content (TAG). Further, A. thaliana
DGAT2
had a greater preference for linoleoyl-CoA and linolenoyl-CoA as acyl donors
relative
to oleoyl-CoA, compared to DGAT1. This substrate preference can be used to
distinguish the two DGAT classes in addition to their amino acid sequences. In
an
embodiment, an exogenous polynucleotide of the invention which encodes a DGAT2
which comprises one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
Examples of DGAT3 polypeptides include proteins encoded by DGAT3 genes
from peanut (Arachis hypogaea, Saha, et al., 2006), as well as variants and/or
mutants
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thereof. A DGAT has little or no detectable MGAT activity, for example, less
than 300
pmol/min/mg protein, preferably less than 200 pmol/min/mg protein, more
preferably
less than 100 pmol/min/mg protein.
In a particularly preferred embodiment, the DGAT has a preference for medium
chain fatty acids. For instance, the DGAT comprises one or more of the
following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in SEQ ID NO:56, or a biologically active fragment thereof, or a
polypeptide whose amino acid sequence is at least 30% identical to SEQ ID
NO:56,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
As used herein, the term "phospholipid:diacylglycerol acyltransferase" (PDAT;
EC 2.3.1.158) or its synonym "phospholipid:1,2-diacyl-sn-glycerol 0-
acyltransferase"
means an acyltransferase that transfers an acyl group from a phospholipid,
typically
PC, to the sn-3 position of DAG to form TAG. This reaction is different to
DGAT and
uses phospholipids as the acyl-donors. Increased expression of PDAT such as
PDAT1,
which may be exogenous or endogenous to the cell or plant of the invention,
increases
the production of TAG from PC. There are several forms of PDAT in plant cells
including PDAT1, PDAT2 or PDAT3 (Ghosal et al., 2007). Sequences of exemplary
PDAT coding regions and polypeptides are provided in Accession Nos:
XM 002462417.1, (Sorghum), NM 001147943 (Zea mays), (Dahlqvist et al., 2000;
Fan et al., 2013a and b; Fan et al., 2014) although any PDAT encoding gene can
be
used. The PDAT may be exogenous or endogenous to the plant or part thereof.
As used herein, the term ''monoacylglycerol acyltransferase" or "MGAT" refers
to a protein which transfers a fatty acyl group from acyl-CoA to a MAG
substrate, for
example sn-2 MAG, to produce DAG. Thus, the term "monoacylglycerol
acyltransferase activity" at least refers to the transfer of an acyl group
from acyl-CoA to
MAG to produce DAG. The term "MGAT" as used herein includes enzymes that act
on sn-1/3 MAG and/or sn-2 MAG substrates to form sn-1,3 DAG and/or sn-1,2/2,3-
DAG, respectively. In a preferred embodiment, the MGAT has a preference for sn-
2
MAG substrate relative to sn-1 MAG, or substantially uses only sn-2 MAG as
substrate. As used herein, MGAT does not include enzymes which transfer an
acyl
group preferentially to LysoPA relative to MAG, such enzymes are known as
LPAATs.
That is, a MGAT preferentially uses non-phosphorylated monoacyl substrates,
even
though they may also have low catalytic activity on LysoPA. A preferred MGAT
does
not have detectable activity in acylating LysoPA. A MGAT may also have DGAT
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function but predominantly functions as a MGAT, i.e., it has greater catalytic
activity
as a MGAT than as a DGAT when the enzyme activity is expressed in units of
nmoles
product/min/mg protein (also see Yen et al., 2002). There are three known
classes of
MGAT, referred to as, MGAT1, MGAT2 and MGAT3, respectively. Examples of
MGAT1, MGAT2 and MGAT3 polypeptides are described in W02013/096993.
As used herein, an "MGAT pathway" refers to an anabolic pathway, different to
the Kennedy pathway for the formation of TAG, in which DAG is formed by the
acylation of either sn-1 MAG or preferably sn-2 MAG, catalysed by MGAT. The
DAG
may subsequently be used to form TAG or other lipids. W02012/000026
demonstrated
firstly that plant leaf tissue can synthesise MAG from G-3-P such that the MAG
is
accessible to an exogenous MGAT expressed in the leaf tissue, secondly MGAT
from
various sources can function in plant tissues, requiring a successful
interaction with
other plant factors involved in lipid synthesis and thirdly the DAG produced
by the
exogenous MGAT activity is accessible to a plant DGAT, or an exogenous DGAT,
to
produce TAG. MGAT and DGAT activity can be assayed by introducing constructs
encoding the enzymes (or candidate enzymes) into Saccharomyces cerevisiae
strain
H1246 and demonstrating TAG accumulation.
Some of the motifs that have been shown to be important for catalytic activity
in
some DGAT2s are also conserved in MGAT acyltransferases. Of particular
interest is
a putative neutral lipid-binding domain with the concensus sequence FLXLVOCN
(SEQ ID NO:6) where each X is independently any amino acid other than proline,
and
N is any nonpolar amino acid, located within the N-terminal transmembrane
region
followed by a putative glycerol/phospholipid acyltransferase domain. The
FLXLXXXN motif (SEQ ID NO:6) is found in the mouse DGAT2 (amino acids 81-88)
and MGAT1/2 but not in yeast or plant DGAT2s. It is important for activity of
the
mouse DGAT2. Other DGAT2 and/or MGAT1/2 sequence motifs include:
1. A highly conserved YFF' tripeptide (SEQ ID NO:2) in most DGAT2
polypeptides and also in MGAT1 and MGAT2, for example, present as amino acids
139-141 in mouse DGAT2. Mutating this motif within the yeast DGAT2 with non-
conservative substitutions rendered the enzyme non-functional.
2. HPHG tetrapeptide (SEQ ID NO:3), highly conserved in MGATs as well as in
DGAT2 sequences from animals and fungi, for example, present as amino acids
161-
164 in mouse DGAT2, and important for catalytic activity at least in yeast and
mouse
DGAT2. Plant DGAT2 acyltransferases have a EPHS (SEQ ID NO:4) conserved
sequence instead, so conservative changes to the first and fourth amino acids
can be
tolerated.
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3. A longer conserved motif which is part of the putative glycerol
phospholipid
domain. An example of this motif is
RXGFX(K/R)XAXXXGXXX(LN)VPXXXFG(E/Q) (SEQ ID NO:5), which is present
as amino acids 304-327 in mouse DGAT2. This motif is less conserved in amino
acid
sequence than the others, as would be expected from its length, but homologs
can be
recognised by motif searching. The spacing may vary between the more conserved
amino acids, i.e., there may be additional X amino acids within the motif, or
less X
amino acids compared to the sequence above.
One important component in glycerolipid synthesis from fatty acids esterified
to
ACP or CoA is the enzyme sn-glycerol-3-phosphate acyltransferase (GPAT), which
is
another of the polypeptides involved in the biosynthesis of non-polar lipids.
This
enzyme is involved in different metabolic pathways and physiological
functions. It
catalyses the following reaction: G3P + fatty acyl-ACP or -CoA --> LPA + free-
ACP or
-CoA. The GPAT-catalyzed reaction occurs in three distinct plant subcellular
compartments: plastid, endoplasmic reticulum (ER) and mitochondria. These
reactions
are catalyzed by three different types of GPAT enzymes, a soluble form
localized in
plastidial stroma which uses acyl-ACP as its natural acyl substrate (PGPAT in
Figure
1), and two membrane-bound forms localized in the ER and mitochondria which
use
acyl-CoA and acyl-ACP as natural acyl donors, respectively (Chen et al.,
2011).
As used herein, the term "glycerol-3-phosphate acyltransferase" (GPAT; EC
2.3.1.15) and its synonym "glycerol-3-phosphate O-acyltransferase" refer to a
protein
which acylates glycerol-3-phosphate (G-3-P) to form LysoPA and/or MAG, the
latter
product forming if the GPAT also has phosphatase activity on LysoPA. The acyl
group
that is transferred is from acyl-CoA if the GPAT is an ER-type GPAT (an "acyl-
CoA:sn-glycerol-3-phosphate 1-0-acyltransferase" also referred to as
"microsomal
GPAT") or from acyl-ACP if the GPAT is a plastidial-type GPAT (PGPAT). Thus,
the
term "glycerol-3-phosphate acyltransferase activity" refers to the acylation
of G-3-P to
form LysoPA and/or MAG. The term "GPAT" encompasses enzymes that acylate G-3-
P to form sn-1 LPA and/or sn-2 LPA, preferably sn-2 LPA. Preferably, the GPAT
which may be over-expressed in the Pull modification is a membrane bound GPAT
that
functions in the ER of the cell, more preferably a GPAT9, and the plastidial
GPAT that
is down-regulated in the Prokaryotic Pathway modification is a soluble GPAT
("plastidial GPAT"). In a preferred embodiment, the GPAT has phosphatase
activity.
In a most preferred embodiment, the GPAT is a sn-2 GPAT having phosphatase
activity which produces sn-2 MAG.
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As used herein, the term "sn-1 glycerol-3-phosphate acyltransferase" (sn-1
GPAT) refers to a protein which acylates sn-glycerol-3-phosphate (G-3-P) to
preferentially form 1-acyl-sn-glycerol-3-phosphate (sn-1 LPA). Thus, the term
"sn-1
glycerol-3-phosphate acyltransferase activity" refers to the acylation of sn-
glycerol-3-
phosphate to form 1-acyl-sn-glycerol-3-phosphate (sn-1 LPA).
As used herein, the term "sn-2 glycerol-3-phosphate acyltransferase" (sn-2
GPAT) refers to a protein which acylates sn-glycerol-3-phosphate (G-3-P) to
preferentially form 2-acyl-sn-glycerol-3-phosphate (sn-2 LPA). Thus, the term
"sn-2
glycerol-3-phosphate acyltransferase activity" refers to the acylation of sn-
glycerol-3-
phosphate to form 2-acyl-sn-glycerol-3-phosphate (sn-2 LTA).
The GPAT family is large and all known members contain two conserved
domains, a plsC acyltransferase domain (PF01553) and a HAD-like hydrolase
(PF12710) superfamily domain and variants thereof. In addition to this, at
least in
Arabidopsis thaliana, GPATs in the subclasses GPAT4-GPAT8 all contain a N-
terminal region homologous to a phosphoserine phosphatase domain (PF00702),
and
GPATs which produce MAG as a product can be identified by the presence of such
a
homologous region. Some GPATs expressed endogenously in leaf tissue comprise
the
conserved amino acid sequence GDLVICPEGTTCREP (SEQ ID NO:7). GPAT4 and
GPAT6 both contain conserved residues that are known to be critical to
phosphatase
activity, specifically conserved amino acids in Motif I (DXDX[T/V][L/V]; SEQ
ID
NO:8) and Motif III (K4G/S][D/S]XXX[D/N]; SEQ ID NO:9) located at the N-
terminus (Yang et at., 2010).
Homologues of Arabidopsis GPAT4 (Accession No. NP_171667.1) and GPAT6
(NP_181346.1) include AAF02784.1 (Arabidopsis thaliana), AAL32544.1
(Arabidopsis thaliana), AAP03413.1 (Oryza sativa), ABK25381.1 (Picea
sitchensis),
ACN34546.1 (Zea Mays), BAF00762.1 (Arabidopsis thaliana), BAH00933.1 (Oryza
sativa), EAY84189.1 (Oryza sativa), EAY98245.1 (Oryza saliva), EAZ21484.1
(Oryza
sativa), EEC71826.1 (Oryza sativa), EEC76137.1 (Otyza sativa), EEE59882.1
(Oryza
sativa), EFJ08963.1 (Selaginella moellendorffii), EFJ11200.1 (Selaginella
moellendorffii), NP 001044839.1 (Oryza sativa), NP 001045668.1 (Oryza sativa),
NP 001147442.1 (Zea mays), NP 001149307.1 (Zea mays), NP 001168351.1 (Zea
mays), AFH02724.1 (Brassica napus) NP_191950.2 (Arabidopsis thaliana),
XP 001765001.1 (Physcomitrella patens), XP_001769671.1 (Physcomitrella
patens),
(Vitis vinifera), XP_002275348.1 (Vitis vinifera), XP_002276032.1 (Vitis
vinifera),
XP 002279091.1 (Vitis vinifera), XP 002309124.1 (Populus trichocarpa),
XP 002309276.1 (Populus trichocarpa), XP 002322752.1 (Populus trichocarpa),
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XP 002323563.1 (Populus trichocarpa), XP_002439887.1 (Sorghum bicolor),
XP 002458786.1 (Sorghum bicolor), XP 002463916.1 (Sorghum bicolor),
XP_002464630.1 (Sorghum bicolor), XP_002511873 .1 (Ricinus communis),
XP_002517438.1 (Ricinus communis), XP 002520171.1 (Ricinus communis),
ACT32032.1 ( Vernicia fordii), NP_001051189.1 (Oryza sativa), AFH02725 .1
(Brassica napus), XP_002320138.1 (Populus trichocarpa), XP_002451377.1
(Sorghum bicolor), XP_002531350.1 (Ricinus communis), and XP_002889361.1
(Arabidopsis lyrata).
In an embodiment, an exogenous polynucleotide of the invention which encodes
a GPAT which comprises one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
In a particularly preferred embodiment, the GPAT, preferablty a GPAT9, has a
preference for utilising medium chain fatty acid substrates. For instance, the
GPAT9
comprises one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of SEQ ID NO:97 to 119, preferably SEQ ID NO:97, or a
biologically active fragment thereof, or a polypeptide whose amino acid
sequence is at
least 30% identical to any one of SEQ ID NO:97 to 119, preferably at least 30%
identical to SEQ ID NO:97,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
The soluble plastidial GPATs (PGPAT, also known as ATS1 in Arabidopsis
thaliana) have been purified and genes encoding them cloned from several plant
species such as pea (Pisum sativum, Accession number: P30706.1), spinach
(Spinacia
oleracea, Accession number: Q43869.1), squash (Cucurbita moschate, Accession
number: P10349.1), cucumber (Cucumis sativus, Accession number: Q39639.1) and
Arabidopsis thaliana (Accession number: Q43307.2). The soluble plastidial GPAT
is
the first committed step for what is known as the prokaryotic pathway of
glycerolipid
synthesis and is operative only in the plastid (Figure 1). The so-called
prokaryotic
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pathway is located exclusively in plant plastids and assembles DAG for the
synthesis of
galactolipids (MGDG and DGMG) which contain C16:3 fatty acids esterified at
the sn-
2 position of the glycerol backbone.
Conserved motifs and/or residues can be used as a sequence-based diagnostic
for the identification of GPAT enzymes. Alternatively, a more stringent
function-based
assay could be utilised. Such an assay involves, for example, feeding labelled
glycerol-
3-phosphate to cells or microsomes and quantifying the levels of labelled
products by
thin-layer chromatography or a similar technique. GPAT activity results in the
production of labelled LPA whilst GPAT/phosphatase activity results in the
production
of labelled MAG.
As used herein, the term "lysophosphatidic acid acyltransferase" (LPAAT; EC
2.3.1.51) and its synonyms "1-acyl-glycerol-3-phosphate acyltransferase",
"acyl-
CoA:1-acyl-sn-glycerol-3-phosphate 2-0-acyltransferase" and "1 -acylglycerol-3-
phosphate 0-acyltransferase" refer to a protein which acylates
lysophosphatidic acid
(LPA) to form phosphatidic acid (PA). The acyl group that is transferred is
from acyl-
CoA if the LPAAT is an ER-type LPAAT or from acyl-ACP if the LPAAT is a
plastidial-type LPAAT (PLPAAT). Thus, the term "lysophosphatidic acid
acyltransferase activity" refers to the acylation of LPA to form PA.
In a particularly preferred embodiment, the LPAAT has a preference for
medium chain fatty acids. For instance, the LPAAT comprises one or more of the
following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in SEQ ID NO:94, or a biologically active fragment thereof, or a
polypeptide whose amino acid sequence is at least 30% identical to SEQ ID
NO:94,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
Oil Body Coating Polypeptides
TAGs are accumulated in plant tissues as subcellular spherical lipid droplets
(LDs, also called oil bodies or lipid bodies) of approximately 0.5-2 m in
diameter. In
seeds, each LD has a matrix of TAGs surrounded by a layer of phospholipids
(PLs) and
structural proteins termed oleosins (Chapman and Ohlrogge, 2012; Hsieh and
Huang,
2004; Murphy, 2012). The small size of LDs provides a large surface area per
unit
TAG, which would facilitate lipase binding and lipolysis during germination
(Huang
and Huang, 2016). Recent proteomics and homology based studies have led to the
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identification of several new protein components involved in the formation,
maintenance, and/or turnover of LDs (Pyc et al., 2017).
Regarding protein structural organization, oleosin comprises an N-terminal
domain, a central hydrophobic domain, and a C-terminal domain (Hsiao and Tzen,
2011). Oleosin-H is distinguished from the other isoform oleosin-L by an extra
18-
residue segment in its C-terminal domain (Tai et al., 2002). Ubiquitin is a
highly-
conserved regulatory protein that attaches to lysine r-amino groups of target
proteins by
its C-terminal glycine residue (Hsiao and Tzen, 2011). Protein ubiquitination
is
integral to many biological pathways such as proteasomal degradation, stress
responses, hormone biosynthesis and signaling, morphogenesis, chromatin
structure,
self-incompatibility, and battling pathogens (Sorokin et al., 2009). Some
studies
suggested that oleosin might be involved in storage lipid degradation after
germination
(Poxleitner et al., 2006). It has been noticed that protein ubiquitination is
involved not
only in the ubiquitin/265 proteasome pathway, but also in various biological
functions
possibly associated with different ubiquitin linkages (Weissman, 2001).
Ectopic
expression of several LD proteins, such as the plant oleosins and SE1PINs as
well as
the human perilipins, was shown to modulate LD morphology and accumulation in
yeast (S. cerevisiae) (Cai et al., 2015). Lipid reserves are metabolized via
the
successive events of lipolysis, fatty acid (FA) transport to glyoxysomes,
activation of
acyl-CoA derivatives, ft-oxidation, glyoxylate cycle, partial tricarboxylic
acid cycle,
and gluconeogenesis (Deruyffelaere et al., 2015).
In an embodiment, the oil body coating polypeptide is non-allergenic, or not
known to be allergenic, such as to humans.
As used herein, the term "Oleosin" refers to an amphipathic protein present in
the membrane of oil bodies in the storage tissues of seeds (see, for example,
Huang,
1996; Tai et al., 2002; Lin et al., 2005; Capuano et at., 2007; Lui et al.,
2009; Shimada
and Hara-Nishimura, 2010) and artificially produced variants (see for example
W02011/053169 and W02011/127118).
Oleosins are of low Mr (15-26,000), corresponding to about 140-230 amino acid
residues, which allows them to become tightly packed on the surface of oil
bodies.
Within each seed species, there are usually two or more oleosins of different
Mr. Each
oleosin molecule contains a relatively hydrophilic, variable N-terminal domain
(for
example, about 48 amino acid residues), a central totally hydrophobic domain
(for
example, of about 70-80 amino acid residues) which is particularly rich in
aliphatic
amino acids such as alanine, glycine, leucine, isoleucine and valine, and an
amphipathic a-helical domain of about 30-40 amino acid residues at or near the
C-
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terminus. The central hydrophobic domain typically contains a proline knot
motif of
about 12 residues at its center. Generally, the central stretch of hydrophobic
residues is
inserted into the lipid core and the amphiphatic N-terminal and/or amphiphatic
C-
terminal are located at the surface of the oil bodies, with positively charged
residues
embedded in a phospholipid monolayer and the negatively charged ones exposed
to the
exterior.
As used herein, the term "Oleosin" encompasses polyoleosins which have
multiple oleosin polypeptides fused together in a head-to-tail fashion as a
single
polypeptide (W02007/045019), for example 2x, 4x or 6x oleosin peptides, and
caleosins which bind calcium and which are a minor protein component of the
proteins
that coat oil bodies in seeds (Froissard et al., 2009), and steroleosins which
bind sterols
(W02011/053169). However, generally a large proportion (at least 80%) of the
oleosins of oil bodies will not be caleosins and/or steroleosins. The term
"oleosin" also
encompasses oleosin polypeptides which have been modified artificially, such
oleosins
which have one or more amino acid residues of the native oleosins artificially
replaced
with cysteine residues, as described in W02011/053169. Typically, 4-8 residues
are
substituted artificially, preferably 6 residues, but as many as between 2 and
14 residues
can be substituted. Preferably, both of the amphipathic N-terminal and C-
teiminal
domains comprise cysteine substitutions. The modification increases the cross-
linking
ability of the oleosins and increases the thermal stability and/or the
stability of the
proteins against degradation by proteases.
A substantial number of oleosin protein sequences, and nucleotide sequences
encoding therefor, are known from a large number of different plant species.
Examples
include, but are not limited to, oleosins from sesame, Arabidposis, canola,
corn, rice,
peanut, castor, soybean, flax, grape, cabbage, cotton, sunflower, sorghum and
barley.
Examples of oleosins (with their Accession Nos) include Brassica napus oleosin
(CAA57545.1.), Brassica napus oleosin S1-1 (ACG69504.1), Brassica napus
oleosin
S2-1 (ACG69503.1), Brassica napus oleosin S3-1 (ACG69513.1), Brassica napus
oleosin S4-1 (ACG69507.1), Brassica napus oleosin S5-1 (ACG69511.1), Arachis
hypogaea oleosin 1 (AAZ20276.1), Arachis hypogaea oleosin 2 (AAU21500.1),
Arachis hypogaea oleosin 3 (AAU21501.1), Arachis hypogaea oleosin 5
(ABC96763.1), Ricinus communis oleosin 1 (EEF40948.1), Ricinus communis
oleosin
2 (EEF51616.1), Glycine max oleosin isoform a (P29530.2), Glycine max oleosin
isoform b (P29531.1), Linum usitatissimum oleosin low molecular weight isoform
(ABB01622.1), Linurn usitatissimum oleosin high molecular weight isoform
(ABB01624.1), Helianthus annuus oleosin (CAA44224.1), Zea mays oleosin
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(NP_001105338.1), Brassica napus steroleosin (ABM30178.1), Brassica napus
steroleosin SLOI -1 (ACG69522.1), Brassica napus steroleosin SL02-1
(ACG69525.1), Sesarnum indicum steroleosin (AAL13315.1), Sesame indicurn
OleosinL (Tai et al., 2002; Accession number AF091840; SEQ ID NO:86), Ficus
purnila var. awkeotsang olcosin L-isoform (Accession No. ABQ57397.1), Cucumis
sativus oleosinL (Accession No. XP 004146901.1), Linum usitatissimum oleosinL
(Accession No. ABB01618.1), Glycine max oleosinL (Accession No.
XP_003556321.2), Ananas comosus oleosinL (Accession No. 0AY72596.1), Se/aria
italica oleosinL (Accession No. XP_004956407.1), Fragaria vesca subsp. vesca
oleosinL (Accession No. XP 004307777.1), Brassica napus oleosinL (Accession
No.
CDY03377.1), Solanum lycopersicum oleosinL (Accession No. XP_004240765.1),
Sesame indicum OleosinH1 (Tai et al., 2002; Accession number AF302807),
Vanilla
planifolia leaf OleosinUl (Huang and Huang, 2016; Accession number SRX648194),
Persea americana mesocarp OleosinM lipid droplet associated protein (Huang and
Huang, 2016; Accession number 5RX627420), Arachis hypogaea Oleosin 3
(Parthibane et al., 2012a and b; Accession number AY722696), A. thaliana
Caleosin3
(Shen et al., 2014; Laibach et al., 2015; Accession number AK317039), A.
thaliana
steroleosin (Accession number AT081653), Zea mays steroleosin (NP
001152614.1),
Brassica napus caleosin CLO-1 (ACG69529.1), Brassica napus caleosin CLO-3
(ACG69527.1), Sesamum indicum caleosin (AAF13743.1), Zea mays caleosin
(NP 001151906.1), Glycine max caleosin (AAB71227). Other lipid encapsulation
polypeptides that are functionally equivalent are plastoglobulins and MLDP
polypeptides (W02011/127118). In an embodiment, an exogenous polynucleotide of
the invention which encodes a oleosin (such as an OleosinL) or steroleosin
which
comprises one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
In an embodiment, the oleosin is oleosinL or an ortholog thereof. OleosinL
lacks the about 18 amino acid H-form insertion towards the C-terminus of other
oleosins (see, for example, Tai et al., 2002). Thus, OleosinL's can readily be
distinguished from other oleosins based on protein alignment.
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As used herein, a "lipid droplet associated protein" or "LDAP" means a
polypeptide which is associated with lipid droplets in plants in tissues or
organs other
than seeds, anthers and pollen, such as fruit tissues including pericarp and
mesocarp.
LDAPs may be associated with oil bodies in seeds, anthers or pollen as well as
in the
tissues or organs other than seeds, anthers and pollen. They are distinct from
oleosins
which are polypeptides associated with the surface of lipid droplets in seed
tissues,
anthers and pollen. LDAPs as used herein include LDAP polypeptides that are
produced naturally in plant tissues as well as amino acid sequence variants
that are
produced artificially. The function of such variants can be tested as
exemplified in
Example 6.
Horn et al. (2013) identified two LDAP genes which are expressed in avocado
pericarp. The encoded avocado LDAP1 and LDAP2 polypeptides were 62% identical
in amino acid sequence and had homology to polypeptide encoded by Arab idopsis
At3g05500 and a rubber tree SRPP-like protein. Gidda et al. (2013) identified
three
LDAP genes that were expressed in oil palm (Elaeis guineensis) mesocarp but
not in
kernels and concluded that LDAP genes were plant specific and conserved
amongst all
plant species. LDAP polypeptides may contain additional domains (Gidda et al.,
(2013). Genes encoding LDAPs are generally up-regulated in non-seed tissues
with
abundant lipid and can be identified thereby, but are thought to be expressed
in all non-
seed cells that produce oil including for transient storage. Horn et al.
(2013) shows a
phylogenetic tree of SRPP-like proteins in plants. Exemplary LDAP polypeptides
are
described in Example 6 and Example 9 herein, such as Rhodococcus opacus TadA
lipid
droplet associated protein (MacEachran et al., 2010; Accession number
HM625859),
Nannochloropsis oceanica LSDP oil body protein (Vieler et al., 2012; Accession
number JQ268559) and Trichoderma reesei HFBI hydrophobin (Linder et al., 2005;
Accession number Z68124). Homologs of LDAPs in other plant species can be
readily
identified by those skilled in the art. In an embodiment, an exogenous
polynucleotide
of the invention which encodes an LDAP which comprises one or more of the
following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
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As used herein, the term a "polypeptide involved in starch biosynthesis"
refers
to any polypeptide, the downregulation of which in a plant cell below normal
(wild-
type) levels results in a reduction in the level of starch synthesis and a
decrease in the
levels of starch. This reduces the flow of carbon from sugars into starch. An
example
of such a polypeptide is AGPase.
As used herein, the term "ADP-glucose phosphorylase" or "AGPase" refers to
an enzyme which regulates starch biosynthesis, catalysing conversion of
glucose-1 -
phosphate and ATP to ADP-glucose which serves as the building block for starch
polymers. The active form of the AGPase enzyme consists of 2 large and 2 small
subunits.
The AGPase enzyme in plants exists primarily as a tetramer which consists of 2
large and 2 small subunits. Although these subunits differ in their catalytic
and
regulatory roles depending on the species (Kuhn et al., 2009), in plants the
small
subunit generally displays catalytic activity. The molecular weight of the
small subunit
is approximately 50-55 kDa. Sequences of exemplary AGPase small subunit
polypeptides are provided in Accession Nos: XM_002462095.1 (Sorghum) and
XM_008666513.1 (Zea mays) (Sanjaya et al., 2011; Zale et al., 2016). The
molecular
weight of the large subunit is approximately 55-60 kDa. The plant enzyme is
strongly
activated by 3-phosphoglycerate (PGA), a product of carbon dioxide fixation;
in the
absence of PGA, the enzyme exhibits only about 3% of its activity. Plant
AGPase is
also strongly inhibited by inorganic phosphate (Pi). In contrast, bacterial
and algal
AGPase exist as homotetramers of 50kDa. The algal enzyme, like its plant
counterpart,
is activated by PGA and inhibited by Pi, whereas the bacterial enzyme is
activated by
fructose-1, 6-bisphosphate (FBP) and inhibited by AMP and Pi.
TAG Lipases and Beta-Oxidation
As used herein, the term "polypeptide involved in the degradation of lipid
and/or
which reduces lipid content" refers to any polypeptide which catabolises
lipid, the
downregulation of which in a plant cell below normal (wild-type) levels
results an
increase in the level of oil, such as fatty acids and/or TAGs, in a cell of a
transgenic
plant or part thereof such as a vegetative part, tuber, beet or a seed.
Examples of such
polypeptides include, but are not limited to, lipases, or a lipase such as a
CGi58
(Comparative Gene identifier-58-Like) polypeptide, a SUGAR-DEPENDENTI (SDP1)
triacylglycerol lipase (see, for example, Kelly et al., 2011) and a lipase
described in
W02009/027335.
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As used herein, the term "TAG lipase" (EC.3.1.1.3) refers to a protein which
hydrolyzes TAG into one or more fatty acids and any one of DAG, MAG or
glycerol.
Thus, the term "TAG lipase activity" refers to the hydrolysis of TAG into
glycerol and
fatty acids.
As used herein, the term "CGi58" refers to a soluble acyl-CoA-dependent
lysophosphatidic acid acyltransferase encoded by the At4g24160 gene in
Arabidopsis
thaliana and its homologs in other plants and "Ictlp" in yeast and its
homologs. The
plant gene such as that from Arabidopsis gene locus At4g24160 is expressed as
two
alternative transcripts: a longer full-length isofonn (At4g24160.1) and a
smaller
isoform (At4g24160.2) missing a portion of the 3' end (see James et al., 2010;
Ghosh et
al., 2009; US 201000221400). Both mRNAs code for a protein that is homologous
to
the human CGI-58 protein and other orthologous members of this a/13 hydrolase
family
(ABHD). In an embodiment, the CGI58 (At4g24160) protein contains three motifs
that
are conserved across plant species: a GXSXG lipase motif (SEQ ID NO:25), a 1-
IX(4)D
acyltransferase motif (SEQ ID NO:26), and VX(3)HGF, a probable lipid binding
motif
(SEQ ID NO:27). The human
CGI-58 protein has lysophosphatidic acid
acyltransferase (LPAAT) activity but not lipase activity. In contrast, the
plant and yeast
proteins possess a canonical lipase sequence motif GXSXG (SEQ ID NO:25), that
is
absent from vertebrate (humans, mice, and zebrafish) proteins, and have lipase
and
phospholipase activity (Ghosh et al., 2009). Although the plant and yeast
CGI58
proteins appear to possess detectable amounts of TAG lipase and phospholipase
A
activities in addition to LPAAT activity, the human protein does not.
Disruption of the homologous CGI-58 gene in Arabidopsis thaliana results in
the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy
of
isolated lipid droplets from cgi-58 loss-of-function mutants showed they
contain
triacylglycerols with common leaf-specific fatty acids. Leaves of mature cgi-
58 plants
exhibit a marked increase in absolute triacylglycerol levels, more than 10-
fold higher
than in wild-type plants. Lipid levels in the oil-storing seeds of cgi-58 loss-
of-function
plants were unchanged, and unlike mutations in 13-oxidation, the cgi-58 seeds
germinated and grew normally, requiring no rescue with sucrose (James et al.,
2010).
Examples of nucleotides encoding CGi58 polypeptides include those from
Arabidopsis thaliana (NM 118548.1 encoding NP 194147.2), Brachypodium
distachyon (XP_003578450.1). Glycine max (XM_003523590.1 encoding
XP 003523638.1), Zea mays (NM 001155541.1 encoding NP 001149013.1),
Sorghum bicolor (XM_002460493.1 encoding XP 002460538.1), Ricinus communis
(XM 002510439.1 encoding XP 002510485.1), Medicago
truncatula
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(XM_O 03603685.1 encoding XP_003603733 .1), and Oryza sativa (encoding
EAZ09782.1). In an embodiment, a genetic modification of the invention down-
regulates endogenous production of CGi58, wherein CGi58 is encoded by one or
more
of the following:
i) nucleotides comprising a sequence set forth a the above mentioned
accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
Other lipases which have lipase activity on TAG include SUGAR-
DEPENDENT1 triacylglycerol lipase (SDP1, see for example Eastmond et al.,
2006;
Kelly et al., 2011) and SDP1-like polypeptides found in plant species as well
as yeast
(TGL4 polypeptide) and animal cells, which are involved in storage TAG
breakdown.
The SDP1 and SDP1-like polypeptides appear to be responsible for initiating
TAG
breakdown in seeds following germination (Eastmond et al., 2006). Plants that
are
mutant in SDP1, in the absence of exogenous WRI1 and DGAT1, exhibit increased
levels of PUFA in their TAG. As used herein, "SDP1 polypeptides" include SDP1
polypeptides, SDP1-like polypeptides and their homologs in plant species. SDP1
and
SDP1-like polypeptides in plants are 800-910 amino acid residues in length and
have a
patatin-like acylhydrolase domain that can associate with oil body surfaces
and
hydrolyse TAG in preference to DAG or MAG. SDP1 is thought to have a
preference
for hydrolysing the acyl group at the sn-2 position of TAG. Arabidopsis
contains at
least three genes encoding SDP1 lipases, namely SDPI (Accession No. NP 196024,
nucleotide sequence SEQ ID NO:37 and homologs in other species), SDP1L
(Accession No. NM 202720 and homologs in other species, Kelly et al., 2011)
and
ATGLL (Atl g33270) (Eastmond et al, 2006). Of particular interest for reducing
gene
activity are SDPI genes which are expressed in vegetative tissues in plants,
such as in
leaves, stems and roots. Levels of non-polar lipids in vegetative plant parts
can
therefore be increased by reducing the activity of SDP1 polypeptides in the
plant parts,
for example by either mutation of an endogenous gene encoding a SDP1
polypeptide or
introduction of an exogenous gene which encodes a silencing RNA molecule which
reduces the expression of an endogenous SDP] gene. Such a reduction is of
particular
benefit in tuber crops such as sugarbeet and potato, and in "high sucrose"
plants such as
sweet sorghum, sugarcane and and sugarbeet.
Genes encoding SDP1 homologues (including SDP1-like homologues) in a plant
species of choice can be identified readily by homology to known SDP1 gene
sequences. Known SDP1 nucleotide or amino acid sequences include Accession
Nos.:
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in Brassica napus, GN078290, GN078281, GN078283; Capsella rubella,
XP 006287072; Theobroma cacao, XP_007028574.1; Populus trichocarpa,
XP 002308909; Prunus persica, XP 007203312; Prunus mume, XP 008240737;
Malus domestica, XP_008373034; Ricinus communis, XP_002530081; Medicago
truncatula, XP_003591425; Solanum lycopersicum, XP_004249208; Phaseolus
vulgaris, XP_007162133; Glycine max, XP 003554141; Solanum tuberosum,
XP_006351284; Glycine max, XP_003521151; Cicer arietinum, XP_004493431;
Cucumis sativus, XP_004142709; Cucumis melo, XP_008457586; Jatropha curcas,
KDP26217; Vitis vinifera, CB130074; Oryza sativa, Japonica Group BAB61223;
Oryza
saliva, Indica Group EAY75912; Oryza sativa, Japonica Group NP_001044325;
Sorghum bicolor, XP 002458531 (SEQ ID NO:38); Brachypodium distachyon,
XP_003567139; Zea mays, AFW85009; Hordeum vulgare, BAK03290; Aegilops
tauschii, EMT32802; Sorghum bicolor, XP_002463665; Zea mays, NP_001168677;
Horde= vulgare, BAK01155; Aegilops tauschii, EMT02623; Triticum urartu,
EMS67257; Physcomitrella patens, XP 001758169. Preferred SDP1 sequences for
use
in genetic constructs for inhibiting expression of the endogenous genes are
from
cDNAs corresponding to the genes which are expressed most highly in the plant
cells,
vegetative plant parts or the seeds, whichever is to be modified. Nucleotide
sequences
which are highly conserved between cDNAs corresponding to all of the SDP1
genes in
a plant species are preferred if it is desired to reduce the activity of all
members of a
gene family in that species. In an embodiment, a genetic modification of the
invention
down-regulates endogenous production of SDP1, wherein SDP1 is encoded by one
or
more of the following:
i) nucleotides comprising a sequence set forth a the above mentioned
accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
As shown in the Examples, reduction of the expression and/or activity of SDP1
TAG lipase in plant leaves greatly increased the TAG content, both in terms of
the
amount of TAG that accumulated and the earlier timing of accumulation during
plant
development, in the context of co-expression of the transcription factor WRI1
and a
fatty acyl acyltransferase. In particular, the increase was observed in plants
prior to
flowering, and was up to about 70% on a weight basis (% dry weight) at the
onset of
senescence. The increase was relative to the TAG levels observed in
corresponding
plant leaves transformed with exogenous polynucleotides encoding the WRI1 and
fatty
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acyl acyltransferase but lacking the modification that reduced SDP1 expression
and/or
activity.
Reducing the expression of other TAG catabolism genes in plant parts can also
increase TAG content, such as the ACX genes encoding acyl-CoA oxidases such as
the
Acxl (At4g16760 and homologs in other plant species) or Acx2 (At5g65110 and
homologs in other plant species) genes. Another polypeptide involved in lipid
catabolism is PXA1 which is a peroxisomal ATP-binding cassette transporter
that is
requires for fatty acid import for 13-oxidation (Zolman et al. 2001).
Export of Fatty Acids from Plastids
As used herein, the term "polypeptide which increases the export of fatty
acids
out of plastids of the cell" refers to any polypeptide which aids in fatty
acids being
transferred from within plastids of plant cells in a plant or part thereof to
outside the
plastid, which may be any other part of the cell such as for example the
endoplasmic
reticulum (ER). Examples of such polypeptides include, but are not limited to,
a C16
or C18 fatty acid thioesterase such as a FATA polypeptide or a FATB
polypeptide, a
C6 to C14 fatty acid thioesterase (which is also a FATB polypeptide), a fatty
acid
transporter such as an ABCA9 polypeptide or a long-chain acyl-CoA synthetase
(LACS).
As used herein, the term "fatty acid thioesterase" or "FAT" or "acyl-ACP
thioesterase" refers to an enzyme which catalyses the hydrolysis of the
thioester bond
between an acyl moiety and acyl carrier protein (ACP) in acyl-ACP and the
release of a
free fatty acid. Such enzymes typically function in the plastids of an
organism which is
synthesizing de novo fatty acids. As used herein, the term "C16 or C18 fatty
acid
thioesterase" refers to an enzyme which catalyses the hydrolysis of the
thioester bond
between a C16 and/or C18 acyl moiety and ACP in acyl-ACP and the release of
free
C16 or C18 fatty acid in the plastid. The free fatty acid is then re-
esterified to CoA in
the plastid envelope as it is transported out of the plastid. The substrate
specificity of
the fatty acid thioesterase (FAT) enzyme in the plastid is involved in
determining the
spectrum of chain length and degree of saturation of the fatty acids exported
from the
plastid. FAT enzymes can be classified into two classes based on their
substrate
specificity and nucleotide sequences, FATA and FATB (EC 3.1.2.14) (Jones et
al.,
1995). FATA polypeptides prefer oleoyl-ACP as substrate, while FATB
polypeptides
show higher activity towards saturated acyl-ACPs of different chain lengths
such as
acting on palmitoyl-ACP to produce free palmitic acid. Examples of FATA
polypeptides useful for the invention include, but are not limited to, those
from
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Arabidopsis thaliana (NP 189147), Arachis hypogaea (GU324446), Helianthus
annuus (AAL79361), Carthamus tinctorius (AAA33020), Morus notabilis
(XP 010104178.1), Brassica napus (CDX77369.1), Ricinus
communis
(XP 002532744.1) and Camelina sativa (AFQ60946.1). Examples of FATB
polypeptides useful for the invention include, but are not limited to, those
from Zea
mays (AIL28766), Brassica napus (ABH11710), Helianthus annuus (AAX19387),
Arabidopsis thaliana (AEE28300), Umbellularia californica (AAC49001), Arachis
hypogaea (AFR54500), Ricinus communis (EEF47013) and Brachypodium sylvaticum
(ABL85052.1). In an embodiment, an exogenous polynucleotide of the invention
which encodes a thioesterase which comprises one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any of the above mentioned accessions, or a biologically
active fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to that set
forth in any of the above mentioned accessions,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
A subclass of FATB polypeptides are fatty acid thioesterases which have
hydrolysis activity on a C6C14 saturated acyl moiety linked by a thioester
bond to
ACP. Such enzymes are also referred to as medium chain fatty acid (MCFA)
thioesterases or MC-FAT enzymes. Such enzymes may also have thioesterase
activity
on C16-ACP, indeed they may have greater thioesterase activity on a C16 acyl-
ACP
substrate than on a MCFA-ACP substrate, nevertheless they are considered
herein to be
an MCFA thioesterase if they produce at least 0.5% MCFA in the total fatty
acid
content when expressed exogenously in a plant cell. Examples of MCFA
thioesterases
are given in Example 10 herein. In a particularly preferred embodiment, the
thioesterase has a preference for hydrolysing medium chain fatty acid
substartes. For
instance, the thioesterease comprises one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in any one of SEQ ID NOs 87 to 93, or a biologically active
fragment
thereof, or a polypeptide whose amino acid sequence is at least 30% identical
to any
one or more of both of SEQ ID NOs 87 to 93,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
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More particularly preferred embodiment, the thioesterease is a C12:0-ACP
thioestersae which comprises one or more of the following:
i) nucleotides encoding a polypeptide comprising amino acids whose sequence
is set forth in SEQ ID NO:93, or a biologically active fragment thereof, or a
polypeptide whose amino acid sequence is at least 30% identical to SEQ ID
NO:93,
ii) nucleotides whose sequence is at least 30% identical to i), and
iii) a polynucleotide which hybridizes to one or both of i) or ii) under
stringent
conditions.
As used herein, the term "fatty acid transporter" relates to a polypeptide
present
in the plastid membrane which is involved in actively transferring fatty acids
from a
plastid to outside the plastid. Examples of ABCA9 (ABC transporter A family
member
9) polypeptides useful for the invention include, but are not limited to,
those from
Arabidopsis thaliana (Q9FLT5), Capsella rubella (XP_006279962.1), Arabis
alpine
(KFK27923.1), Camelina saliva (XP 010457652.1), Brassica napus (CDY23040.1)
and Brassica rapa (XP_009136512.1).
As used herein, the term "acyl-CoA synthetase" or "ACS" (EC 6.2.1.3) means a
polypeptide which is a member of a ligase family that catalyzes the formation
of fatty
acyl-CoA by a two-step process proceeding through an adenylated intermediate,
using
a non-esterified fatty acid, CoA and ATP as substrates to produce an acyl-CoA
ester,
AMP and pyrophosphate as products. As used herein, the term "long-chain acyl-
CoA
synthetase" (LACS) is an ACS that has activity on at least a C18 free fatty
acid
substrate although it may have broader activity on any of C14-C20 free fatty
acids. The
endogenous plastidial LACS enzymes are localised in the outer membrane of the
plastid and function with fatty acid thioesterase for the export of fatty
acids from the
plastid (Schnurr et al., 2002). In Arabidopsis, there are at least nine
identified LACS
genes (Shockey et al., 2002). Preferred LACS polypeptides are of the LACS9
subclass,
which in Arabidopsis is the major plastidial LACS. Examples of LACS
polypeptides
useful for the invention include, but arc not limited to, those from
Arabidopsis thaliana
(Q9CAP8), Camelina sativa (XP 010416710.1), Capsella rubella (XP 006301059.1),
Brassica napus (CDX79212.1), Brassica rapa (XP_009104618.1), Gossypium
raimondii (XP 012450538.1) and Vitis Vinifera (XP 002285853.1). Homologs of
the
above mentioned polypeptides in other species can readily be identified by
those skilled
in the art.
Polypeptides Involved in Diacylglycerol (DAG) Production
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=
Levels of non-polar lipids in, for example, vegetative plant parts can also be
increased by reducing the activity of polypeptides involved in diacylglycerol
(DAG)
production in the plastid in the plant parts, for example by either mutation
of an
endogenous gene encoding such a polypeptide or introduction of an exogenous
gene
which encodes a silencing RNA molecule which reduces the expression of a
target gene
involved in diacylglycerol (DAG) production in the plastid.
As used herein, the term "polypeptide involved in diacylglycerol (DAG)
production in the plastid" refers to any polypeptide in the plastid of plant
cells in a plant
or part thereof that is directly involved in the synthesis of diacylglycerol.
Examples of
such polypeptides include, but are not limited to, a plastidial GPAT, a
plastidial
LPAAT or a plastidial PAP.
GPATs are described elsewhere in the present document. Examples of plastidial
GPAT polypeptides which can be targeted for down-regulation in the invention
include, but are not limited to, those from Arabidopsis thaliana (BAA00575),
Capsella
rubella (XP 006306544.1), Camelina sativa (010499766.1), Brassica napus
(CDY43010.1), Brassica rapa (XP_009145198.1), Helianthus annuus (ADV16382.1)
and Citrus unshiu (BAB79529.1). Homologs in other species can readily be
identified
by those skilled in the art.
LPAATs are described elsewhere in the present document. As the skilled
person would appreciate, plastidial LPAATs to be targeted for down-regulation
for
reducing DAG synthesis in the plastid are not endogenous LPAATs which function
outside of the plastid such as those in the ER, for example being useful for
producing
TAG comprising medium chain length fatty acids. Examples of plastidial LPAAT
polypeptides which can be targeted for down-regulation in the invention
include, but
are not limited to, those from Brassica napus (ABQ42862), Brassica rapa
(XP_009137939.1), Arabidopsis thaliana (NP 194787.2), Camelina saliva
(XP_010432969.1), Glycine max (XP_006592638.1) and Solanum tuberosum
(XP 006343651.1). Homologs in other species of the above mentioned
polypeptides
can readily be identified by those skilled in the art.
As used herein, the term "phosphatidic acid phosphatase" (PAP) (EC 3.1.3.4)
refers to a protein which hydrolyses the phosphate group on 3-sn-phosphatidate
to
produce 1,2-diacyl-sn-glycerol (DAG) and phosphate. Examples of plastidial PAP
polypeptides which can be targeted for down-regulation in the invention
include, but
are not limited to, those from Arabidopsis thaliana (Q6NLA5), Capsella rubella
(XP 006288605.1), Camelina sativa (XP 010452170.1), Brassica napus
(CDY10405.1), Brassica rapa (XP_009122733.1), Glycine max (XP_003542504.1)
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and Solanum tuberosum (XP_006361792.1). Homologs in other species of the above
mentioned polypeptides can readily be identified by those skilled in the art.
Another enzyme that results in DAG production, but in the ER rather than the
plastid, is PDCT. As used herein, the term "phosphatidylcholine:diacylglycerol
cholinephosphotransferase" (PDCT; EC 2.7.8.2) means an
cholinephosphotransferase
that transfers a phospho-choline headgroup from a phospholipid, typically PC,
to
produce DAG, or the reverse reaction to produce PC from DAG. Thus, the two
substrates of the forward reaction are cytidine monophosphate (CMP) and
phosphatidylcholine and the two products are CDP-choline and DAG. PDCT belongs
to the phosphatidic acid phosphatase-related protein family and typically
possesses
lipid phosphatase/phosphotransferase (LPT) domains. In Arabidopsis thaliana,
PDCT
is encoded by the ROD] (At3g15820) and ROD2 (At3g15830) genes (Lu etal.,
2009).
Homologous genes are readily identified in other plant species (Guan et al.,
2015).
Sequences of exemplary PDCT coding regions and polypeptides are provided in,
Accession Nos XM 002437214 and EU973573.1), although any PDCT encoding gene
can be used. In an embodiment, the PDCT is other than A. thaliana PDCT (Lu et
al.,
2009). Increased expression of PDCT, which may be exogenous or endogenous to
the
cell or plant of the invention and which is preferably expressed from an
exogenous
polynucleotide, increases the flow of esterified acyl groups from PC to DAG
and
thereby increases the TTQ in the total fatty acid content and the level of TAG
in
vegetative plant parts or cells of the invention. Alternatively, decreasing
the level of
PDCT activity in the cell or plant by mutation in the gene or by a silencing
RNA
molecule reduces the production of PC from DAG, the reverse PDCT reaction.
Import of Fatty Acids into Plastids
Levels of non-polar lipids in vegetative plant parts can also be increased by
reducing the activity of TGD polypeptides in the plant parts, for example by
either
mutation of an endogenous gene encoding a TGD polypeptide or introduction of
an
exogenous gene which encodes a silencing RNA molecule which reduces the
expression of an endogenous TGD gene. As used herein, a
"Trigalactosyldiacylglycerol
(TGD) polypeptide" is one which is involved in the ER to chloroplast lipid
trafficking
(Xu et al., 2010; Fan et al.. 2015) and involved in forming a protein complex
which has
permease function for lipids. Four such polypeptides are known to form or be
associated with a TGD permease, namely TGD-1 (Accession No. At1g19800 and
homologs in other species), TGD-2 (Accession No At2g20320 and homologs in
other
species), TGD-3 (Accession No. NM-105215 and homologs in other species) and
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=
TGD-4 (At3g06960 and homologs in other species) (US 20120237949). TGD5 is also
involved in ER to choroplast lipid trafficking, and down-regulation of TGD5 is
associated with increased oil production (US2015/337017; Fan et al., 2015).
Sequences
of exemplary TGD5 polypeptides are provided in Accession Nos XM_002442154 and
EU972796.1). TGD-1, -2 and -3 polypeptides are thought to be components of an
ATP-
Binding Cassette (ABC) transporter associated with the inner envelope membrane
of
the chloroplast. TGD-2 and TGD-4 polypeptides bind to phosphatidic acid
whereas
TGD-3 polypetide functions as an ATPase in the chloroplast stroma. As used
herein,
an "endogenous TGD gene" is a gene which encodes a TGD polypeptide in a plant.
Mutations in TGD-1 gene in A. thaliana caused accumulation of
triacylglycerols,
oligogalactolipids and phosphatidic acid (PA) (Xu et al., 2005). Mutations in
TGD
genes or SDP1 genes, or indeed in any desired gene in a plant, can be
introduced in a
site-specific manner by artificial zinc finger nuclease (ZEN), TAL effector
(TALEN) or
CRISPR technologies (using a Cas9 type nuclease) as known in the art.
Preferred
exogenous genes encoding silencing RNAs are those encoding a double-stranded
RNA
molecule such as a hairpin RNA or an artificial microRNA precursor.
Sucrose Metabolism
The TAG levels and/or the TTQ of the total fatty content in the cells, plants
and
plant parts of the invention can also be increased by modifying sucrose
metabolism,
particularly in the stems of plants such as sugarcane, Sorghum and Zea mays.
In an
embodiment, this is achieved by increasing expression of a sucrose metabolism
polypeptide such as invertase or sucrose synthase, or of a sucrose transport
polypeptide
such as SUSI, SUS4, SUT2, SUT4, or SWEET. The effect of these polypeptides is
to
increase the supply of sucrose and its monosaccharide components in the
cytosol of the
cells and/or to decrease the transfer and/or storage of sucrose in the
vacuoles of the
cells, particularly in stem cells. Sequences of examples of these polypeptides
are
provided in SEQ ID NOs:274-292 of WO 2016/004473. Invertase such as bCIN, INV2
or INV3 acts to convert sucrose into hexoses which can be exported from the
vacuoles
into the cytoplasm (McKinley et al., 2016). Increased expression of SUSI or
SUS4
breaks down cytosolic sucrose into hexoses for glycolysis and de novo fatty
acid
synthesis rather than transfer of the sucrose into vacuoles, such as in stem
parenchyma
cells (McKinley et al., 2016). Increased expression of sugar transport
polypeptides
such as tonoplast sucrose exporter, for example SUT2 or SUT4. or SWEET
polypeptide releases vacuolar sucrose for cytosolic glycolysis and increases
de novo
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fatty acid biosynthesis (Bihmidine et al., 2016; Qazi et al., 2012; Schneider
et al., 2012;
Hedrich et al., 2015; Klemens et al., 2013).
The TAG levels and/or the TTQ of the total fatty content in the cells, plants
and
plant parts of the invention can also be increased by reducing the level of
TST
polypeptides such as TST1 or TST2, particularly in the stems of plants such as
sugarcane, Sorghum and Zea mays. TST polypeptide can be decreased by mutation
of
the endogenous genes encoding the polypeptide, or by introduction of an
exogenous
polynucleotide that encodes a silencing RNA molecule. Sequences of exemplary
TST
cDNAs and polypeptides are provided as SEQ ID NOs:266-273 of WO 2016/004473.
Fatty Acid Modifying Enzymes
As used herein, the term "FAD2" refers to a membrane bound delta-12 fatty acid
desturase that desaturates oleic acid (C18:1 9) to produce linoleic acid
(C18:29'12).
As used herein, the term ''epoxygenase" or "fatty acid epoxygenase" refers to
an
enzyme that introduces an epoxy group into a fatty acid resulting in the
production of
an epoxy fatty acid. In preferred embodiment, the epoxy group is introduced at
the
12th carbon on a fatty acid chain, in which case the epoxygenase is a Al2-
epoxygenase,
especially of a C16 or C18 fatty acid chain. The epoxygenase may be a A9-
epoxygenase, a Al5 epoxygenase, or act at a different position in the acyl
chain as
known in the art. The epoxygenase may be of the P450 class. Preferred
epoxygenases
are of the mono-oxygenase class as described in W098/46762. Numerous
epoxygenases or presumed epoxygenases have been cloned and are known in the
art.
Further examples of expoxygenases include proteins comprising an amino acid
sequence provided in SEQ ID NO:21 of WO 2009/129582, polypeptides encoded by
genes from Crepis pakastina (CAA76156, Lee et al., 1998), Stokesia laevis
(AAR23815) (monooxygenase type), Euphorbia lagascae (AAL62063) (P450 type),
human CYP2J2 (arachidonic acid epoxygenase, U37143); human CYPIA1 (arachidonic
acid epoxygenase, K03191), as well as variants and/or mutants thereof.
As used herein, the term, "hydroxylase" or "fatty acid hydroxylase" refers to
an
enzyme that introduces a hydroxyl group into a fatty acid resulting in the
production of
a hydroxylated fatty acid. In a preferred embodiment, the hydroxyl group is
introduced
at the 2nd, 12th and/or 17th carbon on a C18 fatty acid chain. Preferably, the
hydroxyl
group is introduced at the 12th carbon, in which case the hydroxylase is a Al2-
hydroxylase. In another preferred embodiment, the hydroxyl group is introduced
at the
15th carbon on a C16 fatty acid chain. Hydroxylases may also have enzyme
activity as
a fatty acid desaturase. Examples of genes encoding Al2-hydroxylases include
those
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from Ricinus communis (AAC9010, van de Loo 1995); Physaria lindheimeri,
(ABQ01458, Dauk et al., 2007); Lesquerella fendleri, (AAC32755, Broun et al.,
1998);
Daucus carota, (AAK30206); fatty acid hydroxylases which hydroxylate the
terminus
of fatty acids, for example: A, thaliana CYP86A1 (P48422, fatty acid co-
hydroxylase);
Vicia sativa CYP94A1 (P98188, fatty acid co-hydroxylase); mouse CYP2E1
(X62595,
lauric acid to-1 hydroxylase); rat CYP4A1 (M57718, fatty acid co-hydroxylase),
as well
as as variants and/or mutants thereof.
As used herein, the term "conjugase" or "fatty acid conjugase" refers to an
enzyme capable of forming a conjugated bond in the acyl chain of a fatty acid.
Examples of conjugases include those encoded by genes from Calendula
officinalis
(AF343064, Qiu et al., 2001); Vernicia fordii (AAN87574, Dyer et al., 2002);
Punica
granatum (AY178446, lwabuchi et al., 2003) and Trichosanthes kirilowii
(AY178444,
Iwabuchi et al., 2003); as well as as variants and/or mutants thereof.
As used herein, the term "acetylenase" or "fatty acid acetylenase" refers to
an
enzyme that introduces a triple bond into a fatty acid resulting in the
production of an
acetylenic fatty acid. In a preferred embodiment, the triple bond is
introduced at the
2nd, 6th, 12th and/or 17th carbon on a C18 fatty acid chain. Examples
acetylenases
include those from Helianthus annuus (AA038032, ABC59684), as well as as
variants
and/or mutants thereof.
Examples of such fatty acid modifying genes include proteins according to the
following Accession Numbers which are grouped by putative function, and
homologues from other species: Al2-acetylenases ABC00769, CAA76158,
AA038036, AA038032; Al2 conjugases AAG42259, AAG42260, AAN87574; Al2-
desaturases P46313, ABS18716, AAS57577, AAL61825, AAF04093, AAF04094; Al2
epoxygenases XP_001840127, CAA76156, AAR23815; Al2-hydroxylases ACF37070,
AAC32755, ABQ01458, AAC49010; and Al2 P450 enzymes such as AF406732.
Silencing Suppressors
In an embodiment, a transgenic plant or part thereof of the invention may
comprise a silencing suppressor.
As used herein, a "silencing suppressor" enhances transgene expression in a
plant or part thereof of the invention. For example, the presence of the
silencing
suppressor results in higher levels of a polypeptide(s) produced an exogenous
polynucleotide(s) in a plant or part thereof of the invention when compared to
a
corresponding plant or part thereof lacking the silencing suppressor. In an
embodiment, the silencing suppressor preferentially binds a dsRNA molecule
which is
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21 base pairs in length relative to a dsRNA molecule of a different length.
This is a
feature of at least the p19 type of silencing suppressor, namely for p19 and
its
functional orthologs. In another embodiment, the silencing suppressor
preferentially
binds to a double-stranded RNA molecule which has overhanging 5' ends relative
to a
corresponding double-stranded RNA molecule having blunt ends. This is a
feature of
the V2 type of silencing suppressor, namely for V2 and its functional
orthologs. In an
embodiment, the dsRNA molecule, or a processed RNA product thereof, comprises
at
least 19 consecutive nucleotides, preferably whose length is 19-24 nucleotides
with 19-
24 consecutive basepairs in the case of a double-stranded hairpin RNA molecule
or
processed RNA product, more preferably consisting of 20, 21, 22, 23 or 24
nucleotides
in length, and preferably comprising a methylated nucleotide, which is at
least 95%
identical to the complement of the region of the target RNA, and wherein the
region of
the target RNA is i) within a 5' untranslated region of the target RNA, ii)
within a 5'
half of the target RNA, iii) within a protein-encoding open-reading frame of
the target
RNA, iv) within a 3' half of the target RNA, or v) within a 3' untranslated
region of the
target RNA.
Further details regarding silencing suppressors are well known in the art and
described in WO 2013/096992 and WO 2013/096993.
Polynucleotides
The terms "polynucleotide", and "nucleic acid" are used interchangeably. They
refer to a polymeric form of nucleotides of any length, either
deoxyribonucleotides or
ribonucleotides, or analogs thereof. A polynucleotide of the invention may be
of
genomic, cDNA, semisynthetic, or synthetic origin, double-stranded or single-
stranded
and by virtue of its origin or manipulation: (1) is not associated with all or
a portion of
a polynucleotide with which it is associated in nature, (2) is linked to a
polynucleotide
other than that to which it is linked in nature, or (3) does not occur in
nature. The
following are non-limiting examples of polynucleotides: coding or non-coding
regions
of a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA
(tRNA), ribosomal RNA (rRNA), ribozymes, cDNA, recombinant polynucleotides,
plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence,
chimeric DNA of any sequence, nucleic acid probes, and primers. For in vitro
use, a
polynucleotide may comprise modified nucleotides such as by conjugation with a
labeling component.
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As used herein, an "isolated polynucleotide" refers to a polynucleotide which
has been separated from the polynucleotide sequences with which it is
associated or
linked in its native state, or a non-naturally occurring polynucleotide.
As used herein, the term "gene" is to be taken in its broadest context and
includes the deoxyribonucleotide sequences comprising the transcribed region
and, if
translated, the protein coding region, of a structural gene and including
sequences
located adjacent to the coding region on both the 5' and 3' ends for a
distance of at least
about 2 kb on either end and which are involved in expression of the gene. In
this
regard, the gene includes control signals such as promoters, enhancers,
termination
and/or polyadenylation signals that are naturally associated with a given
gene, or
heterologous control signals, in which case, the gene is referred to as a
"chimeric gene".
The sequences which are located 5' of the protein coding region and which are
present
on the mRNA are referred to as 5' non-translated sequences. The sequences
which are
located 3' or downstream of the protein coding region and which are present on
the
mRNA are referred to as 3' non-translated sequences. The term "gene"
encompasses
both cDNA and genomic forms of a gene. A genomic form or clone of a gene
contains
the coding region which may be interrupted with non-coding sequences termed
"introns", "intervening regions", or "intervening sequences." Introns are
segments of a
gene which are transcribed into nuclear RNA (nRNA). Introns may contain
regulatory
elements such as enhancers. Introns are removed or "spliced out" from the
nuclear or
primary transcript; introns are therefore absent in the mRNA transcript. A
gene which
contains at least one intron may be subject to variable splicing, resulting in
alternative
mRNAs from a single transcribed gene and therefore polypeptide variants. A
gene in its
native state, or a chimeric gene may lack introns. The mRNA functions during
translation to specify the sequence or order of amino acids in a nascent
polypeptide.
The term "gene" includes a synthetic or fusion molecule encoding all or part
of the
proteins of the invention described herein and a complementary nucleotide
sequence to
any one of the above.
As used herein, "chimeric DNA" refers to any DNA molecule that is not
naturally found in nature; also referred to herein as a "DNA construct" or
"genetic
construct". Typically, a chimeric DNA comprises regulatory and transcribed or
protein
coding sequences that are not naturally found together in nature. Accordingly,
chimeric DNA may comprise regulatory sequences and coding sequences that are
derived from different sources, or regulatory sequences and coding sequences
derived
from the same source, but arranged in a manner different than that found in
nature. The
open reading frame may or may not be linked to its natural upstream and
downstream
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regulatory elements. The open reading frame may be incorporated into, for
example,
the plant genome, in a non-natural location, or in a replicon or vector where
it is not
naturally found such as a bacterial plasmid or a viral vector. The term
"chimeric DNA"
is not limited to DNA molecules which are replicable in a host, but includes
DNA
capable of being ligated into a replicon by, for example, specific adaptor
sequences.
A "transgene" is a gene that has been introduced into the genome by a
transformation procedure. The term includes a gene in a progeny plant or part
thereof
such as a vegetative plant part which was introducing into the genome of a
progenitor
cell thereof. Such progeny cells etc may be at least a 3rd or 4th generation
progeny from
the progenitor cell which was the primary transformed cell, or of the
progenitor
transgenic plant (referred to herein as a TO plant). Progeny may be produced
by sexual
reproduction or vegetatively such as, for example, from tubers in potatoes or
ratoons in
sugarcane. The term "genetically modified", "genetic modification" and
variations
thereof, is a broader term that includes introducing a gene into a cell by
transformation
or transduction, mutating a gene in a cell and genetically altering or
modulating the
regulation of a gene in a cell, or the progeny of any cell modified as
described above.
A "genomic region" as used herein refers to a position within the genome where
a transgene, or group of transgenes (also referred to herein as a cluster),
have been
inserted into a cell, or predecessor thereof. Such regions only comprise
nucleotides that
have been incorporated by the intervention of man such as by methods described
herein.
A "recombinant polynucleotide" of the invention refers to a nucleic acid
molecule which has been constructed or modified by artificial recombinant
methods.
The recombinant polynucleotide may be present in a cell of a plant or part
thereof in an
altered amount or expressed at an altered rate (e.g., in the case of mRNA)
compared to
its native state. In one embodiment, the polynucleotide is introduced into a
cell that
does not naturally comprise the polynucleotide. Typically an exogenous DNA is
used
as a template for transcription of mRNA which is then translated into a
continuous
sequence of amino acid residues coding for a polypeptide of the invention
within the
transformed cell. In another embodiment, the polynucleotide is endogenous to
the
plant or part thereof and its expression is altered by recombinant means, for
example,
an exogenous control sequence is introduced upstream of an endogenous gene of
interest to enable the transformed plant or part thereof to express the
polypeptide
encoded by the gene, or a deletion is created in a gene of interest by ZFN,
Talen or
CRISPR methods.
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A recombinant polynucleotide of the invention includes polynucleotides which
have not been separated from other components of the cell-based or cell-free
expression system, in which it is present, and polynucleotides produced in
said cell-
based or cell-free systems which are subsequently purified away from at least
some
other components. The polynucleotide can be a contiguous stretch of
nucleotides or
comprise two or more contiguous stretches of nucleotides from different
sources
(naturally occurring and/or synthetic) joined to form a single polynucleotide.
Typically, such chimeric polynucleotides comprise at least an open reading
frame
encoding a polypeptide of the invention operably linked to a promoter suitable
of
driving transcription of the open reading frame in a cell of interest.
With regard to the defined polynucleotides, it will be appreciated that %
identity
figures higher than those provided above will encompass preferred embodiments.
Thus, where applicable, in light of the minimum % identity figures, it is
preferred that
the polynucleotide comprises a polynucleotide sequence which is at least 60%,
more
preferably at least 65%, more preferably at least 70%, more preferably at
least 75%,
more preferably at least 80%, more preferably at least 85%, more preferably at
least
90%, more preferably at least 91%, more preferably at least 92%, more
preferably at
least 93%, more preferably at least 94%, more preferably at least 95%, more
preferably
at least 96%, more preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, more preferably at least 99.1%, more preferably at
least 99.2%,
more preferably at least 99.3%, more preferably at least 99.4%, more
preferably at least
99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more
preferably
at least 99.8%, and even more preferably at least 99.9% identical to the
relevant
nominated SEQ ID NO.
A polynucleotide of, or useful for, the present invention may selectively
hybridise, under stringent conditions, to a polynucleotide defined herein. As
used
herein, stringent conditions are those that: (1) employ during hybridisation a
denaturing
agent such as formamide, for example, 50% (v/v) formamide with 0.1% (w/v)
bovine
serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate
buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 C; or (2) employ
50%
formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate
(pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon
sperm
DNA (50 g/m1), 0.1% SDS and 10% dextran sulfate at 42 C in 0.2 x SSC and 0.1%
SDS, and/or (3) employ low ionic strength and high temperature for washing,
for
example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50 C.
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Polynucleotides of the invention may possess, when compared to naturally
occurring molecules, one or more mutations which are deletions, insertions, or
substitutions of nucleotide residues. Polynucleotides which have mutations
relative to
a reference sequence can be either naturally occurring (that is to say,
isolated from a
natural source) or synthetic (for example, by performing site-directed
mutaRenesis or
DNA shuffling on the nucleic acid as described above).
Polynucleotides for Reducing Expression of Genes
RNA Interference
RNA interference (RNAi) is particularly useful for specifically reducing the
expression of a gene, which results in reduced production of a particular
protein if the
gene encodes a protein. Although not wishing to be limited by theory,
Waterhouse et
al. (1998) have provided a model for the mechanism by which dsRNA (duplex RNA)
can be used to reduce protein production. This technology relies on the
presence of
dsRNA molecules that contain a sequence that is essentially identical to the
mRNA of
the gene of interest or part thereof. Conveniently, the dsRNA can be produced
from a
single promoter in a recombinant vector or host cell, where the sense and anti-
sense
sequences are flanked by an unrelated sequence which enables the sense and
anti-sense
sequences to hybridize to form the dsRNA molecule with the unrelated sequence
forming a loop structure. The design and production of suitable dsRNA
molecules is
well within the capacity of a person skilled in the art, particularly
considering
Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619, WO 99/53050, WO
99/49029, and WO 01/34815.
In one example, a DNA is introduced that directs the synthesis of an at least
partly double stranded RNA product(s) with homology to the target gene to be
inactivated such as, for example, a SDP], TGD, plastidial GPAT, plastidial
LPAAT,
plastidial PAP, AGPase gene. The DNA therefore comprises both sense and
antisense
sequences that, when transcribed into RNA, can hybridize to form the double
stranded
RNA region. In one embodiment of the invention, the sense and antisense
sequences
are separated by a spacer region that comprises an intron which, when
transcribed into
RNA, is spliced out. This arrangement has been shown to result in a higher
efficiency
of gene silencing (Smith et al., 2000). The double stranded region may
comprise one
or two RNA molecules, transcribed from either one DNA region or two. The
presence
of the double stranded molecule is thought to trigger a response from an
endogenous
system that destroys both the double stranded RNA and also the homologous RNA
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transcript from the target gene, efficiently reducing or eliminating the
activity of the
target gene.
The length of the sense and antisense sequences that hybridize should each be
at
least 19 contiguous nucleotides, preferably at least 50 contiguous
nucleotides, more
preferably at least 100 or at least 200 contiguous nucleotides. Generally, a
sequence of
100-1000 nucleotides corresponding to a region of the target gene mRNA is
used. The
full-length sequence corresponding to the entire gene transcript may be used.
The
degree of identity of the sense sequence to the targeted transcript (and
therefore also the
identity of the antisense sequence to the complement of the target transcript)
should be
at least 85%, at least 90%, or 95-100%. The RNA molecule may of course
comprise
unrelated sequences which may function to stabilize the molecule. The RNA
molecule
may be expressed under the control of a RNA polymerase II or RNA polymerase
III
promoter. Examples of the latter include tRNA or snRNA promoters.
Preferred small interfering RNA ("siRNA") molecules comprise a nucleotide
sequence that is identical to about 19-25 contiguous nucleotides of the target
mRNA.
Preferably, the siRNA sequence commences with the dinucleotide AA, comprises a
GC-content of about 30-70% (preferably, 30-60%, more preferably 40-60% and
more
preferably about 45%-55%), and does not have a high percentage identity to any
nucleotide sequence other than the target in the genome of the organism in
which it is
to be introduced, for example, as determined by standard BLAST search.
microRNA
MicroRNAs (abbreviated miRNAs) are generally 19-25 nucleotides (commonly
about 20-24 nucleotides in plants) non-coding RNA molecules that are derived
from
larger precursors that form imperfect stem-loop structures. miRNAs bind to
complementary sequences on target messenger RNA transcripts (mRNAs), usually
resulting in translational repression or target degradation and gene
silencing. Artificial
miRNAs (amiRNAs) can be designed based on natural miRNAs for reducing the
expression of any gene of interest, as well known in the art.
In plant cells, miRNA precursor molecules are believed to be largely processed
in the nucleus. The pri-miRNA (containing one or more local double-stranded or
"hairpin" regions as well as the usual 5' "cap" and polyadenylated tail of an
mRNA) is
processed to a shorter miRNA precursor molecule that also includes a stem-loop
or
fold-back structure and is termed the "pre-miRNA". In plants, the pre-miRNAs
are
cleaved by distinct DICER-like (DCL) enzymes, yielding miRNA:miRNA* duplexes.
Prior to transport out of the nucleus, these duplexes are methylated.
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In the cytoplasm, the miRNA strand from the miRNA:miRNA duplex is
selectively incorporated into an active RNA-induced silencing complex (RISC)
for
target recognition.The RISC- complexes contain a particular subset of
Argonaute
proteins that exert sequence-specific gene repression (see, for example,
Millar and
Waterhouse, 2005; F'asquinelli et al., 2005; Almeida and Allshire, 2005).
Cosuppression
Genes can suppress the expression of related endogenous genes and/or
transgenes already present in the genome, a phenomenon termed homology-
dependent
gene silencing. Most of the instances of homologydependent gene silencing fall
into
two classes - those that function at the level of transcription of the
transgene, and those
that operate post-transcriptionally.
Post-transcriptional homology-dependent gene silencing (i.e., cosuppression)
describes the loss of expression of a transgene and related endogenous or
viral genes in
transgenic plants. Cosuppression often, but not always, occurs when transgene
transcripts are abundant, and it is generally thought to be triggered at the
level of
mRNA processing, localization, and/or degradation. Several models exist to
explain
how cosuppression works (see in Taylor, 1997).
Cosuppression involves introducing an extra copy of a gene or a fragment
thereof into a plant in the sense orientation with respect to a promoter for
its
expression. The size of the sense fragment, its correspondence to target gene
regions,
and its degree of sequence identity to the target gene can be determined by
those skilled
in the art. In some instances, the additional copy of the gene sequence
interferes with
the expression of the target plant gene. Reference is made to WO 97/20936 and
EP
0465572 for methods of implementing co-suppression approaches.
Recombinant Vectors
One embodiment of the present invention includes a recombinant vector, which
comprises at least one polynucleotide defined herein and is capable of
delivering the
polynucleotide into a host cell. Recombinant vectors include expression
vectors.
Recombinant vectors contain heterologous polynucleotide sequences, that is,
polynucleotide sequences that are not naturally found adjacent to a
polynucleotide
defined herein, that preferably, are derived from a different species. The
vector can be
either RNA or DNA, and typically is a viral vector, derived from a virus, or a
plasmid.
Plasmid vectors typically include additional nucleic acid sequences that
provide for
easy selection, amplification, and transformation of the expression cassette
in
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prokaryotic cells, e.g., pUC-derived vectors, pGEM-derived vectors or binary
vectors
containing one or more T-DNA regions. Additional nucleic acid sequences
include
origins of replication to provide for autonomous replication of the vector,
selectable
marker genes, preferably encoding antibiotic or herbicide resistance, unique
multiple
cloning sites providing for multiple sites to insert nucleic acid sequences or
genes
encoded in the nucleic acid construct, and sequences that enhance
transformation of
prokaryotic and eukaryotic (especially plant) cells.
"Operably linked" as used herein, refers to a functional relationship between
two
or more nucleic acid (e.g., DNA) segments. Typically, it refers to the
functional
relationship of a transcriptional regulatory element (promoter) to a
transcribed
sequence. For example, a promoter is operably linked to a coding sequence of a
polynucleotide defined herein, if it stimulates or modulates the transcription
of the
coding sequence in an appropriate cell. Generally, promoter transcriptional
regulatory
elements that are operably linked to a transcribed sequence are physically
contiguous to
the transcribed sequence, i.e., they are cis-acting. However, some
transcriptional
regulatory elements such as enhancers need not be physically contiguous or
located in
close proximity to the coding sequences whose transcription they enhance.
When there are multiple promoters present, each promoter may independently
be the same or different.
Recombinant vectors may also contain one or more signal peptide sequences to
enable an expressed polypeptide defined herein to be retained in the
endoplasmic
reticulum (ER) in the cell, or transfer into a plastid, and/or contain fusion
sequences
which lead to the expression of nucleic acid molecules as fusion proteins.
Examples of
suitable signal segments include any signal segment capable of directing the
secretion
or localisation of a polypeptide defined herein.
To facilitate identification of transformants, the recombinant vector
desirably
comprises a selectable or screenable marker gene. By "marker gene" is meant a
gene
that imparts a distinct phenotype to cells expressing the marker gene and
thus, allows
such transformed cells to be distinguished from cells that do not have the
marker. A
selectable marker gene confers a trait for which one can "select" based on
resistance to
a selective agent (e.g., a herbicide, antibiotic). A sereenable marker gene
(or reporter
gene) confers a trait that one can identify through observation or testing,
that is, by
"screening" (e.g., 13-glucuronidase, lueiferase, GFP or other enzyme activity
not present
in untransformed cells). Exemplary selectable markers for selection of plant
transformants include, but are not limited to, a hyg gene which encodes
hygromycin B
resistance; a neomycin phosphotransferase (nptIl) gene conferring resistance
to
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kanamycin, paromomycin; a glutathione-S-transferase gene from rat liver
conferring
resistance to glutathione derived herbicides as for example, described in EP
256223; a
glutamine synthetase gene conferring, upon overexpression, resistance to
glutamine
synthetase inhibitors such as phosphinothricin as for example, described in WO
87/05327; an acetyltransferase gene from Streptomyces viridochromogenes
conferring
resistance to the selective agent phosphinothricin as for example, described
in EP
275957; a gene encoding a 5-enolshikimate-3-phosphate synthase (EPSPS)
conferring
tolerance to N-phosphonomethylglycine as for example, described by Hinchee et
al.
(1988); a bar gene conferring resistance against bialaphos as for example,
described in
W091/02071; a nitrilase gene such as bxn from Klebsiella ozaenae which confers
resistance to bromoxynil (Stalker et al., 1988); a dihydrofolate reductase
(DHFR) gene
conferring resistance to methotrexate (Thillet et al., 1988); a mutant
acetolactate
synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea,
or other
ALS-inhibiting chemicals (EP 154,204): a mutated anthranilate synthase gene
that
confers resistance to 5-methyl tryptophan; or a dalapon dehalogenase gene that
confers
resistance to the herbicide.
Preferably, the recombinant vector is stably incorporated into the genome of
the
cell such as the plant cell. Accordingly, the recombinant vector may comprise
appropriate elements which allow the vector to be incorporated into the
genome, or into
a chromosome of the cell.
Expression Vector
As used herein, an "expression vector" is a DNA vector that is capable of
transforming a host cell and of effecting expression of one or more specified
polynucleotides. Expression vectors of the present invention contain
regulatory
sequences such as transcription control sequences, translation control
sequences,
origins of replication, and other regulatory sequences that are compatible
with the host
cell and that control the expression of polynucleotides of the present
invention. In
particular, expression vectors of the present invention include transcription
control
sequences. Transcription control sequences are sequences which control the
initiation,
elongation, and termination of transcription. Particularly important
transcription
control sequences are those which control transcription initiation such as
promoter,
enhancer, operator and repressor sequences. The choice of the regulatory
sequences
used depends on the target organism such as a plant and/or target organ or
tissue of
interest. Such regulatory sequences may be obtained from any eukaryotic
organism
such as plants or plant viruses, or may be chemically synthesized. A number of
vectors
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suitable for stable transfection of plant cells or for the establishment of
transgenic
plants have been described in for example, Pouwels et al., Cloning Vectors: A
Laboratory Manual, 1985, supp. 1987, Weissbach and Weissbach, Methods for
Plant
Molecular Biology, Academic Press, 1989, and Gelvin et al., Plant Molecular
Biology
Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors
include for example, one or more cloned plant genes under the transcriptional
control
of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant
expression vectors also can contain a promoter regulatory region (e.g., a
regulatory
region controlling inducible or constitutive, environmentally- or
developmentally-
regulated, or cell- or tissue-specific expression), a transcription initiation
start site, a
ribosome binding site, a transcription termination site, and/or a
polyadenylation signal.
A number of constitutive promoters that are active in plant cells have been
described. Suitable promoters for constitutive expression in plants include,
but are not
limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort
mosaic
virus (FMV) 35S, the light-inducible promoter from the small subunit (SSU) of
the
ribulose-1,5-bis-phosphate carboxylase, the rice cytosolic triosephosphate
isomerase
promoter, the adenine phosphoribosyltransferase promoter of Arabidopsis, the
rice
actin 1 gene promoter, the mannopine synthase and octopine synthase promoters,
the
Adh promoter, the sucrose synthase promoter, the R gene complex promoter, and
the
chlorophyll a/P binding protein gene promoter. These promoters have been used
to
create DNA vectors that have been expressed in plants, see for example, WO
84/02913.
All of these promoters have been used to create various types of plant-
expressible
recombinant DNA vectors.
For the purpose of expression in source tissues of the plant such as the leaf,
seed, root or stem, it is preferred that the promoters utilized in the present
invention
have relatively high expression in these specific tissues. For this purpose,
one may
choose from a number of promoters for genes with tissue- or cell-specific, or -
enhanced
expression. Examples of such promoters reported in the literature include, the
chloroplast glutamine synthetase GS2 promoter from pea, the chloroplast
fructose-1,6-
biphosphatase promoter from wheat, the nuclear photosynthetic ST-LS1 promoter
from
potato, the serine/threonine kinase promoter and the glucoamylase (Cl-IS)
promoter
from Arabidopsis thaliana. Also reported to be active in photosynthetically
active
tissues are the ribulose-1,5-bisphosphate carboxylase promoter from eastern
larch
(Larix laricina), the promoter for the Cab gene, Cab6, from pine, the promoter
for the
Cab-1 gene from wheat, the promoter for the Cab-1 gene from spinach, the
promoter
for the Cab 1R gene from rice, the pyruvate, orthophosphate dikinase (PPDK)
promoter
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from Zea mays, the promoter for the tobacco Lhcbl*2 gene, the Arabidopsis
thaliana
Suc2 sucrose-H3 symporter promoter, and the promoter for the thylakoid
membrane
protein genes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS).
Other promoters for the chlorophyll a/3-binding proteins may also be utilized
in the
present invention such as the promoters for LhcB gene and PsbP gene from white
mustard (Sinapis alba).
A variety of plant gene promoters that are regulated in response to
environmental, hormonal, chemical, and/or developmental signals, also can be
used for
expression of RNA-binding protein genes in plant cells, including promoters
regulated
by (1) heat, (2) light (e.g., pea RbcS-3A promoter, maize RbcS promoter), (3)
hormones such as abscisic acid, (4) wounding (e.g., WunI), or (5) chemicals
such as
methyl jasmonate, salicylic acid, steroid hormones, alcohol, Safeners (WO
97/06269),
or it may also be advantageous to employ (6) organ-specific promoters.
As used herein, the term "plant storage organ specific promoter" refers to a
promoter that preferentially, when compared to other plant tissues, directs
gene
transcription in a storage organ of a plant. For the purpose of expression in
sink tissues
of the plant such as the tuber of the potato plant, the fruit of tomato, or
the seed of
soybean, canola, cotton, Zea mays, wheat, rice, and barley, it is preferred
that the
promoters utilized in the present invention have relatively high expression in
these
specific tissues. The promoter for fl-conglycinin or other seed-specific
promoters such
as the napin, zein, linin and phaseolin promoters, can be used. Root specific
promoters
may also be used. An example of such a promoter is the promoter for the acid
chitinase
gene. Expression in root tissue could also be accomplished by utilizing the
root
specific subdomains of the CaMV 35S promoter that have been identified.
In a particularly preferred embodiment, the promoter directs expression in
tissues and organs in which lipid biosynthesis takes place. Such promoters may
act in
seed development at a suitable time for modifying lipid composition in seeds.
Preferred promoters for seed-specific expression include: 1) promoters from
genes
encoding enzymes involved in lipid biosynthesis and accumulation in seeds such
as
desaturases and elongases, 2) promoters from genes encoding seed storage
proteins,
and 3) promoters from genes encoding enzymes involved in carbohydrate
biosynthesis
and accumulation in seeds. Seed specific promoters which are suitable are, the
oilseed
rape napin gene promoter (US 5,608,152), the Vicia faba USP promoter (Baumlein
et
al., 1991), the Arabidopsis oleosin promoter (WO 98/45461), the Phaseolus
vulgaris
phaseolin promoter (US 5,504,200), the Brassica Bce4 promoter (WO 91/13980),
or
the legumin B4 promoter (Baumlein et al., 1992), and promoters which lead to
the
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seed-specific expression in monocots such as maize, barley, wheat, rye, rice
and the
like. Notable promoters which are suitable are the barley 1pt2 or 1ptl gene
promoter
(WO 95/15389 and WO 95/23230), or the promoters described in WO 99/16890
(promoters from the barley hordein gene, the rice glutelin gene, the rice
oryzin gene,
the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the
maize zein
gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene).
Other
promoters include those described by Broun et al. (1998). Potenza et al.
(2004), US
20070192902 and US 20030159173. In an embodiment, the seed specific promoter
is
preferentially expressed in defined parts of the seed such as the cotyledon(s)
or the
endosperm. Examples of cotyledon specific promoters include, but are not
limited to,
the FPI promoter (Ellerstrom et al., 1996), the pea legumin promoter (Perrin
et al.,
2000), and the bean phytohemagglutnin promoter (Perrin et al., 2000). Examples
of
endosperm specific promoters include, but are not limited to, the maize zein-1
promoter
(Chikwamba et al., 2003), the rice glutelin-1 promoter (Yang et al., 2003),
the barley
D-hordein promoter (Horvath et al., 2000) and wheat HMW glutenin promoters
(Alvarez et al., 2000). In a further embodiment, the seed specific promoter is
not
expressed, or is only expressed at a low level, in the embryo and/or after the
seed
germinates.
In another embodiment, the plant storage organ specific promoter is a fruit
specific promoter. Examples
include, but are not limited to, the tomato
polygalacturonase, E8 and Pds promoters, as well as the apple ACC oxidase
promoter
(for review, see Potenza et al., 2004). In a preferred embodiment, the
promoter
preferentially directs expression in the edible parts of the fruit, for
example the pith of
the fruit, relative to the skin of the fruit or the seeds within the fruit.
In an embodiment, the inducible promoter is the Aspergillus nidulans ale
system. Examples of inducible expression systems which can be used instead of
the
Aspergillus nidulans ale system are described in a review by Padidam (2003)
and
Corrado and Karali (2009). In another embodiment, the inducible promoter is a
safener
inducible promoter such as, for example, the maize 1n2-1 or 1n2-2 promoter
(Hershey
and Stoner, 1991), the safener inducible promoter is the maize GST-27 promoter
(Jepson et al., 1994), or the soybean QH2/4 promoter (Ulmasov et al., 1995).
In another embodiment, the inducible promoter is a senescence inducible
promoter such as, for example, senescence-inducible promoter SAG (senescence
associated gene) 12 and SAG 13 from Arabidopsis (Gan, 1995; Gan and Amasino,
1995) and LSC54 from Brassica napus (Buchanan-Wollaston, 1994). Such promoters
=
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show increased expression at about the onset of senescence of plant tissues,
in
particular the leaves.
For expression in vegetative tissue leaf-specific promoters, such as the
ribulose
biphosphate carboxylase (RBCS) promoters, can be used. For example, the tomato
RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and light grown
seedlings
(Meier et al., 1997). A ribulose bisphosphate carboxylase promoters expressed
almost
exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels,
described
by Matsuoka et al. (1994), can be used. Another leaf-specific promoter is the
light
harvesting chlorophyll alb binding protein gene promoter (see, Shiina et al.,
1997). The
Arabidopsis thaliana myb-related gene promoter (Atmyb5) described by Li et al.
(1996), is leaf-specific. The Atmyb5 promoter is expressed in developing leaf
trichomes, stipules, and epidermal cells on the margins of young rosette and
cauline
leaves, and in immature seeds. A leaf promoter identified in maize by Busk et
al.
(1997), can also be used.
In some instances, for example when LEC2 or BBM is recombinantly
expressed, it may be desirable that the transgene is not expressed at high
levels. An
example of a promoter which can be used in such circumstances is a truncated
napin A
promoter which retains the seed-specific expression pattern but with a reduced
expression level (Tan et al., 2011).
The 5' non-translated leader sequence can be derived from the promoter
selected
to express the heterologous gene sequence of the polynucleotide of the present
invention, or may be heterologous with respect to the coding region of the
enzyme to
be produced, and can be specifically modified if desired so as to increase
translation of
mRNA. For a review of optimizing expression of transgenes, see Koziel et al.
(1996).
The 5' non-translated regions can also be obtained from plant viral RNAs
(Tobacco
mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus, Alfalfa mosaic
virus,
among others) from suitable eukaryotic genes, plant genes (wheat and maize
chlorophyll a/b binding protein gene leader), or from a synthetic gene
sequence. The
present invention is not limited to constructs wherein the non-translated
region is
derived from the 5' non-translated sequence that accompanies the promoter
sequence.
The leader sequence could also be derived from an unrelated promoter or coding
sequence. Leader sequences useful in context of the present invention comprise
the
maize Hsp70 leader (US 5,362,865 and US 5,859,347), and the TMV omega element.
The termination of transcription is accomplished by a 3' non-translated DNA
sequence operably linked in the expression vector to the polynucleotide of
interest.
The 3' non-translated region of a recombinant DNA molecule contains a
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polyadenylation signal that functions in plants to cause the addition of
adenylate
nucleotides to the 3' end of the RNA. The 3' non-translated region can be
obtained
from various genes that are expressed in plant cells. The nopaline synthase 3'
untranslated region, the 3' untranslated region from pea small subunit Rubisco
gene, the
3' untranslated region from soybean 7S seed storage protein gene are commonly
used in
this capacity. The 3' transcribed, non-translated regions containing the
polyadenylate
signal of Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable.
Recombinant DNA technologies can be used to improve expression of a
transformed polynucleotide by manipulating, for example, the efficiency with
which
the resultant transcripts are translated by codon optimisation according to
the host cell
species or the deletion of sequences that destabilize transcripts, and the
efficiency of
post-translational modifications.
Transfer Nucleic Acids
Transfer nucleic acids can be used to deliver an exogenous polynucleotide to a
cell and comprise one, preferably two, border sequences and one or more
polynucleotides of interest. The transfer nucleic acid may or may not encode a
selectable marker. Preferably, the transfer nucleic acid forms part of a
binary vector in
a bacterium, where the binary vector further comprises elements which allow
replication of the vector in the bacterium, selection, or maintenance of
bacterial cells
containing the binary vector. Upon transfer to a eukaryotic cell, the transfer
nucleic
acid component of the binary vector is capable of integration into the genome
of the
eukaryotic cell or, for transient expression experiments, merely of expression
in the
cell.
As used herein, the term "extrachromosomal transfer nucleic acid" refers to a
nucleic acid molecule that is capable of being transferred from a bacterium
such as
Agrobacterium sp., to a plant cell such as a plant leaf cell. An
extrachromosomal
transfer nucleic acid is a genetic element that is well-known as an element
capable of
being transferred, with the subsequent integration of a nucleotide sequence
contained
within its borders into the genome of the recipient cell. In this respect, a
transfer
nucleic acid is flanked, typically, by two "border" sequences, although in
some
instances a single border at one end can be used and the second end of the
transferred
nucleic acid is generated randomly in the transfer process. A polynucleotide
of interest
is typically positioned between the left border-like sequence and the right
border-like
sequence of a transfer nucleic acid. The polynucleotide contained within the
transfer
nucleic acid may be operably linked to a variety of different promoter and
terminator
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regulatory elements that facilitate its expression, that is, transcription
and/or translation
of the polynucleotide. Transfer DNAs (T-DNAs) from Agrobacterium sp. such as
Agrobacterium tumefaciens or Agrobacterium rhizogenes, and man made
variants/mutants thereof are probably the best characterized examples of
transfer
nucleic acids. Another example is P-DNA ("plant-DNA") which comprises 1-DNA
border-like sequences from plants.
As used herein, "T-DNA" refers to a T-DNA of an Agrobacterium turnefaciens
Ti plasmid or from an Agrobacterium rhizogenes Ri plasmid, or variants thereof
which
function for transfer of DNA into plant cells. The T-DNA may comprise an
entire T-
DNA including both right and left border sequences, but need only comprise the
minimal sequences required in cis for transfer, that is, the right T-DNA
border
sequence. The T-DNAs of the invention have inserted into them, anywhere
between
the right and left border sequences (if present), the polynucleotide of
interest. The
sequences encoding factors required in trans for transfer of the T-DNA into a
plant cell
such as vir genes, may be inserted into the T-DNA, or may be present on the
same
replicon as the T-DNA, or preferably are in trans on a compatible replicon in
the
Agrobacterium host. Such "binary vector systems" are well known in the art. As
used
herein. "P-DNA" refers to a transfer nucleic acid isolated from a plant
genome, or man
made variants/mutants thereof, and comprises at each end, or at only one end,
a T-DNA
border-like sequence.
As used herein, a "border" sequence of a transfer nucleic acid can be isolated
from a selected organism such as a plant or bacterium, or be a man made
variant/mutant thereof. The border sequence promotes and facilitates the
transfer of the
polynucleotide to which it is linked and may facilitate its integration in the
recipient
cell genome. In an embodiment, a border-sequence is between 10-80 bp in
length.
Border sequences from 1-DNA from Agrobacterium sp. are well known in the art
and
include those described in Lacroix et al. (2008).
Whilst traditionally only Agrobacterium sp. have been used to transfer genes
to
plants cells, there are now a large number of systems which have been
identified/developed which act in a similar manner to Agrobacterium sp.
Several non-
Agrobacterium species have recently been genetically modified to be competent
for
gene transfer (Chung et al., 2006; Broothaerts et al., 2005). These include
Rhizobium
sp. NGR234, Sinorhizobium meliloti and Mezorhizobium loti.
As used herein, the terms "transfection", "transformation" and variations
thereof
are generally used interchangeably. "Transfected" or "transformed" cells may
have
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been manipulated to introduce the polynucleotide(s) of interest, or may be
progeny
cells derived therefrom.
Plants
The invention also provides a plant or part thereof comprising two or more
exogenous polynucleotides and/or genetic modifications as described herein.
The term
"plant" when used as a noun refers to whole plants, whilst the term "part
thereof' refers
to plant organs (e.g., leaves, stems, roots, flowers, fruit), single cells
(e.g., pollen), seed,
seed parts such as an embryo, endosperm, scutellum or seed coat, plant tissue
such as
vascular tissue, plant cells and progeny of the same. As used herein, plant
parts
comprise plant cells.
As used herein, the terms "in a plant" and -in thc plant" in the context of a
modification to the plant means that the modification has occurred in at least
one part
of the plant, including where the modification has occurred throughout the
plant, and
does not exclude where the modification occurs in only one or more but not all
parts of
the plant. For example, a tissue-specific promoter is said to be expressed "in
a plant",
even though it might be expressed only in certain parts of the plant.
Analogously, "a
transcription factor polypeptide that increases the expression of one or more
glycolytic
and/or fatty acid biosynthetic genes in the plant" means that the increased
expression
occurs in at least a part of the plant.
As used herein, the term "plant" is used in it broadest sense, including any
organism in the Kingdom Plantae. It also includes red and brown algae as well
as
green algae. It includes, but is not limited to, any species of flowering
plant, grass, crop
or cereal (e.g., oilseed, maize, soybean), fodder or forage, fruit or
vegetable plant, herb
plant, woody plant or tree. It is not meant to limit a plant to any particular
structure. It
also refers to a unicellular plant (e.g., microalga). The term "part thereof'
in reference
to a plant refers to a plant cell and progeny of same, a plurality of plant
cells, a
structure that is present at any stage of a plant's development, or a plant
tissue. Such
structures include, but are not limited to, leaves, stems, flowers, fruits,
nuts, roots, seed,
seed coat, embryos. The term "plant tissue" includes differentiated and
undifferentiated
tissues of plants including those present in leaves, stems, flowers, fruits,
nuts, roots,
seed, for example, embryonic tissue, endosperm, dermal tissue (e.g.,
epidermis,
periderm), vascular tissue (e.g., xylem, phloem), or ground tissue (comprising
parenchyma, collenchyma, and/or sclerenchyma cells), as well as cells in
culture (e.g.,
single cells, protoplasts, callus, embryos, etc.). Plant tissue may be in
plan/a, in organ
culture, tissue culture, or cell culture.
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As used herein, the term "vegetative tissue" or "vegetative plant part" is any
plant tissue, organ or part other than organs for sexual reproduction of
plants. The
organs for sexual reproduction of plants are specifically seed bearing organs,
flowers,
pollen, fruits and seeds. Vegetative tissues and parts include at least plant
leaves, stems
(including bolts and tillers but excluding the heads), tubers and roots, but
excludes
flowers, pollen, seed including the seed coat, embryo and endosperm, fruit
including
mesocarp tissue, seed-bearing pods and seed-bearing heads. In one embodiment,
the
vegetative part of the plant is an aerial plant part. In another or further
embodiment,
the vegetative plant part is a green part such as a leaf or stem.
A ''transgenic plant" or variations thereof refers to a plant that contains a
transgene not found in a wild-type plant of the same species, variety or
cultivar.
Transgenic plants as defined in the context of the present invention include
plants and
their progeny which have been genetically modified using recombinant
techniques to
cause production of at least one polypeptide defined herein in the desired
plant or part
thereof. Transgenic plant parts has a corresponding meaning. The plant and
plant parts
of the invention may comprise genetic modifications, for example gene
mutations, and
be considered as "non-transgenic" provided they lack transgenes.
The terms "seed" and "grain" are used interchangeably herein. "Grain" refers
to
mature grain such as harvested grain or grain which is still on a plant but
ready for
harvesting, but can also refer to grain after imbibition or germination,
according to the
context. Mature grain commonly has a moisture content of less than about 18%.
In a
preferrd embodiment, the moisture content of the grain is at a level which is
generally
regarded as safe for storage, preferably between 5% and 15%, between 6% and
8%,
between 8% and 10%, or between 10% and 15%. "Developing seed" as used herein
refers to a seed prior to maturity, typically found in the reproductive
structures of the
plant after fertilisation or anthesis, but can also refer to such seeds prior
to maturity
which are isolated from a plant. Mature seed commonly has a moisture content
of less
than about 12%.
As used herein, the term "plant storage organ" refers to a part of a plant
specialized to store energy in the form of for example, proteins,
carbohydrates, lipid.
Examples of plant storage organs are seed, fruit, tuberous roots, and tubers.
A
preferred plant storage organ of the invention is seed.
As used herein, the term "phenotypically normal" refers to a genetically
modified plant or part thereof, for example a plant such as a tragsenic plant,
or a
storage organ such as a seed, tuber or fruit of the invention not having a
significantly
reduced ability to grow and reproduce when compared to an unmodified plant or
part
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thereof. Preferably, the biomass, growth rate, germination rate, storage organ
size, seed
size and/or the number of viable seeds produced is not less than 90% of that
of a plant
lacking said genetic modifications or exogenous polynucleotides when grown
under
identical conditions. This term does not encompass features of the plant which
may be
different to the wild-type plant but which do not effect the usefulness of the
plant for
commercial purposes such as, for example, a ballerina phenotype of seedling
leaves. In
an embodiment, the genetically modified plant or part thereof which is
phenotypically
normal comprises a recombinant polynucleotide encoding a silencing suppressor
operably linked to a plant storage organ specific promoter and has an ability
to grow or
reproduce which is essentially the same as a corresponding plant or part
thereof not
comprising said polynucleotide.
Plants go through a series of growing stages from sowing of a seed,
germination
and emergence of a seedling, through to flowering, seed setting, physiological
maturity
and ultimately senescence. These stages are well known and readily defined,
for
example for Sorghum plants as follows. Taking the day the seedling first
emerges
above the soil as day 0, the vegetative stage of growth is defined herein as
from 10 days
to initiation of flowering at about 60-70 days, and physiogical maturity is
reached at
about 100 days, depending on the environmental conditions. The vegetative
stage
includes the boot leaf stage from about 45 days until the first flowering. The
boot leaf is
the last leaf formed on the plant, from which the panicle (head) emerges. The
"boot leaf
stage" is defined as from when the boot leaf has fully emerged to initiation
of
flowering.
As used herein, the term "commencement of flowering" or "initiation of
flowering" with respect to a plant refers to the time that the first flower on
the plant
opens, or the time of onset of anthesis.
As used herein, the term "seed set" with respect to a seed-bearing plant
refers to
the time when the first seed of the plant reaches maturity. This is typically
observable
by the colour of the seed or its moisture content, well known in the art.
As used herein, the term "mature" as it relates to a plant leaf means that it
has
reached full size but has not begun to show signs of ageing or death such as
yellowing
and/or sensensce. The skilled person can readily determine whether a leaf of a
particular plant can be considered as mature.
As used herein, the term "senescence" with respect to a whole plant refers to
the
final stage of plant development which follows the completion of growth,
usually after
the plant reachesµ maximum aerial biomass or height. Senescence begins when
the plant
aerial biomass reaches its maximum and begins to decline in amount and
generally
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ends with death of most of the plant tissues. It is during this stage that the
plant
mobilises and recycles cellular components from leaves and other parts which
accumulated during growth to other parts to complete its reproductive
development.
Senescence is a complex, regulated process which involves new or increased
gene
expression of some genes. Often, some plant parts are senescing while other
parts of
the same plant continue to grow. Therefore, with respect to a plant leaf or
other green
organ, the term "senescence" as used herein refers to the time when the amount
of
chlorophyll in the leaf or organ begins to decrease. Senescence is typically
associated
with dessication of the leaf or organ, mostly in the last stage of senescence.
Senescence
is usually observable by the change in colour of the leaf from green towards
yellow and
eventually to brown when fully dessicated. It is believed that cellular
senescence
underlies plant and organ senescence.
Plants provided by or contemplated for use in the practice of the present
invention include both monocotyledons and dicotyledons. In preferred
embodiments,
the plants of the present invention are crop plants (for example, cereals and
pulses,
maize, wheat, potatoes, rice, sorghum, millet, cassava, barley) or legumes
such as
soybean, beans or peas. The plants may be grown for production of edible
roots,
tubers, leaves, stems, flowers or fruit. The plants may be vegetable plants
whose
vegetative parts are used as food. The plants of the invention may be:
Acrocomia
aculeata (macauba palm), Arabidopsis thaliana, Aracinis hypogaea (peanut),
Astrocaryum murumuru (murumuru), Astrocaryum vulgare (tucuma), Attalea
geraensis
(Indaid-rateiro), Attalea hum//is (American oil palm), Attalea oleifera
(andaia), Attalea
phalerata (uricuri), Attalea speciosa (babassu), Avena sativa (oats), Beta
vulgaris
(sugar beet), Brassica sp. such as Brassica carinata, Brassica juncea,
Brassica
napobrassica, Brassica napus (canola), Camelina sativa (false flax), Cannabis
sativa
(hemp), Carthamus tinctorius (safflower), Caryocar brasiliense (pequi), Cocos
nucifera (Coconut), Crambe abyssinica (Abyssinian kale), Cucumis melo (melon),
Elaeis guineensis (African palm), Glycine max (soybean), Gossypium hirsutum
(cotton), Helianthus sp. such as Helianthus annuus (sunflower), Hordeum
vulgare
(barley), Jatropha curcas (physic nut), Joannesia princeps (arara nut-tree),
Lemna .sp.
(duckweed) such as Lemna aequinoctialis, Lemna disperma, Lemna ecuadoriensis,
Lemna gibba (swollen duckweed), Lemna japonica, Lemna minor, Lemna minuta,
Lemna obscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemna
trisulca,
Lemna turionifera, Lemna valdiviana, Lemna yungensis, Licania rigida
(oiticica),
Linum usitatissimum (flax), Lupinus angustifolius (lupin), Mauritia flexuosa
(buriti
palm). Maximiliana mar/pa (inaja palm), Miscanthus sp. such as Miscanthus x
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giganteus and Miscanthus sinensis, Nicotiana sp. (tabacco) such as Nicotiana
tabacum
or Nicotiana benthamiana, Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus
bataua
(pataud), Oenocarpus distichus (bacaba-de-leque), Oryza sp. (rice) such as
Oryza sativa
and Oryza glaberrima, Panicum virgatum (switchgrass), Paraqueiba paraensis
(man),
Persea amencana (avocado), Pongamia pinnata (Indian beech), Populus
trichocarpa,
Ricinus communis (castor), Saccharum sp. (sugarcane), Sesamum indicum
(sesame),
Solanum tuberosum (potato), Sorghum sp. such as Sorghum bicolor, Sorghum
vulgare,
Theobrom grandiforum (cupuassu), Trifolium sp., Trithrinax brasiliensis
(Brazilian
needle palm), Triticum sp. (wheat) such as Triticum aestivum, Zea mays (corn),
alfalfa
(Medicago sativa), rye (Secale cerale), sweet potato (Lopmoea batatus),
cassava
(Manihot esculenta), coffee (Cofea spp.), pineapple (Anana comosus), citris
tree
(Citrus spp.), cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa
spp.),
avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango
(Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew
(Anacardium occidentak), macadamia (Macadamia intergrifalia) and almond
(Prunus
amygdalus).
In an embodiment, the plant is not a Nicotiana sp.
Other preferred plants include C4 grasses such as, in addition to those
mentioned above, Andropogon gerardi, Bouteloua curtipendula, B. gracilis,
Buchloe
dactyloides, Schizachyrium scoparium, Sorghastrum nutans, Sporobolus
cryptandrus;
C3 grasses such as Elymus canadensis, the legumes Lespedeza capitata and
Petalostemum villosum, the forb Aster azureus; and woody plants such as
Quercus
ellipsoidalis and Q. macrocarpa. Other preferred plants include C3 grasses.
In a preferred embodiment, the plant is an angiosperm.
In an embodiment, the plant is an oilseed plant, preferably an oilseed crop
plant.
As used herein, an "oilseed plant" is a plant species used for the commercial
production
of lipid from the seeds of the plant. The oilseed plant may be, for example,
oil-seed
rape (such as canola), maize, sunflower, safflower, soybean, sorghum, flax
(linseed) or
sugar beet. Furthermore, the oilseed plant may be other , Brassicas, cotton,
peanut,
poppy, rutabaga, mustard, castor bean, sesame, safflower, Jatropha curcas or
nut
producing plants. The plant may produce high levels of lipid in its fruit such
as olive,
oil palm or coconut. Horticultural plants to which the present invention may
be applied
are lettuce, endive, or vegetable Brassicas including cabbage, broccoli, or
cauliflower.
The present invention may be applied in tobacco, cucurbits, carrot,
strawberry, tomato,
or pepper.
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In a preferred embodiment, the plant is a member of the family Fabaceae (or
Leguminosae) such as alfalfa, clover, peas, lucerne, beans, lentils, lupins,
mesquite,
carob, soybeans, and peanuts, or a member of the family Poaceae such as corn,
sorghum, wheat, barley and oats. In a particularly preferred embodiment, the
plant is
alfalfa, clover, corn or sorghum, each of which are particularly useful for
forage or
fodder for animals.
In a preferred embodiment, the transgenic plant is homozygous for each and
every gene that has been introduced (transgene) so that its progeny do not
segregate for
the desired phenotype. The transgenic plant may also be heterozygous for the
introduced transgene(s), preferably uniformly heterozygous for the transgene
such as
for example, in Fl progeny which have been grown from hybrid seed. Such plants
may
provide advantages such as hybrid vigour, well known in the art.
Transformation of plants
Transgenic plants can be produced using techniques known in the art, such as
those generally described in Slater et al., Plant Biotechnology - The Genetic
Manipulation of Plants, Oxford University Press (2003), and Christou and Klee,
Handbook of Plant Biotechnology, John Wiley and Sons (2004).
As used herein, the terms "stably transforming", "stably transformed" and
variations thereof refer to the integration of the polynucleotide into the
genome of the
cell such that they are transferred to progeny cells during cell division
without the need
for positively selecting for their presence. Stable transformants, or progeny
thereof,
can be identified by any means known in the art such as Southern blots on
chromosomal DNA, or in situ hybridization of genomie DNA, enablimg their
selection.
Agrobacterium-mediated transfer is a widely applicable system for introducing
genes into plant cells because DNA can be introduced into cells in whole plant
tissues,
plant organs, or explants in tissue culture, for either transient expression,
or for stable
integration of the DNA in the plant cell genome. For example, floral-dip (in
planta)
methods may be used. The use of Agrobacterium-mediated plant integrating
vectors to
introduce DNA into plant cells is well known in the art. The region of DNA to
be
transferred is defined by the border sequences, and the intervening DNA (T-
DNA) is
usually inserted into the plant genome. It is the method of choice because of
the facile
and defined nature of the gene transfer.
Acceleration methods that may be used include for example, microprojectile
bombardment and the like. One example of a method for delivering transforming
nucleic acid molecules to plant cells is microprojectile bombardment. This
method has
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been reviewed by Yang et al., Particle Bombardment Technology for Gene
Transfer,
Oxford Press, Oxford, England (1994). Non-biological particles
(microprojectiles) that
may be coated with nucleic acids and delivered into cells, for example of
immature
embryos, by a propelling force. Exemplary particles include those comprised of
tungsten, gold, platinum, and the like.
In another method, plastids can be stably transformed. Methods disclosed for
plastid transformation in higher plants include particle gun delivery of DNA
containing
a selectable marker and targeting of the DNA to the plastid genome through
homologous recombination (US 5,451,513, US 5,545,818, US 5,877,402, US
5,932479,
and WO 99/05265). Other methods of cell transformation can also be used and
include
but are not limited to the introduction of DNA into plants by direct DNA
transfer into
pollen, by direct injection of DNA into reproductive organs of a plant, or by
direct
injection of DNA into the cells of immature embryos followed by the
rehydration of
desiccated embryos.
The regeneration, development, and cultivation of plants from single plant
protoplast transformants or from various transformed explants is well known in
the art
(Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press,
San
Diego. Calif., (1988)). This regeneration and growth process typically
includes the
steps of selection of transformed cells, culturing those individualized cells
through the
usual stages of embryonic development through the rooted plantlet stage.
Transgenic
embryos and seeds are similarly regenerated. The resulting transgenic rooted
shoots
are thereafter planted in an appropriate plant growth medium such as soil.
The development or regeneration of plants containing the foreign, exogenous
gene is well known in the art. Preferably, the regenerated plants are self-
pollinated to
provide homozygous transgenic plants. Otherwise, pollen
obtained from the
regenerated plants is crossed to seed-grown plants of agronomically important
lines.
Conversely, pollen from plants of these important lines is used to pollinate
regenerated
plants. A transgenic plant of the present invention containing a desired
polynucleotide
is cultivated using methods well known to one skilled in the art.
To confirm the presence of the transgenes in transgenic cells and plants, a
polymerase chain reaction (PCR) amplification or Southern blot analysis can be
performed using methods known to those skilled in the art. Expression products
of the
transgenes can be detected in any of a variety of ways, depending upon the
nature of
the product, and include Northern blot hybridisation, Western blot and enzyme
assay.
Once transgenic plants have been obtained, they may be grown to produce plant
tissues
or parts having the desired phenotype. The plant tissue or plant parts, may be
harvested,
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and/or the seed collected. The seed may serve as a source for growing
additional plants
with tissues or parts having the desired characteristics. Preferably, the
vegetative plant
parts are harvested at a time when the yield of non-polar lipids are at their
highest. In
one embodiment, the vegetative plant parts are harvested about at the time of
flowering,
or after flowering has initiated. Preferably, the plant parts are harvested at
about the
time senescence begins, usually indicated by yellowing and drying of leaves.
Transgenic plants formed using Agrobacterium or other transformation methods
typically contain a single genetic locus on one chromosome. Such transgenic
plants
can be referred to as being hemizygous for the added gene(s). More preferred
is a
transgenic plant that is homozygous for the added gene(s), that is, a
transgenic plant
that contains two added genes, one gene at the same locus on each chromosome
of a
chromosome pair. A homozygous transgenic plant can be obtained by self-
fertilising a
hemizygous transgenic plant, germinating some of the seed produced and
analyzing the
resulting plants for the gene of interest.
It is also to be understood that two different transgenic plants that contain
two
independently segregating exogenous genes or loci can also be crossed (mated)
to
produce offspring that contain both sets of genes or loci. Selfing of
appropriate Fl
progeny can produce plants that are homozygous for both of the exogenous genes
or
loci. Back-crossing to a parental plant and out-crossing with a non-transgenic
plant are
also contemplated, as is vegetative propagation. Similarly, a transgenic plant
can be
crossed with a second plant comprising a genetic modification such as a mutant
gene
and progeny containing both of the transgene and the genetic modification
identified.
Descriptions of other breeding methods that are commonly used for different
traits and
crops can be found in Fehr, In: Breeding Methods for Cultivar Development,
Wilcox J.
ed., American Society of Agronomy, Madison Wis. (1987).
TILLING
In one embodiment, TILLING (Targeting Induced Local Lesions IN Genomes)
can be used to produce plants in which endogenous genes comprise a mutation,
for
example genes encoding an SDP1 or TGD polypeptide, TST, a plastidial GPAT,
plastidial LPAAT, phosphatidic acid phosphatase (PAP), or a combination of two
or
more thereof. In a first step, introduced mutations such as novel single base
pair
changes are induced in a population of plants by treating seeds (or pollen)
with a
chemical mutagen, and then advancing plants to a generation where mutations
will be
stably inherited. DNA is extracted, and seeds are stored from all members of
the
population to create a resource that can be accessed repeatedly over time. For
a
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TILLING assay, heteroduplex methods using specific endonucleases can be used
to
detect single nucleotide polymorphisms (SNPs). Alternatively, Next Generation
Sequencing of DNA from pools of mutagenised plants can be used to identify
mutants
in the gene of choice. Typically, a mutation frequency of one mutant per 1000
plants in
the mutagenised population is achieved. Using this approach, many thousands of
plants
can be screened to identify any individual with a single base change as well
as small
insertions or deletions (1-30 bp) in any gene or specific region of the
genome.
TILLING is further described in Slade and Knauf (2005), and Henikoff et al.
(2004).
In addition to allowing efficient detection of mutations, high-throughput
TILLING technology is ideal for the detection of natural polymorphisms.
Therefore,
interrogating an unknown homologous DNA by heteroduplexing to a known sequence
reveals the number and position of polymorphic sites. Both nucleotide changes
and
small insertions and deletions are identified, including at least some repeat
number
polymorphisms. This has been called Ecotilling (Comai et al., 2004).
Genome editing using site-specific nucleases
Genome editing uses engineered nucleases such as RNA guided DNA
endonucleases or nucleases composed of sequence specific DNA binding domains
fused to a non-specific DNA cleavage module. These engineered nucleases enable
efficient and precise genetic modifications by inducing targeted DNA double
stranded
breaks that stimulate the cell's endogenous cellular DNA repair mechanisms to
repair
the induced break. Such mechanisms include, for example, error prone non-
homologous end joining (NHEJ) and homology directed repair (HDR).
In the presence of donor plasmid with extended homology arms, 11DR can lead
to the introduction of single or multiple transgenes to correct or replace
existing genes.
In the absence of donor plasmid, NHEJ-mediated repair yields small insertion
or
deletion mutations of the target that cause gene disruption.
Engineered nucleases useful in the methods of the present invention include
zinc
finger nucleases (ZENs), transcription activator-like (TAL) effector nucleases
(TALEN) and CRISPR/Cas9 type nucleases, and related nucleases.
Typically nuclease encoded genes are delivered into cells by plasmid DNA,
viral vectors or in vitro transcribed mRNA.
A zinc finger nuclease (ZFN) comprises a DNA-binding domain and a DNA-
cleavage domain, wherein the DNA binding domain is comprised of at least one
zinc
finger and is operatively linked to a DNA-cleavage domain. The zinc finger DNA-
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binding domain is at the N-terminus of the protein and the DNA-cleavage domain
is
located at the C-terminus of said protein.
A ZFN must have at least one zinc finger. In a preferred embodiment, a ZFN
would have at least three zinc fingers in order to have sufficient specificity
to be useful
for targeted genetic recombination in a host cell or organism. Typically, a
ZFN having
more than three zinc fingers would have progressively greater specificity with
each
additional zinc finger.
The zinc finger domain can be derived from any class or type of zinc finger.
In
a particular embodiment, the zinc finger domain comprises the Cis2His2 type of
zinc
finger that is very generally represented, for example, by the zinc finger
transcription
factors TFIIIA or Sp 1. In a preferred embodiment, the zinc finger domain
comprises
three Cis2His2 type zinc fingers. The DNA recognition and/or the binding
specificity of
a ZFN can be altered in order to accomplish targeted genetic recombination at
any
chosen site in cellular DNA. Such modification can be accomplished using known
molecular biology and/or chemical synthesis techniques. (see, for example,
Bibikova et
al., 2002).
The ZFN DNA-cleavage domain is derived from a class of non-specific DNA
cleavage domains, for example the DNA-cleavage domain of a Type II restriction
enzyme such as FokI (Kim et al., 1996). Other useful endonucleases may
include, for
example, Hhal, HindIII, Nod, BbvCI, EcoRI, Bgll, and Alwl.
A transcription activator-like (TAL) effector nuclease (TALEN) comprises a
TAL effector DNA binding domain and an endonuclease domain.
TAL effectors are proteins of plant pathogenic bacteria that are injected by
the
pathogen into the plant cell, where they travel to the nucleus and function as
transcription factors to turn on specific plant genes. The primary amino acid
sequence
of a TAL effector dictates the nucleotide sequence to which it binds. Thus,
target sites
can be predicted for TAL effectors, and TAL cffectors can be engineered and
generated
for the purpose of binding to particular nucleotide sequences.
Fused to the TAL effector-encoding nucleic acid sequences are sequences
encoding a nuclease or a portion of a nuclease, typically a nonspecific
cleavage domain
from a type II restriction endonuclease such as Fokl (Kim et al., 1996). Other
useful
endonucleases may include, for example, Hhal, Hindu, Nod, BbvCI, EcoRI, Bgil,
and
A/wI. The fact that some endonucleases (e.g., Fokl) only function as dimers
can be
capitalized upon to enhance the target specificity of the TAL effector. For
example, in
some cases each Fokl monomer can be fused to a TAL effector sequence that
recognizes a different DNA target sequence, and only when the two recognition
sites
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are in close proximity do the inactive monomers come together to create a
functional
enzyme. By requiring DNA binding to activate the nuclease, a highly site-
specific
restriction enzyme can be created.
A sequence-specific TALEN can recognize a particular sequence within a
preselected target nucleotide sequence present in a cell. Thus, in some
embodiments, a
target nucleotide sequence can be scanned for nuclease recognition sites, and
a
particular nuclease can be selected based on the target sequence. In other
cases, a
TALEN can be engineered to target a particular cellular sequence.
Genome editing using programmable RNA-guided DNA endonucleases
Distinct from the site-specific nucleases described above, the clustered
regulatory interspaced short palindromic repeats (CRISPR)/Cas system provides
an
alternative to ZFNs and TALENs for inducing targeted genetic alterations, via
RNA-
guided DNA cleavage.
CRISPR systems rely on CRISPR RNA (crRNA) and transactivating chimeric
RNA (tracrRNA) for sequence-specific cleavage of DNA. Three types of
CRISPR/Cas
systems exist: in type II systems, Cas9 serves as an RNA-guided DNA
endonuclease
that cleaves DNA upon crRNA¨tracrRNA target recognition. CRISPR RNA base pairs
with tracrRNA to form a two-RNA structure that guides the Cas9 endonuclease to
complementary DNA sites for cleavage.
The CRISPR system can be portable to plant cells by co-delivery of plasmids
expressing the Cas endonuclease and the necessary crRNA components. The Cas
endonuclease may be converted into a nickase to provide additional control
over the
mechanism of DNA repair (Cong et al., 2013).
CRISPRs are typically short partially palindromic sequences of 24-40bp
containing inner and terminal inverted repeats of up to 11 bp. Although
isolated
elements have been detected, they are generally arranged in clusters (up to
about 20 or
more per genome) of repeated units spaced by unique intervening 20-58bp
sequences.
CRISPRs are generally homogenous within a given genome with most of them being
identical. However, there are examples of heterogeneity in, for example, the
Archaea
(Mojica et al., 2000).
Feedstuffs
The present invention includes compositions which can be used as feedstuffs.
For purposes of the present invention, "feedstuffs" include any food or
preparation for
animal (including human) consumption and which serves to nourish or build up
tissues
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or supply energy, and/or to maintain, restore or support adequate nutritional
status or
metabolic function. Feedstuffs of the invention include nutritional
compositions for
babies and/or young children.
As used herein, the term "animal" refers to any eukaryotic organism capable of
ingesting plant derived material. In an embodiment, the animal is a ruminant
animal
(cattle, sheep, goats etc). Alternatively, the animal is a non-ruminant
animal. In one
embodiment, the animal is a mammal. In an embodiment, the animal is a human.
In an
embodiment, the animal is a livestock animal such, but not limited to, as
cattle, goats,
sheep, pigs, horses, poultry such as chickens and the like. In an embodiment,
the cattle
are diary cattle or beef cattle. In another embodiment, the animal is a fish,
for instance
fish bred using aquaculture including, but not limited to, salmon, trout,
carp, bass,
bream, turbot, sole, milkfish, grey mullet, grouper, flounder, sea bass, cod,
haddock,
Japanese flounder, catfish, char, whitefish, sturgeon, tench, roach, pike,
pike-perch,
yellowtail, tilapia, eel or tropical fish (such as the fresh, brackish, and
salt water
tropical fish). The animal may be a crustacean such as, but not limited to,
krill, clams,
shrimp (including prawns), crab, and lobster.
Feedstuffs of the invention may comprise for example, a plant or part thereof
such as a vegetative plant part of the invention along with a suitable
carrier(s). The
term "carrier" is used in its broadest sense to encompass any component which
may or
may not have nutritional value. As the person skilled in the art will
appreciate, the
carrier must be suitable for use (or used in a sufficiently low concentration)
in a
feedstuff, such that it does not have deleterious effect on an organism which
consumes
the feedstuff. Feedstuffs may comprise plant parts which have been harvested
and
subsequently processed or treated, for example, by chopping, cutting, drying,
pressing
or pelleting the plant parts, into a form that is suitable for consumption by
the animal,
or altered by processes such as drying or fermentation to produce hay or
silage.
The feedstuff of the present invention comprises a lipid and/or protein
produced
directly or indirectly by use of the methods, plants or parts thereof
disclosed herein.
The composition may either be in a solid or liquid form. Additionally, the
composition
may include edible macronutrients, vitamins, and/or minerals in amounts
desired for a
particular use. The amounts of these ingredients will vary depending on
whether the
composition is intended for use with normal individuals or for use with
individuals
having specialized needs such as individuals suffering from metabolic
disorders and the
like.
Examples of suitable carriers with nutritional value include, but are not
limited
to, macronutrients such as edible fats, carbohydrates and proteins. Examples
of such
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edible fats include, but are not limited to, coconut oil, borage oil, fungal
oil, black
current oil, soy oil, and mono- and di-glycerides. Examples of such
carbohydrates
include, but are not limited to, glucose, edible lactose, and hydrolyzed
starch.
Additionally, examples of proteins which may be utilized in the nutritional
composition
of the invention include, but are not limited to, soy proteins,
electrodialysed whey,
electrodialysed skim milk, milk whey, or the hydrolysates of these proteins.
With respect to vitamins and minerals, the following may be added to the
feedstuff compositions of the present invention, calcium, phosphorus,
potassium,
sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine,
and
vitamins A, E, D. C, and the B complex. Other such vitamins and minerals may
also be
added.
A feedstuff composition of the present invention may also be added to food
even
when supplementation of the diet is not required. For example, the composition
may
be added to food of any type, including, but not limited to, margarine,
butter, cheeses,
milk, yogurt, chocolate, candy, snacks, salad oils, cooking oils, cooking
fats, meats,
fish and beverages.
Additionally, material produced in accordance with the present invention may
also be used as animal food supplements to alter an animal's tissue or milk
fatty acid
composition to one more desirable for human or animal consumption, or to
reduce
methane production in ruminant animals. Furthermore, feedstuffs of the
invention can
be used in aquaculture to increase the levels of fatty acids and nutrition in
fish for
human or animal consumption.
Preferred feedstuffs of the invention are the plants, seed and other plant
parts
such as leaves, fruits and stems which may be used directly as food or feed
for humans
or other animals. For example, animals may graze directly on such plants grown
in the
field, or be fed more measured amounts in controlled feeding. The invention
includes
the use of such plants and plant parts as feed for increasing the
polyunsaturated fatty
acid levels in humans and other animals.
For consumption by non-human animals the feedstuff may be in any suitable
form for such as, but not limited to, silage, hay or pasture growing in a
field. In an
embodiment, the feedstuff for non-human consumption is a leguminous plant, or
part
thereof, which is a member of the family Fabaceae family (or Leguminosae) such
as
alfalfa, clover, peas, lucerne, beans, lentils, lupins, mesquite, carob,
soybeans, and
peanuts.
In embodiment, the animal is in a feedlot and/or a shed.
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In an embodiment, the plant or fraction thereof comprises at least about 5%,
at
least about 10%, at least about 50%, at least about 75%, at least about 90% or
all of the
feedstuff.
Silage
As used herein, "silage" is a relatively high-moisture fodder which has been
produced and stored in a process called ensilage and which is typically fed to
cattle,
sheep or other ruminants. During the storage time, carbohydrates, lipids and
proteins in
the plant material ferment, producing organic acids, or are broken down
oxidatively, or
both. The plant material upon harvest and the post-fermentation plant
materials are both
included in silage as the term is used herein. Silage is typically made from
grass crops
such as maize, sorghum, oats or other cereals, or from mixed pasture grasses
and
legumes such as alfalfa or clover, using the green, above-ground parts of the
plants.
Silage is made either by placing cut vegetation (usually the whole above-
ground plant
biomass which can include reproductive tissues) in a pit or silo or other
means for
storage, and compressing it down so as to leave as little air as possible with
the plant
material. Oxygen is excluded to some extent by covering it with a plastic
sheet or by
wrapping the plant material tightly within plastic film (baling) to reduce air
inflow.
Silage is made from plant material with a suitable moisture content, generally
about
50% to 60% of the fresh weight, depending on the means of storage and the
degree of
compression used and the amount of water that will be lost in storage, but not
exceeding 75%. For sorghum and corn, harvest begins when the whole-plant
moisture
is at a suitable level, ideally a few days before it is ripe. For pasture-type
crops, the
plants are mowed and allowed to wilt for a day or so until the moisture
content drops to
a suitable level. Ideally the crop is mowed when in full flower and deposited
in the pit
or silo on the day of its cutting. At harvesting, or after, the plant material
is shredded or
chopped by the harvester into pieces typically about 1-5 cm long. The plant
material
may be placed in large heaps on the ground and compressed to reduce the amount
of
air, then covered with plastic, or into a silo. Alternatively, the plant
material may be
baled in plastic wrapping to exclude air, which typically requires a lower
moisture
content of about 30-40%, but still too damp to be stored as dry hay.
The cut or chopped, stored plant material undergoes mostly anaerobic
fermentation, which starts about 48 hours after the pit or silo is filled. The
fermentation
process converts sugars and other carbohydrates such as hemicellulose to
organic acids,
mostly acetic, propionic, lactic and butyric acids. Fermentation starts after
the trapped
oxygen is consumed and is essentially complete after about two weeks of
storage, or
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may continue for longer periods. When the plant material is closely packed,
the supply
of oxygen is limited and the fermentation results in the decomposition of the
carbohydrates, some lipids and proteins in the material into the organic
acids. This
product is named sour silage. If, on the other hand, the fodder is more
loosely packed,
the main reaction is oxidation which proceeds more rapidly and the temperature
rises.
If the mass is compressed when the temperature is 60-75C, the reaction ceases
and
sweet silage results. Fermentation may be aided by inoculation with specific
microorganisms such as lactic acid bacteria to speed fermentation or improve
the
resulting silage, e.g. with Lactobacillus plantarum.
Bulk silage is commonly fed to dairy cattle, while baled silage tends to be
used
for beef cattle, sheep and horses. The advantages of silage as animal feed are
several.
During fermentation, the silage bacteria act on the cellulose and other
carbohydrates in
the forage to produce the organic fatty acids, thereby lowering the pH. This
inhibits
competing bacteria that might cause spoilage and the organic acids thereby act
as
natural preservatives, improve digestibility and palatability. This
preservative action is
particularly important during winter in temperate regions, when green forage
is
unavailable.
Silage can be produced using techniques known in the art such as those
described in CN 101940272 CN 103461658 CN 101946853, CN 101946853, CN
104381743, US3875304 and US 6224916. Pellets for animal feed can be produced
using techniques known in the art such as those described in US 3035920,
US3573924
and US 5871802.
Plant Biomass
An increase in the total lipid content of plant biomass equates to greater
energy
content, making its use as a feed or forage or in the production of biofuel
more
economical.
The main components of naturally occurring plant biomass are carbohydrates
(approximately 75%, dry weight) and lignin (approximately 25%), which can vary
with
plant type. The carbohydrates are mainly cellulose or hemicellulose fibers,
which
impart strength to the plant structure, and lignin, which holds the fibers
together. Plant
biomass typically has a low energy density as a result of both its physical
form and
moisture content. This also makes it inconvenient and inefficient for storage
and
transport without some kind of pre-processing. There are a range of processes
available
to convert it into a more convenient form including: 1) physical pre-
processing (for
example, grinding) or 2) conversion by thermal (for example, combustion,
gasification,
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pyrolysis) or chemical (for example, anaerobic digestion, fewientation,
composting,
transesterification) processes. In this way, the biomass is converted into
what can be
described as a biomass fuel.
Combustion
Combustion is the process by which flammable materials are allowed to burn in
the presence of air or oxygen with the release of heat. The basic process is
oxidation.
Combustion is the simplest method by which biomass can be used for energy, and
has
been used to provide heat This heat can itself be used in a number of ways: 1)
space
heating, 2) water (or other fluid) heating for central or district heating or
process heat,
3) steam raising for electricity generation or motive force. When the
flammable fuel
material is a form of biomass the oxidation is of predominantly the carbon (C)
and
hydrogen (H) in the cellulose, hemicellulose, lignin, and other molecules
present to
form carbon dioxide (CO2) and water (1420). The plants of the invention
provide
improved fuel for combustion by virtue of the increased lipid content.
Gasification
Gasification is a partial oxidation process whereby a carbon source such as
plant
biomass, is broken down into carbon monoxide (CO) and hydrogen (H2), plus
carbon
dioxide (CO2) and possibly hydrocarbon molecules such as methane (CH4). If the
gasification takes place at a relatively low temperature, such as 700 C to
1000 C, the
product gas will have a relatively high level of hydrocarbons compared to high
temperature gasification. As a result it may be used directly, to be burned
for heat or
electricity generation via a steam turbine or, with suitable gas clean up, to
run an
internal combustion engine for electricity generation. The combustion chamber
for a
simple boiler may be close coupled with the gasifier, or the producer gas may
be
cleaned of longer chain hydrocarbons (tars), transported, stored and burned
remotely. A
gasification system may be closely integrated with a combined cycle gas
turbine for
electricity generation (IGCC - integrated gasification combined cycle). Higher
temperature gasification (1200 C to 1600 C) leads to few hydrocarbons in the
product
gas, and a higher proportion of CO and H2. This is known as synthesis gas
(syngas or
biosyngas) as it can be used to synthesize longer chain hydrocarbons using
techniques
such as Fischer-Tropsch (FT) synthesis. If the ratio of H2 to CO is correct
(2:1) FT
synthesis can be used to convert syngas into high quality synthetic diesel
biofuel which
is compatible with conventional fossil diesel and diesel engines.
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Pyrolysis
As used herein, the term "pyrolysis" means a process that uses slow heating in
the absence of oxygen to produce gaseous, oil and char products from biomass.
Pyrolysis is a thermal or thermo-chemical conversion of lipid-based,
particularly
triglyceride-based, materials. The products of pyrolysis include gas, liquid
and a sold
char, with the proportions of each depending upon the parameters of the
process. Lower
temperatures (around 400 C) tend to produce more solid char (slow pyrolysis),
whereas
somewhat higher temperatures (around 500 C) produce a much higher proportion
of
liquid (bio-oil), provided the vapour residence time is kept down to around is
or less.
Temperatures of about 275 C to about 375 C can be used to produce liquid bio-
oil
having a higher proportion of longer chain hydrocarbons. Pyrolysis involves
direct
thermal cracking of the lipids or a combination of thermal and catalytic
cracking. At
temperatures of about 400-500 C, cracking occurs, producing short chain
hydrocarbons
such as alkanes, alkenes, alkadienes, aromatics, olefins and carboxylic acid,
as well as
carbon monoxide and carbon dioxide.
Four main catalyst types can be used including transition metal catalysts,
molecular sieve type catalysts, activated alumina and sodium carbonate (Maher
and
Bressler, 2007). Examples are given in US 4102938. Alumina (A1203) activated
by acid
is an effective catalyst (US 5233109). Molecular sieve catalysts are porous,
highly
crystalline structures that exhibit size selectivity, so that molecules of
only certain sizes
can pass through. These include zeolite catalysts such as ZSM-5 or HZSM-5
which are
crystalline materials comprising A104 and SiO4 and other silica-alumina
catalysts. The
activity and selectivity of these catalysts depends on the acidity, pore size
and pore
shape, and typically operate at 300-500 C. Transition metal catalysts arc
described for
example in US 4992605. Sodium carbonate catalyst has been used in the
pyrolysis of
oils (Dandik and Aksoy, 1998).
As used herein, "hydrothermal processing", "HTP", also referred to as "theimal
depolymerisation" is a form of pyrolysis which reacts the plant-derived
matter,
specifically the carbon-containing material in the plant-derived matter, with
hydrogen
to produce a bio-oil product comprised predominantly of paraffinic
hydrocarbons along
with other gases and solids. A significant advantage of HTP is that the
vegetative plant
material does not need to be dried before forming the composition for the
conversion
reaction, although the vegetative plant material can be dried beforehand to
aid in
transport or storage of the biomass. The biomass can be used directly as
harvested from
the field. The reactor is any vessel which can withstand the high temperature
and
pressure used and is resistant to corrosion. The solvent used in the HTP
includes water
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or is entirely water, or may include some hydrocarbon compounds in the form of
an oil.
Generally, the solvent in IITP lacks added alcohols. The conversion reaction
may occur
in an oxidative, reductive or inert environment. "Oxidative" as used herein
means in the
presence of air, "reductive" means in the presence of a reducing agent,
typically
hydrogen gas or methane, for example 10-15% H2 with the remainder of the gas
being
1\12, and "inert" means in the presence of an inert gas such as nitrogen or
argon. The
conversion reaction is preferably carried out under reductive conditions. The
carbon-
containing materials that are converted include cellulose, hemi-cellulose,
lignin and
proteins as well as lipids. The process uses a conversion temperature of
between 270 C
and 400 C and a pressure of between 70 and 350 bar, typically 300 C to 350 C
and a
pressure between 100-170bar. As a result of the process, organic vapours,
pyrolysis
gases and charcoal are produced. The organic vapours are condensed to produce
the
bio-oil. Recovery of the bio-oil may be achieved by cooling the reactor and
reducing
the pressure to atmospheric pressure, which allows bio-oil (organic) and water
phases
to develop and the bio-oil to be removed from the reactor.
The yield of the recovered bio-oil is calculated as a percentage of the dry
weight
of the input biomass on a dry weight basis. It is calculated according to the
formula:
weight of bio-oil x 100/dry weight of the vegetative plant parts. The weight
of the bio-
oil does not include the weight of any water or solids which may be present in
a bio-oil
mixture, which are readily removed by filtration or other known methods.
The bio-oil may then be separated into fractions by fractional distillation,
with
or without additional refining processes. Typically, the fractions that
condense at these
temperatures are termed: about 370 C, fuel oil; about 300 C, diesel oil; about
200 C,
kerosene; about 150 C, gasoline (petrol). Heavier fractions may be cracked
into lighter,
more desirable fractions, well known in the art. Diesel fuel typically is
comprised of
C13-C22 hydrocarbon compounds.
Transesterification
"Transesterification" as used herein is the conversion of lipids, principally
triacylglycerols, into fatty acid methyl esters or ethyl esters by reaction
with short chain
alcohols such as methanol or ethanol, in the presence of a catalyst such as
alkali or
acid. Methanol is used more commonly due to low cost and availability, but
ethanol,
propanol or butanol or mixtures of the alcohols can also be used. The
catalysts may be
homogeneous catalysts, heterogeneous catalysts or enzymatic catalysts.
Homogeneous
catalysts include ferric sulphate followed by KOH. Heterogeneous catalysts
include
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CaO, K3PO4, and W03/ZrO2. Enzymatic catalysts include Novozyme 435 produced
from Candida antarctica.
Transesterification can be carried out on extracted oil, or preferably
directly in
situ in the vegetative plant material. The vegetative plant parts may be dried
and milled
prior to being used to prepare the composition for the conversion reaction,
but does not
need to be. The advantage of direct conversion to fatty acid esters,
preferably FAME, is
that the conversion can use lower temperatures and pressures and still provide
good
yields of the product, for example, comprising at least 50% FAME by weight.
The
yield of recovered bio-oil by transesterification is calculated as for the HTP
process.
Production of Non-Polar Lipids
Techniques that are routinely practiced in the art can be used to extract,
process,
purify and analyze the lipids such as the TAG produced by plants or parts
thereof of the
instant invention. Such techniques are described and explained throughout the
literature in sources such as, Fereidoon Shahidi, Current Protocols in Food
Analytical
Chemistry, John Wiley & Sons, Inc. (2001) D1.1.1-D1.1.11, and Perez-Vich et
al.
(1998).
Production of oil from vegetative plant parts or seed
Typically, vegetative plant parts or plant seeds are cooked, pressed, and/or
extracted to produce crude vegetative oil or seedoil, which is then degummed,
refined,
bleached, and deodorized. Generally, techniques for crushing seed are known in
the
art. For example, oilseeds can be tempered by spraying them with water to
raise the
moisture content to, for example, 8.5%, and flaked using a smooth roller with
a gap
setting of 0.23 to 0.27 mm. Depending on the type of seed, water may not be
added
prior to crushing. Application of heat deactivates enzymes, facilitates
further cell
rupturing, coalesces the lipid droplets, and agglomerates protein particles,
all of which
facilitate the extraction process. Vegetative plant parts can be similarly
treated,
depending on the moisture content.
In an embodiment, the majority of the vegetative oil or seedoil is released by
passage through a screw press. Cakes (vegetative plant meal, seedmeal)
expelled from
the screw press may then be solvent extracted for example, with hexane, using
a heat
traced column, or not be solvent treated, in which case it may be more
suitable as
animal feed. Alternatively, crude vegetative oil or seedoil produced by the
pressing
operation can be passed through a settling tank with a slotted wire drainage
top to
remove the solids that are expressed with the vegetative oil or seedoil during
the
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pressing operation. The clarified vegetative oil or seedoil can be passed
through a plate
and frame filter to remove any remaining fine solid particles. Once the
solvent is
stripped from the crude oil, the pressed and extracted portions are combined
and
subjected to normal lipid processing procedures (i.e., degumming, caustic
refining,
bleaching, and deodorization).
Extraction of the lipid from vegetative plant parts of the invention uses
analogous methods to those known in the art for seedoil extraction. One way is
physical
extraction, which often does not use solvent extraction. Expeller pressed
extraction is a
common type, as are the screw press and ram press extraction methods.
Mechanical
extraction is typically less efficient than solvent extraction where an
organic solvent
(e.g., hexane) is mixed with at least the plant biomass, preferably after the
biomass is
dried and ground. The solvent dissolves the lipid in the biomass, which
solution is then
separated from the biomass by mechanical action (e.g., with the pressing
processes
above). This separation step can also be performed by filtration (e.g., with a
filter press
or similar device) or centrifugation etc. The organic solvent can then be
separated from
the non-polar lipid (e.g., by distillation). This second separation step
yields non-polar
lipid from the plant and can yield a re-usable solvent if one employs
conventional
vapor recovery. In an embodiment, the oil and/or protein content of the plant
part or
seed is analysed by near-infrared reflectance spectroscopy as described in
Horn et al.
(2007) prior to extraction.
If the vegetative plant parts are not to be used immediately to extract the
lipid it
is preferably processed to ensure the lipid content is retained as much as
possible (see,
for example, Christie, 1993), such as by drying the vegetative plant parts.
Degumming
Degumming is an early step in the refining of oils and its primary purpose is
the
removal of most of the phospholipids from the oil, which may be present as
approximately 1-2% of the total extracted lipid. Addition of ¨2% of water,
typically
containing phosphoric acid, at 70-80 C to the crude oil results in the
separation of most
of the phospholipids accompanied by trace metals and pigments. The insoluble
material
that is removed is mainly a mixture of phospholipids and triacylglycerols and
is also
known as lecithin. Degumming can be performed by addition of concentrated
phosphoric acid to the crude oil to convert non-hydratable phosphatides to a
hydratable
form, and to chelate minor metals that are present. Gum is separated from the
oil by
centrifugation. The oil can be refined by addition of a sufficient amount of a
sodium
hydroxide solution to titrate all of the fatty acids and removing the soaps
thus formed.
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Alkali refining
Alkali refining is one of the refining processes for treating crude oil,
sometimes
also referred to as neutralization. It usually follows degumming and precedes
bleaching. Following degumming, the oil can treated by the addition of a
sufficient
amount of an alkali solution to titrate all of the fatty acids and phosphoric
acids, and
removing the soaps thus formed. Suitable alkaline materials include sodium
hydroxide,
potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide,
calcium carbonate and ammonium hydroxide. This process is typically carried
out at
room temperature and removes the free fatty acid fraction. Soap is removed by
centrifugation or by extraction into a solvent for the soap, and the
neutralised oil is
washed with water. If required, any excess alkali in the oil may be
neutralized with a
suitable acid such as hydrochloric acid or sulphuric acid.
Bleaching
Bleaching is a refining process in which oils are heated at 90-120 C for 10-30
minutes in the presence of a bleaching earth (0.2-2.0%) and in the absence of
oxygen
by operating with nitrogen or steam or in a vacuum. This step in oil
processing is
designed to remove unwanted pigments (carotenoids, chlorophyll, gossypol etc),
and
the process also removes oxidation products, trace metals, sulphur compounds
and
traces of soap.
Deodorization
Deodorization is a treatment of oils and fats at a high temperature (200-260
C)
and low pressure (0.1-1 mm Hg). This is typically achieved by introducing
steam into
the oil at a rate of about 0.1 ml/minute/100 ml of oil. Deodorization can be
performed
by heating the oil to 260 C under vacuum, and slowly introducing steam into
the oil at
a rate of about 0.1 ml/minute/100 ml of oil. After about 30 minutes of
sparging, the oil
is allowed to cool under vacuum. The oil is typically transferred to a glass
container
and flushed with argon before being stored under refrigeration. If the amount
of oil is
limited, the oil can be placed under vacuum for example, in a Parr reactor and
heated to
260 C for the same length of time that it would have been deodorized. This
treatment
improves the colour of the oil and removes a majority of the volatile
substances or
odorous compounds including any remaining free fatty acids, monoacylglycerols
and
oxidation products.
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Winterisation
Winterization is a process sometimes used in commercial production of oils for
the separation of oils and fats into solid (stearin) and liquid (olein)
fractions by
crystallization at sub-ambient temperatures. It was applied originally to
cottonseed oil
to produce a solid-free product. It is typically used to decrease the
saturated fatty acid
content of oils.
Algae
Algae can produce 10 to 100 times as much mass as terrestrial plants in a year
and can be cultured in open-ponds (such as raceway-type ponds and lakes) or in
photobioreactors. The most common oil-producing algae can generally include
the
diatoms (bacillariophytes), green algae (chlorophytes), blue-green algae
(cyanophytes),
and golden-brown algae (chrysophytes). In addition a fifth group known as
haptophytes may be used. Groups include brown algae and heterokonts. Specific
non-
limiting examples algae include the Classes: Chlorophyceae, Eustigmatophyceae,
Prymnesiophyceae, Bacillariophyceae. Bacillariophytes capable of oil
production
include the genera Amphipleura, Amphora, Chaetoceros, Cyclotella, Cymbella,
Fragilaria, Hantzschia, Navicula, Nitzschia, Phaeodactylum, and Thalassiosira.
Specific non-limiting examples of chlorophytes capable of oil production
include
Ankistrodesmus, Botryococcus, Chlorella, Chlorococcum, Dunaliella,
Monoraphidium,
Oocystis, Scenedesmus, and Tetraselmis. In one aspect, the chlorophytes can be
Chlorella or Dunaliella. Specific non-limiting examples of cyanophytes capable
of oil
production include Oscillatoria and Synechococcus. A
specific example of
chrysophytes capable of oil production includes Boekelovia. Specific non-
limiting
examples of haptophytes include Isochysis and Pleurochysis.
Specific algae useful in the present invention include, for example,
Chlamydomonas sp. such as Chlamydomonas reinhardtii, Dunaliella sp. such as
Dunaliella sauna, Dunaliella tertiolecta, D. acidophila, D. Lateralis.
D.martima. D.
parva, D. polmorpha, D. primolecta, D. pseudosalina, D. quartolecta. D.
viridis,
Haematococcus sp., Chlorella .sp. such as Chlorella vulgaris, Chlorella
sorokiniana or
Chlorella prototheco ides, Thraustochytrium sp., Schizochytrium sp., Volvox
sp,
Nannochloropsis sp., Botryococcus braunii which can contain over 60wt% lipid,
Phaeodactylum tricornutum, Thalassiosira pseudonana, Isochrysis sp., Pavlova
sp.,
Chlorococcum sp, Ellipsoidion sp., Neochloris sp., Scenedesmus sp.
Algae of the invention can be harvested using microscreens, by centrifugation,
by flocculation (using for example, chitosan, alum and ferric chloride) and by
froth
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flotation. Interrupting the carbon dioxide supply can cause algae to
flocculate on its
own, which is called "autoflocculation". In froth flotation, the cultivator
aerates the
water into a froth, and then skims the algae from the top. Ultrasound and
other
harvesting methods are currently under development.
Lipid may be extracted from the algae by mechanical crushing. When algal
mass is dried it retains its lipid content, which can then be "pressed" out
with an oil
press. Osmotic shock may also be used to release cellular components such as
lipid
from algae, and ultrasonic extraction can accelerate extraction processes.
Chemical
solvents (for example, hexane, benzene, petroleum ether) are often used in the
extraction of lipids from algae. Enzymatic extraction using enzymes to degrade
the cell
walls may also be used to extract lipids from algae. Supercritical CO2 can
also be used
as a solvent. In this method, CO2 is liquefied under pressure and heated to
the point
that it becomes supercritical (having properties of both a liquid and a gas),
allowing it
to act as a solvent.
Uses of Plant Lipids
The lipids produced by the methods described have a variety of uses. In some
embodiments, the lipids are used as food oils. In other embodiments, the
lipids are
refined and used as lubricants or for other industrial uses such as the
synthesis of
plastics. In some preferred embodiments, the lipids are refined to produce
biodiesel.
Biodiesel can be made from oils derived from the plants, algae and fungi of
the
invention. Use of plant triacylglycerols for the production of biofuel is
reviewed in
Durrett et at. (2008). The resulting fuel is commonly referred to as biodiesel
and has a
dynamic viscosity range from 1.9 to 6.0 mm2s-I (ASTM D6751). Bioalcohol may
produced from the fermentation of sugars or the biomass other than the lipid
left over
after lipid extraction. General methods for the production of biofuel can be
found in,
for example, Maher and Bressler (2007), Greenwell et al. (2010), Karmakar et
al.
(2010), Alonso et al. (2010), Liu et al. (2010). Gong and Jiang (2011),
Endalew et al.
(2011) and Semwal et al. (2011).
The present invention provides methods for increasing oil content in
vegetative
tissues. Plants of the present invention have increased energy content of
leaves and/or
stems such that the whole above-ground plant parts may be harvested and used
to
produce biofuel. Furthermore, the level of oleic acid is increased
significantly while the
polyunsaturated fatty acid alpha linolenic acid (ALA) was reduced. The plants.
algae
and fungi of the present invention thereby reduce the production costs of
biofuel.
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Biodiesel
The production of biodiesel, or alkyl esters, is well known. There are three
basic routes to ester production from lipids: 1) Base catalysed
transesterification of the
lipid with alcohol; 2) Direct acid catalysed esterification of the lipid with
methanol; and
3) Conversion of the lipid to fatty acids, and then to alkyl esters with acid
catalysis.
Any method for preparing fatty acid alkyl esters and glyceryl ethers (in which
one, two
or three of the hydroxy groups on glycerol are etherified) can be used. For
example,
fatty acids can be prepared, for example, by hydrolyzing or saponifying TAG
with acid
or base catalysts, respectively, or using an enzyme such as a lipase or an
esterase. Fatty
acid alkyl esters can be prepared by reacting a fatty acid with an alcohol in
the presence
of an acid catalyst. Fatty acid alkyl esters can also be prepared by reacting
TAG with
an alcohol in the presence of an acid or base catalyst. Glycerol ethers can be
prepared,
for example, by reacting glycerol with an alkyl halide in the presence of
base, or with
an olefin or alcohol in the presence of an acid catalyst. The alkyl esters can
be directly
blended with diesel fuel, or washed with water or other aqueous solutions to
remove
various impurities, including the catalysts, before blending.
Aviation Fuel
For improved performance of biofuels, thermal and catalytic chemical bond-
breaking (cracking) technologies have been developed that enable converting
bio-oils
into bio-based alternatives to petroleum-derived diesel fuel and other fuels,
such as jet
fuel.
The use of medium chain fatty acid source, such produced by a cell of the
invention, a plant or part thereof of the invention, a seed of of the
invention, or a
transgenic version of any one thereof, precludes the need for high-energy
fatty acid
chain cracking to achieve the shorter molecules needed for jet fuels and other
fuels with
low-temperature flow requirements. This method comprises cleaving one or more
medium chain fatty acid groups from the glycerides to form glycerol and one or
more
free fatty acids. In addition, the method comprises separating the one or more
medium
chain fatty acids from the glycerol, and decarboxylating the one or more
medium chain
fatty acids to form one or more hydrocarbons for the production of the jet
fuel.
Compositions
The present invention also encompasses compositions, particularly
pharmaceutical compositions, comprising one or more plants, plant parts,
lipids,
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proteins, nitrogen containing molecules, or carbon containing molecules,
produced
using the methods of the invention.
A pharmaceutical composition may additionally comprise an active ingredient
and a standard, well-known, non-toxic pharmaceutically-acceptable carrier,
adjuvant or
vehicle such as phosphate-buffered saline, water, ethanol, polyols, vegetable
oils, a
wetting agent, or an emulsion such as a water/oil emulsion. The composition
may be in
either a liquid or solid form. For example, the composition may be in the form
of a
tablet, capsule, ingestible liquid, powder, topical ointment or cream. Proper
fluidity
can be maintained for example, by the maintenance of the required particle
size in the
case of dispersions and by the use of surfactants. It may also be desirable to
include
isotonic agents for example, sugars, sodium chloride, and the like. Besides
such inert
diluents, the composition can also include adjuvants such as wetting agents,
emulsifying and suspending agents, sweetening agents, flavoring agents and
perfuming
agents.
A typical dosage of a particular fatty acid is from 0.1 mg to 20 g, taken from
one
to five times per day (up to 100 g daily) and is preferably in the range of
from about 10
mg to about 1, 2, 5, or 10 g daily (taken in one or multiple doses). As known
in the art,
a minimum of about 300 mg/day of fatty acid, especially polyunsaturated fatty
acid, is
desirable. However, it will be appreciated that any amount of fatty acid will
be
beneficial to the subject.
Possible routes of administration of the pharmaceutical compositions of the
present invention include for example, enteral and parenteral. For example, a
liquid
preparation may be administered orally. Additionally, a homogenous mixture can
be
completely dispersed in water, admixed under sterile conditions with
physiologically
acceptable diluents, preservatives, buffers or propellants to form a spray or
inhalant.
The dosage of the composition to be administered to the subject may be
determined by one of ordinary skill in the art and depends upon various
factors such as
weight, age, overall health, past history, immune status, etc., of the
subject.
Additionally, the compositions of the present invention may be utilized for
cosmetic purposes. The compositions may be added to pre-existing cosmetic
compositions, such that a mixture is formed, or a fatty acid produced
according to the
invention may be used as the sole "active" ingredient in a cosmetic
composition.
Polypeptides
The terms "polypeptide" and "protein" are generally used interchangeably
herein.
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A polypeptide or class of polypeptides may be defined by the extent of
identity
(% identity) of its amino acid sequence to a reference amino acid sequence, or
by
having a greater % identity to one reference amino acid sequence than to
another. The
% identity of a polypeptide to a reference amino acid sequence is typically
determined
by GAP analysis (Needleman and Wunsch, 1970; GCG program) with parameters of a
gap creation penalty = 5, and a gap extension penalty = 0.3. The query
sequence is at
least 100 amino acids in length and the GAP analysis aligns the two sequences
over a
region of at least 100 amino acids. Even more preferably, the query sequence
is at least
250 amino acids in length and the GAP analysis aligns the two sequences over a
region
of at least 250 amino acids. Even more preferably, the GAP analysis aligns two
sequences over their entire length, and the extent of identity is determined
over the full
length of the reference sequence. The polypeptide or class of polypeptides may
have
the same enzymatic activity as, or a different activity than, or lack the
activity of, the
reference polypeptide. Preferably, the polypeptide has an enzymatic activity
of at least
10% of the activity of the reference polypeptide.
As used herein a "biologically active fragment" is a portion of a polypeptide
of
the invention which maintains a defined activity of a full-length reference
polypeptide
for example. DGAT activity. Biologically active fragments as used herein
exclude the
full-length polypeptide. Biologically active fragments can be any size portion
as long
as they maintain the defined activity. Preferably, the biologically active
fragment
maintains at least 10% of the activity of the full length polypeptide.
With regard to a defined polypeptide or enzyme, it will be appreciated that %
identity figures higher than those provided herein will encompass preferred
embodiments. Thus, where applicable, in light of the minimum % identity
figures, it is
preferred that the polypeptide/enzyme comprises an amino acid sequence which
is at
least 60%, more preferably at least 65%, more preferably at least 70%, more
preferably
at least 75%, more preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, more preferably at least 91%, more preferably at
least 92%,
more preferably at least 93%, more preferably at least 94%, more preferably at
least
95%, more preferably at least 96%, more preferably at least 97%, more
preferably at
least 98%, more preferably at least 99%, more preferably at least 99.1%, more
preferably at least 99.2%, more preferably at least 99.3%, more preferably at
least
99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more
preferably
at least 99.7%, more preferably at least 99.8%, and even more preferably at
least 99.9%
identical to the relevant nominated SEQ ID NO.
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Amino acid sequence mutants of the polypeptides defined herein can be
prepared by introducing appropriate nucleotide changes into a nucleic acid
defined
herein, or by in vitro synthesis of the desired polypeptide. Such mutants
include for
example, deletions, insertions, or substitutions of residues within the amino
acid
sequence. A combination of deletions, insertions and substitutions can be made
to
arrive at the final construct, provided that the final polypeptide product
possesses the
desired characteristics.
Mutant (altered) polypeptides can be prepared using any technique known in the
art, for example, using directed evolution or rathional design strategies (see
below).
Products derived from mutated/altered DNA can readily be screened using
techniques
described herein to determine if they possess transcription factor, fatty acid
acyltransferase or OBC activities.
In designing amino acid sequence mutants, the location of the mutation site
and
the nature of the mutation will depend on characteristic(s) to be modified.
The sites for
mutation can be modified individually or in series for example, by (1)
substituting first
with conservative amino acid choices and then with more radical selections
depending
upon the results achieved, (2) deleting the target residue, or (3) inserting
other residues
adjacent to the located site.
Amino acid sequence deletions generally range from about 1 to 15 residues,
more preferably about 1 to 10 residues and typically about 1 to 5 contiguous
residues.
Substitution mutants have at least one amino acid residue in the polypeptide
removed and a different residue inserted in its place. The sites of greatest
interest for
substitutional mutagcnesis to inactivate enzymes include sites identified as
the active
site(s). Other sites of interest are those in which particular residues
obtained from
various strains or species are identical. These positions may be important for
biological
activity. These sites, especially those falling within a sequence of at least
three other
identically conserved sites, are preferably substituted in a relatively
conservative
manner. Such conservative substitutions are shown in Table 1 under the heading
of
"exemplary substitutions".
Table 1. Exemplary substitutions.
Original Exemplary
Residue Substitutions
Ala (A) val; leu; ile; gly
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Arg (R) lys
Asn (N) gin; his
Asp (D) glu
Cys (C) ser
Gin (Q) asn; his
Glu (E) asp
Gly (G) pro, ala
His (H) asn; gin
Ile (I) leu; val; ala
Leu (L) ile; val; met; ala; phe
Lys (K) arg
Met (M) leu; phe
Phe (F) leu; val; ala
Pro (P) gly
Ser (S) thr
Thr (T) ser
Trp (W) tyr
Tyr (Y) trp; phe
Val (V) lie; leu; met; phe, ala
In a preferred embodiment a mutant/variant polypeptide has only, or not more
than, one or two or three or four conservative amino acid changes when
compared to a
naturally occurring polypeptide. Details of conservative amino acid changes
are
provided in Table 1. As the skilled person would be aware, such minor changes
can
reasonably be predicted not to alter the activity of the polypeptide when
expressed in a
transgenic plant or part thereof. Mutants with desired activity may be
engineered using
standard procedures in the art such as by performing random mutagenesis,
targeted
mutagenesis, or saturation mutagenesis on known genes of interest, or by
subjecting
different genes to DNA shuffling.
EXAMPLES
Example 1. General Materials and Methods
Expression of genes in plant cells in a transient expression system
Genes were expressed in plant cells using a transient expression system
essentially as described by Voinnet et al. (2003) and Wood et al. (2009).
Binary
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vectors containing the coding region to be expressed by a strong constitutive
e35S
promoter containing a duplicated enhancer region were introduced into
Agrobacterium
tumefaciens strain AGL1. A chimeric binary vector, 35S:p19, for expression of
the p19
viral silencing suppressor was separately introduced into AGL1, as described
in
W02010/057246. A chimeric binary vector, 35S:V2, for expression of the V2
viral
silencing suppressor was separately introduced into AGL1. The recombinant
cells
were grown to stationary phase at 28 C in LB broth supplemented with 50 mg/L
kanamycin and 50 mg/L rifampicin. The bacteria were then pelleted by
centrifugation
at 5000 g for 5 min at room temperature before being resuspended to 0D600 =
1.0 in
an infiltration buffer containing 10 mM MES pH 5.7, 10 mM MgCl2 and 100 uM
acetosyringone. The cells were then incubated at 28 C with shaking for 3 hours
after
which the 0D600 was measured and a volume of each culture, including the viral
suppressor construct 35S:p19 or 35S:V2, required to reach a final
concentration of
0D600 = 0.125 added to a fresh tube. The final volume was made up with the
above
buffer. Leaves were then infiltrated with the culture mixture and the plants
were
typically grown for a further three to five days after infiltration before
leaf discs were
recovered for either purified cell lysate preparation or total lipid
isolation.
Transformation of Sorghum bicolor L.
Plant Material
Sorghum plants of the inbred cultivar TX-430 (Miller, 1984) were grown in a
plant growth chamber (Conviron, PGC-20 flex) at 28 1 C "day" temperature and
20
1 C "night" temperature, with a 16 hr photoperiod at a light intensity during
the "day"
of 900-1000 LUX. Panicles were covered with white translucent paper bags
before
flowering. Immature embryos were harvested from panicles 12-15 days after
anthesis.
Panicles were washed several times with water and developing seeds that were
uniform
in size were isolated and surface-sterilized using 20% commercial bleach mixed
with
0.1% Tween-20 for 15-20 min. They were then washed with sterile distilled
water 3
times each for 20 min, and blotted dry in a laminar flow hood. Immature
embryos (IEs)
ranging from 1.4 to 2.5 mm in length were aseptically isolated in the laminar
flow hood
and used as the starting tissue for preparation of green regenerative tissue.
Base Cultivation Media
Media used for plant transformation were based on MS (Murashige and Skoog.
1962). supplied by PhytoTechnology Laboratories (M519). The pH of the media
was
adjusted to 5.8 before sterilization at 121 C for 15 min. Heat sensitive plant
growth
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'
regulators and other additives such as Geneticin (G418, Sigma) used as a
selection
agent, were filter sterilized (0.2 I'm) and added to the media after
sterilization when the
media had cooled to about 55 C. The optimized culture medium composition for
the
different stages of plant transformation from callus induction to plant
regeneration from
green tissue induced from immature embryos is presented in Table 2.
Cultivation Methods and Materials
The isolated lEs ranging from 1.4 to 2.5 mm in length were placed onto callus
induction media-osmotic medium (CIM-osmotic medium, Table 2) with their
scutellum
facing upward. The CIM base medium was modified to improve callus quality and
induction frequency from immature embryos, as well as callus regeneration
media, by
including a-Lipoic acid (1 to 5 mg/1), Melatonin (5 to 10 mg/I) and 2-
Aminoidan-2-
phosphonic acid HCl (1 to 2 mg/1) unless otherwise stated. For the development
of
green tissue, immature embryos were incubated under fluorescent light of
approximately 45-501.1mol s-1 m-2 (16 h/day) in a tissue culture room at 24
2 C. After
three days of culture, the root and shoot poles of the immature embryos were
aseptically separated and re-inoculated on to the same CIM and maintained
under the
same conditions as described above. They were subcultured every two weeks onto
the
same CIM for 6 weeks and evaluated for callus quality, callus induction
efficiency and
transformation efficiency.
Table 2. Media used in DEC tissue induction and transformation of sorghum
Name of the Composition Culture
medium duration
CIM- MS medium powder with vitamins, 4.33 g/1; 2,4-D, 1 3-4
hrs before
Osmotic mg/1; BAP, 0.5 mg/1; L-proline, 0.7 g/I; L-Lipoic
bombardment;
Medium acid, 1 mg/I; peptone, 0.82 g/1; Myo-inositol, 150 o/n
post
mg/1; Copper sulfate. 0.8 mg/I; Manitol, 36.4 g/I; bombardment
Sorbitol, 36.4 g/1; Agar, 8.5 g/1, pH 5.8
CIM- pre MS medium powder with vitamins, 4.33 g/1; 2,4-D, 1 3-4
days
selection mg/1; BAP, 0.5 mg/1; L-proline, 0.7 g/I; L-Lipoic
medium acid, 1 mg/1; peptone, 0.82 g/1; Myo-inosito,1 150
mg/1; Copper sulfate, 0.8 mg/1; Maltose, 30 g/1; L-
cysteine, 50 mg/I; Ascorbic acid, 15 mg/1; Agar, 9
g/l, pH 5.8
CIM-callus MS medium powder with vitamins, 4.33 g/1; 2,4-D, 1 4
weeks
induction mg/1; BAP, 0.5 mg/I; L-proline, 0.7 g/I; L-Lipoic
medium/G25 acid. 1 mg/1; peptone, 0.82 g/I; Myo-inositol, 150
mg/1; Copper sulfate, 0.8 mg/1; Maltose, 30 g/I;
Geneticin, 25 mg/1; Agar, 9 g/1, pH 5.8
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.*
Name of the Composition Culture
medium duration
SIM-shoot MS medium powder with vitamins, 4.33 g/1; BAP, 2 weeks
induction 1.0 mg/1; 2,4-D, 0.5 mg/I; L-proline, 0.7 g/I; L-Lipoic
medium/G25 acid, 1 mg/1; peptone, 0.82 g/1; Myo-inositol, 150
mg/1; Copper sulfate, 0.8 mg/1; Maltose, 30 g/1;
Geneticin, 25 mg/1; Agar, 9 g/l, pH 5.8
SRM- shoot MS medium powder with vitamins, 4.33 g/1; BAP, 2 weeks
regeneration 1.0 mg/1; TDZ, 0.5 mg/1; L-proline, 0.7 g/1; L-Lipoic
medium/G25 acid, 1 mg/1; peptone. 0.82 g/I; Myo-inositol, 150
mg/1; Copper sulfate, 0.8 mg/1; Maltose, 30 g/1;
Geneticin, 25 mg/1; Agar, 9 g/1, pH 5.8
SOG-shoot MS medium powder with vitamins, 2.2 g/1; L- 2 weeks
out growth proline, 0.7 g/1; L-Lipoic acid, 1 mg/1; peptone, 0.82
medium/G30 g/I; Myo-inositol, 150 mg/1; Copper sulfate, 0.8 mg/1;
Sucrose, 15 g/1; Geneticin, 30 mg/1; Agar, 9 g/l, pH
5.8
RIM-root MS medium powder with vitamins, 4.33 g/l; L- 4 weeks
induction proline, 0.7 g/1; L-Lipoic acid, 1 mg/1; peptone, 0.82
medium/G15 g/l; Myo-inositol, 150 mg/1; Copper sulfate, 0.8 mg/I;
sucrose, 15 g/I; IAA, 1 mg/1; IBA, 1 mg/1; NAA, 1
mg/1; PVP, 2 g/1; Geneticin, 15 mg/I; Agar 9 g/l, pH
5.8
Callus initiated from lEs in the first 3-4 weeks on CIM were mostly
embryogenic
and slowly differentiated into embryogenic callus with nodular structures
which were
coloured from pale to darker green. Embryogenic calli with green nodular
structures
were selected and maintained on the same medium (CIM) by subculturing every 2
weeks for up to 6 months or more, for use as explants for transformation. This
type of
tissue is termed herein as "differentiating embryogenic callus" tissue or
"DEC" tissue,
since this tissue forms nodular structures of differentiating cells which
maintain
embryogenic and organogenic potential, even though the tissues were really a
mixture
of callus cells, cells forming nodular structures and granular structures, and
intermediate cells which the inventors understood were on the developmental
pathway
somewhere between callus (which is undifferentiated cells) and the nodular
structures.
Sometimes, the tissues included early stage (globular) somatic embryos.
Particle-bombardment of green regenerative DEC tissues
Plasmids containing a selectable marker gene encoding the neomycin
phosphotransferase II (NptII) providing resistance to the antibiotic
Geneticin, under the
control of the pUbi promoter and terminated by the nos 3' region, were made or
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a.
obtained for experiments to achieve stable transformation or for co-
bombardment with
other plasmids. Plasmid DNAs were isolated using a ZymopureTM Maxiprep kit
(USA)
according to the manufacturer's instructions. As a control vector for
transformation, a
genetic vector was obtained which contained uidA (GUS) and bar genes designed
for
expression in plant cells. The uidA gene was under the regulatory control of a
maize
polyubiquitin promoter (pUbi) and an Agrobacteriurn tumefaciens octopine
synthase
polyadenylation/terminator (ocs 3') sequence. The sequence between the
promoter and
the protein coding region included the 5' UTR and first intron of the Ubi
gene. The
uidA reporter gene also contained, within its protein coding region, an intron
from a
castor bean catalase gene which prevented translation of functional GUS
protein in
Agrobacterium, thereby reducing the background GUS gene expression in
inoculated
plant tissues. Therefore, any GUS expression would be due to expression of the
uidA
gene in the plant cells. The bar gene was also under the regulatory control of
a pUbi
promoter and terminated with an Agrobacterium nopaline synthase 3' regulatory
sequence (nos 3'). The uidA/bar vector was initially used in experiments to
detect
transient gene expression in the sorghum DEC tissues.
Uniform healthy, green regenerative DEC tissues (4-5 mm in size), produced
using methods described above and having been cultured for 6 weeks to 6 months
from
initiation, were used for mieroprojectile-mediated transformation
(bombardment) with
the plasmids. Approximately 15 uniform green DEC tissues (each 4-5 mm) were
placed
at the centre of a petri dish (90 mm diameter) containing C1M-osmotic medium
(Table
2) and incubated in the dark for about 4 hrs prior to bombardment. Bombardment
was
performed with a PDS-1000 He device (Biorad, Hercules, CA) as described by Liu
et
al. (2014). Post bombardment, the tissues were kept on the same osmotic medium
overnight and transferred to pre-selection medium the next morning
Green DEC tissues bombarded with the genetic vector plasmid having a
selectable marker encoding NptII were transferred to CIM-PS medium for 3-4
days
before any selection, with addition to the medium of two compounds as
antioxidants,
L-cysteine (50 mg/1) and ascorbic acid (15 mg/1) (Table 2). Without the
addition of
these antioxidants in pre-selection medium, many of the bombarded tissues
turned
brown, some quite dark brown in colour, and many lost any ability to grow
further.
After 3-4 days on pre-selection medium, some of the bombarded tissues were
subjected
to GUS staining and viewed under a microscope to count the distinctive blue
(GUS
positive) spots, to check that genes had been transferred and could be
expressed. The
inclusion of the two antioxidants in the pre-selection medium improved the
efficiency
of the transformation as shown by the transient expression of the GUS gene.
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Selection and regeneration of transgenic plants with optimised conditions
Following bombardment and 3-4 days culture on pre-selection medium without
selective agent (Geneticin), the bombarded tissues had increased in size from
4-5 mm
to about 6-7 mm. These tissues were transferred to selective medium CIM/G25
containing 25 mg/1 Geneticin (Table 2) and cultured for a further 4 weeks.
When
possible, the bombarded tissues were split into 2-6 pieces each, increasing
the recovery
of independent transformants. All of the tissues were cultured on the media as
described in Table 2 and maintained in order to regenerate putative transgenic
plants.
Plants were regenerated efficiently upon growth on these media. Each bombarded
tissue and the shoots obtained from it were subcultured and maintained
separately for
calculation of the transformation efficiency. Positive transformation was
confirmed by
PCR on plant genomic DNA isolated from shoot samples, showing the presence of
the
selectable marker gene. The number of transformants was calculated per input
DEC
tissue. Transformation efficiencies of about 50% were obtained, expressed as
independent transformants per input bombarded tissue.
Agrobacterium-mediated transformation of green regenerative DEC tissues
Uniform healthy, green regenerative DEC tissues (4-5 mm in size) produced
using methods described in the foregoing examples and which have been cultured
for 6
weeks to 6 months from initiation, are used for Agrobacterium-mediated
transformation.
Genetic vectors having T-DNA regions containing the genes for transformation
were designed and made for transformation of green regenerative DEC tissues
using
Agrobacterium-mediated transformation. A control binary vector contained uidA
(GUS) and bar genes designed for expression in plant cells. The uidA gene was
under
the regulatory control of a maize polyubiquitin promoter (pUbi) and an
Agrobacterium
tumefaciens octopine synthase polyadenylation/terminator (ocs 3') sequence.
The
sequence between the promoter and the protein coding region included the 5'
UTR and
first intron of the Ubi gene. The uidA reporter gene also contained, within
its protein
coding region, an intron from a castor bean catalase gene which prevented
translation
of functional GUS protein in Agrobacterium, thereby reducing the background
GUS
gene expression in inoculated plant tissues. Therefore, any GUS expression was
due to
expression of the uidA gene in the plant cells. The bar gene was also under
the
regulatory control of a pUbi promoter and terminated with an Agrobacterium
nopaline
synthase 3' regulatory sequence (nos 3').
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A suitable Agrobacterium tumefaciens strain was obtained e.g., AGL1 as
described in Lazo et at. (1991) and the genetic vector is introduced into the
Agrobacterium tumefaciens strain by heat shock method.
Agrobacterium cultures harboring the genetic construct are grown in suitable
medium e.g., LB medium, and under appropriate conditions to produce an
Agrobacterium inoculum, after which time the uniform healthy, green
regenerative
DEC tissues are infected with Agrobacterium inoculum. The infected DEC tissues
are
blotted on sterile filter paper to remove excess Agrobacterium and transferred
to co-
cultivation medium, optionally supplemented with antioxidants, and incubated
in the
dark at approximately 22-24 C for 2-4 days. Following incubation, the DEC
tissues are
treated with an appropriate agent to kill the Agrobacterium, washed in sterile
water,
transferred to an appropriate medium and allowed to grow. After 4-6 weeks,
shoots are
excised and cultured on shoot elongation medium, after which time putative
transgenic
shoots are then detected using appropriate assays.
Brassica napus transformation
Brassica napus seeds were sterilized using chlorine gas as described by
Kereszt
et al. (2007) and germinated on tissue culture medium. Cotyledonary petioles
with 2-4
mm stalk were isolated as described by Belide et al. (2013) and used as
explants. A.
tumefaciens AGL1 (Lazo et al., 1991) cultures containing the binary vector
were
prepared and cotyledonary petioles inoculated with the cultures as described
by Belide
et al. (2013). Infected cotyledonary petioles were cultured on MS medium
supplemented with 1 mg/L TDZ + 0.1 mg/L NAA + 3 mg/L AgNO3 + 250 mg/L
cefotaxime, 50 mg/L timentin and 25 mg/L kanamycin and cultured for 4 weeks at
24 C with 16hr/8hr light-dark photoperiod with a biweekly subculture on to the
same
medium. Explants with green callus were transferred to shoot initiation medium
(MS +
1 mg/L kinetin + 3 mg/L AgNO3 + 250 mg/L cefotaxime + 50 mg/L timentin + 25
mg/L kanamycin) and cultured for another 2-3 weeks. Small shoots (-1 cm) were
isolated from the resistant callus and transferred to shoot elongation medium
(MS
medium with 0.1 mg/L gibberelic acid + 3 mg/L AgNO3 + 250 mg/L cefotaxime + 25
mg/L kanamycin) and cultured for another two weeks. Healthy shoots with one or
two
leaves were selected and transferred to rooting media (1/2 MS with 1 mg/L NAA
+ 20
mg/L ADS + 3 mg/L AgNO3 + 250 mg/L cefotaxime) and cultured for 2-3 weeks.
DNA was isolated from small leaves of resistant shoots using the plant DNA
isolation
kit (Bioline, Alexandria, NSW, Australia) as described by the manufacturer's
protocol.
The presence of T-DNA sequences was tested by PCR amplification on genomic
DNA.
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Positive, transgenic shoots with roots were transferred to pots containing
seedling
raising mix and grown in a glasshouse at 24 C daytime/16 C night-time
(standard
conditions).
Purified leaf lysate ¨ enzyme assays
Nicotiana benthamiana leaf tissues previously infiltrated as described above
were ground in a solution containing 0.1 M potassium phosphate buffer (pH 7.2)
and
0.33 M sucrose using a glass homogenizer. Leaf homogenate was centrifuged at
20,000 g for 45 minutes at 4 C after which each supernatant was collected.
Protein
content in each supernatant was measured according to Bradford (1976) using a
Wallac1420 multi-label counter and a Bio-Rad Protein Assay dye reagent (Bio-
Rad
Laboratories, Hercules, CA USA). Acyltransferase assays used 100 p.2 protein
according to Cao et al. (2007) with some modifications. The reaction medium
contained 100 mM Tris-HC1 (pH 7.0), 5 mM MgCl2, 1 mg/mL BSA (fatty acid-free),
200 mM sucrose, 40 mM cold oleoyl-CoA, 16.4 1,1M sn-2 monooleoylglycerol[14C1
(55mCi/mmol, American Radiochemicals, Saint Louis, MO USA) or 6.0 M
,14
Cliglycerol-3-phosphate (G-3-P) disodium salt (150 mCi/mmol, American
Radiochemicals). The assays were carried out for 7.5, 15, or 30 minutes.
Lipid analysis
Analysis of oil content in seeds
When seed oil content or total fatty acid composition was to be determined in
small seeds such as Arabidopsis seeds, fatty acids in the seeds were directly
methylated
without crushing of seeds. Seeds were dried in a desiccator for 24 hours and
approximately 4 mg of seed was transferred to a 2 ml Wass vial containing a
Teflon-
lined screw cap. 0.05 mg triheptadecanoin (TAG with three C17:0 fatty acids)
dissolved in 0.1 ml toluene was added to the vial as internal standard. Seed
fatty acids
were methylated by adding 0.7 ml of 1N methanolic HC1 (Supelco) to the vial
containing seed material. Crushing of the seeds was not necessary for complete
methylation with small seeds such as Arab idopsis seeds. The mixture was
vortexed
briefly and incubated at 80 C for 2 hours. After cooling the mixtures to room
temperature, 0.3 ml of 0.9% NaCl (w/v) and 0.1 ml hexane was added to the vial
and
mixed well for 10 minutes in a Heidolph Vibramax 110. The FAME were collected
into a 0.3 ml glass insert and analysed by GC with a flame ionization detector
(FID) as
described below.
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The peak area of individual FAME were first corrected on the basis of the peak
area responses of a known amount of the same FAMEs present in a commercial
standard GLC-411 (NU-CHEK PREP, INC., USA). GLC-411 contains equal amounts
of 31 fatty acids (% by weight), ranging from C8:0 to C22:6. In case of fatty
acids
which were not present in the standard, the peak area responses of the most
similar
FAME was taken. For example, the peak area response of FAMEs of 16:1d9 was
used
for 16:1d7 and the FAME response of C22:6 was used for C22:5. The corrected
areas
were used to calculate the mass of each FAME in the sample by comparison to
the
internal standard mass. Oil is stored mainly in the form of TAG and its weight
was
calculated based on FAME weight. Total moles of glycerol was determined by
calculating moles of each FAME and dividing total moles of FAMEs by three. TAG
content was calculated as the sum of glycerol and fatty acyl moieties using a
relation:
% oil by weight = 100x ((41x total mol FAME/3)+(total g FAME- (15x total mol
FAME)))/g seed, where 41 and 15 are molecular weights of glycerol moiety and
methyl
group, respectively.
Analysis of fatty acid content in larger seeds
To determine fatty acid composition in single seeds that were larger, such as
canola and Camelina seeds. or Sorghum or corn seeds, direct methylation of
fatty acids
in the seed was performed as for Arabidopsis seeds except with breaking of the
seed
coats. This method extracted sufficient oil from the seed to allow fatty acid
composition analysis. To determine the fatty acid composition of total
extracted lipid
from seeds, seeds were crushed and lipids extracted with CHC13/Me0H. Aliquots
of the
extracted lipid were methylated and analysed by GC. Pooled seed-total lipid
content
(seed oil content) of canola was determined by two extractions of lipid using
CFIC13/Me0H from a known weight of desiccated seeds after crushing, followed
by
methylation of aliquots of the lipids together with the 17:0 fatty acids as
internal
standard. In the case of larger seeds such as Camelina, the lipid from a known
amount
of seeds was methylated together with known amount of 17:0 fatty acids as for
the
Arabidopsis oil analysis and FAME were analysed by GC. For TAG quantitation,
TAG
was fractionated from the extracted lipid using TLC and directly methylated in
silica
using 17:0 TAG as an internal standard. These methods are described more fully
as
follows.
After harvest at plant maturity, seeds were desiccated by storing the seeds
for
24 hours at room temperature in a desiccator containing silica gel as
desiccant.
Moisture content of the seeds was typically 6-8%. Total lipids were extracted
from
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known weights of the desiccated seeds by crushing the seeds using a mixture of
chloroform and methanol (2/1 v/v) in an eppcndorf tube using a Reicht tissue
lyser (22
frequency/seconds for 3 minutes) and a metal ball. One volume of 0.1M KC1 was
added and the mixture shaken for 10 minutes. The lower non-polar phase was
collected
after centrifuging the mixture for 5 minutes at 3000 rpm. The remaining upper
(aqueous) phase was washed with 2 volumes of chloroform by mixing for 10
minutes.
The second non-polar phase was also collected and pooled with the first. The
solvent
was evaporated from the lipids in the extract under nitrogen flow and the
total dried
lipid was dissolved in a known volume of chloroform.
To measure the amount of lipid in the extracted material, a known amount of
17:0-TAG was added as internal standard and the lipids from the known amount
of
seeds incubated in 1 N methanolic-HC1 (Supelco) for 2 hours at 80 C. FAME thus
made were extracted in hexane and analysed by GC. Individual FAME were
quantified
on the basis of the amount of 17:0 TAG-FAME. Individual FAME weights, after
subtraction of weights of the esterified methyl groups from FAME, were
converted into
moles by dividing by molecular weights of individual FAME. Total moles of all
FAME
were divided by three to calculate moles of TAG and therefore glycerol. Then,
moles
of TAG were converted in to weight of TAG. Finally, the percentage oil content
on a
seed weight basis was calculated using seed weights, assuming that all of the
extracted
lipid was TAG or equivalent to TAG for the purpose of calculating oil content.
This
method was based on Li et al. (2006). Seeds other than Camelina or canola
seeds that
are of a similar size can also be analysed by this method.
Canola and other seed oil content can be measured by nuclear magnetic
resonance techniques (Rossell and Pritchard, 1991) by a pulsed wave NMS 100
Minispec (Bruker Pty Ltd Scientific Instruments, Germany). The NMR method can
simultaneously measured moisture content. Seed oil content can also be
measured by
near infrared reflectance (NIR) spectroscopy such as using a NIRSystems Model
5000
monochromator. Moisture content can also be measured on a sample from a batch
of
seeds by drying the seeds in the sample for 18 hours at about 100 C, according
to Li et
al. (2006).
Analysis of lipids from leaf lysaie assays
Lipids from the lysate assays were extracted using chloroform:methano1:0.1 M
KC1 (2:1:1) and recovered. The different lipid classes in the samples were
separated on
Silica gel 60 thin layer chromatography (TLC) plates (MERCK, Dermstadt,
Germany)
impregnated with 10% boric acid. The solvent system used to fractionate TAG
from
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the lipid extract was chloroform/acetone (90/10 v/v). Individual lipid classes
were
visualized by exposing the plates to iodine vapour and identified by running
parallel
authentic standards on the same TLC plate. The plates were exposed to phosphor
imaging screens overnight and analysed by a Fujifilm FLA-5000 phosphorimager
before liquid scintillation counting for DPM quantification.
Total lipid isolation andfractionation of lipids from vegetative tissues
Fatty acid composition of total lipid in leaf and other vegetative tissue
samples
was determined by direct methylation of the fatty acids in freeze-dried
samples. For
total lipid quantitation, fatty acids in a known weight of freeze-dried
samples, with 17:0
FFA, were directly methylated. To determine total TAG levels in leaf samples,
TAG
was fractionated by TLC from extracted total lipids, and methylated in the
presence of
17:0 TAG internal standard, because of the presence of substantial amounts of
polar
lipids in leaves. This was done as follows. Tissues including leaf samples
were freeze-
dried, weighed (dry weight) and total lipids extracted as described by Bligh
and Dyer
(1959) or by using chloroform:methano1:0.1 M KCl (CMK; 2:1:1) as a solvent.
Total
lipids were extracted from N. benthamiana leaf samples, after freeze dying, by
adding
9004 of a chloroform/methanol (2/1 v/v) mixture per 1 cm diameter leaf sample.
0.8
DAGE was added per 0.5 mg dry leaf weight as internal standard when TLC-FID
analysis was to be performed. Samples were homogenized using an IKA ultra-
turrax
tissue lyser after which 500 I., 0.1 M KC1 was added. Samples were vortexed,
centrifuged for 5 mm and the lower phase was collected. The remaining upper
phase
was extracted a second time by adding 600 ItL chloroform, vortexing and
centrifuging
for 5 min. The lower phase was recovered and pooled into the previous
collection.
Lipids were dried under a nitrogen flow and resuspended in 2 pt chloroform per
mg
leaf dry weight. Total lipids of N. tabacum leaves or leaf samples were
extracted as
above with some modifications. If 4 or 6 leaf discs (each approx 1 cm2 surface
area)
were combined, 1.6 ml of CMK solvent was used, whereas if 3 or less leaf discs
were
combined, 1.2 ml CMK was used. Freeze dried leaf tissues were homogenized in
an
eppendorf tube containing a metallic ball using a Reicht tissue lyser (Qiagen)
for 3
minutes at 20 frequency/sec.
Separation of neutral lipids via TLC and transmethylation
Known volumes of total leaf extracts such as, for example, 30 tit were loaded
on a TLC silica gel 60 plate (1x20 cm) (Merck KGaA, Germany). The neutral
lipids
were fractionated into the different types and separated from polar lipids via
TLC in an
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equilibrated development tank containing a hexane/DEE/acetic acid (70/30/1
v/v/v/)
solvent system. The TAG bands were visualised by primuline spraying, marked
under
UV, scraped from the TLC plate, transferred to 2 mL GC vials and dried with
N2. 750
[IL of 1N methanolic-HC1 (Supelco analytical, USA) was added to each vial
together
with a known amount of C17:0 TAG as an internal standard, depending on the
amount
of TAG in each sample. Typically, 30 jig of the internal standard was added
for low
TAG samples whilst up to 200 [tg of internal standard was used in the case of
high
TAG samples.
Lipid samples for fatty acid composition analysis by GC were transmethylated
by incubating the mixtures at 80 C for 2 hours in the presence of the
methanolic-HCl.
After cooling samples to room temperature, the reaction was stopped by adding
350 IA
FLO. Fatty acyl methyl esters (FAME) were extracted from the mixture by adding
350
ill hexane, vortexing and centrifugation at 1700 rpm for 5 mm. The upper
hexane
phase was collected and transferred into GC vials with 300 [11 conical
inserts. After
evaporation, the samples were resuspended in 30 [t1 hexane. One ill was
injected into
the GC.
The amount of individual and total fatty acids (TFA) present in the lipid
fractions was quantified by GC by determining the area under each peak and
calculated
by comparison with the peak area for the known amount of internal standard.
TAG
content in leaf was calculated as the sum of glycerol and fatty acyl moieties
in the TAG
fraction using a relation: % TAG by weigh = 100x ((41x total mol
FAME/3)+(total g
FAME- (15x total mol FAME)))/g leaf dry weight, where 41 and 15 are molecular
weights of glycerol moiety and methyl group, respectively.
Capillary gas-liquid chromatography (GC)
FAME were analysed by GC using an Agilent Technologies 7890A GC (Palo
Alto, California, USA) equipped with an SGE BPX70 (70% cyanopropyl
polysilphenylene-siloxane) column (30 m x 0.25 mm i.d., 0.25 [tm film
thickness), an
FID, a split/splitless injector and an Agilent Technologies 7693 Series auto
sampler and
injector. Helium was used as the carrier gas. Samples were injected in split
mode
(50:1 ratio) at an oven temperature of 150 C. After injection, the oven
temperature was
held at 150 C for 1 min, then raised to 210 C at 3 C.min-1 and finally to 240
C at
50 C.min-1. Peaks were quantified with Agilent Technologies ChemStation
software
(Rev B.04.03 (16), Palo Alto, California, USA) based on the response of the
known
amount of the external standard GLC-411 (Nucheck) and C17:0-Me internal
standard.
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Quantification of TAG via Iatroscan
One 41_, of lipid extract was loaded on one Chromarod-SII for TLC-FID
IatroscanTm (Mitsubishi Chemical Medience Corporation ¨ Japan). The Chromarod
rack was then transferred into an equilibrated developing tank containing 70
mL of a
hexane/CHC13/2-propanol/formic acid (85/10.716/0.567/0.0567 v/v/v/v) solvent
system. After 30 mm of incubation, the Chromarod rack was dried for 3 min at
100 C
and immediately scanned on an Iatroscan MK-6s TLC-FID analyser (Mitsubishi
Chemical Medience Corporation ¨ Japan). Peak areas of DAGE internal standard
and
TAG were integrated using SIC-48011 integration software (Version:7.0-E SIC
System
instruments Co., LTD ¨ Japan).
TAG quantification was carried out in two steps. First, DAGE was scanned in
all samples to correct the extraction yields after which concentrated TAG
samples were
selected and diluted. Next, TAG was quantified in diluted samples with a
second scan
according to the external calibration using glyceryl trilinoleate as external
standard
(Sigma-Aldrich).
Quantification of TAG in leaf samples by GC
The peak area of individual FAME were first corrected on the basis of the peak
area responses of known amounts of the same FAMEs present in a commercial
standard GLC-411 (NU-CHEK PREP, Inc., USA). The corrected areas were used to
calculate the mass of each FAME in the sample by comparison to the internal
standard.
Since oil is stored primarily in the form of TAG, the amount of oil was
calculated based
on the amount of FAME in each sample. Total moles of glycerol were determined
by
calculating the number of moles of FAMEs and dividing total moles of FAMEs by
three. The amount of TAG was calculated as the sum of glycerol and fatty acyl
moieties using the formula: % oil by weight = 100x ((41x total mol
FAME/3)+(total g
FAME-(15x total mol FAME)))/g leaf dry weight, where 41 and 15 were the
molecular
weights of glycerol moiety and methyl group, respectively.
Total Lipid Extraction and Fatty Acid Profile Analysis
Total lipids were extracted from freeze-dried N benthamiana leaves. During the
extraction of total lipids, TAG 51:0 (tri-C17:0) was added as the internal
standard for
the quantification of both the TAG and total fatty acid (TFA) contents. Freeze
dried
leaf tissue was ground to powder in a microcentrifuge tube containing a
metallic ball
using Reicht tissue lyser (Qiagen) for 3 mm. at 20 frequency/s.
Chloroform:methanol
(2:1, v/v) was added and mixed for a further 3 mm. on the tissue lyser before
the
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addition of 1:3 (v/v) of 0.1 M KC1. The sample was then mixed for a further 3
min.
before centrifugation (5 min. at 14,000 g), after which the lower lipid phase
was
collected. The remaining phase was washed once with chloroform, and the lower
phase
extracted and pooled with the earlier extract. Lipid phase solvent was then
evaporated
completely using 1\1/ gas flow and the lipids resuspended in 5 [IL chloroform
per mg of
original dry leaf weight.
Fatty acid methyl esters (FAMEs) of total lipids (equivalent to 10mg dry
weight) were produced by incubating extracted lipid in 1 N methanolic-HC1
(Supelco,
Bellefonte, PA) at 80 C for 3 hours. FAMEs were analyzed by an Agilent 7890A
gas
ehromatograph coupled with flame ionisation detector (GC-FID, Agilent
Technologies,
Palo Alto, CA), on a BPX70 column (30m, 0.25 mm inner diameter, 0.25 nrn film
thickness, SGE) essentially as described previously (Zhou et al., 2011),
except the
column temperature program. The column temperature was programmed as an
initial
temperature at 100 C holding for 3 min, ramping to 240 C at a rate of 7 C/min
and
holding for 1 min. NuChek GLC-426 was used as the external reference standard.
Peaks were integrated with Agilent Technologies ChemStation software (Rev
B.04.03
(16)).
TLC Analysis
From the total lipid extracts (equivalent to 10mg dry weight of plant tissue),
TAG and polar lipids were fractionated by TLC (Silica gel 60, MERCK) using
hexane:diethylether:acetic acid (70:30:1 v/v/v) and visualized by spraying
Primuline
(Sigma, 5 mg/100 ml acetone:water (80:20 v/v)) and exposing plate under UV.
TLC
analysis was primarily used for the identification of fatty acid composition
of TAG and
phospholipids from lipid extraction samples. This also enabled the
determination of the
total TAG content for each sample. The TAG and phospholipid fractions were
scraped
from the TLC plates and methylated according to the FAME preparation protocol
described previously.
LC-MS Analysis
Lipids extracted from 1 mg dry leaf weight were dissolved and diluted to 1
mg/ml in mL butanol:methanol (1:1, v/v) and analyzed by liquid chromatography-
mass
spectrometry (LC-MS), based on previously described methods (Petrie et al.,
2012).
Briefly, lipids were chromatographically separated using a Waters BEH C8 (100
mm x
2.1 mm, 2.7 lam) fitted to an Agilent 1290 series LC and 6490 triple
quadrupole LC-
MS with Jet Stream ionisation with a binary gradient flow rate of 0.2 mL/min.
The
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mobile phases were: A. H20:acetonitrile (10:90, v/v) with 10 mM ammonium
formate
and 0.2 % acetic acid; B. H20:acetonitrile:isopropanol (5:15:80, v/v) with 10
mM
ammonium formate and 0.2 % acetic acid. For the phosphatidylcholine (PC) and
lysophosphatidylcholine (LPC) species hydrogen adducts were quantified by the
characteristic 184 m/z phosphatidyl head group ion under positive ionisation
mode. The
ammonium adducts of monogalactosyl diacylglycerol (MGDG), digalactosyl
diacylglycerol (DGDG), diacylglycerol (DAG) and TAG lipid species were
analyzed
by the neutral loss of singular fatty acids C12 to C18. Multiple reaction
monitoring
(MRM) lists were based on the following major fatty acids: 12:0, 14:0, 16:0,
16:3, 18:0,
18:1, 18:2, 18:3, using a collision energy of 28 V for all lipid classes
except for DAG
where a collision energy of 14 V was used. Individual MRM TAG was identified
based
on ammoniated precursor ion and product ion from neutral loss.
Example 2. Modifying traits in vegetative parts of monocotyledonous plants
Chimeric DNA constructs were designed to increase oil content in
monocotyledonous plants, for example the C4 plant S. bicolor (sorghum), by
expressing a combination of genes encoding WRI1, Z. mays LEC1 (Accession
number
AAK95562; SEQ ID NO:32), DGAT and Oleosin in the transgenic plants. Several
pairs of constructs for biolistic co-transformation were designed and produced
by
restriction enzyme-ligation cloning, as follows.
The genetic construct pOIL136 was a binary vector containing three monocot
expression cassettes, namely a selectable marker gene encoding
phosphinothricin
acetyltransferase (PAT) for plant selection, a second cassette for expressing
DGAT and
a third for expressing Oleosin. pJP136 was first produced by amplifying an
Actin-1
gene promoter from Oryza sativa (McElroy et al., 1990) and inserting it as a
blunt-C/al
fragment into pORE04 (Coutu et al., 2007) to produce pOIL094. pOIL095 was then
produced by inserting a version of the Sesamum indicum Oleosin L gene which
had
been codon optimised for monocot expression into pOIL094 at the Kpnl site.
pOIL093
was produced by cloning a monocot (Triticum aestivum) codon optimised version
of
the Umbelopsis ramanniana DGAT2a gene (Lardizabal et al., 2008) as a Smal-Kpni
fragment into a vector already containing a Zea mays Ubiquitin gene promoter.
pOIL134 was then produced by cloning the Notl DGAT2a expression cassette from
pOIL093 into pOIL095 at the Notl sites. pOIL141 was produced by inserting the
selectable marker gene coding for PAT as a BamHI-SacI fragment into a vector
containing the Z. mays Ubiquitin-1 promoter. Finally, pOIL136 was produced by
cloning the Z mays Ubiquitin::PAT expression cassette as a blunt-AscI fragment
into
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the Zral-Ascl of p0IL096. The genetic construct p0IL136 therefore contained
the
following expression cassettes: promoter 0. sativa Actin::S. indicum Oleosin,
promoter
Z. mays Ubiquitin:: U. ramanniana DGAT2a and promoter Z. mays Ubiquitin::PAT.
A similar vector pOIL197, containing NPTII instead of PAT was constructed by
subcloning of the Z. mays Ubiquitin::NPTII cassette from pUKN (Liu and Godwin,
2012) as a HindlII-Smal fragment into the Ascl (blunted) and HindlII sites of
pJP3343.
The resulting vector, pOIL196, was then digested with Hind111 (blunted) and
Agel. The
resulting 3358bp fragment was cloned into the Zral - Agel sites of pOIL134,
yielding
pOIL197.
A set of constructs containing genes encoding the Z. mays WRI1 (ZmWRI) or
the LEC1 (ZmLEC1) transcription factors under the control of different
promoters were
designed and produced for biolistic co-transformation in combination with
pOIL136 or
pOIL197 to test the effect of promoter strength and cell specificity on the
function of
WRII or LEC1, or both if combined, when expressed in vegetative tissues of a
C4 plant
such as sorghum. This separate set of constructs did not contain a selectable
marker
gene, except for p0IL333 which contained NPTII as selectable marker. The
different
promoters tested were as follows. The Z. mays Ubiquitin gene promoter (pZmUbi)
was
a strong constitutive monocot promoter while the enhanced CaMV 35S promoter
(e35S) having a duplicated enhancer region was reported to result in lower
transgene
expression levels (reviewed in Girijashankar and Swathisree, 2009). Whilst the
Z. mays
phosphoenolpyruvate carboxylase (pZmPEPC) gene promoter was active in leaf
mesophyl cells (Matsuoka and Minami, 1989), the site of photosynthesis in C4
plant
species, the Z. mays Rubisco small subunit (pZmSSU) gene promoter was specific
for
the bundle sheath cell layer (Nomura et al., 2000; Lebrun et al., 1987), the
cells where =
carbon fixation takes place in C4 plants.
The expression of the Z. mays gene encoding the SEE1 cysteine protease
(Accession number AJ494982) was identified as similar to that of the A.
thaliana
SAG12 senescence-specific promoter during plant development. Therefore a
1970bp
promoter from the SEE1 gene (SEQ ID NO:53) was also selected to drive
expression of
the genes encoding the Z. mays WRI1 and LEC1 transcription factors. Further,
the
promoter from the gene encoding Aeluropus littoralis zinc finger protein AlSAP
(Ben
Saad et al., 2011; Accession number DQ885219; SEQ ID NO:54), the promoter from
the gene encoding the Saccharum hybrid DIRIGENT (DIR16) (Damaj et al., 2010;
Accession number GU062718; SEQ ID NO:82), the promoter from the gene encoding
the Saccharum hybrid 0-Methyl transferase (OMT) (Damaj et al., 2010; Accession
number GU062719; SEQ ID NO:83), the Al promoter allel from the gene encoding
the
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Saccharum hybrid RIMYB1 (Mudge etal., 2013; Accession number JX514703.1; SEQ
ID NO:84), the promoter from the gene encoding the Saccharum hybrid Loading
Stem
Gene 5 (LSG5) (Moyle and Birch, 2013; Accession number JX514698.1; SEQ ID
NO:85) and the promoter from the sucrose-responsive ArRoIC gene from A.
rhizogenes
(Yokoyama et al., 1994; Accession number DQ160187; SEQ ID NO:55) were also
selected for expression of ZmWRI1 expression in stem tissue. Therefore, each
of these
promoters was individually joined upstream of the ZmWRI1 or ZmLEC1 coding
regions, as follows.
An intermediate vector, pOIL100, was first produced by cloning the Z. mays
WRI1 coding sequence and a Glycine max lectin gene transcription
terminator/polyadenylation region, flanked by AscI-Nco1 sites, into the same
sites in the
binary vector pJP3343. The WRI1 coding sequence was codon optimized using T.
aestivum codon preferences. The different versions of the constructs for WRI1
expression were based on pOIL100 and were produced by cloning the various
promoters into pOIL100, pOIL101 was produced by cloning a XhoI-SalI fragment
containing the e35S promoter with duplicated enhancer region into the XhoI
site of
pOIL100. pOIL102 was produced by cloning a Hind111-AvrII fragment containing
the
Z. mays Ubiquitin gene promoter (Christensen et al., 1992) into the HindIII-
Xba1 sites
of pOIL100. pOIL103 was produced by cloning a HindIII-Nco1 fragment containing
a
Z. mays PEPC gene promoter (Matsuoka and Minami, 1989) into the HindIII-Nco1
sites
of pOIL100. pOIL104 was produced by cloning a HindIII-A-vr11 fragment
containing a
Z. mays SSU gene promoter into the HindIII-AvrII sites of pOIL100.
A synthetic fragment containing the Z. mays SEE1 promoter region flanked by
HindIII-Xho1 unique sites was synthesized. This fragment was cloned upstream
of the
Z. mays WRI1 protein coding region using the HindIII-Xhol sites in pOIL100.
The
resulting vector was designated pOIL329. A synthetic fragment containing the
A.
littoralis A1SAP promoter region flanked by XhoI-Xba1 unique sites was
synthesized.
This fragment was cloned upstream of the Z. mays WRI1 coding region using the
XbaI-
Xhol sites in pOIL100. The resulting vector was designated pOIL330. A
synthetic
fragment containing the A. rhizogenes ArRolC promoter region flanked by
P.spOMI-
Xho1 unique sites was synthesized. This fragment was cloned upstream of the Z.
mays
WRI1 coding region using the PspOMI-XhoI sites in pOIL100. The resulting
vector
was designated pOIL335. Finally, a binary vector (pOIL333) containing the Z.
mays
SEE1::ZmLEC1 expression cassette was obtained in three steps. First, a
355::GUS
expression vector was constructed by amplifying the GUS coding region with
flanking
primers containing Avr11 and KpnI sites. The resulting fragment was
subsequently
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cloned into the Spel-Kpnl sites of pJP3343. The resulting vector was
designated
pTV111. Next, the 35S promoter region of pTV111 was replaced by the Z. mays
SEE1
promoter. To this end, the Z mays SEE1 sequence was amplified using flanking
primers containing HindlIl and Xhol unique sites. The resulting fragment was
cut with
the respective restriction enzymes and subcloned into the Sall-HindIII sites
of pTV111.
The resulting vector was designated pOIL332. Next the ZmLEC1 coding sequence
was
amplified using flanking primers containing Notl and EcoRV sites. This
resulting
fragment was subcloned into the respective sites of pOIL332, yielding pOIL333.
A 2673bp synthetic fragment containing the Saccharum D1R16 promoter region
flanked by HindIII-Xbal sites was synthesized. This fragment was cloned
upstream of
the Z. mays WRI1 protein coding region using the HindIII-Xbal sites in
pOIL100. The
resulting vector was designated pOIL337. A 2947bp synthetic fragment
containing the
Saccharum OMT promoter region flanked by Xhol-Xbal sites was synthesized. This
fragment was cloned upstream of the Z. mays WRI1 protein coding region using
the
Xhol-Xbal sites in pOIL100. The resulting vector was designated pOIL339. A 118
lbp
synthetic fragment containing the Saccharum R1MYB1 promoter region flanked by
HindIII-Xhol sites was synthesized. This fragment was cloned upstream of the
Z. mays
WRI1 protein coding region using the HindIII-Xhol sites in pOIL100. The
resulting
vector was designated pOIL341. A 4482bp synthetic fragment containing the
Saccharum LSG5 promoter region flanked by XbaIll-Smal sites was synthesized.
This
fragment was cloned as an Xballl-Smal fragment upstream of the Z. mays WRI1
protein coding region using the Stul-Nhel sites in pOIL100. The resulting
vector was
designated pOIL343.
Two putative S. bicolor SDP1 genes were identified by a BLASTn search using
an A. thaliana SD?] nucleotide sequence (Accession number NM 120486; SEQ ID
NO:37) as query. The Accession numbers of the two S. bicolor SDP1 homologs are
XM 002458486 (SEQ ID NO:38) and XM_002463620 (SEQ ID NO:73). A 7991bp
synthetic fragment was synthesized and contained the following genetic
components in
order: a matrix association region (MAR), a Z. mays promoter, a TMV 5' UTR
sequence, a 2198bp hairpin RNA encoding region (SEQ ID NO:75) directed against
both S. bicolor SDP] genes. an OCS gene polyadenylation/transcription
terminator, an
0. sativa Actin-1 gene promoter, TMV 5' UTR sequence, and a NOS gene
polyadenylation/transcription terminator. The hairpin RNA encoding region
contained
a Pdk intron (Wesley et al., 2001) and a Cat intron, the second in reverse
orientation.
The entire fragment was synthesized and inserted into an E. coli expression
vector. The
resulting vector was designated pOIL385.
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Whole plasmid DNA was prepared from pOIL101, pOIL102, pOIL103,
pOIL104, pOIL197, pOIL136, pOIL329 and pOIL385 for biolistic transformation.
pOIL197 DNA was then mixed with DNA from either pOIL101, pOIL102, pOIL103,
pOIL104, pOIL329 or pOIL385 and introduced by biolistics into S. bicolor
(TX430)
differentiating embryonic calli (DEC) cells to produced transformed plants as
described
in Example 1. Alternatively, constructs for expression of the same
combinations of
genes are introduced separately or co-transformed by Agrobacierium-mediated
methods (Gurel et at., 2009; Wu et al., 2014) into DEC tissues.
Between 9 and 47 transgenic plants were regenerated and selected by antibiotic
resistance for the pairs of constructs including pOIL197 with each of pOIL101
(p35SSWRI1); pOIL102 (pZmUbi::WRI1), pOIL103 (pZmPEPC::WRI1), pOIL104
(pSSU::WRI1) and pOIL329 (pSEE1::WRI1). Transformations were also carried out
with pOIL197 or pOIL102 alone, and for the transformation vector without an
insert
(empty vector control). The presence of the introduced transgenes in plants
that were
resistant to the selective agent was demonstrated by PCR. The copy number of
each
transgene was also determined by digital droplet PCR (ddPCR).
Total leaf lipids were quantified in a first subset of transgenic S. bicolor
plants
prior to their transfer from MS medium to soil. This preliminary screening
suggested
slightly elevated total lipid levels in leaf tissue of some events at this
very early stage.
The line with the highest total lipid content, pOIL136 (2), was further
analyzed by thin
layer chromatography (TLC) to determine the effect of transgene expression on
TAG
accumulation. Leaf tissue of this particular line was sampled at vegetative
stage
following transfer to soil in the glasshouse. When compared to the wildtype
and empty
vector negative controls, pOIL136 (2) exhibited increased TAG levels in leaf
tissue
after TLC separation and iodine staining. Subsequent quantification revealed
10-fold
increased TAG in the transgenic line compared to the negative controls. The
TAG
profile was dominated by the polyunsaturated fatty acids linoleic and cc-
linolenic acid.
The presence or absence of all three transgenes was determined by digital PCR
analysis. Of note, up to 30% mortality rate was observed for plantlets at
rooting stage
during tissue culture following transformation with the pOIL103 and pOIL197
combination due to unknown reasons.
Confirmed transgenic plants were transferred to soil in pots in the glasshouse
and leaves were sampled from primary transformants at vegetative stage of
growth (i.e.
prior to the appearance of the boot leaf), at the boot leaf stage (defined as
when the
boot leaf has fully emerged, the boot leaf is the last leaf formed on the
plant and from
which the panicle (head) emerges) and at the mature seed-setting stage. Total
fatty acid
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(TFA) and triacylglycerol (TAG) contents (% leaf dry weight) were quantified
by TLC-
GC as described in Example 1.
TFA levels in wild-type and empty vector negative controls decreased during
plant development and were in the range 0.05-2.9% (weight/dry weight). The
highest
TFA levels were detected prior to the appearance of the boot leaf (termed the
vegetative stage of growth) and were below 3%. TAG levels in the same plants
were
consistently low in the range 0-0.2% during the entire plant life cycle. Both
the TFA
content and the TAG content had fatty acid compositions of predominantly
C16:0,
C18:2A912 (LA) and C18:3A9'12'15(ALA). In particular, ALA was present at about
>70%
of the TFA content, reflecting the use of this fatty acid in wild-type plastid
membranes.
ALA also was the predominant fatty acid in the small amount of TAG present in
the
wild-type leaves.
27 confirmed transgenic plants which had been transformed with pOIL197 or
pOIL136, comprising both pZmUbi:DGAT and pZmUbi:Oleosin genes in addition to
the selectable marker genes, were analysed at the vegetative, boot leaf and
mature seed
setting stages. Some data are presented in Table 5. Generally, the pOIL197 and
pOIL136 primary transformants displayed increased TFA and TAG accumulation
compared to the negative control lines, but only to about triple for the TFA
level
compared to the controls. The highest TFA levels were detected at the
vegetative stage
of growth. Similar to the wild-type and negative control lines, TFA levels
decreased as
the plants grew and developed. Maximum TFA levels at vegetative, boot leaf and
mature seed setting stages equalled 4.3%, 3.3% and 2.2%, respectively. The
highest
TAG levels detected varied between 0.8 and 1.4% depending on the age of the
plant at
the time of sampling (Table 3), so were increased up to 7-fold relative to the
very low
levels in the wild-type leaves. The TFA composition remained largely unchanged
at the
different stages and was dominated by ALA. The TAG composition displayed a
higher
degree of variation between the different transgenic lines. Compared to the
fatty acid
composition of the TFA content, the level of LA (18:2A912) was consistently
increased
in TAG throughout all plant stages investigated.
Nine primary regenerated plants made by transformation with the single vector
pOIL102 (pZmUbi:WRI1) were generated by co-bombardment of pOIL102 and
pUKN, containing the NPTH selectable marker gene. Table 4 shows some of the
data
for TFA and TAG contents and fatty acid compositions were measured at the
three
growth stages. When compared to the plants transformed with the constructs
encoding
DGAT2 and Oleosin (pOIL197 or pOIL136), TFA and TAG levels in the pOIL102
transgenic events were generally lower. Indeed, levels of TFA and TAG were
similar to
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the levels in the wild-type and negative control plants at vegetative stage.
Maximum
TFA levels at vegetative, boot leaf and mature seed setting stages were 2.6%,
2.5% and
2.0%, respectively (Table 4). Maximum TAG levels observed were 0.2%, 0.4% and
0.9% at vegetative, boot leaf and mature seed setting stages, respectively.
Thirty-seven primary regenerated plants were obtained after co-bombardment
with both pOIL197 (pZmUbi:DGAT and pZmUbi:Oleosin) and pOIL102
(pZmUbi:WRI1). Four of the regenerated events were found to be non-transgenic.
In
addition, 2 plants did not contain pOIL102 while 2 other plants did not
contain the
DGAT2 transgene. All of the transgenic plants were analysed for TFA and TAG
contents and fatty acid composition at the three growth stages, as above.
Representative
data are presented in Table 5. Some of the plants exhibited greatly increased
TFA and
TAG levels compared to the plants transfomred with single vectors pOIL197,
pOIL136
or pOIL102. The maximum TFA levels at vegetative, boot leaf and mature seed
setting
stages in the pOIL102+p0I1,197 transformed plants equalled 7.2%, 6.4% and 8.7%
(w/dry weight), respectively. Importantly, the maximum observed TAG levels
increased during plant development from 2.7% (vegetative stage) to 3.5% (boot
leaf
stage) and 6.1% (mature seed setting stage). Compared with the data obtained
for the
separate transformations with the DGAT and WRI1 transgenes, this exemplified
the
synergism for co-expressing DGAT and WRI1 transgenes to increase non-polar
lipid
accumulation in vegetative plant tissues. High levels of TAG and TFA were in
most
cases associated with a substantial reduction in the C18:3 9'12'15 content,
which was
reduced by about 50% in the lines with the highest levels of TAG.
Forty-seven primary transformants were obtained following transformation with
both pOIL197 (pZmUbi:DGAT and pZmUbi:Oleosin) and pOIL103
(pZmPEPC:WRI1). Copy number analysis by ddPCR revealed one non-transgenic
plant and 3 plants that did not contain DGAT2 and/or OLEOSIN transgenes. All
events
were subsequently analysed for TFA and TAG contents and fatty acid composition
during the three stages of plant development. Some plants with this gene
combination
exhibited the highest TFA and TAG levels detected in this experimental series.
TFA
levels were observed at vegetative, boot leaf and mature seed setting stages
in the
pOIL103+pOIL197 population at 8.3%, 8.3% and 9.7%, respectively. Maximum TAG
levels observed at vegetative, boot leaf and mature seed setting stages were
at 2.3%,
6.6% and 7.6%, respectively. Of note, the highest TAG (6.6%) and TFA (8.3%)
levels
amongst all transgenic lines were detected in event TX-03-31 at mature seed
setting
stage. While C18:3A9'12'15 typically dominated the TFA fraction other than
TAG, the
TAG in this population of transgenic plants displayed a high degree of
variability in
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fatty acid composition. Of note, some plants exhibited increases in levels of
palmitic
acid (C16:0) and/or linoleic acid (LA, C18:29'12) at the expense of ALA.
Indeed, the
ALA level in both TFA and TAG contents was reduced below 40% in some plants as
a
percentage of the total fatty acid content, while less than 30% in other
selected events.
The ALA level in TAG was even less than 20% in some selected plants, as a
percentage of the total fatty acid content.
Due to the use of biolistic transformation in this experiment, many of the
transgenic sorghum plants contained high transgene copy numbers as determined
by
digital PCR. In addition, varying degrees of male and female sterility were
observed
amongst the transgenic lines, likely a result of the multiple transgene
insertions. The
inventors therefore did not pursue homozygosity of the transgenes in
subsequent
generations but rather performed a detailed analysis on vegetative progeny
plants
obtained from selected primary transformants. To this end, tillers were
propagated
allowing for triplicate analyses of TAG and TFA levels. Furthermore, the
analyses
focussed on the boot leaf stage of growth as this was a distinct and easily
identified
time point during development that allowed for good comparison between the
different
transgenic lines, grown under the same environmental conditions. Plants
containing the
higher levels of TFA and TAG were propagated by separating tillers and
transplanting
them into soil in new pots. The tillers produced new roots and continued to
grow.
Quantitation of the total lipid content in triplicate leaves from established
tillers
confirmed elevated TAG and TFA contents in several independent lines co-
transformed
with either pOIL102+pOIL197 or pOIL103+pOIL197. The highest levels were
observed in progeny plants of line 03-31, confirming the earlier results.
Leaves of this
line contained on average 6.9% TFA and 4.6% TAG (% DW) at boot leaf stage.
This
corresponded to an 89.4-fold increase in TAG content compared to wild-type
control
leaves at the same developmental stage.
Twenty primary regenerated plants were obtained following transformation
with both pOIL197 (pZmUbi:DGAT and pZmUbi:Oleosin) and pOIL104
(pSSU:WRI1). Five plants were found to be non-transgenic and four other plants
had
only the gene(s) from one of the genetic constructs. All plants were analysed
for TFA
and TAG contents and fatty acid composition. Leaves of primary transformants
containing both pOIL197 and pOIL104 T-DNA regions, sampled at vegetative,
mature
and seed setting stages of growth contained up to 4.1%, 5.9% and 5.89% TFA,
respectively. Surprisingly, the highest TFA levels detected in this population
were
accompanied by a relatively low TAG content. TAG levels in pOIL104+pOIL197
transgenic plants at vegetative, boot leaf and seed setting stages reached
only to 0.7%,
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2.8% and 3.4%. Increased TAG levels were typically associated with a reduction
in
C18:3 9"2'15 and an increase in both palmitic acid and LA.
Forty-three primary regenerated plants were obtained following transformation
with both pOIL197 (pZmUbi:DGAT and pZmUbi:Oleosin) and pOIL101
(p35S:WRI1). One plant was non-transgenic, another lacked the WRI1 transgene
and
another lacked the DGAT1 transgene. All plants were analysed for TFA and TAG
contents and fatty acid composition at boot leaf stage. Leaves of primary
transformants
containing both pOIL197 and pOIL104 T-DNA regions contained up to 4% TFA while
TAG levels were low with a maximum of 1.4%. Increased TAG levels were
associated
with a reduction in C18:3A912.15as a percentage of the total fatty acid
content.
Twenty primary transformants were obtained following transfoiniation with
both pOIL197 (pZmUbi:DGAT and pZmUbi:Oleosin) and pOIL329 (pSEEI :WRI1).
All plants were confirmed to be transgenic by ddPCR. TFA and TAG levels in
leaves
of 10 plants at vegetative growth stage were increased up to 3.6% and 0.3%,
respectively. Maximum TFA and TAG levels at boot leaf stage equalled 3.8% and
1.5%, respectively. The low TFA and TAG levels were likely the result of the
senescence-specific expression patterns of the SEE1 promoter used to drive
WR11
transgene expression. Increased TAG levels were typically associated with a
reduction
in C18:3 9'12'15 as a percentage of the total fatty acid content.
Thirty-six primary regenerated plants were obtained following transformation
with both pOIL197 (pZmUbi:DGAT and pZmUbi:Oleosin) and pOIL385
(SDP1hpRNAi). Two plants lacked pOIL197 and another two lacked pOIL385. The
highest TFA level detected in transgenic leaves at the vegetative growth stage
was
4.2%. TAG levels in this particular event at the same growth stage was only
1.0%. TFA
and TAG levels in leaves sampled at boot leaf stage were increased up to 3.9%
and
1.6%, respectively. The lower TFA and TAG levels could be due to the absence
of a
WRI1 transgene in this transgenic population. No changes in TAG or TFA fatty
acid
composition were detected relative to the wild-type plants.
Transgene expression levels were determined in propagated tillers of selected
lines by RT-PCR. In the majority of transgenic lines, the DGAT2a transgene was
typically expressed at a higher levels than the WRI1 transgene. Oleosin L gene
expression was either low or not detected. Total lipid and TAG contents at the
boot leaf
stage were used to calculate correlation coefficients with gene expression.
Both WRI1
and DGAT2a gene expression showed a significant positive correlation with TAG
levels amongst pOIL102+pOIL197 and pOIL103+pOIL197 transgenic populations.
Significant, albeit slightly weaker, correlation was observed for TFA content
and WRI1
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or DGAT2a expression. Olcosin L expression was not correlated with either TAG
or
TFA accumulation in transgenic leaf tissues. It was observed that plant TX-03-
31
which had a relatively high TTQ had the highest level of expression of DGAT
amongst
the tested plants. It was concluded that high levels of DGAT expression were
beneficial
for increasing the TAG level and also the TTQ.
The most surprising and unexpected observation made in these experiments was
the relatively high level of TFA accompanied by the low levels of TAG in most
of the
transformed sorghum plants, except in a few exceptional plants such as plant
TX-03-
31. That is, although fatty acid synthesis and accumulation were significantly
increased, much of the fatty acid was appearing as TFA but not as TAG. This
observation was the opposite of what had been seen with the WRI1 + DGAT
transgenic
plants for Nicotiana including tobacco. To quantitate this in the sorghum
plants, the
quotient of the TAG to TFA level was calculated for all of the above mentioned
transgenic sorghum populations (Tables 3-6). The TAG/TFA Quotient (TTQ)
parameter was calculated as the level of TAG (%) divided by the level of TFA
(%),
each as a percentage of the dry weight of the plant material (leaf in this
case). It was
observed that for many of the sorghum lines, the TTQ was in the range of 0.01
to 0.6,
i.e. less than 60% of the TFA was present as TAG. Addition of one or more
further
genetic modifications to the combination of WRI1 and DGAT genes such as, for
example, which provide for a reduction in the expression of endogenous
SDP1,TGD or
TST genes, or an increase in the levels of one or more of PDAT, PDCT or CPT
polypeptides increases the TTQ to above 0.6 for a larger proportion of the
plant lines.
In particular, reduction in TAG lipase in combination with at least WRI1 and
DGAT
increases the TTQ to up to 0.95.
Due to the large difference in absolute TFA and TAG levels in many transgenic
lines, the inventors selected two pOIL102+pOIL197 events (02-10, 02-19) and
two
pOIL103+pOIL197 events (03-31 and 03-48) for quantitation of the major neutral
and
polar lipid classes, to determine the type of lipid other than TAG in which
the high
level of fatty acids was present. The types of lipid were separated by TLC and
quantitated. The propagated tillers were smaller compared to tillers obtained
from wild-
type controls plants grown under the same conditions with the exception of
line 03-48.
Quantitation by GC-FID of TAG and TFA levels in triplicate leaves confirmed
increases in both lipid fractions. Maximum average TAG levels in triplicate
leaves (%
DW) of lines 02-19 and 03-31 sampled at boot leaf stage were 2.8% and 5.2%,
respectively. For all of the transgenic lines, linoleic acid was increased at
the expense
of a-linolenic acid. However, differences were observed in the levels of
palmitic acid
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and oleic acid. Lines 02-10 and 02-19 contained increased proportions of oleic
acid,
whereas palmitic acid was elevated in the TFA and TAG fractions of 03-31 and
03-48
leaves. Lipid quantitation in leaf and stem tissues at seed setting stage
revealed
considerable leaf-to-leaf variation. Lower TFA and TAG contents were observed
in
older leaves of wild-type and transgenic propagated tillers. The TFA and TAG
levels in
the flag leaf of line 03-31 at seed setting equalled 9.9% and 8.4% on a DW
basis,
respectively. Transgenic stem tissues contained up to about 3% total lipids on
a dry
weight basis compared to 0.3% in wild-type stems.
Total lipid extracts from the wild-type and transgenic leaves sampled at boot
leaf stage were subjected to LC-MS to analyse different neutral and polar
lipid classes
in more detail. Plants of all four transgenic lines exhibited elevated TAG,
amounting to
a 100-fold increase in line 03-31 compared to the wild-type control leaves.
Small
increases in levels of PC were detected in plants of the 03-31 and 03-48
transgenic lines
while levels of the plastidial galactolipids MGDG and DGDG were variable,
increased
in some, decreased in other plants. Both LPC and DAG constituted minor lipid
classes.
TAG molecular species in plants of lines 03-31 and 03-48 were enriched in
palmitic
acid and linoleic acid. Major TAG species included TAG (50:2) and TAG (50:3)
which
contained two palmityl groups and TAG (52:4) and TAG (52:5) which contained
palmitoyl and linoloyl groups. In contrast, plants of lines 02-10 and 02-19
exhibited
distinctly different TAG profiles. Leaf tissues of both lines preferentially
accumulated
TAG comprising one or more linolyl chains such as TAG (52:3-5) and TAG (54:4-
8).
The distinct differences in TAG profiles between the two transgenic
populations were
consistent with earlier GC-FID results.
Changes in TAG compositions were also reflected in the precursor DAG.
Dominant DAG (34:2) and DAG (34:3) molecular species in plants 03-31 and 03-48
were enriched in palmitic acid while both 02-10 and 02-19 plants had DAG
molecules
containing two C18 acyl chains (DAG 36:2-6). Abundant eukaryotic galactolipid
species such as MGDG (36:6) and DGDG (36:6) were either reduced or not
significantly affected. Two prokaryotic galactolipid species, MGDG (34:3) and
DGDG
(34:2) were increased slightly in plants 03-31 and 03-48. The dominant
prokaryotic
DGDG species (34:3) was either unchanged or reduced in transgenic leaves. PC
molecular species containing palmitic or linoleic acid including PC (34:1-2)
and PC
(36:4) were elevated, particularly in lines 03-31 and 03-48. Di-palmitoyl PC
(32:0) was
increased in line 03-31, reflecting the higher levels of palmitic acid as
detected by GC-
FID.
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Taken together, these results indicated an increased flux of acyl chains into
TAG from PC in the transgenic lines whilst galactolipid biosynthesis mainly
occurred
via the eukaryotic pathway. These data also led the inventors to understand
that
reduction of TGD activity or increases in PDCT and/or CPT in the plants in
addition to
the present transgenes would likely enhance the TFA and TAG levels.
TAG accumulation affects starch and amino acid content
Transitory starch levels in transgenic leaves of lines 03-31 and 03-48 were
reduced 7.4- and 15.3-fold on average, respectively. In contrast, starch
levels in leaves
of 02-10 and 02-19 plants were not significantly affected. Sucrose constituted
the
dominant leaf soluble sugar in all plants. Sucrose levels were 2-fold lower in
line 03-48
while similar to the wild-type control in line 02-19. Raffinose was reduced by
19.6-fold
in line 03-48 while monosaccharides such as glucose, fructose and galactose
displayed
smaller reductions.
A metabolite quantitation by GC-MS identified 36 compounds that were
significantly different in leaves of wild-type and transgenic plants. Twenty
metabolites
were detected at higher levels in TAG-accumulating leaves, including multiple
amino
acids, urea and the citric acid cycle (TCA) intermediate, ct-ketoglutarate.
Several
dicarboxylic acids, sugar alcohols, fructose, xylose and shikimate were
amongst the
metabolites that were less abundant in transgenic leaves. Principle component
analysis
revealed clear separations of both transgenic events and the wild-type
control.
Sorghum leaves accumulate TAG as cytosolic lipid droplets
To examine transgenic leaves microscopically to see whether the increased
TAG was accumulated in oil droplets, flag leaves of re-established side
tillers from
transgenic S. bicolor plants were harvested at the beginning of flowering and
kept on
ice until sections were prepared for imaging. Fresh, thin hand sections were
stained for
10 mm with a solution of 50 mM PIPES pH7 supplemented with 2 ug/m1 of BODIPY
505-515 (4,4-
Difluoro-1,3,5,7-Tetramethy1-4-Bora-3a,4a-Diaza-s-Indacene,
ThemloFisher Scientific). They were then rinsed in a solution of PIPES pH7 and
imaged right away. Control sections were placed directly in PIPES pH7 for 10
min
before being mounted on slides and imaged.
All samples were imaged with a confocal laser scanning microscope (Leica
TCS SP8) equipped with a white light laser and a 40x water immersion objective
([NA]=1.1), and controlled by the LAS X software (Leica Microsystems). Imaging
was
done in a sequential manner: BODIPY was excited at 505 nm and its emission was
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collected at 520-540 nm, while in a separate track, chloroplasts were excited
at 633 nm
and their auto-fluorescence was collected at 650-690 nm. Maximum projections
were
generated with the LAS X software. Confocal imaging settings were optimized to
distinguish cell types in which oil accumulated by minimizing chloroplast auto-
fluorescence in the bundle sheath cells as opposed to the surrounding
mesophyll cells.
Leaf cross sections of line 03-10 revealed an abundance of small lipid
droplets
that preferentially accumulated in the cytosol of mesophyll cells. The unequal
distribution likely reflected the tissue specificity of the PEPC promoter used
to generate
this particular transgenic line. Some lipid accumulation was also visible in
the bundle
sheath cells of transgenic lines and the wild-type control. Line 02-10
contained an
intermediate number of lipid droplets, confirming previous LC-MS and GC-F1D
TAG
quantitation results. Transmission electron micrographs showed densely packed
small
lipid droplets in the cytosol of mesophyll cells in line 03-31. Mesophyll
cells of the
wild-type control plants were largely devoid of cytosolic oil droplets.
The chimeric DNA constructs for Agrobacterium-mediated transformation are
used to transform Zea mays (corn) as described by Gould et al. (1991).
Briefly, shoot
apex explants are co-cultivated with transgenic Agrobacteriurn for two days
before
being transferred onto a MS salt media containing kanamycin and carbenicillin.
After
several rounds of sub-culture, transformed shoots and roots spontaneously form
and are
transplanted to soil. The constructs are similarly used to transform Hordeum
vulgare
(barley) and Avena saliva (oats) using transformation methods known for these
species.
Briefly, for barley, the Agrobacterium cultures are used to transform cells in
immature
embryos of barley (cv. Golden Promise) according to published methods (Tingay
et al.,
1997; Bartlett et at., 2008) with some modifications in that embryos between
1.5 and
2.5 mm in length are isolated from immature caryopses and the embryonic axes
removed. The resulting explants are co-cultivated for 2-3 days with the
transgenic
Agrobacterium and then cultured in the dark for 4-6 weeks on media containing
timentin and hygromycin to generate embryogenic callus before being moved to
transition media in low light conditions for two weeks. Calli are then
transferred to
regeneration media to allow for the regeneration of shoots and roots before
transfer of
the regenerated plantlets to soil. Transformed plants are obtained and grown
to
maturity in the glasshouse.
Table 3. TFA and TAG levels, fatty acid composition and 1-1Q in sorghum leaves
transformed with pOIL197 or pOIL136 (pZmUbi:DGAT; pZmUbi:Oleosin) during the
boot leaf stage of growth. The lines are listed in order of increasing TFA
levels.
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TAG =
or C16: C18:3
Line TFA 0 C18:0 C18:1 C18:2 n3 Other TFA TAG TTQ
TX-197-14 TFA 12.7 5.2 2.0 14.4 57.7 8.1 1.2
TX-197-14 TAG 8.8 7.1 3.1 22.7 54.7 3.6 0.3 0.266
TX-197-15 TFA 14.5 5.0 2.3 14.7 55.8 7.7 1.2
TX-197-15 TAG 12.7 7.1 3.2 21.0 51.7 4.3 0.3 0.262
TX-197-19 TFA 13.1 3.2 2.0 14.3 60.9 6.4 1.2 ____________________
TX-197-19 TAG 10.6 4.3 3.4 24.4 54.0 3.2 0.2 0.203
TX-136-03 TFA 14.1 1.8 1.7 12.6 65.0 4.8 1.2
TX-136-03 TAG 14.5 4.3 4.5 32.9 42.2 1.6 0.1 0.045
TX-197-08 TFA 14.4 3.5 1.3 14.2 62.2 4.4 1.2
TX-197-08 TAG 13.7 5.2 2.7 22.4 50.5 5.5 0.3 0.211
TX-197-11 TFA 14.1 3.8 2.0 15.0 57.0 _______ 8.2 1.3
TX-197-11 TAG 10.3 4.8 3.0 22.8 55.9 3.1 0.3 0.267
TX-136-24 TFA 15.5 2.2 2.2 16.9 58.1 5.2 1.3
TX-136-24 TAG 14.7 3.3 4.0 32.4 42.9 2.7 0.2 0.164
TX-136-02 TFA 12.3 1.5 1.4 14.7 65.7 4.4 1.5
TX-136-02 TAG 13.9 2.7 3.0 28.7 46.6 5.1 0.7 0.444
TX-197-30 TFA 13.1 2.3 1.3 9.3 65.1 8.8 2.0
TX-197-30 TAG 10.0 3.0 2.2 15.0 65.3 4.5 0.4 0.223
TX497-46 TFA 13.2 2.5 0.8 7.9 71.2 4.5 2.0
TX-197-46 TAG 17.3 18.6 3.2 14.7 42.5 3.7 0.1 0.033
TX-197-45 TFA 13.6 2.7 0.6 6.7 71.7 4.5 2.1
TX-197-45 TAG 22.7 17.7 4.4 12.9 38.6 3.6 0.1 0.030
TX-197-39 TFA 12.6 3.6 1.1 9.0 66.2 7.4 2.1
TX-197-39 TAG 9.5 4.0 1.6 12.8 66.7 5.5 0.6 0.291
TX-197-22 TFA 13.6 2.0 0.8 7.3 71.3 4.9 2.1
Tx-197-22 TAG 13.8 3.3 1.8 14.2 64.6 2.3 0.1 0.056
TX-197-34 TFA 12.0 3.2 1.2 9.6 67.9 5.9 2.2
TX-197-34 TAG 9.1 4.6 2.3 18.4 63.2 2.3 0.4 0.190
Tx-197-50 TFA 13.0 2.5 1.1 9.1 66.8 7.5 2.5
TX-197-50 TAG 11.4 4.6 2.1 15.3 59.8 6.9 0.5 0.183
TX-197-43 TFA 12.4 2.3 0.7 8.0 71.9 4.7 2.5
TX-197-43 TAG 11.0 4.4 1.8 15.7 62.3 4.8 0.2 0.065
TX-197-32 TFA 12.5 2.1 1.1 9.0 70.0 5.3 2.5
TX-197-32 TAG 12.8 3.7 2.1 16.1 60.3 5.0 0.6 0.220
Tx-197-33 TFA 12.1 2.7 0.7 7.9 71.0 5.6 2.5
TX-197-33 TAG 11.1 4.8 1.4 15.4 62.4 4.9 0.3 0.130
TX-197-41 TFA 12.8 1.9 0.7 8.1 72.8 3.7 2.6
TX-197-41 TAG 15.1 5.9 2.4 16.7 53.7 6.3 0.2 0.065
TX-197-36 TFA 12.2 2.0 0.8 7.7 71.6 5.6 2.6
TX-197-36 TAG 11.4 3.4 1.6 13.9 65.6 4.1 0.4 0.158
TX-197-42 TFA 12.4 2.1 0.8 8.2 70.3 6.3 2.7
TX-197-42 TAG 12.4 5.4 2.3 17.8 57.1 5.0 0.2 0.060
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TX-197-51 TFA 13.6 2.1 1.0 9.9 66.8 6.6 2.7
TX-197-51 TAG 13.1 4.6 3.0 18.8 53.4 7.0 0.5 0.175
TX-197-49 TFA 15.2 2.9 1.0 9.3 65.3 6.3 2.7
TX-197-49 TAG 17.3 5.0 2.0 16.7 52.7 6.3 0.5 0.192
TX-197-48 TFA 13.0 2.3 1.0 8.8 68.5 6.4 2.8
TX-197-48 TAG 13.0 4.7 2.2 16.1 58.0 6.0 0.4 0.144
TX-197-38 TFA 12.2 2.0 1.0 7.7 72.1 5.0 2.9
TX-197-38 TAG 11.2 3.4 2.2 14.9 63.8 4.5 0.5 0.160
TX-197-35 TFA 12.8 1.8 0.9 8.5 69.4 6.6 2.9
TX-197-35 TAG 12.7 2.9 1.7 14.5 63.3 4.9 0.7 0.227
TX-197-40 TFA 12.7 1.9 0.7 7.7 73.9 3.1 2.9
TX-197-40 TAG 16.3 4.7 3.3 20.8 52.4 2.6 0.1 0.031
TX-197-47 TFA 13.9 2.4 0.6 6.9 72.2 3.9 2.9
TX-197-47 TAG 24.6 19.8 5.2 10.7 34.8 4.9 0.0 0.017
TX-136-01 TFA 11.6 1.4 1.3 14.1 67.2 4.3 3.3
TX-136-01 TAG 14.6 2.9 3.0 29.5 44.1 5.9 0.7 0.199
TX-197-44 TFA 13.5 2.1 1.4 14.7 63.1 5.1 3.4
TX-197-44 TAG 14.4 4.3 3.1 25.0 45.0 8.2 0.8 0.245
TX-136-25 TFA 13.6 2.2 0.7 10.8 67.4 5.2 3.4
TX-136-25 TAG 16.6 4.2 1.4 20.1 51.5 6.1 1.0 0.286
TX-197-28 TFA 11.5 1.3 0.4 7.8 75.3 3.6 3.4
TX-197-28 TAG 17.4 4.5 1.6 19.5 50.2 6.9 0.1 0.035
TX-197-37 TFA 12.6 3.4 6.3 17.4 54.1 6.2 4.5
TX-197-37 TAG 13.4 5.0 10.1 27.4 40.2 3.9 1.9 0.426
Table 4. TFA and TAG levels, fatty acid composition and TTQ in sorghum leaves
transformed with pOIL102 (pZmUbi:WRI1) during the boot leaf stage of growth.
TAG or C16: C18:3
Line TFA 0 C18:0 C18:1
C18:2 n3 Other TFA TAG TTO
TX-102-8 TFA 16.9 4.2 2.3 12.3 57.7 6.5 0.9
TX-102-8 TAG 14.5 6.2 13.5 25.7 36.8 3.4 0.2 0.243
TX-102-4 TFA 17.1 4.2 2.0 12.5 57.5 6.7 0.9
TX-102-4 TAG 10.5 4.4 3.0 20.0 59.6 2.6 0.2 0.182
TX-102-1 TFA 16.6 4.3 3.9 15.4 50.7 9.1 1.1
TX-102-1 TAG 10.7 4.4 5.3 21.9 54.1 3.6 0.3 0.273
TX-102-5 TFA 16.7 4.1 1.7 11.6 60.2 5.8 1.1
TX-102-5 TAG 11.7 5.5 2.8 21.4 56.1 2.5 0.1 0.118
TX-102-6 TFA 17.8 3.8 15.9 17.0 38.8 6.6 1.5
TX-102-6 TAG 19.6 7.0 29.4 25.4 13.9 4.7 0.4 0.267
TX-102-2 TFA 15.0 1.9 1.7 19.1 56.5 5.9 1.7
TX-102-2 TAG 10.6 1.9 2.7 30.2 51.2 3.4 0.4 0.258
TX-102-7 TFA 15.0 3.1 7.0 13.9 56.1 4.9 2.4
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TX-102-7 TAG 16.1 6.5 20.5 28.0 24.4 4.5 0.3 0.111
TX-102-3 TFA 14.4 3.5 9.5 13.4 50.9 8.2 2.5
TX-102-3 TAG 16.9 6.7 23.9 24.7 22.5 5.2 0.4 0.150
Table 5. TFA and TAG levels, fatty acid composition and TTQ in sorghum leaves
transformed with pOIL102 (pZmUbi:WRI1) and pOIL197 (pZmUbi:DGAT and
pZmUbi:Oleosin) during the boot leaf stage of growth. The lines are listed in
order of
increasing TFA levels.
TAG
or C16 C18:3
Line TFA :0 C18:0 C18:1 C18:2 n3 Other TFA TAG TTQ
TX-02-27 TFA 17.3 3.8 1.4 10.1 60.1 7.2 1.0
TX-02-27 TAG 11.9 4.4 2.1 19.4 61.2 0.8 0.2 0.164
TX-02-21 TFA 15.9 2.3 2.0 19.3 53.3 7.3 1.2
TX-02-21 TAG 12.6 3.7 2.7 27.0 51.0 3.0 0.4 0.318
Tx-02-01 TFA 15.2 4.2 5.1 14.7 53.2 7.5 1.3
TX-02-01 TAG 11.7 5.6 9.3 26.1 42.9 4.5 0.3 0.199
TX-02-12 TFA 15.3 3.2 2.0 13.6 58.9 6.9 1.3
TX-02-12 TAG 13.7 4.2 3.6 25.1 50.4 EM 0.1 0.111
TX-02-33 TFA 15.9 4.3 1.0 10.1 59.7 9.1 1.4
TX-02-33 TAG 14.3 5.4 2.7 18.9 54.7 4.0 0.1 0.107
TX-02-13 TFA 15.4 5.1 11.4 19.4 39.1 9.5 1.4
TX-02-13 TAG 12.9 6.5 20.3 25.2 28.6 6.4 0.5 0.389
TX-02-36 TFA 16.2 3.4 1.8 12.3 58.5 7.8 1.4
TX-02-36 TAG 15.4 5.8 3.3 21.5 48.9 5.1 0.3 0.209
TX-02-37 TFA 3.3 3.5 1.3 9.9 65.3 6.7 1.4
Tx-02-37 TAG 9.6 3.6 3.8 20.4 60.6 2 0.2 0.137
TX-02-18 TFA 14.6 3.0 1.4 9.8 65.5 5.7 1.4
TX-02-18 TAG 12.5 5.6 4.3 20.6 54.8 2.3 0.1 0.077
TX-02-34 TFA 16.6 2.2 2.2 17.6 54.7 6.7 1.4
TX-02-34 TAG 14 2.8 4.1 30.3 44.7 4 0.7 0,231
TX-02-31 TFA 13.3 3.1 1.8 10.1 64.7 7.0 1.5
TX-02-31 TAG 5.4 1.8 3.2 17.8 71.1 0.7 0.3 0.171
TX-02-29 TFA 13.2 3.2 1.1 8.2 68.6 5.6 1.6
TX-02-29 TAG 10.5 4.7 2.9 18.1 62.0 1.8 0.1 0.082
TX-02-35 TFA 17.8 3.4 6.5 14.0 50.3 8.0 1.6
TX-02-35 TAG 18.8 5.3 19.1 28.4 22.4 6.1 0.2 0.108
TX-02-09 TFA 14.0 3.3 0.9 9.9 66.0 6.0 1.6
TX-02-09 TAG 11.2 4.7 1.9 19.6 58.7 3.9 0.1 0.036
TX-02-24 TFA 12.9 3.5 0.6 7.9 67.3 7.7 1.8
TX-02-24 TAG 10.7 3.5 1.6 11.8 69.0 3.4 0.1 0.044
TX-02-126 TFA 13.8 2.7 1.1 9.9 66.4 6.0 1.8
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TX-02-126 TAG 12.8 4.3 2.1 17.0 58.6 5.2 0.5 0.247
______ TX-02-23 TFA 13.6 2.7 0.7 8.9 68.3 5.8 1.9
TX-02-23 TAG 10.0 3.3 2.2 18.2 63.9 2.4 0.1 0.047
TX-02-07 TFA 17.5 2.3 10.9 17.5 44.5 7.3 1.9
TX-02-07 TAG 21.0 3.9 24.5 27.4 15.2 8.0 0.4
0.225 I
TX-02-28 TFA 12.8 2.9 0.5 7.7 68.4 7.8 2.0
TX-02-28 TAG 13.0 5.5 1.2 11.1 64.3 4.8 0.1 0.063
TX-02-04 TFA 13.6 2.9 1.2 12.1 65.3 4.9 2.1
'1'X-02-04 TAG 12.0 4.4 2.4 21.6 55.9 3.6 0.4 0.206
TX-02-25 TFA 12.2 2.8 0.5 9.4 68.8 6.3 2.5
TX-02-25 TAG 10.3 4.2 1.0 15.4 62.5 6.6 0.4
0.159 I
TX-02-05 TFA 13.6 3.6 3.2 14.7 59.8 5.1 2.5
TX-02-05 TAG 12.2 5.5 7.0 26.8 43.4 5.1 0.6 0.220
TX-02-14 TFA 15.9 5.7 30.9 12.7 26.0 8.9 2.8
TX-02-14 TAG 17.9 8.5 42.6 14.9 7.8 8.4 1.4 0.514
TX-02-131 TFA 12.6 1.4 0.6 8.3 73.1 3.9 2.9
TX-02-131 TAG 16.0 3.9 1.9 18.0 53.9 6.3 0.2 0.061
TX-02-129 TFA 12.1 1.6 1.0 10.4 70.5 4.3 2.9
TX-02-129 TAG 12.8 3.6 2.5 22.0 53.6 5.5 0.3 0.106
TX-02-08 TFA 17.6 2.6 5.6 17.2 51.2 5.8 3.0
TX-02-08 TAG 24.4 5.9 15.8 29.3 15.8 8.8 0.6 0.183
TX-02-02 TFA 17.9 3.1 7.2 15.5 49.6 6.7 3.1
TX-02-02 , TAG 23.7 6.5 17.7 22.8 19.6 9.7 0.6
0.194
TX-02-11 TFA 25.1 4.1 9.0 16.3 36.3 9.1 3.2
Tx-02-11 TAG 33.3 6.6 13.9 20.9 16.0 9.3 1.1 0.341
TX-02-127 TFA 11.4 1.6 0.3 8.9 75.4 2.4 3.5
TX-02-127 TAG 21.0 5.8 1.4 20.6 47.4 3.9 0.1 0.016
TX-02-30 TFA 16.4 3.1 3.7 17.1 53.8 5.9 4.0
TX-02-30 TAG 21.3 5.0 7.6 27.1 30.5 8.5 0.9 I
0.236
TX-02-19 TFA 13.5 2.7 25.4 22.6 30.8 5.0 4.2
TX-02-19 TAG 14.0 3.3 34.3 27.0 16.6 4.8 2.3 0.548
TX-02-06 TFA 24.0 4.8 14.3 19.6 29.7 7.7 4.8 __
Tx-02-06 TAG 29.7 6.9 19.2 23.0 13.4 7.7 2.7 0.555
TX-02-10 TFA 22.0 3.3 10.3 22.7 33.7 7.9 6.3
TX-02-10 TAG 24.8 4.1 12.9 27.0 22.4 8.8 3.5 0.551
TX-02-38 TFA 24.8 4.4 13.9 24.5 23.7 8.7 6.4
TX-02-38 TAG 21.5 5.3 8.6 25.2 39.3 0.0 2.5 0.392
Table 6. TFA and TAG levels, fatty acid composition and TTQ in pOILI03+pOIL197
primary transformants at boot leaf stage.
Line TFA C16 C18:0 C18:1 C18:2 C18:3n Other TFA TAG TTQ
or :0 3
TAG
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TX-03-20 'WA 12.2 2.6 1.7 10.3 67.5 5.7 2.1
TX-03-20 TAG 9.4 3.6 3.3 18.1 63.0 2.5 0.4 0.217
TX-03-54 TFA 13.6 3.5 3.0 12.1 61.5 6.4 2.1
TX-03-54 TAG 14.1 6.9 7.0 22.5 43.5 6.0 0.4 0.207
TX-03-61 TFA 23.9 3.1 1.7 19.0 43.9 8.3 2.2
TX-03-61 TAG 31.4 6.6 3.4 28.3 19.6 10.8 0.4 0.159
TX-03-02 TFA 14.9 3.0 2.8 12.1 60.6 6.6 2.2
TX-03-02 TAG 14.8 5.5 5.6 20.6 46.7 6.8 0.5 0.222
TX-03-53 TFA 18.5 3.7 8.9 15.4 43.1 10.4 2.3
TX-03-53 TAG 20.1 6.8 16.7 24.5 23.3 8.6 0.6 0.275
TX-03-01 TFA 13.4 3.0 3.0 12.5 61.8 6.4 2.3
TX-03-01 TAG 13.9 5.5 7.5 23.0 42.6 7.4 0.4 0.164
TX-03-47 TFA 12.8 2.1 1.6 7.5 70.7 5.3 2.4
TX-03-47 TAG 14.8 5.1 5.0 19.3 52.1 3.7 0.1 0.050
TX-03-07 TFA 18.4 2.8 7.6 15.6 47.1 8.5 2.5
TX-03-07 TAG 25.8 6.4 18.7 25.5 15.2 8.5 0.3 0.127
TX-03-05 TFA 21.4 2.3 1.4 9.7 59.1 6.1 2.6
TX-03-05 TAG 36.4 5.6 3.9 17.1 28.4 8.6 0.4 0.168
TX-03-49 TFA 18.1 3.7 8.2 13.2 52.0 4.9 2.6
TX-03-49 TAG 24.1 8.2 18.3 20.9 18.8 9.7 0.5 0.212
TX-03-34 TFA 19.0 2.7 6.0 15.4 50.6 6.4 2.6
TX-03-34 TAG 24.8 10.5 10.9 23.9 20.6 9.3 0.8 0.287
TX-03-32 TFA 18.2 2.2 1.6 12.4 60.2 5.4 2.8
TX-03-32 TAG 20.8 14.6 3.2 21.4 31.5 8.5 0.6 0.204
TX-03-04 TFA 18.8 3.1 5.8 13.4 50.3 8.6 2.9
TX-03-04 TAG 26.7 7.5 14.6 23.1 19.0 9.1 0.3 0.118
TX-03-23 TFA 18.9 1.7 1.0 7.9 63.2 7.3 2.9
TX-03-23 TAG 25.0 4.6 2.5 18.1 39.6 10.2 0.2 0.070
TX-03-25 TFA 14.5 1.8 0.4 6.4 73.5 3.4 3.0
TX-03-25 TAG 20.3 5.1 1.0 12.3 53.6 7.7 0.3 0.110
TX-03-18 TFA 21.1 2.9 1.2 17.8 46.3 10.7 3.0
TX-03-18 TAG 22.6 5.9 4.5 31.1 22.6 13.3 0.4 0.143
TX-03-50 TFA 16.5 2.6 6.1 12.9 53.9 8.0 3.0
TX-03-50 TAG 20.2 19.9 12.9 19.6 20.6 6.8 0.7 0.217
TX-03-60 TFA 20.2 2.9 0.8 14.1 55.7 6.2 3.1
TX-03-60 TAG 30.5 6.2 1.6 21.6 30.2 9.9 0.6 0.202
TX-03-21 TFA 12.3 1.7 0.5 6.8 74.4 4.4 3.2
TX-03-21 TAG 16.1 4.7 1.6 13.1 57.0 7.5 0.2 0.067
TX-03-40 TFA 17.1 1.4 0.4 8.0 68.2 4.9 3.2
TX-03-40 TAG 34.5 4.4 0.9 14.5 39.8 5.9 0.4 0.112
TX-03-62 TFA 25.3 2.9 1.7 14.7 47.9 7.6 3.3
TX-03-62 TAG 40.3 5.6 3.5 22.3 18.7 9.5 0.6 0.171
TX-03-36 TFA 19.5 2.0 2.0 11.4 58.3 6.8 3.5
TX-03-36 TAG 31.2 4.0 4.4 20.0 29.4 11.0 0.6 0.160
TX-03-63 TFA 25.4 3.6 2.6 18.2 42.0 8.2 3.5
TX-03-63 TAG 33.1 6.1 3.8 24.9 21.6 10.4 1.4 0.383
TX-03-45 TFA 16.4 1.4 0.5 8.1 69.1 4.5 3.5
TX-03-45 TAG 30.8 4.6 1.4 16.2 40.7 6.3 0.2 0.058
TX-03-17 TFA 14.2 1.8 0.8 6.9 71.2 5.2 3.6
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TX-03-17 TAG 18.7 4.5 2.2 13.5 52.8 8.3 0.4 0.120
TX-03-57 TFA 18.7 3.4 1.5 13.8 55.8 6.8 3.6
TX-03-57 TAG 23.4 6.3 3.0 21.0 36.2 10.1 1.2 0.330
TX-03-11 TFA 29.1 6.4 2.1 22.4 33.0 7.1 3.6
TX-03-11 TAG 30.6 8.5 2.8 27.0 19.7 11.4 1.9 0.510
TX-03-48 TFA 27.1 3.7 3.7 20.6 37.2 7.6 3.7
TX-03-48 TAG 31.2 5.0 5.5 27.1 23.0 8.1 2.1 0.569
TX-03-29 TFA 20.1 2.3 1.7 13.4 55.5 7.1 3.7
TX-03-29 TAG 33.0 5.0 4.1 24.3 26.4 7.2 0.4 0.104
TX-03-26 TFA 15.3 1.6 0.4 5.9 71.3 5.5 3.9
TX-03-26 TAG 25.2 4.6 1.7 13.3 49.7 5.5 0.3 0.074
TX-03-10 TFA 28.6 6.8 2.1 21.8 33.0 7.7 3.9
TX-03-10 TAG 31.0 8.5 2.9 26.7 18.6 12.2 1.9 0.491
TX-03-58 TFA 16.3 2.6 1.3 14.5 60.3 5.0 4.1
TX-03-58 TAG 20.4 5.2 2.8 24.3 39.2 8.2 1.1 0.278
TX-03-08 TFA 19.8 2.0 0.7 6.6 64.9 5.9 4.1
TX-03-08 TAG 34.8 5.2 2.7 14.3 34.5 8.5 0.2 0.051
- TX-03-33 TFA 27.4 2.4 1.5 16.3 46.0
6.4 4.2
- TX-03-33 TAG 39.2 5.4 2.3 21.9 20.8
10.5 1.6 0.386
- TX-03-22 TFA 19.8 2.8 3.1 11.8 53.4
9.1 4.2
- TX-03-22 TAG 28.4 5.3 5.4 19.4 38.3 3.2 1.2 0.287
TX-03-41 TFA 18.1 2.6 3.1 11.1 58.0 7.1 4.8
TX-03-41 TAG 27.8 6.0 6.8 19.3 34.9 5.3 0.7 0.139
TX-03-46 TFA 24.6 2.0 0.6 7.9 57.4 7.4 4.9
TX-03-46 TAG 44.7 4.2 1.3 13.4 31.4 5.0 1.1 0.220
TX-03-28 , TFA 28.5 2.1 1.3 23.4 33.7 11.0 .. 6.2
TX-03-28 TAG 36.0 2.9 3.1 29.6 18.5 10.0 3.7 0.596
TX-03-31 TFA 33.4 2.9 4.3 28.6 25.5 5.5 8.3
TX-03-31 TAG 38.0 3.6 4.9 30.6 14.8 8.1 6.6 0.789
Example 3. Increasing expression of thioesterase in plant cells
De novo fatty acid synthesis takes place in the plastids of eukaryotic cells
where
the fatty acids are synthesized while bound to acyl carrier protein as acyl-
ACP
conjugates. Following chain elongation to C16:0 and C18:0 acyl groups and then
desaturation to C18:1 while linked to ACP, the fatty acids are cleaved from
the ACP by
thioesterases and enter the eukaryotic pathway by export from the plastids and
transport
to the ER where they participate in membrane and storage lipid biogenesis. in
chloroplasts, the export process has two steps: firstly, acyl chains are
released as free
fatty acids by the enzymatic activity of acyl-ACP thioesterases (fatty acyl
thioesterase;
FAT), secondly by reaction with CoA to form acyl-CoA esters which is catalysed
by
long chain acyl-CoA synthetases (LACS). A. thaliana contains 3 fatty acyl
thioesterases which can be distinguished based on their acyl chain
specificity. FATA1
and FATA2 preferentially hydrolyze unsaturated acyl-ACPs while saturated acyl-
ACP
chains are typically cleaved by FATB.
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To explore the effect upon total fatty acid content, TAG content, and fatty
acid
composition of the co-expression of a thioesterase and genes encoding the WRI1
and/or
DGAT polypeptides, chimeric genes were made for each of the three A. thaliana
thioesterases by insertion of the coding regions into the pJP3343 binary
expression
vector for transient expression in N. benthamiana leaf cells from the 35S
promoter.
Protein coding regions for the A. thaliana FATA1 (Accession No. NP 189147.1,
SEQ
ID NO:43) and FATA2 (Accession No. NP 193041.1, SEQ ID NO:44) thioesterases
were amplified from silique cDNA using primers containing EcoRI and PstI sites
and
subsequently cloned into pJP3343 using the same restriction sites. The
resulting
expression vectors were designated pOIL079 and pOlL080, respectively. The
protein
coding region of the A. thaliana FATB gene (Accession No. NP 172327.1, SEQ ID
NO:45) was amplified using primers containing NotI and Sad flanking sites and
cloned
into the corresponding restriction sites of pJP3343, resulting in pOIL081.
Constructs
pOIL079, pOIL080 and pOIL081 are infiltrated into N. benthamiana leaf tissue,
either
individually or in combination with constructs containing the genes for the A.
thaliana
WRI1 transcription factor (AtWRI1) (pW3414) and/or DGAT1 acyltransferase
(AtDGAT1) (pJP3352). For comparison, chimeric genes encoding the Cocos
nucifera
FatB1 (CnFATB1) (pJP3630), C. nucifera FatB2 (CnFATB2) (pJP3629) were
introduced into N. benthamiana leaf tissue in parallel with the Arabidopsis
thioesterases, to compare the effect of the FatB polypeptides having MCFA
specificity
to the Arabidopsis thioesterases which do not have MCFA specificity. All of
the
infiltrations included a chimeric gene for expression of the p19 silencing
suppressor as
described in Example 1. The negative control infiltrated only the p19 T-DNA.
A synergistic effect was observed between thioesterase expression and WRI1
and/or DGAT over-expression on TAG levels in N. benthamiana leaves. Expression
of
the thioesterase genes without the WRI1 or DGAT genes significantly increased
TAG
levels above the low level in the negative control (p19 alone). For example,
expression
of the coconut FATB2 thioesterase resulted in an 8.2-fold increase in TAG
levels in the
leaves compared to the negative control. Co-expression of the A. thaliana WRI1
transcription factor with each of the thioesterases further increased TAG
levels
compared to the AtWRI1 control. Co-expression of each of the coconut
thioesterases
CnFATB1 and CnFATB2 with WRI1 resulted in higher TAG levels than each of the
three A. thaliana thioesterases with WRI1. Interestingly, the converse was
observed
when the A. thaliana DGAT1 acyltransferase was co-expressed in combination
with a
thioesterase and WRI1. This suggested a better match in acyl-chain specificity
of the A.
thaliana thioestcrases and the A. thaliana DGAT1 acyltransferase, resulting in
a greater
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flux of acyl-chains from the acyl-ACP into TAG. The non-MCFA thioesterases
were
also considerably more effective in elevating the percentage of oleic acid in
the total
fatty acid content in the leaves. Co-expression of the AtWRI1, AtDGAT1 and
AtFATA2 resulted in the greatest level of TAG in the leaves, providing a level
which
was 1.6-fold greater than when AtWRI1 and AtDGAT1 were co-expressed without
the
thioesterase. In another experiment, transient overexpression of FATA2 in
combination
with WRI1 and DGAT1 led to a 2.5-fold increase in TAG level relative to a
p19+WRI1+DGAT1 control, which represented a 50-fold increase in TAG levels
relative to p19 alone. Addition of FATA1 increased TAG levels 2-fold compared
to
p19+WRI1+DGAT1, a 40-fold increase compared to p19 alone. Addition of FATB
increased TAG levels by 1.6-fold relative to p19+WRI1+DGAT1, a 32-fold
increase
relative to p19 control.
Co-expression of thioesterase FATA or FATB together with WRI1 and DGATl
resulted in modified leaf fatty acid composition relative to WRI1 and DGAT1
without
thioesterase. Addition of FATA1 increased the percentages of C16:0 and C18:0
at the
expense of saturated fatty acids. Addition of FATA2 also increased the
proportion of
C18:0 but did not have as great an effect on C16:0. In contrast, addition of
FATB
increased C16:0 but not C18:0 levels. In each case, addition of FATA1, FATA2
and
FATB reduced C18:1 levels. Notably, the C16:0 percentage increased from 28.4%
in
p19+WRIl+DGAT1 without thioesterase to 43.8% with the addition of FATA1, to
34.4% with the addition of FATA2 and to 46.3% with the addition of FATB.
These experiments confirmed the synergistic increase in oil synthesis and
accumulation when both WRI1 and DGAT were co-expressed as well as showing the
further synergistic increase obtained by adding a thioesterase to the
combination.
Effect of transient thioesterase expression in a high oil background
The three A. thaliana thioesterase genes were also tested by transient
expression
in leaves of N. benthamiana plants (transgenic line AT001) which were
transgenic for
and stably expressing WRI1, DGAT1 and OLEOSIN genes (El Tahchy et al., 2017).
Thirty plants from homozygous, T2 generation, transgenic AT001 seeds were
grown in
a randomised design alongside wild-type (WT) controls. At a vegetative stage
of
growth, 53 days after sowing (DAS), the transgenic leaves contained about 8.7%
(DW)
TAG compared to about 0.03% (DW) TAG in the wild-type plants. After further
growth of the transgenic plants, TAG levels increased from about 11.2% to
about
21.3% (DW) during flowering stages. They continued to increase, reaching about
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31.4% (DW) TAG at maturity (late seed development stage). As the plants
senesced,
the TAG level in at least some plants decreased to about 19.6% DW.
The genes encoding the thioesterases were introduced into leaves of young
plants (49 DAS) when the leaves typically had about 3.1% (DW) TAG, and sampled
5
days after infiltration with the Agrobacterium strains. Leaf samples were
harvested and
analyzed for TAG content. FATA2 overexpression in AT001 N. benthamiana leaves
significantly increased TAG to 4.4% (DW) compared to the p19 control (3.1%
TAG).
FATA1 increased TAG content to 3.9% (DW). FATB transient expression did not
appear to increase TAG accumulation in this experiment.
Samples were also used in radiolabel feeding assays with [14q-acetate. [14CJI-
acetate was added in a 10 minute pulse to leaf discs of AT001 leaves,
infiltrated
previously with genes encoding p19 and one of FATA I , FATA2 and FATB. This
pulse
was followed by a 20 minute chase. Lipid extracts were prepared at each time
point
followed by separation of labelled lipid classes on TLC. Quantitation of the
labelled
reaction products showed increases in the rate of TAG production in the AT001
leaves
transiently expressing FATA I (602 DPM), FATA2 (762 DPM) and FATB (559 DPM)
compared to the p19 control (283 DPM).
Three different binary expression vectors were constructed to test the effect
of
co-expression of genes encoding VVR11, DGAT1 and FATA on TAG levels and fatty
acid composition in stably transformed N. tabacum leaves. The vector pOIL121
contained an SSU::AtWRI1 gene for expression of AtWRI1 from the SSU promoter,
a
35S::AtDGAT1 gene for expression of AtDGAT from the 35S promoter, and an
enTCUP2::AtFATA2 gene for expression of AtFATA2 from the enTCUP2 promoter
which is a constitutive promoter. These genetic constructs were derived from
pOIL38
by first digesting the DNA with NotI to remove the gene coding for the S.
indicum
oleosin. The protein coding region of the A. thaliana FATA2 gene was amplified
and
flanked with Notl sites using pOIL80 DNA as template. This fragment was then
inserted into the ArotI site of pOIL38. p011,121 then served as a parent
vector for
pOIL122 which contained an additional enTCUP2::SDP1 hairpin RNA cassette for
RNAi-mediated silencing of the endogenous SDP1 gene in the transgenic plants.
To do
this, the entire N. benthamiana SDP1 hairpin cassette was isolated from pOIL51
(Vanhercke et al., 2017) as an Sfol-SmaI fragment and cloned into the *I site
of
pOIL121, producing pOIL122 (Figure 2). A third vector, pOIL123, containing the
SSU::WRI1 and 355::DGAT1 genes and the enTCUP2::SDP1 hairpin RNA gene was
obtained in a similar way by cloning the enTCUP2::SDP I hairpin RNA cassette
as a
Sfol-Smal fragment into the SfoI site of pOIL36.
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In summary, the vectors contained the gene combinations:
pOIL121: SSU::AtWRIL 35S::AtDGAT1, enTCUP2::AtFATA2.
pOIL122: SSU::AtWRIL 35S: :AtDGAT1,
enTCUP2::AtFATA2,
enTCUP2::SDP1 hairpin.
pOIL123: SSU::AtWRIL 35S::AtDGAT1, enTCUP2::SDP1 hairpin.
The three constructs were each used to produce transformed N. tabacum plants
(cultivar Wi38) by Agrobacterium-mediated transformation. Co-expression of the
A.
thaliana FATA2 thioesterase or silencing of the endogenous SDP1 TAG lipase in
combination with AtWRI1 and AtDGAT1 "expression each resulted in further
elevated
TAG levels compared to expression of AtWRI1 and AtDGAT1 in the absence of both
of the thioesterase gene and the SDP1-silencing gene. The greatest TAG yields
were
obtained using pOIL122 by the combined action of all four chimeric genes. In
absence
of SDP1, pOIL121 lines yielded 13.3% TAG which was included increased
palmitate
(16:0) levels (36%) and reduced ALA (18:3w3) levels (7%).
It is noted that N. benthamiana is an 18:3 plant. The same constructs pOIL079,
pOIL080 and pOIL081 are used to transform A. thaliana, a 16:3 plant.
The inventors conceived of the model that increasing plastidial fatty acid
export
such as by increased fatty acyl thiocsterase activity reduces acyl-ACP
accumulation in
the plastids, thereby increasing fatty acid biosynthesis as a result of
reduced feedback
inhibition on the acetyl-CoA carboxylase (ACCase) (Andre et al., 2012; Moreno-
Perez
et al., 2012). Thioesterase over-expression increases export of acyl chains
from the
plastids into the ER, thereby providing an efficient link between so-called
'Push' and
'Pull' metabolic engineering strategies.
=
Example 4. The effect of different transcription factor polypeptides on plant
traits
Previously reported experiments with WRI1 and DGAT (Vanhercke et al.,
2013) used a synthetic gene encoding A. thaliana AtWRI1 (Accession No.
AAP80382.1) and a synthetic gene encoding AtDGAT1, also from A. thaliana
(Accession No. AAF19262; SEQ ID NO: 1). To compare other WRI polypeptides with
AtWRI1 for their ability to combine with DGAT to increase oil content, other
WRI
coding sequences were identified and used to generate constructs for
expression in N.
benthamiana leaves. Nucleotide sequences encoding the A. thaliana WRI3
(Accession
No. AAM91814.1, SEQ ID NO:46) and WRI4 (Accession No. NP 178088.2, SEQ ID
NO:47) transcription factors (To et al., 2012) were synthesized and inserted
as Ecold
fragments into pJP3343 under the control of the 35S promoter. The resulting
binary
expression vectors were designated pOIL027 and pOIL028, respectively. The
coding
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sequence for the oat (Avena sativa) WRI1 (AsWRI1, SEQ ID NO:48) was PCR
amplified from a vector provided by Prof. Sten Stymne (Swedish University of
Agricultural Sciences) using flanking primers containing additional EcoRI
sites. The
amplified fragment was inserted into pJP3343 resulting in pOIL055. A WRI1
candidate
sequence from S. bicolor (Accession No. XP_002450194.1, SEQ ID NO:49) was
identified by a BLASTp search on the NCBI server using the Zea mays WRI1 amino
acid sequence (Accession No. NP_001137064.1, SEQ ID NO:50) as query. The
protein
coding region of the S. bicolor WRI1 gene (SbWRI1) was synthesized and
inserted as an
EcoRI fragment into pJP3343, yielding pOIL056. A gene candidate encoding a
WRI1
was identified from the Chinese tallow (Triadica sebifera; TsWRIL SEQ ID
NO:51)
transcriptome (Uday et al., submitted). The protein coding region was
synthesized and
inserted as an EcoRI fragment into pJP3343 resulting in pOIL070. The pJP3414
and
pJP3352 binary vectors containing the coding sequences for expression of the
A.
thaliana WRI1 and DGAT1 polypeptides were as described by Vanhercke et al.
(2013).
Plasmids containing the various WRI coding sequences were introduced into N.
benthamiana leaf tissue for transient expression using a gene encoding the p19
viral
suppressor protein in all inoculations as described in Example 1. The genes
encoding
the WRI polypeptides were either tested alone or in combination with the DGAT1
acyltransferase gene, the latter to provide greater TAG biosynthesis and
accumulation.
The positive control in this experiment was the combination of the genes
encoding A.
thaliana WRI1 transcription factor and AtDGAT1. All infiltrations were done in
triplicate using three different plants and TAG levels were analyzed as
described in
Example 1. Expression of most of the individual WRI polypeptides in the
absence of
exogenously added DGAT1 resulted in increased, yet still low, TAG levels
(<0.23%
on dry weight basis) in infiltrated leaf spots, compared to the control which
had only
the p19 construct (Figure 3). The exception was TsWRI1 which, by itself, did
not
appear to increase TAG levels significantly. In addition, differences in TAG
levels
produced by expression of the different WRI transcription factors on their own
were
not great. Both AsWRI1 and SbWRI1 yielded TAG levels similar to AtWRI1 on its
own. Analysis of the TAG fatty acid composition revealed only minor changes
except
for increased C18:1A9 levels from expression of AtWRI3 in the infiltrated leaf
tissues
(Table 7).
In contrast, differences in0 TAG yields from expression of the different WRI
polypeptides were more pronounced upon co-expression with the AtDGAT1
acyltransferase. This again demonstrated the synergistic effect of WRI1 and
DGAT co-
expression on TAG biosynthesis in infiltrated N. benthamiana leaf tissue, as
reported
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=
by Vanhercke et al. (2013). Intermediate TAG levels were observed upon co-
expression of DGAT1 with AtWRI3, AtWRI4 and TsWRI1 expressing vectors while
levels obtained with the AsWRI1 and AtWRI1 were significantly lower. In a
result that
could not have been predicted beforehand, the highest TAG yields were obtained
with
co-expression of DGAT with SbWRI1, even though the assay was done in
dicotyledonous cells. TAG fatty acid composition analysis revealed increased
levels of
C18:1 9 and decreased levels of C18:3 9'12'15 (ALA) in the case of SbWRIL
AsWRI1
and the AtWRI1 positive control. Unlike AtWRI1, however, expression of AsWR11
and SbWRI1 both displayed increased C16:0 levels compared to the p19 negative
control. Interestingly, AtWRI3 infiltrated leaf samples exhibited a distinct
TAG profile
with C18:1 9 being enriched while C16:0 and ALA were only slightly affected.
This experiment showed that the S. bicolor WRI1 transcription factor, SbWRI1,
was superior to AtWRI1 when co-expressed with DGAT to increase TAG levels in
vegetative plant parts. The inventors also concluded that a transcription
factor, for
example a WRI1, from a monocotyledonous plant could function well in a
dicotyledonous plant cell, indeed might even have superior activity compared
to a
corresponding transcription factor from a dicotyledonous plant. Likewise, a
transcription factor from a dicotyledonous plant could function well in a
monocotyledonous plant cell.
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Table 7. TAG fatty acid composition in X benthamiana leaf samples infiltrated
with different chimeric genes for expression of WRI (n=3).
co All samples were also infiltrated with the P19 construct. The TAG
samples also contained 0.1-0.4% C14:0; 0.5-1.2% C16:3 and; 0.1-0.7%
C18:1A11.
co
Infiltrated C16:0 C16:1 C18:0 C18:1 C18:2
C18:3n3 C20:0 C20:1 C22:0 C24:0
0
genes
1-`
Control (P19) 33.6 4.7 0.5 0.4 8.9 2.2
4.7 + 0.6 16.9 1.0 32.2 + 7.8 1.1 + 0.2 0.8 1.5 0.0
0.0
WRI1 35.5 3.4 0.7 0.2 5.2 0.8
5.4 1.3 17.1 1.0 33.1 2.7 0.8 0.1 0.5 0.6 0.3
0.0 0.0
WRI3 27.3 1.6 0.9 0.2 4.8 + 0.3
10.2 1.5 16.1 1.0 37.8 1.2 0.8 0.1 0.6 0.7 0.1 0.2
0.0
WRI4 30.1 0.4 1.0 0.4 5.2 0.8
4.6 0.6 17.2 + 0.4 38.1 1.6 0.8 0.1 1.3+1.3 0.0 0.0
AsVVRI 35.7 + 3.0 1.7 + 0.4 5.3 0.7
6.5 0.3 15.4 0.4 31.6 1.6 0.8 0.1 0.4 0.7 0.3 + 0.1
0.0
SbWRI 37.4 0.8 1.9 0.3 4.8 + 0.3
7.0 1.2 15.2 0.3 30.8 + 0.3 0.8 0.1 0.4 + 0.6 0.3 +
0.0 0.0
TsWRI 34.5 4.8 0.0 9.4 8.2 5.9 1.7
16.0 0.7 29.3 0.0 n.d. 0.0 0.0
12.4
Control (P19) 31.0 2.1 0.9 + 0.1 8.7 + 1.3
8.0 + 2.3 24.9 1.5 22.1 + 4.7 2.0 0.1 0.0 0.6 0.6 0.2
+ 0.4
WRIl+DGAT 27.7 0.1 0.3 0.0 7.0 0.1
17.2 0.7 27.9 + 0.9 14.7 0.3 2.4 0.2 0.3 + 0.0 1.1 0.1
0.8 0.2
WRI3+DGAT 30.0 + 0.8 0.6 0.1 5.9 + 0.4
13.9 2.9 21.5 1.1 21.3 0.8 2.8 0.1 0.2 0.0 1.8 0.1
1.0 0.2
WRI4+DGAT 27.0 0.5 0.2 0.1 8.5 0.2
5.8 0.7 23.9 0.8 25.2 1.3 3.5 + 0.1 0.2 + 0.0 2.1
0.2 1.7 0.2
AsWRI+DGAT 33.8 + 0.5 1.1 + 0.1 5.5 0.9
12.2 1.6 26.0 1.9 16.3 1.3 2.2 0.2 0.2 + 0.0 1.2 + 0.1
0.8 0.1
SbWRI+DGAT 34.6 0.5 1.3 0.1 5.6 0.4
13.9 1.6 23.6 1.3 15.8 + 0.6 2.2 + 0.1 0.2 0.0 1.2 0.1
0.9 0.1
TsWRI+DGAT 25.4 0.5 0.2 0.0 9.4 0.1 7.7+ 1.0
27.0 1.3 22.1 2.4 3.6 0.2 0.2 0.0 1.8 0.2 1.3 0.2
186
Use of other transcription factors
Genetic constructs were prepared for expression of each of 24 different
transcription factors in plant cells to test their ability to function for
increasing TAG
levels in combination with other genes involved in TAG biosynthesis and
accumulation. These transcription factors were candidates as alternatives for
WRI1 or
for addition to combinations including one or more of WRI1, LEC1 and LEC2
transcription factors for use in plant cells, particularly in vegetative plant
parts. Their
selection was largely based on their reported involvement in embryogenesis
(reviewed
in Baud and Lepiniec (2010), and Ikeda et al. (2006)), similar to LEC2, or
plant
storage lipid metabolism. Experiments were therefore carried out to assay
their
function, using the N. benthamiana expression system (Example 1), as follows.
Nucleotide sequences of the protein coding regions of the following
transcription factors were codon optimized for expression in N. benthamiana
and N.
tabacum, synthesized and subcloned as Notl-Sacl fragments into the respective
sites
of pJP3343: A. thaliana FUS3 (pOIL164) (Luerssen et al., 1998; Accession
number
AAC35247; SEQ ID NO:34), A. thaliana LEC1L (pOIL165) (Kwong et al. 2003;
Accession number AAN15924; SEQ ID NO:33), A. thaliana LEC1 (pOIL166) (Lotan
et al., 1998; Accession number AAC39488; SEQ ID NO:31), G. max MYB73
(pOIL167) (Liu et al., 2014; Accession number ABH02868; SEQ ID NO:57), A.
thaliana bZIP53 (pOIL168) (Alonso et al., 2009; Accession number AAM14360;
SEQ ID NO:58), A. thaliana AGL15 (pOIL169) (Zheng et al., 2009; Accession
number NP 196883; SEQ ID NO:59), A. thaliana MYB118 (Accession number
AAS58517; pOIL170; SEQ ID NO:60), MYB115 (Wang et al., 2002; Accession
number AAS10103; pOIL171; SEQ ID NO:61), A. thaliana TANMEI (p011,172)
(Yamagishi et al., 2005; Accession number BAE44475; SEQ ID NO:62), A. thaliana
WUS (pOIL173) (Laux et al., 1996; Accession number NP_565429; SEQ ID NO:63),
A. thaliana BBM (pOIL174) (Boutilier et al., 2002; Accession number AAM33893,
SEQ ID NO:64), B. napus GFR2a1 (Accession number AFB74090; pOIL177; SEQ
ID NO:64), GFR2a2 (Accession number AFB74089; pOIL178; SEQ ID NO:65) (Liu
et al. (2012)), E. guineensis NF-YB1 (pOIL405) (Geurin et al., 2016; Accession
number XM 010907896; SEQ ID NO:143 , E. guineensis ZFP1 (pOIL406) (Geurin
et al., 2016; Accession number XM 010930940; SEQ ID NO:144), A. thaliana NF-
YB2 (pOIL407) (Geurin et al., 2016; Accession number NM_124138; SEQ ID
NO:145), A. thaliana NF-YB3 (pOIL408) (Geurin et al., 2016; Accession number
NM 117534; SEQ ID NO:146), A. thaliana ZFP2 (pOIL409) (Geurin et al, 2016;
Accession number NM 125133; SEQ ID NO:147), E. guineensis ABI5 (pOIL410)
(Yeap et al., 2017; Accession number XM_010909282; SEQ ID NO:148), E.
guineensis NF-YC2 (p0IL411) (Yeap et al,, 2017; Accession number
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XM 010911913; SEQ ID NO:149), and E. guineensis NE-YA3 (pOIL412) (Yeap et
at., 2017; Accession number XM_010941630; SEQ ID NO:150). In addition, a codon
optimized version of the A. thaliana PHR1 transcription factor involved in
adaptation
to high light phosphate starvation conditions was similarly subcloned into
pJP3343
(pOIL189) (Nilsson et al (2012); Accession number AAN72198; SEQ ID NO:221).
The sequence coding for the G. max DOF4 (Wang et al., 2007; Accession number
DQ857254; SEQ ID NO:151) was codon optimized for expression in N. benthamiana
and N. tabacum, synthesized as a Notl-Spel fragment and subcloned into
pJP3343.
The resulting vector was designated pOIL379. Finally, the gene coding for the
G. max
ZE351 transcription factor (Li et al., 2017; Accession number XM_003526219;
SEQ
ID NO:152) was synthesized as a Notl-EcoRI fragment and cloned into 0133343,
resulting in pOIL420. These transcription factors are summarised in Table 8.
As a screening assay to determine the function of these transcription factors,
the genetic constructs and a gene encoding DGAT1were co-infiltrated into N.
benthamiana leaf cells as described in Example 1, either with or without a
gene
encoding WRIL Total lipid content and fatty acid composition of the leaf cells
were
analysed 5 days post-infiltration. Among the various embryogenic transcription
factors tested, only overexpression of FUS3 resulted in significantly
increased TAG
levels in N. benthamiana leaf tissue when compared to DGAT and DGAT1+WRI1
control infiltrations (Table 9).
Table 8. Additional transcription factors and the genetic constructs for their
expression
Plasmid Transcription Species Length Accession
factor (amino acid) number
pOIL164 FUS3 A. thaliana 312 AAC35247
pOIL165 LEC1L A. thaliana 234 AAN15924
pOIL166 LEC I A. thaliana 208 AAC39488
pOIL167 MYB73 G. max 74 ABI102868
pOIL168 bZIP53 A. thaliana 146 AAM14360
pOIL169 AGL15 A. thaliana 268 NP 196883
pOIL170 MYB118 A. thaliana 437 AAS58517
pOIL171 MYB115 A. thaliana 359 AAS10103
pOIL172 TANMEI A. thaliana 386 BAE44475
pOIL173 WUS A. thaliana 292 NP 565429
pOIL174 BBM A. thaliana 584 AAM33803
pOIL177 GFR2a1 B. napus 453 AFB74090
pOIL178 GER2a2 B. napus 461 AFB74089
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pOIL189 PHR1 A. thaliana 409 AAN72198
pOIL379 DOF4 G. max 300 DQ857254
pOIL405 NF-YB1 E. guineensis 215 XM 010907896
pOIL406 ZFP1 E. guineensis 142 XM 010930940
pOIL407 NF-YB2 A. thaliana 190 NM 124138
pOIL408 NF-YB3 A. thaliana 161 NM 117534
pOIL409 ZFP2 A. thaliana 150 NM 125133
pOIL410 ABI5 E. guineensis 398 XM 010909282
pOIL411 NF-YC2 E. guineensis 272 XM 010911913
pOIL412 NF-YA3 E. guineensis 352 XM 010941630
pOIL420 ZF351 G. max 351 003526219
Table 9. TAG level (% leaf dry weight) and fatty acid profile of infiltrated
N.
benthamiana leaves.
C16:0 C16:1 , C18:0 C18:1 C18:2 C18:3 TAG
P19 27.1 0.3 9.6 + 4.4 22.4 30.5 0.0
1.5 0.1 1.7 1.2 4.0 0.9
P19+DGAT1 26.3 + 0.1 10.7 3.7 26.1 26.4 0.2
1.0 0.0 0.6 0.7 1.6 1.4 0.0
P19+DGAT1+FUS3 24.1 + 0.1 6.3 + 5.2 + 27.9 30.0 + 0.6
1.0 0.0 0.4 1.6 1.8 1.8 0.1
P19+DGAT1+LEC1L 26.0 + 0.1 + 10.3 3.9 26.6 26.4 0.2 +
1.4 0.0 0.8 1.0 2.1 0.7 0.0
P19 30.3 + 0.0 12.4 6.8 22.9 26.0 + 0.0
0.7 0.7 , 0.9 0.2 0.9
P19 DGAT1 25.8 0.0 10.1 4.4 26.1 26.2 + 0.2
1.1 0.4 0.9 1.3 1.4 0.0
P19+DGAT I +WR11 22.7 0.0 10.1 + 14.9 27.9 + 18.5 + 0.3 +
0.9 0.4 0.5 1.3 0.8 0.1 ,
P19 DGAT1+FUS3 23.9 0.2 7.6 5.3 + 29.1 26.8 0.4
0.7 0.1 0.4 0.7 0.8 0.7 0.1
P19 DGAT1+LEC1 24.9 0.1 11.1 4.0 25.9 26.1 0.1
, 0.4 0.2 0.2 0.1 0.5 0.6 0.0
P19+DGAT1+MYB 73 25.8 0.0 10.9 4.3 + 26.2 25.2 + 0.1
0.3 0.7 1.0 0.8 1.8 0.0
P19 34.2 0.0 10.6 8.3 19.5 23.2 0.1
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4.9 3.1 4.1 1.4 0.8 0.1
P19+DGAT1 27.7 0.3 + 9.9 4.2 26.4 22.5 0.2
0.1 0.1 1.1 0.3 L8 0.4 0.1
P19+DGAT1+WRI1 24.8 0.2 8.8 14.7 1 27.6 17.2 0.4 +
1.0 0.0 1.0 0.6 1.0 0.3 0.1
P19+DGAT1+bZIP53 29.3 + 0.1 + 8.7 2.9 22.0 25.9 0.1
0.8 0.2 0.4 0.3 0.5 0.5 0.1
P19+DGAT1+AGL15 29.2 + 0.2 4.9 + 7.0 + 19.8 30.0 0.3
1.4 0.0 0.9 1.9 0.8 1.3 0.1
P19+DGAT1+MYB118 31.6 0.2 5.8 1 4.8 + 20.7 1 28.2 + 0.2
1.7 0.1 1.2 0.8 0.3 1.6 0.1
P19 27.4 0.0 6.9 4.8 20.0 + 39.0 0.1
1.2 1.0 2.6 1.5 4.1 0.0
P19+DGAT I 26.0 1 0.0 8.0 4.2 + 22.3 1 33.9 + 0.2
1.1 0.6 1.6 2.4 4.3 0.0
P19+DGAT1+WRI1 23.4 0.1 8.5 17.0 + 23.3 23.3 0.5 +
0.8 0.1 0.6 2.4 1.8 4.3 0.1
P19+DGAT1+MYB115 26.3 0.1 6.6 2.8 22.5 35.7 0.2
0.4 0.1 0.3 0.4 1.8 2.9 0.0
P19+DGAT1+TANMEI 25.6 0.1 8.5 + 2.6 + 21.9 35.3 0.2
0.9 0.2 1.2 0.5 2.0 3.8 0.0
P19+DGAT1+WUS 24.3 0.1 5.5 1.7 16.8 47.9 0.2
0.9 0.1 0.6 0.2 1.6 3.3 0.0
P19 30.5 0.0 8.1 8.2 21.8 28.3 0.1
1.3 0.9 6.0 1.2 7.3 0.1
P19+DGAT I +WRI1 25.9 0.2 8.3 19.9 24.5 16.0 0.8
________________________ 1.7 0.0 0.7 2.8 1.1 0.6 0.1
P19+DGAT1+WRI1+BBM 27.7 0.2 6.7 21.2 19.8 18.5 0.5
0.7 0.0 0.2 0.7 0.5 0.6 0.1
P19+DGAT1+WRI1+GFR2a1 29.2 0.4 6.1 12.9 + 24.3 20.9 + 0.4 +
1.3 0.0 0.1 1.5 0.4 0.5 0.1
P19+DGAT1+WRI1+GFR2a2 29.9 0.4 5.5 13.5 23.0 + 21.3 0.5
2.4 0.1 0.6 2.7 0.5 1.2 0.1
P19+DGAT1+WRI1 +PHR1 26.2 0.2 4.9 7.6 19.2 36.0 1 0.3
0.3 0.0 0.0 0.2 0.3 0.7 0.0
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P19 32.0 1.6 11.1 5.5 23.3 1 25.4 1 0.0
1.9 2.7 2.7 2.2 1.1 3.3
P19+DGAT1+WR11 27.5 + 0.7 6.8 16.6 26.7 16.5 1.2 +
1.2 0.8 0.4 2.1 0.8 0.3 0.2
P19+DGAT1+WRIl+FUS3 23.6 2.1 6.5 13.3 32.1 15.6 1.6
1.1 3.5 0.5 0.9 2.6 1.5 0.1
P19+GFP 35.8 0.0 + 8.5 2.0 19.7 32.1 0.03
1.8 0.0 0.8 1.3 1.2 2.2 +0.0
P19+GFP+DGAT1+WRI1 24.6 0.2 10.3 22.7 + 23.0 + 14.0 + 0.99
1.4 0.0 0.5 2.7 1.7 0.6 0.2
P19+GFP+DGAT1+NF-YB2 27.6 + 0.1 + 10.2 3.0 24.1 27.1 0.25
0.6 0.0 0.2 0.2 1.1 1.2 0.0
P19+GFP+DGAT1+NF-YB3 27.4 0.1 + 10.8 + 3.1 24.6 26.0 0.27
0.5 0.0 0.5 1.0 0.9 0.7 0.1
P19+GFP+DGAT1+NF-YA3 28.9 0.2 8.3 3.6 22.7 + 29.2 0.17
0.8 0.0 0.4 0.5 1.0 0.9 0.0
P19+GFP 38.3 0.0 11.1 + 2.9 21.3 26.4 0.0 +
1.3 0.0 1.2 1.4 1.0 3.8 0.0
P19+GFP+DGAT1+WRI1 29.8 0.3 7.6 + 18.3 + 23.9 + 15.0 + 1.1
1.1 0.0 1.7 0.6 1.4 0.7 0.5
P19+GFP+DGAT1+DOF4 32.5 0.0 5.1 + 3.6 20.5 32.6 + 0.2 +
0.5 0.0 0.7 0.2 0.9 1.2 0.1
P19+GFP+DGAT1+NF-YB1 27.9 0.0 10.8 2.9 27.0 23.7 0.3
0.7 0.0 0.5 0.5 1.3 1.4 0.1
P19+GFP+DGAT1+ZFP1 25.4 0.1 4.1 5.2 22.8 + 36.2 0.3
1.4 0.2 0.3 1.2 0.8 0.8 0.1
P19+GFP 37.7 0.0 + 11.5 + 2.6 + 22.2 24.1 + 0.0
1.7 0.0 1.5 2.3 1.9 4.7 0.0
P19+GFP+DGAT1+WRI1 28.0 0.2 9.3 17.2 27.3 + 13.0 + 0.8 +
2.1 0.0 1.0 3.1 1.2 0.3 0.3
P19+GFP+DGAT1+ZF351 30.8 0.2 9.5 2.6 25.4 25.4 0.2
0.5 0.1 0.7 1.5 1.1 2.1 0.0
P19 18.9 0.4 5.6 6.1 18.3 + 45.8 0.4 +
2.9 0.3 1.7 4.8 1.7 9.5 0.1
P19+DGAT1+WRI1 21.4 + 0.2 9.9 + 19.4 + 20.5 23.8 1.7
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2.3 0.0 0.8 0.9 0.9 2.7 0.6
P19+DGAT1+WRI1+ZFP2 23.1 + 0.3 5.3 9.3 16.2 40.5 1.0
1.2 0.1 0.5 1.7 0.7 4.1 0.4
P19+DGAT1+WRIl+ABI5 21.4 0.2 8.4 11.4 + 23.2 29.9 + 1.2 +
1.1 0.0 0.7 1.3 1.4 2.9 0.4
P19+DGAT1+WR11+NF- 20.5 0.2 9.6 18.1 21.2 25.4 + 1.6 +
YC2 0.7 0.1 0.4 0.6 0.6 1.5 0.4
For stable transformation of plants using genes encoding the alternative
transcription factors, the following binary constructs are made. The genes for
expression of the transcription factors use either the SSU promoter or the
SAG12
promoter. Over-expression of embryogenic transcription factors such as LEC1
and
LEC2 has been shown to induce a variety of pleotropic effects, undesirable in
the
present context, including somatic embryogenesis (Feeney et al. (2012); Santos-
Mendoza et al. (2005); Stone et al. (2008); Stone et al. (2001); Shen et al.
(2010)). To
minimize possible negative impact on plant development and biomass yield,
tissue or
developmental-stage specific promoters are preferred over constitutive
promoters to
drive the ectopic expression of master regulators of embryogenesis.
Example 5. Stem-specific expression of a gene encoding a transcription factor
Leaves of N. tabacum plants expressing transgenes encoding WRI1, DGAT
and Oleosin contain about 16% TAG at seed setting stage of development.
However,
the TAG levels were much lower in stems (1%) and roots (1.4%) of the plants
(Vanhercke et al., 2014a and b). The inventors considered whether the lower
TAG
levels in stems and roots were due to poor promoter activity of the Rubisco
SSU
promoter used to express the gene encoding WRI1 in the transgenic plants. The
DGAT transgene in the T-DNA of pJP3502 was expressed by the CaMV35S
promoter which is expressed more strongly in stems and roots and therefore was
unlikely to be the limiting factor for TAG accumulation in stems and roots.
In an attempt to increase TAG biosynthesis in stem tissue, a construct was
designed in which the gene encoding WRI1 was placed under the control of an A.
thaliana SDP1 promoter. A 3.156kb synthetic DNA fragment was synthesized
comprising 1.5kb of the A. thaliana SDP1 promoter (SEQ 1D NO:41) (Kelly et
al.,
2013a and b), followed by the coding region for the A. thaliana WRI1
polypeptide
and the G. max lectin terminator/polyadenylation region. This fragment was
inserted
between the Sad l and Not' sites of pJP3303. The resulting vector was
designated
pOIL050, which was then used to transform cells from the N. tabacum plants
homozygous for the T-DNA from pJP3502 by Agrobacterium-mediated
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transformation. Transgenic plants were selected for hygromycin resistance and
a total
of 86 independent transgenic plants were grown to maturity in the glasshouse.
Samples were taken from transgenic leaf and stem tissue at seed setting stage
and
contain increased TAG levels compared to the N. tabacum parental plants
transformed
with pJP3502.
Example 6. Effect of oil body protein expression on plant traits
N. tabacum plants transformed with the T-DNA of pJP3502 and expressing
transgenes encoding A. thaliana WRI1, DGAT1 and S. indicum Oleosin had
increased
TAG levels in vegetative tissues. As shown in Example 2 above, when the
endogenous gene encoding SDP1 TAG lipase was silenced in those plants, the
leaf
TAG levels further increased, which indicated to the inventors that
substantial TAG
turnover was occurring in the plants that retained SDP I activity. Therefore,
the level
of expression of the transgenes in the plants was determined. While Northern
hybridisation blotting confirmed strong WRII and DGAT1 expression and some
oleosin mRNA expression, expression analysis by digital PCR and qRT-PCR
detected
only very low levels of oleosin transcripts. The expression analysis revealed
that the
gene encoding the Oleosin was poorly expressed compared to the WRII and DGAT1
transgenes. From these experiments, the inventors concluded that the oil
bodies in the
leaf tissue were not completely protected from TAG breakdown because of
inadequate production of Oleosin protein when encoded by the T-DNA in pJP3502.
To improve stable accumulation of TAG throughout plant development, several
pJP3502 modifications were designed in which the Oleosin gene was substituted.
These modified constructs were as follows.
1. pJP3502 contains a gene (SEQ ID NO:42 provides the sequence of its
complement) encoding the S. indicum oleosin which was poorly expressed.
That gene has an internal UBQ10 intron which might be reducing the
expression level. To test this, a 502bp synthetic DNA fragment containing the
S. indicum oleosin gene and lacking the internal UBQ10 intron was
synthesized and inserted into pJP3502 as a Notl fragment, to substitute the
oleosin gene containing the intron in pJP3502. The resultant plasmid was
designated pOIL040.
2. The Rubisco small subunit (SSU) promoter driving expression of the oleosin
gene in pJP3502 was replaced by the constitutive enTCUP2 promoter. To this
end, a 2321bp fragment containing the enTCUP2 promoter, Oleosin protein
coding region, G. max lectin terminator/polyadenylation region and the first
643bp of the downstream SSU promoter driving wril expression was
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synthesized and subcloned into the AscI and Spel sites of pJP3502 resulting in
pOIL038.
3. A similar strategy was followed for the expression of an engineered version
of
the S. indicum oleosin gene containing 6 introduced cysteine residues (o3-3)
under the control of the enTCUP2 promoter (Winichayakul et al., 2013). A
2298bp fragment containing the enTCUP2 promoter, Oleosin o3-3 protein
coding region, G. max lectin terminator/polyadenylation region and the first
643bp of the downstream SSU promoter driving wri/ expression was
synthesized and subcloned into the Asc.' and Spel sites of pJP3502 resulting
in
p0IL037.
4. The Notl sites flanking the S. indicum oleosin gene in pJP3502 were used to
exchange the protein coding region for one encoding peanut 01eosin3
(Accession No. AAU21501.1) (Parthibane et at., 2012a and b). A 528bp
fragment containing the oleosin3 gene, flanked by Notl sites, was synthesized
and subcloned into the respective site of pJP3502. The resulting vector was
designated pOIL041.
5. Similarly, a 1077bp Nod flanked fragment containing the gene coding for the
A. thaliana steroleosin (Arab-1) (Accession No. AAM10215.1) (Jolivet et al.,
2014) was synthesized and subcloned into the Notl site of pJP3502, resulting
in p0IL043.
6. The Nannochloropsis oceanic lipid droplet surface protein (LDSP) (Accession
No. AFB75402.1) (Vieler et al., 2012) was synthesized as a 504bp Non-
flanked fragment and subcloned into the Notl site of pJP3502, yielding
pOIL044.
7. Finally, the A. thaliana caleosin (CL03) (Accession No. 022788.1) (Shimada
et al., 2014) was synthesized as a 612bp Notl flanked fragment and subcloned
into pJP3502, resulting in p0IL042.
Each of these constructs was introduced into N. benthamiana leaf cells as
described in Example 1. Transient expression of both pJP3502 and p0IL040 in N.
benthamiana leaf tissue resulted in elevated TAG levels and similar changes in
the
TAG fatty acid profile but p0IL040 increased the TAG level more (1.3% compared
to
0.9%). Each of the constructs p0IL037, p0IL038, p0IL041, p0IL042 and p0IL043
were used to stably transform N. tabacum plants (cultivar W38) by
Agrobacterium-
mediated methods. Transgenic plants were selected on the basis of kanamycin
resistance and are grown to maturity in the glasshouse. Samples are taken from
transgenic leaf tissue at different stages during plant development and
contain
increased TAG levels compared to wild-type N. tabacum and N. tabacum plants
transformed with pJP3502.
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Cloning and characterisation of LDAP polypeptides from Sapium sebifera
Oleosins are not highly expressed in non-seed oil accumulating plant tissues
such as the mesocarp of olive, oil palm, and avocado (Murphy, 2012). Instead,
lipid
droplet associated proteins (LDAP) have been identified in these tissues that
may play
a similar role to that of oleosin in seed tissues (Horn et al., 2013). The
inventors
therefore considered it possible that oleosin might not be the optimal
packaging
protein to protect the accumulated oil from TAG lipase or other cytosolic
enzyme
activities in vegetative tissues of plants. LDAP polypeptides were therefore
identified
and evaluated for enhancement of TAG accumulation, as follows.
The fruit of Chinese tallow tree, Sapium sebifera, a member of the family
Euphorbiaceae, was of particular interest to the inventors as it contains an
oil-rich
tissue outside of the seed. A recent study (Divi et al, submitted for
publication)
indicated that this olcoginous tissue, called a tallow layer, might be derived
from the
mesocarp of its fruit. Therefore, the inventors queried the transcriptome of
S. sebifera
for LDAP sequences. A comparative analysis of expressed genes in the fruit
coat and
seed tissues revealed a group of three previously unidentified LDAP genes
which
were highly expressed in the tallow layer.
Nucleotide sequences encoding the three LDAPs were obtained by RT-PCR
using RNAs derived from tallow tissue using three pairs of primers. The primer
sequences were based on the DNA sequences flanking the entire coding region of
each of the three genes. The primer sequences were: for LDAP1, 5'-
TTTTAACGATATCCGCTAAAGG-3' (SEQ ID NO:76) and 5' -
AATGAATGAACAAGAATTAAGTC-3 ' (SEQ ID NO:77) AT-3'; LDAP2, 5'-
CTTTTCTCACACCGTATCTCCG-3' (SEQ ID NO:78) and 5'-AGCATGATATA
CTTGTCGAGAAAGC-3' (SEQ ID NO:79); LDAP3, 5' -
GC GACAGTGTAGCGTTTT-3 ' (SEQ ID NO:80) and 5' -
ATACATAAAATGAAAACTATTGTGC-3' (SEQ ID NO: 81).
Analysis of the S. sebifera transcriptome revealed multiple orthologs for each
of the LDAP genes, including eight LDAP1, six LDAP2, and six LDAP3 genes, with
less than 10% sequence divergence within each gene family. The putative
peptide
sequences were aligned and a phylogenetic tree was constructed using Genious
software (Figure 4), together with LDAPs homologs from other plant species,
including two from avocado (Pam), one from oil palm, one from Partheniutn
argentatum (Par), two from Arabidopsis(Ath), five from Taraxacum
brevicorniculatum (Tbr), three from Hevea brasiliensis (Hbr), as presented in
Figure
4. The phylogenetic tree was revealed that the SsLDAP3 shared greater amino
acid
sequence identity to the LDAP1 and LDAP2 polypeptides from avocado and the
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LDAP from oil palm, while the SsLDAP1 and SsLDAP2 polypeptides were more
divergent.
Genetic constructs for over-expression of LDAP
In order to test the function of the LDAPs from S. sebiftra, expression
vectors
were made to express each of these polypeptides under the control of the 35S
promoter in leaf cells. The full length SsLDAP cDNA sequences were inserted
into
the pDONR207 destination vector by recombination reactions, replacing the CcdB
and Cm(R) regions of the destination vector with the SsLDAP cDNA fragments.
Following confirmation by restriction digestion analysis and DNA sequencing,
the
constructs were introduced into Agrobacterium tumefaciens strain AGL1 and used
for
both transient expression in N. benthamiana leaf cells and stable
transformation of N.
tabacurn.
The expression of each of the three SsLDAP genes under the transcriptional
control of the 35S promoter in N. benthamiana leaves in combination with the
expression of 35S::AtDGAT1 and 35S::AtWRI1 yielded substantially higher levels
of
TAG accumulation relative to the cells infiltrated with the 35S::AtDGAT1 and
35S::AtWRI1 genes without the LDAP construct. The TAG level was increased
about
2-fold above the TAG level in the control cells. A significant increase in the
level of
a-linolenic acid (ALA) and a reduced level of saturated fatty acids was
observed in
the cells receiving the combination of genes, relative to the control cells.
Co-localisation of YFP-fused LDAP polypeptides with lipid droplets in leaf
cells
In order to characterise SsLDAPs in vivo and observe their dynamic
behaviour, expression constructs were made for expression of fusion
polypeptides
consisting of the LDAP polypeptides fused to yellow fluorescent protein (YFP).
For
each fusion polypeptide, the YFP was fused in-frame to the C-terminus of the
SsLDAP. The full open reading frame of each of the three LDAP genes without a
stop
codon, at its 3' end, was fused to the YFP sequence and the chimeric genes
inserted
into pDONR207. Following confirmation of the resultant constructs by
restriction
digestion and DNA sequencing, the constructs were introduced into A.
tumefaciens
strain AGL1 and used for both transient expression in N. benthamiana leaf
cells and
stable transformation of N. tabacum. Three days following infiltration of the
leaf cells
with the LDAP-YFP constructs, leaf discs from the infiltrated zones were
stained with
Nile Red, which positively stained lipid droplets, and observed under a
confocal
microscope to detect both the red stain (lipid droplets) and fluorescence from
the YFP
polypeptide. Co-localisation of LDAP-YFP with the lipid droplets was observed,
indicating that the LDAP associated with the lipid droplets in the leaf cells.
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Example 7. Modifying traits in monocotyledonous plants - Expression in leaves
and stems
A series of binary expression vectors was designed for Agrobacterium-
mediated transformation of sorghum (S. bicolor) and wheat (Triticum aestivum)
to
increase the oil content in vegetative tissues. The starting vectors for the
constructions
were pOIL093-095, pOIL134 and pOIL100-104 (see Example 5 of WO
2016/004473). Firstly, a DNA fragment encoding the Z. mays WRI1 polypeptide
was
amplified by PCR using pOIL104 as a template and primers containing Kpnl
restriction sites. This fragment was subcloned downstream of the constitutive
Oryza
saliva Actinl promoter of pOIL095, using the KpnI site. The resulting vector
was
designated p011,154. The DNA fragment encoding the Umbelopsis ramanniana
DGAT2a under the control of the Z mays ubiquitin promoter (pZmUbi) was
isolated
from pOIL134 as a NotI fragment and inserted into the Non site of pOIL154,
resulting
in pOIL155. An expression cassette consisting of the PAT coding region under
the
control of the pZmUbi promoter and flanked at the 3' end by the A. tumefaciens
NOS
terminator/polyadenylation region was constructed by amplifying the PAT coding
region using pJP3416 as a template. Primers were designed to incorporate Band-
A and
Sad restriction sites at the 5' and 3' ends, respectively. After BamHI + Sad
double
digestion, the PAT fragment was cloned into the respective sites of
pZLUbilcasNK.
The resulting intermediate was designated pOIL141. Next, the PAT selectable
marker
cassette was introduced into the pOIL155 backbone. To this end, pOIL141 was
first
cut with Nod, blunted with Klenow fragment of DNA polymerase I and
subsequently
digested with AscI. This 2622bp fragment was then subcloned into the ZraI ¨
AscI
sites of pOIL155, resulting in pOIL156. Finally, the Actinl promoter driving
WRI1
expression in pOIL156 was exchanged for the Z. mays Rubisco small subunit
promoter (pZmSSU) resulting in pOIL157. This vector was obtained by PCR
amplification of the Z. mays SSU promoter using pOIL104 as a template and
flanking
primers containing AsiSI and PmlI restriction sites. The resulting amplicon
was then
cut with ,S'pel + Mild and subcloned into the respective sites of pOIL156.
These vectors therefore contained the following expression cassettes:
pOIL156: promoter 0. sativa Actin1::Z. mays WRI1, promoter Z. mays
Ubiquitin:: U. rammaniana DGAT2a and promoter Z. mays Ubiquitin::PAT
pOIL157: promoter Z. mays SSU::Z. mays WR11, promoter Z. mays
Ubiquitin:: U. rammaniana DGAT2a and Z. mays Ubiquitin::PAT.
A second series of binary expression vectors containing the Z. mays SEE1
senescence promoter (Robson et al., 2004, see Example 5 of WO 2016/004473), Z.
mays LEC1 transcription factor (Shen et al., 2010) and a S. bicolor SDP1
hpRNAi
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fragment were constructed as follows. First, a matrix attachment region (MAR)
was
introduced into pORE04 by AatII+SnaBI digest of pDCOT and subcloning into the
AatII+EcoRV sites of pORE04. The resulting intermediate vector was designated
pOIL158. Next, the PAT selectable marker gene under the control of the Z. mays
Ubiquitin promoter was subcloned into pOIL158. To this end, pOIL141 was first
digested with Noll, treated with Klenow fragment of DNA polymerase 1 and
finally
digested with AscI. The resulting fragment was inserted into the AscI+Zra1
sites of
pOIL158, resulting in pOIL159. The original RK2 oriV origin of replication in
pOIL159 was exchanged for the RiA4 origin by Swal+SpeI restriction digestion
of
pJP3416, followed by subcloning into the Swa1+AvrII sites of pOIL159. The
resulting
vector was designated pOIL160. A 10.019kb `Monocot senescence partl' fragment
containing the following expression cassettes was synthesized: 0. sativa
Actin1::A.
thaliana DGAT1, codon optimized for Z. mays expression, Z. mays SEE1::Z. mays
WRIL Z. mays SEE!: :Z. mays LEC1. This fragment was subcloned as a SpeI-EcoRV
fragment into the SpeI-Stul sites of pOIL160, resulting in pOIL161. A second
7.967kb
`Monocot senescence part2' fragment was synthesized and contains the following
elements: MAR, Z. mays Ubiquitin::hpRNAi fragment targeted against S.
bicolorIT.
aestivum SDP], empty cassette under the control of the 0. saliva Actinl
promoter.
The sequences of two S. bicolor SDP1 TAG lipases (Accession Nos.
XM 002463620; SEQ ID NO:73 and XM 002458486; SEQ ID NO:38) and one T
aestivum SDP1 sequence (Accession No. AK334547) (SEQ ID NO:74) were obtained
by a BLAST search with the A. thaliana SDP1 sequence (Accession No.
NM 120486). A synthetic hairpin construct (SEQ ID NO:75) was designed
including
four fragments (67bp, 90bp, 50bp, 59bp) of the S. bicolor XM_002458486
sequence
that showed highest degree of identity with the T. aestivum SDP1 sequence. In
addition, a 278bp fragment originating from the S. bicolor XM_002463620 SDP1
lipase was included to increase silencing efficiency against both S. bicolor
SDP1
sequences. The `Monocot senescence part2' fragment is subcloned as a BsiWf-
EcoRV
fragment into the BsiWI-FspI sites of pOIL161. The resulting vector is
designated
pOIL162.
The genetic constructs pOIL156 pOIL157, pOIL161 and pOIL162 are used to
transform S. bicolor and T. aestivum using Agrobacterium-mediated
transformation.
Transgenic plants are selected for hygromycin resistance and contain elevated
levels
of TAG and TFA in vegetative tissues compared to untransformed control plants.
Such plants are useful for providing feed for animals as hay or silage, as
well as
producing grain, or may be used to extract oil.
Further genetic constructs are made for expression of combinations of
polypeptides in leaves and stems of monocotyledonous plants, including the C4-
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a
photosynthesis plants S. bicolor and Z. mays. Several constructs are made
containing
genes for expression of WM, DGAT and oleosin, with each gene under the control
of a constitutive promoter such as a maize Ubiquitin gene promoter or a rice
actin
gene promoter, and containing an NPTII gene as selectable marker gene. In one
particular construct, the WRI1 is sorghum WRII. In another, the oleosin is
SiOleosinL (see Example 9). In other particular constructs, the oleosin gene
is
replaced with a gene encoding either LDAP2 or LDAP3 from S. sebifera (Example
6).
These constructs are used as the "core constructs" for transformation of S.
bicolor and
Z. mays and are deployed on their own or in combination with genetic
constructs for
expression of a hairpin RNA targeting one or more SDP1 genes in sorghum or
maize
(see above), a construct encoding Lec2 under the control of a SEEI promoter
(senescence specific), or both. Another construct is made comprising three
genes,
namely for expression of a hairpin RNA targeting the endogenous TGD5 gene to
reduce its expression, a FatA fatty acyl thioesterase and a PDAT, which is
used to
increase the level of TAG and/or the TTQ parameter for plants transformed with
this
construct.
Example 8. Extraction of oil
Extraction of hpidfrom leaves
Transgenic tobacco leaves which had been transformed with the T-DNA from
pJP3502 were harvested from plants grown in a glasshouse during the summer
months. The leaves were dried and then ground to 1-3mm sized pieces prior to
extraction. The ground material was subject to soxhlet (refluxing) extraction
over 24
hours with selected solvents, as described below. 5 g of dried tobacco leaf
material
and 250m1 of solvent was used in each extraction experiment.
Hexane solvent extraction
Hexane is commonly used as a solvent commercially for oil extraction from
pressed oil seeds such as canola, extracting neutral (non-polar) lipids, and
was
therefore tried first. The extracted lipid mass was 1.47g from 5 g of leaf
material, a
lipid recovery of 29% by weight. IH NMR analysis of the hexane extracted lipid
in
DMSO was preformed. The analysis showed typical signals for long chain
triglyceride fatty acids, with no aromatic products being present. The lipid
was then
subjected to GCMS for identification of major components. Direct GCMS analysis
of
the hexane extracted lipid proved to be difficult as the boiling point was too
high and
the material decomposed in the GCMS. In such situations, a common analysis
technique is to first make methyl esters of the fatty acids, which was done as
follows:
18mg lipid extract was dissolved in 1 mL toluene, 3mL of dry 3N methanolic HCL
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was added and stirred overnight at 60 C. 5mL of 5% NaC1 and 5mL of hexane
were
added to the cooled vial and shaken. The organic layer was removed and the
extraction was repeated with another 5mL of hexane. The combined organic
fractions
were neutralized with 8mL of 2% KHCO3, separated and dried with Na2SO4. The
solvent was evaporated under a stream of N2 and then made up to a
concentration of
lmg/mL in hexane for GCMS analysis. The main fatty acids present were 16:0
(palmitic, 38.9%) and 18:1 (oleic, 31.3%) (Table 10).
Table 10. Fatty acid composition in transgenic tobacco leaves
__________
FA 16:0 16:1 18:0 18:1 18:2 20:0 22:0
% wt 38.9 4.6 6.4 31.3 2.5 1.5 0.6
Acetone solvent extraction
Acetone was used as an extraction solvent because its solvent properties
should
extract almost all lipid from the leaves, i.e. both non-polar and polar
lipids. The
acetone extracted oil looked similar to the hexane extracted lipid. The
extracted lipid
mass was 1.59g from 5 g of tobacco leaf, i.e. 31.8% by weight. 1H NMR analysis
of
the lipid in DMSO was performed. Signals typical of long chain triglyceride
fatty
acids were observed, with no signal for aromatic products.
Hot water solvent extraction
Hot water was attempted as an extraction solvent to see if it was suitable to
obtain oil from the tobacco leaves. The water extracted material was gel like
in
appearance and gelled when cooled. The extracted mass was 1.9 g, or 38% by
weight.
This material was like a thick gel and was likely to have included polar
compounds
from the leaves such as sugars and other carbohydrates. The 1H NMR analysis of
the
material in DMSO was preformed. The analysis showed typical signals for long
chain
triglyceride fatty acids, with no aromatic products being extracted. The left
over solid
material was extracted with hexane, yielding 20% of lipid by weight,
indicating that
the water extraction had not efficiently extracted non-polar lipids.
Ethanol solvent extraction
Ethanol was used as an extraction solvent to see if it was suitable to obtain
oil
from the tobacco leaves. The ethanol extracted lipid was similar in appearance
to
both the water- and hexane-extracted lipid, being yellow-red in colour, had a
gel-like
.. appearance and gelled when cooled. The extracted lipid mass was 1.88g from
5 g
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tobacco, or 37.6% by weight. The ethanol solvent would also have extracted
some of
the polar compounds in the tobacco leaves.
Ether solvent extraction
Diethyl ether was attempted as an extraction solvent since it was thought that
it
might extract less impurities than other solvents. The extraction yielded 1.4
g, or 28%
by weight. The ether extracted lipid was similar to the hexane extracted
material in
appearance, was yellowish in colour, and it did appeared a little cleaner than
the
hexane extract. While the diethyl ether extraction appeared to have given the
cleanest
oil, the NMR analysis showed a mixture of more organic compounds.
Example 9. Expression of oil body proteins in plant vegetative tissues
A protein coding region encoding a Rhodococcus opacus TadA lipid droplet
associated protein (MacEachran et al. 2010; Accession number HM625859), codon
optimized for expression in dicotyledonous plants such as Nicotiana
benthamiana,
was synthesized as a NotI-SpeI DNA fragment. The fragment was inserted
downstream of the 355 promoter in pJP3343 using the NotI-SpeI sites. The
resultant
plasmid was designated pOIL380. A protein coding region encoding a Sesame
indicum OleosinL lipid droplet associated protein (Tai et al. 2002; Accession
number
AF091840; SEQ ID NO:86) was synthesized as a Nod-Sad DNA fragment and
inserted downstream of the 35S promoter in pJP3343 using the same sites. The
resultant plasmid was designated pOIL382. A protein coding region encoding a
Sesame indicum OleosinH1 lipid droplet associated protein (Tai et al., 2002;
Accession number AF302807) was synthesized as a NotI-SacI DNA fragment and
cloned downstream of the 35S promoter in pJP3343 using the same sites. The
resultant plasmid was designated pOIL383. A variant of the protein coding
region
encoding S. indicum OleosinH1 having three amino acid substitutions to remove
ubiquitination sites (K130R, K143R, K145R) (Hsiao and Tzen, 2011) was
generated
by targeted mutagenesis. The coding region was inserted downstream of the 35S
promoter in pJP3343 as a NotI-SacI fragment. The resultant plasmid was
designated
pOIL384. A protein coding region encoding a Vanilla planifblia leaf OleosinUl
lipid
droplet associated protein (Huang and Huang, 2016; Accession number SRX648194)
was codon optimized for expression in N benthamiana, synthesized as a SpeI-
EcoRI
DNA fragment and inserted downstream of the 35S promoter in pJP3343 using the
same sites. The resultant plasmid was designated pOIL386. A protein coding
region
encoding a Persea americana mesocarp OleosinM lipid droplet associated protein
(Huang and Huang 2016; Accession number SRX627420) was codon optimized for
expression in N. benthamiana, synthesized as a SpeI-EcoRI DNA fragment and
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inserted downstream of the 35S promoter in pJP3343 using the same restriction
sites.
The resultant plasmid was designated pOIL387. A protein coding region encoding
an
Arachis hypogaea Oleosin 3 lipid droplet associated protein (Parthibane et
al., 2012a;
Accession number AY722696) was codon optimized for expression in N.
benthamiana, flanked by NotI sites and inserted into the binary expression
vector
pJP3502. The resulting plasmid, pOIL041, was digested with NotI and the
resultant
520 bp DNA fragment was inserted downstream of the 35S promoter of pJP3343.
The
resultant plasmid was designated pOIL190. Similarly, the protein coding region
for
the A. thaliana Caleosin3 lipid droplet associated protein (Shen et al., 2014;
Laibach
et al., 2015; Accession number AK317039) was codon optimized for expression in
N.
benthamiana, flanked by NotI sites and inserted into pJP3502. The resulting
plasmid,
pOIL042, was digested with NotI and the resulting 604 bp DNA fragment was
inserted downstream of the 35S promoter of pJP3343. The resultant plasmid was
designated pOIL191. A protein coding region encoding an A. thaliana
steroleosin
lipid droplet associated protein (Accession number AT081653) was codon
optimized
for expression in N. benthamiana, flanked by NotI sites and inserted into
pJP3502.
The resultant plasmid, p011,043, was digested with Notl and the resultant 1069
bp
DNA fragment was inserted downstream of the 35S promoter of pJP3343. The
resultant plasmid was designated pOIL192. A protein coding region encoding a
Nannochloropsis oceanica LSDP oil body protein (Vieler et al., 2012; Accession
number JQ268559) was codon optimized for expression in N. benthamiana, flanked
by NotI sites and inserted into the pJP3502 binary expression vector. The
resultant
plasmid, pOIL044, was digested with NotI and the 496 bp DNA fragment was
inserted downstream of the 35S promoter of pJP3343. The resultant plasmid was
designated pOIL193. A protein coding region encoding a Trichoderma reesei
IIFBI
hydrophobin (Linder et al., 2005; Accession number Z68124) was codon optimized
for expression in N. benthamiana, flanked by NotI sites and inserted into
pJP3502.
The resultant plasmid, pOIL045, was digested with Not1 and the 313 bp DNA
fragment was inserted downstream of the 35S promoter of pJP3343. The resultant
plasmid was designated pOIL194. An ER-targeted variant of the Trichoderma
reesei
HFBI hydrophobin was created by amending the KDEL ER retention peptide to the
C-terminus (Gutierrew et al., 2013). This variant was codon optimized for
expression
in N. benthamiana and cloned as a NotI fragment into pJP3502, resulting in
pOIL046.
Subsequently, pOIL046 was digested with NotI and the 325 bp fragment was
inserted
into pJP3343. The resulting vector was designated pOIL195.
Each of the genetic constructs encoding the lipid droplet associated
polypeptides were introduced into N benthamiana leaves in combination with
genetic
constructs encoding WRI1, DGAT1 and p19 as described in Example 1 with some
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minor modifications. Agrobacterium tumefaciens cultures containing the gene
coding
for the p19 silencing suppressor protein and the chimeric genes of interest
were mixed
such that the final 0D600 of each culture was equal to 0.125 prior to
infiltration.
Samples being compared were located on the same leaf. After infiltration, N.
benthamiana plants were grown for a further five days before leaf discs were
harvested, pooled across three leaves from the same plant, freeze-dried,
weighed and
stored at ¨80 C. Total lipids were extracted from freeze-dried tissues using
chloroform:methano1:0.1 M KC1 (2:1:1 v/v/v) and aliquots loaded on a thin
layer
chromatography (TLC) plate and developed in hexane:diethyl ether:acetic acid
(70:30:1, v/v/v). TAG was recovered, converted to FAME in the presence of a
known
amount of triheptadecanoin (Nu-Chek PREP, Inc. USA) as internal standard for
lipid
quantitation, and analyzed by GC-FID.
The assays showed a range of TAG levels compared to the WR11 + DGAT1
control. Some constructs encoding lipid droplet associated polypeptides
increased the
TAG level relative to the control in some assays whereas others did not. A
consistent
and statistically significant increase in TAG content was observed when the
construct
expressing SiOleosinL (pOIL382) was introduced (Figure 5); this construct was
superior to all the others tested in these assays. In one experiment, the
increase was
2.27-fold compared to p19+WRI+DGAT and 121.7-fold compared to the p19 control.
An increase in the levels of C18:2 and C18:1 (about 22% increased) and a
decrease in
C16:0 (about 23% decreased) was also observed in the TAG for this construct,
relative to the p19+WRI1+DGAT1 control (Figure 5). Microscopic analyses to
visualise lipid droplets in the leaf cells expressing SiOleosinL showed a
decrease in
lipid droplet size and an increase in abundance compared to the control.
The lipid droplets in leaf cells transiently expressing the genes encoding
SiOleosinL together with p19 + WRII + DGAT1 were examined by microscopy. N.
benthamiana treated leaf discs were collected 4 days after infiltration. Each
leaf
sample was prepared, stained and imaged within 30-45 minutes, to ensure the
samples
were imaged fresh. More specifically, immediately after collection, the
abaxial
epidermis was peeled off in 50 mM PIPES pH7. One half of each disc was stained
for
10 minutes in 2 g/ml BODIPY505/515 in 50 mM PIPES pH7, followed by 2-3
washes in 50 mM PIPES pH7. During this time, the other disc half was kept in
50
mM PIPES pH7. Leaf tissue was mounted in 50 mM PIPES pII7 and imaged
immediately, using a Leica SP8 Laser-Scanning Confocal Microscope, a 20x
objective (NA = 0.75), and the LAS X software. Lipid droplets and chloroplasts
were
imaged by exciting the leaf discs with a 505 rim laser. BODIPY 505/515 signal
was
collected between 510 and 540 nm, while chloroplast signal was collected
between
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650 nm and 690 nm. Unstained half discs were imaged to determine tissue auto-
fluorescence.
Microscopy of cells in the leaf discs having the introduced SiOleosinL showed
an accumulation of smaller lipid droplets compared to the discs having the p19
+
WRI1 + DGAT1 without SiOleosinL. In contrast, leaf cells expressing genes
encoding the p19 + WRI1 + DGAT1 + SiOleosinH combination showed larger lipid
droplets which looked about the same as those observed in leaves expressing
p19 +
WRI1 + DGAT1 without an oleosin. Finally, when genes encoding both SiOleosinH
and SiOlcosinL were co-expressed with p19 + WRI1 + DGAT1, the lipid droplets
were smaller and looked similar to those observed in leaves expressing p19 +
WRI1 +
DGAT1 + SiOleosinL. Interestingly, expression of the vanilla leaf oleosin
(pOIL386)
resulted in a different pattern in which lipid droplets appeared compacted in
a smear
form.
Further assays were carried out using radiolabelled [Ng-acetate to measure
the rate of TAG synthesis for the different gene combinations including each
of the
lipid droplet associated polypeptides. The [Ng-acetate was infiltrated into
the same
leaf tissues at 3 days post-infiltration of the genetic constructs i.e. after
the genes had
been expressed for three days. Leaf discs were sampled after 5 min, 10 min and
3 hr
after addition of the radiolabel, and total lipids in the tissues were
extracted and
fractionated by TLC. The amount of radioactivity in different lipid types was
quantitated using a Fujifilm FLA-5000 phosphorirnager or using a Beckman-
Coulter
LS 6500 Multipurpose Scintillation Counter.
These assays demonstrated an increase in TAG synthesis rates in the leaves
expressing SiOleosinL (pOIL382) as well as an increase in PC and PA synthesis
rates
over the three hours in leaves expressing SiOleosinL. SiOleosinL expression
increased TAG accumulation already at 15 minutes (789 dpm) compared to p19
(198
dpm). In N. benthamiana leaf cells expressing genes encoding the
p19+WRI1+DGAT1 combination, TAG accumulated rapidly, reaching 3865 dpm
after 5 min of [Ng-acetate incorporation compared to 293 dpm in the p19
control.
This accumulation reached a maximum at 10 minutes after [Ng-acetate addition
(4519 dpm). However, the radiolabel in TAG quickly reduced thereafter to reach
1013
dpm at 15 minutes, indicating TAG catabolism. When the gene encoding
SiOleosinL
was added, the TAG was stabilised, indicating protection (i.e. TAG packaging)
in the
leaf cells. TAG rapidly accumulated at 5 minutes of infiltration (2855 dpm)
and the
level remained the same at 10 and 15 mm after [Ng-acetate addition. At the 15
min
timepoint, TAG accumulation was equivalent to 2690 dpm for the
p19+WRI1+DGAT1+SiOleosinL combination compared to 1013 dpm for the
p19+WRI1+DGAT1 combination.
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TAG degradation was not correlated with free fatty acid (FFA) levels,
presumably because of further catabolism or of incorporation into lipids other
that
TAG. In order to study TAG degradation and chase the resulting derivatives,
[14Q-
acetate incorporation into TAG and and its stability at 3 hr post-addition was
studied.
.. This experiment showed an increase in [mg in PC (2579 dpm) and PA (1270
dpm) in
leaf cells expressing the SiOleosinL construct compared to 1495 dpm PC and 899
PA
in both p19 and p19+WRI1+DGAT1 controls.
In another experiment, pOIL191 (AtCaleosin 3) was transiently expressed in
N. benthamiana leaves. The expression of this gene increased TAG content by
3.6
.. fold (Figure 6) compared to p19 control. The expression of AtCaleosin3 with
WRI1
and DGAT1 resulted in a further increase in TAG content by up to 15.3 fold
compared p19 control, and up to 1.6 fold compared to WRI1 and DGAT1 control.
TAG yields are comparable with SiOleosin co-expression with WRI1 and DGAT1.
Example 10. Medium-chain fatty acid production in vegetative plant cells
Eccleston et al. (1996) studied the accumulation of C12:0 and C14:0 fatty
acids in both seeds and leaves of transgenic Brassica napus plants transformed
with a
constitutively expressed gene encoding California Bay Laurel 12:0-ACP
thioesterase
(Umbellularia californica). That study reported that substantial levels of
C12:0
accumulated in mature B. napus seeds but only very low levels of C12:0 were
observed in leaf tissue, despite high levels of 12:0-ACP thioesterase
expression and
activity. The same results were obtained when the gene was transformed into A.
thaliana (Voelker et al., 1992). That research was extended by the co-
expression of
the Cocos nucifera LPAAT and Umbellularia californica thioesterase which
resulted
.. in an increased accumulation of total C12:0 as well as an increased
fraction of
trilaurin in the seeds of B. napus (Knutzon et al., 1999). The prior art
therefore
indicated that medium chain fatty acids (MCFA) synthesis in vegetative plant
cells
was problematic.
To test the effect of introducing thioesterases having specificity for MCFA in
.. combination with other genes described herein, chimeric DNAs for expressing
several
different thioesterases were synthesized and introduced into plant cells
either singly or
in combinations. The protein coding regions for thioesterases from organisms
known
to produce MCFAs (Jing et al., 2011) were synthesised and inserted as EcoRI
fragments into the binary vector pJP3343 which contained a 35S-promoter
expression
cassette (Vanhercke et al., 2013). The thioesterases were: Cinnamomum camphora
14:0-ACP thioesterase (referred to as Cinca-TE) (Yuan et al., 1995; Accession
No.
Q39473.1; SEQ ID NO:43), Cocos nucifera acyl-ACP thioesterase FatB1 (Cocnu-
TE1; Accession No. AEM72519.1; SEQ ID NO:88), Cocos nucifera acyl-ACP
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thioesterase FatB2 (Cocnu-TE2; Accession No. AEM72520.1; SEQ ID NO: 89),
Cocos nucifera acyl-ACP thioesterase FatB3 (Cocnu-TE3; Accession No.
AEM72521.1; SEQ ID NO: 90), Cuphea lanceolata acyl-(ACP) thioesterase type B
(Cupla-TE) (Topfer et al., 1995; Accession No. CAB60830.1; SEQ ID NO: 91),
Cuphea viscosissima FatB1 (Cupvi-TE; Accession No. AEM72522.1; SEQ ID NO:
92) and Umbellularia californica 12:0-ACP thioesterase (Umbca-TE) (Voelker et
al.,
1992; Accession No. Q41635.1; SEQ ID NO: 93). These thioesterases were all in
the
FATB class and had specificity for MCFA. The protein coding regions for C.
nucifera
LPAAT (Cocnu-LPAAT, MCFA type) (Knutzon et al., 1995; Accession No.
Q42670.1; SEQ ID NO:94) and A. thaliana plastidial LPAAT1 (Arath-PLPAAT:
Accession No. AEE85783.1; SEQ ID NO:95), were also cloned. Cocnu-LPAAT had
previously been shown to increase MCFA incorporation on the sn-2 position of
TAG
in seeds (Knutzon et al., 1995) whilst A. thaliana plastidial LPAAT (Arath-
PLPAAT)
(Kim etal., 2014) was used as a control LPAAT to determine the effect of any
MCFA
specificity that the Cocnu-LPAAT might have. The former LPAAT uses acyl-CoA as
one substrate and operates in the ER in its native context, whereas the latter
PLPAAT
uses acyl-ACP as substrate and works in the plastid.
The thioesterase genes were introduced into Nicotiana benthamiana leaves by
Agrobacteriurn-mediated infiltration as described in Example 1 along with the
gene
for co-expression of the p19 silencing suppressor and either the Cocnu-LPAAT
or
Arath-PLPAAT to determine whether MCFA could be produced in N. benthamiana
leaf tissue. Infiltrated leaf zones were harvested and freeze-dried five days
after
infiltration with the Agrobacterium mixtures, after which the total fatty acid
content
and composition were determined by GC as described in Example 1 (Table 11).
For
the data shown in Table 11, errors are the standard deviation of triplicate
infiltrations.
The infiltrated zones of control leaves contained only trace (<0.1%) or zero
levels of
fatty acids C12:0 and C14:0 whereas C16:0 was present at 14.9% 0.6 of the II A
in
the total leaf lipids. C12:0 levels were only increased significantly by
expression of
the Cocnu-TE3 (1.2% 0.1) and Umbca-TE (1.6% 0.1). Expression of each of the
tested thioesterases resulted in the accumulation of C14:0 in the N.
benthamiana
leaves, with Cinca-TE giving the highest level of 11.3% 1Ø Similarly,
expression of
each of the thioesterases with the exception of Umbca-TE resulted in increased
C16:0
levels. The highest level of C16:0 accumulation (35.4% 4.7) was observed with
expression of Cocnu-TEL Substantial necrosis of the infiltrated zones was
observed
in the leaves when the FATB genes were expressed alone, which appeared to
correlate
with the level of MCFA production. The inventors considered that the necrosis
was
probably due to levels of free fatty acids (FFA) greater than optimum, and
also due to
the extensive accumulation of MCFA in phospholipid lipid pools rather than in
TAG.
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Table 11. Total leaf fatty acid composition (% total leaf fatty acid) of
selected fatty
acids in Nicotiana benthamiana leaves infiltrated with various thioesterases
(TE) and
LPAATs. Results are grouped by the co-infiltrated gene (single genes (other
than p19
present in all samples), Arath-LPAAT + various TE, Cocnu-LPAAT + various TE).
'Control' denotes uninfiltrated N. benthamiana leaf whereas 1119 only'
contains the
silencing suppressor gene alone. 16:3 is 16:3 7'1 13; 18:3 is 18:3 912'15.
Gene identities
are defined in the text.
12:0 14:0 16:0 16:3 18:3
Control 0.2 0 0.1 0 14.0 0.2 8.1 0.1 57.2 0
p19 only 0.2 0 0.1-10 14.9 0.6 7.0 0.8 53.1 0.7
Cinca-TE 0.4 0 11.3 1.0 21.9 0.7 5.0 0.2 38.5 1.0
Cocnu-TE1 0.2+0 6.3 0.6 35.4 4.7 4.2 1.4 29.915.5
Cocnu-TE2 0.2+0 7.1 0.3 31.9 2.2 4.7 0.5 32.9 2.8
Cocnu-TE3 1.2 0.1 7.2 1.3 19.6 1.6 5.7 0.5 44.8 2.9
z Cupla-TE 0.2 0 1.1 0.2 21.8 2.9 6.0 0.6 48.2 3.1
Cupvi-TE 0.2 0 0.6 0.1 17.3 1.3 6.4 0.4 52.9 2.1
Umbca-TE 1.6 0.1 1.1 0.2 14.4 0.8 6.5 0.3 52.7 0.1
tip Arath- 0.2 0 0.4 0.5 17.4 1.0 6.2 0.3 51.4 1.3
.11) LPAAT
Cocnu- 0.1 0.1 0.1 0 15.1 1.5 6.7 0.5 52.2 4.2
'1D LPAAT
Cinca-TE 0.2+0 7.8 0.1 24.6 0.4 5.3 0.2 39.2 1.5
õeh Cocnu-TE1 0.2+0 4.6 1.3 35.3 1.4 4.410.7 32.712.0
Cocnu-TE2 0.2+0 6.1 0.4 32.5 1.8 4.7 0.1 34.1 0.6
Cocnu-TE3 0.9 0.2 8.5 0.4 21.4 1.9 5.6+0.2 41.7+0.6
Cupla-TE 0.2 0 1.0 0.1 23.4 2.7 5.9 0.5 47.3 1.2
Cupvi-TE 0.2 0 0.6 0 19.0 0.2 6.310.1 51.4+1.0
+ Umbca-TE 1.210.2 1.110.1 15.410.2 6.5 0.2 52.3 1.3
Cinca-TE 0.7 0.2 14.9 1.6 23.0 3.7 4.8 1.4 35.413.3
E-4
Cocnu-TE1 5.4 0.9 40.2 2.8 3.3 0 27.8 1.1
Cocnu-TE2 0.2+0 6.6 1.0 38.311.1 3.710.2 28.2 1.1
Cocnu-TE3 2.0 0.3 10.9 1.0 24.4 1.8 4.9 0.5 37.7+0.9
Cupla-TE 0.5 0.1 1.6 0.3 22.210.6 6.0 0.3 46.9+2.0
0 Cupvi-TE 0.5 0 1.1 0 19.6 0.8 6.0 0.2 49.8 0.3
+ Umbca-TE 3.3 0.5 1.2 0.1 13.9 0.4 6.4+0.2 51.3+1.7
Co-infiltration of the chimeric gene for expressing Arath-PLPAAT with the
thioesterases tended to reduce the accumulation of both C12:0 and C14:0
compared to
the absence of the LPAAT, whilst slightly increasing the accumulation of
C16:0. hl
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contrast, co-infiltration of the genes for expressing Cocnu-LPAAT or Umbca-TE
increased the accumulation of C12:0 to 3.3% 0.5 whilst C14:0 was found to
accumulate to 14.9% 1.6 in the Cinca-TE + Cocnu-LPAAT sample. The highest
C16:0 levels were observed after co-expression of Coenu-TE1 and Cocnu-LPAAT
(40.2% 2.8). Addition of an LPAAT to each inoculated zone decreased the degree
of
necrosis of the leaf tissue. Surprisingly, both C8:0 and C10:0 fatty acids
were also
produced in the plant cells in the transient expression studies. The
accumulation of
C8:0 and C10:0 was not observed when the thioesterase was expressed alone.
However, when thioesterase expression was combined with the co-expression of
CuphoFatB with CnLPAAT and AtWRI1, C8:0 was found to be present at a
concentration of 0.27+0.09% of the total fatty acid content in the plant
cells.
Similarly, when CuplaFatB was co-expressed with CnLPAAT and AtWRIL C10:0
was found to be present at 0.54+0.16% of the total fatty acid content.
These results indicated that the previously-reported acyl specificities of the
.. thioesterases, observed from seed expression, were essentially maintained
in N.
bentharniana leaves and that this expression system was a valid system for
testing
acyl specificity. The addition of the plastidial A. thaliana PLPAAT did not
increase
the accumulation of MCFAs although it did result in slightly increased
accumulation
of C16:0 in A. thaliana cells. In contrast, the C. nucifera LPAAT increased
the
accumulation of C12:0, C14:0 and C16:0 in N. benthannana leaves, which fatty
acids
are found in C. nucifera oil (Laureles et al., 2002). This indicated that the
native N.
bentharniana LPAAT was either not highly expressed in leaf tissue or did not
have
high activity on C12:0, C14:0 and C16:0 substrates.
Medium-chain fatty acid production in vegetative plant cells accumulating high
levels
of TAG
The inventors previously obtained the production of 15% TAG in N. tabacum
leaves by the coordinate expression of chimeric genes encoding A. thaliana
WRIL A.
thaliana DGAT1 and S. indicum Oleosin (Vanhercke et al., 2014a and b). To test
whether the accumulation of MCFA that was observed after expression of
thioesterases in combination with an LPAAT would also occur or be increased in
plant cells producing high levels of TAG (Vanhercke et al., 2013), these genes
were
co-expressed. The best performing C12:0, C14:0 and C16:0 thioesterase/LPAAT
combinations (Cocnu-LPAAT plus Umbca-TE, Cinca-TE and Cocnu-TE2
thioesterases, respectively) were infiltrated with and without the Arath-
WRI1+DGAT
combinations previously described (Vanhercke et al., 2013). The data are shown
in
Figure 7.
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The accumulation of the relevant MCFA (C12:0 for Umbca-TE, C14:0 for
Cinca-TE and C16:0 for Cocnu-TE2) was consistently and substantially increased
most by the addition of Arath-WRI1 to the combinations: C12:0 comprised 9.5%
0.9
of total leaf fatty acids in the 1Jmbca-TE+Cocnu-LPAAT+Arath-WRI1 samples, the
C14:0 level was 18.5% 2.6 in the Cinca-TE+Cocnu-LPAAT+Arath-WRT1 samples
and the C16:0 level was 38.3% 3.0 in the Cocnu-TE2+Cocnu-LPAAT+Arath-WRI1
samples. Thioesterase plus Arath-WRI1 infiltrations were found to have a
significantly greater effect on C12:0 in the presence of Umbca-TE, C14:0 in
the
presence of Cinca-TE and C16:0 in the presence of Cocnu-TE2 relative to
infiltration
with thioesterase plus Cocnu-LPAAT in the absence of WRI1 (Figure 8). The
addition
of the Cocnu-LPAAT to the thioesterase plus Arath-WRI1 mixtures did have an
effect
on the fatty acid composition with relatively small increases in C12:0 and
C14:0
observed in the Umbca-TE and Cinca-TE sets and a small decrease in C16:0 in
the
Cocnu-TE2 set. The maximum levels observed were: 8.8% 1.1 of C12:0 in total
leaf
fatty acids observed in the Umbca-TE + Arath-WRI1 + Cocnu-LPAAT samples.
14.1% 3.5 of C14:0 in the Cinca-TE + Arath-WRI1 + Cocnu-LPAAT samples and
48.6% 3.7 of C16:0 in the Cocnu-TE2 + Arath-WRI1 sample.
Interestingly, the only thioesterase in which the Arath-WRI1 did not increase
MCFA accumulation as much was the Cocnu-TE2, although it still increased
significantly. The addition of this gene alone resulted in the increased
accumulation of
C16:0 from 16.0% 0.4 to 37.3% 0.6 whereas the further addition of Arath-WRI1
only increased this to 48.6%11.7. This may have been due to the C12:0 and
C14:0
intermediates being relatively transient during plastidial fatty acid
synthesis compared
to C16:0.
Other effects that were noted included the increase in C16:0 and C18:1 9 and
decrease in C18:3 9'12'15 levels in the presence of Arath-WRI1. The further
addition of
the Cinca-TE and Cocnu-TE2 decreased C18:3 932'15 levels further still. In
contrast,
the extra C12:0 produced following the addition of Arath-WRI1 to Umbca-TE
appeared to come at the cost of C16:0 rather than additional C18:3 91215
(Figure 9).
A subset of samples were also analysed by LC-MS to gain a better
understanding of MCFA accumulation. The plastidial galactolipids
monogalactosyl
diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) contained only
low
levels of C12:0 and C14:0 and reduced levels of C16:0 relative to the p19
control
infiltration. The major C12:0-containing MGDG species in the Umbca-TE samples
was 30:3 indicating that one C18:3 and one C12:0 were co-located on the
monogalactosyl backbone. The other main C12:0-containing MGDG species was
28:0, indicating that the second fatty acid was C16:0. The major C14:0-
containing
MGDG species in the Cinca-TE samples were 28:0 and 30:0, indicating that a
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significant proportion of the C14:0 in MGDG was either di-C14:0 or with C16:0.
The
C12:0-containing and C14:0-containing MGDG species were not detected in the
p19
control sample. In contrast, C16:0-containing MGDG species tended to be
reduced in
the Cocnu-TE2 samples. The major MGDG species in the wildtype samples (C16:3-
containing 34:6, C18:3-containing 34:6, and C18:3-containing 36:6) all tended
to be
reduced by the expression of the transgenes. This reduction was greatest in
the
presence of the WRI+DGAT combination.
Only trace levels of C12:0-containing DGDG species were observed in the
Umbca-TE samples. The major C14:0-containing species observed in the Cinca-TE
samples were 28:0 and 30:0, both of which were absent in the control. These
species
were also observed at elevated levels in the Cocnu-TE2 samples but only at
trace
levels in the Umbca-TE samples. The major DGDG species in the wildtype samples
(C16:0-containing 34:3, C18:3-containing 34:3, and C18:3-containing 36:6) all
tended
to be reduced by the expression of the transgenes. This reduction was greatest
in the
presence of WRI.
Similarly. TAG species were generally increased considerably in all the
samples containing WRI + DGAT as previously described (Vanhercke et al.,
2013).
C12:0 species were found to be dominant in the high TAG Umbca-TE sample, C14:0
in the high TAG Cinca-TE sample and C16:0 in the high TAG Cocnu-TE2 sample.
LC-MS analysis of the TAG fraction showed that the C12:0-containing 36:0 was
found to be the dominant TAG species, twice the level of TAG species
containing
C18:3, in all Umbca-TE samples containing the WRI transcription factor.
Similarly,
C14:0-containing 42:0 was the dominant TAG species in the Cinca-TE samples co-
transformed with either LPAAT, DGAT, WRI or WRI+DGAT, although the response
was considerably higher in the case of the samples containing WRI. Several
C16:0-
containing TAG species were significantly elevated in both the high TAG Cinca-
TE
(e.g. 44:0 and 50:3) and Cocnu-TE2 (e.g. 46:0, 48:0, 50:2 and 50:3) samples.
Again,
the greatest C16:0 increases were observed in the presence of WRI.
Stable transformation for production qf MCFA in vegetative tissues.
A series of genetic constructs were made in a binary vector in order to stably
transform plants such as tobacco with combinations of genes for production of
MCFA
in vegetative tissues, to identify optimal combinations of genes. These
constructs
included a gene for expression of WRII under the control of either the SSU
promoter
(see Example 3, pOIL121) or the senescence-specific SAG12 promoter, a gene
encoding an oil palm DGAT (below), a gene encoding the coconut LPAAT
(CocnuLPAAT, see above) under the control of an enTCUP promoter and several
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genes expressing a variety of fatty acyl thioesterases (FATB) expressed from
either a
35S promoter or a SAG12 promoter. These are described below.
Cloning of a gene encoding Elaeis guineensis (oil palm) DGAT
In order to firstly test different DGAT enzymes, including representative
DGAT1, DGAT2 and DGAT3 enzymes, candidate oil palm DGAT sequences were
identified from the published transcriptome (Dussert et al., 2013) and codon
optimised
for expression in Nicotiana tabacum. The protein coding regions were then each
cloned individually into binary expression vectors under the control of the
35S
promoter for testing in transient N. benthamiana leaf assays as described in
Example
1. The gene combinations tested were as follows:
1 P19 (negative control)
2 P19+CnLPAAT+WRI1
3 P19+CnLPAAT+AtWRI1+AtDGAT1
4 P19+CnLPAAT+AtWRII+EgDGAT1
5 P19+CnLPAAT+AtWRIl+EgDGA T2
6 P19+CnLPAAT+AtWRIl+EgDGAT3
7 P19+CincaFatB
8 P19+CincaFatB+CnLPAAT+WR11
9 P19+CincaFatB+CnLPAAT+AtWRI1+AtDGAT1
10 P19+CincaFatB+CnLPAAT+AtWRIl+EgDGAT1
11 P19+CincaFatB+CnLPAAT+AtWRI1+EgDGAT2
12 P19+C incaFatB+CnLPAAT+AtWRIl+EgD GAT3
The results for the TFA and TAG levels, and the levels of total MCFA in the
TFA or the TAG contents, are shown in Figure 10. Compared to AtDGAT1, the
expression of EgDGAT1 led to greater accumulation of total fatty acids and
increased
TAG levels. The total MCFA content in the total fatty acid content was reduced
with
.. the expression of EgDGAT I relative to AtDGAT1, but the levels of MCFA
present in
TAG remained about the same (Figure 10).
Preparation of genetic constructs
Genetic constructs for stable transformation (Table 12) were assembled
through the sequential insertion of gene cassettes through the use of
compatible
restriction enzyme sites. The four gene constructs (Table 12) each contained a
gene
encoding the oil palm DGAT1 (EgDGAT1) expressed from the 35S promoter, a gene
encoding the C. nucifera LPAAT (CnLPAAT) expressed from the constitutive
enTCUP2 promoter, and a gene encoding AtWRI1 expressed from either the SSU
promoter or the SAG12 promoter in addition to one of a series of genes
encoding
FATB enzymes.
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The five gene constructs also contained a gene for expression of a hairpin RNA
for reducing expression of an endogenous gene encoding acyl-activating enzyme
(AAE). The hairpin was constructed based on sequence similarity with the
identified
AAE15 from Arabidopsis lyrata (EFH44575.1) and the N benthamiana genome.
AAE has been shown to be involved in the reactivation of MCFA, and hence
further
elongation. It was considered that silencing of AAE might increase MCFA
accumulation. The hairpin cassette was constructed in the vector pKANNIBAL and
then subcloned into the expression vector pWBVec2 with the expression of the
hairpin being driven by the 35S promoter.
Table 12. Summary of assembled genetic constructs.
Construct Gene Combination
pKR1 35S: :UmbcaFATB
Q.) pl(R2 35S: :CincaFATB
d)
C.7 E pl(R3 35S : :CocnuFATB2
731') pOIL115 SAG12::CincaFATB
Ef5 pOIL116 SAG12::UmbcaFATB
pOIL117 SAG12::CocnuFATB2
pOIL300 355::EgDGAT1
pOIL301 enTCUP::CnLPAAT inFATBrmediaFATB construct
g pOIL302 35S::EgDGAT1 + enTCUP::CnLPAAT
pOIL303 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWR11
pOIL304 35S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWRI1
pOIL305 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 +
35S : :UmbcaFATB
pOIL306 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 +
35 S::CincaFATB
pOIL307 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWR11 +
35S::CocnuFATB2
pOIL3 08 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWR11 +
SAG12::UmbcaFATB
pOIL309 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWR11 +
c.J
SAG12::CincaFATB
pOIL310 355::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWR11 +
SAG12::CocnuFATB2
pOIL3 I 1 355: :EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWRI1 +
35S: :UmbcaFATB
pOIL312 35S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWRI1 +
35S::CincaFATB
pOIL313 355::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWR11 +
35 S::CocnuFATB2
pOIL314 35S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWRI1 +
SAG12::UmbcaFATB
pOIL315 355::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWR11 +
SAG12::CincaFATB
pOIL316 35S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWR11 +
SAG12::CocnuFATB2
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pOIL317 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 +
35S::UmbcaFATB + 35S::hpNbAAE
pOIL318 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 +
35S::CincaFATB + 35S::hpNbAAE
pOIL319 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 +
35S::CocnuFATB2 + 35S::hpNbAAE
pOIL320 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 +
SAG12::UmbcaFA TB + 35S ::hpNbAAE
pOIL321 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 +
___________________ SAG12::CincaFATB + 35S::hpNbAAE
pOIL322 35S::EgDGAT1 + enTCUP::CnLPAAT + SSU:AtWRI1 +
e
SAG I 2::CocnuFATB2 + 35S::hpNbAAE
pOIL323 35S::EgDGAT1+ enTCUP::CnLPAAT + SAG12:AtWRI1 +
35S::UmbcaFATB +35S::hpNbAAE
0.4 pOIL324 35S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWRI1 +
e.
35S::CincaFATB + 35S::hpNbAAE
pOIL325 35S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWRII +
35S::CocnuFATB2+35S::hpNbAAE
pOIL326 35S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWR11 +
SAG12::UmbcaFATB +35S::hpNbAAE
pOIL327 35S::EgDGAT1 + enTCUP::CnLPAAT + SAG12:AtWRI1 +
SAG12::CincaFATB +35S::hpNbAAE
pOIL328 35S::EgDGAT1+ enTCUP::CnLPAAT + SAG12:AtWRI1 +
SAG12::CocnuFATB2+355::hpNbAAE
These genetic constructs were used to produce transformed tobacco plants of
cultivars Wisconsin 38 and a high oil line transformed with the T-DNA from
pJP3502. It was observed that plants transformed with the single gene FATB
constructs expressed from the 35S promoter were significantly smaller than
those
transformed with the corresponding FATB construct expressed from the SAG12
promoter or from the four gene constructs. The smaller plant size was
considered to
be caused by a buildup of MCFA which was not incorporated efficiently into
TAG.
Discussion
The present study found that C12:0 production in leaf cells was only about
1.6% of the total fatty acid content after expression of Umbca-TE alone (Table
11).
The addition of a gene for expression of Arath-WRI had a much stronger effect
on
C12:0 and C14:0 accumulation in leaf tissue than the addition of the coconut
LPAAT
(Figures 7 and 9). This indicated that WRI1 in combination with the
thioesterase
greatly increased MCFA accumulation in leaf cells, acting synergistically.
Importantly, much of the C12:0, C14:0 and C16:0 was found to accumulate in the
leaves in TAG, which lipid does not accumulate at substantial levels in wild-
type
leaves. These experiments showed that the cells in the vegetative parts of
plants could
be modified to produce MCFA, particularly C12:0 and C14:0 in TAG at high
levels.
C16:0 levels were also increased substantially.
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Example 11: Gene selection and vector construction
Fatty acyl thioesterases were identified from Cinnamomum camphora 14:0-
ACP thioesterase (referred to as `CcTE', Accession No. Q39473.1, (Yuan et al.,
1995)), Umbellularia californica 12:0-ACP thioesterase (UcTE, Accession No.
Q41635.1, (Voelker et al., 1992)), and Cocos nucifera acyl-ACP thioesterase
FatB2
(CnTE2, Accession No. AEM72520.1, (Jing et al., 2011)). A C. nucifera LPAAT
(CnLPAAT, Accession No. Q42670.1, (Knutzon et al., 1995)) was also identified.
Coding regions were synthesized using codon optimised nucleotide sequences for
expression in Nicotiana plant cells. Expression vectors encoding WRI1 and DGAT
were produced as previously described by Vanhercke et al. (2013).
Three DGAT candidate sequences were identified in the transcriptome of
African oil palm (Elaeis guineensis) (Dussert et al., 2013) and selected to be
tested in
their utilisation of MCFA for the assembly of leaf lipids. The DGATs from oil
palm
were selected based on the fatty acid compositions of palm oil and palm kernel
oil
(Edem, 2002), being high in MCFA content.
A gene encoding glycerol-3-phosphate acyltransferase 9 (GPAT9) from C.
nucifera (coconut, CnGPAT9) was identified from a transcriptome. A genetic
construct to express this enzyme was made from RNA isolated from developing
coconut endosperm, as described below.
Each gene was cloned into the EcoRI site of the binary vector pJP3343 which
contained a constitutive 35S promoter with duplicated enhancer region
(Vanhercke et
al., 2013) for expression in plant cells. Agrobacterium tumefaciens strain
AGL1 was
transformed with each of the constructs.
Example 12: Increasing medium chain fatty acid production in vegetative plant
cells
GPAT9 has recently been identified as functioning in Arabidopsis thaliana
seed to transfer acyl groups from acyl-CoA to the sn-1 position of glycerol-3-
phosphate (G3P) (Shockey et al., 2016; Singer et al., 2016). The inventors
hypothesized that a GPAT9 from coconut might assist in increasing the MCFA
content of transgenic oils produced in vegetative plant cells. A GPAT9 gene
from
coconut was identified by searching an assembled coconut endosperm
transcriptome
using the Arabidopsis thaliana GPAT9 nucleotide sequence (AtGPAT9) (Shockey et
al., 2016) as the BLAST query. A candidate for GPAT9 from coconut was
identified,
namely NCBI Accession number KX235871. High fidelity PCR was used to amplify
the full length CnGPAT9 cDNA sequence from coconut. Following isolation and
sequencing of the full length transcript of interest, the open reading frame
for the
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predicted CnGPAT9 was identified. The predicted amino acid sequence was
aligned
with the sequence of AtGPAT9, revealing that the sequences were 78% identical.
Sequence alignment with other annotated GPAT nucleotide sequences showed that
the
identified CnGPAT9 nucleotide sequence clustered with other GPAT9 sequences
(Figurell).
A nucleotide sequence encoding the candidate CnGPAT9 was synthesized and
inserted into pJP3343 in order to test its enzymatic function using the
transient N.
benthamiana infiltration assay as described in Example 1, in particular to
test its
ability to increase TAG content. AtGPAT9 was used as a positive control. Total
lipids
were extracted from infiltrated leaf zones and analysed to determine the
effect of the
GPAT9s on TAG content (Figure 12). From comparison with the samples where p19
alone was infiltrated, which provided a TAG level of about 0.1%, expression of
either
AtGPAT9 or CnGPAT9 provided significant increases in the TAG content in the
leaf,
to 0.5 0.2% and 0.7 0.1% on a dry weight basis, respectively. There was no
significant difference in the TAG levels between the two GPAT9s. It was
concluded
from these data and the phylogeny (Figure 11) that the isolated CnGPAT9
sequence
from coconut encoded a functional GPAT9.
Example 13: DGAT1 promotes production of MCFA-enriched oils
It has been previously demonstrated that MCFA-containing oils could be
produced in the leaves of N. benthamiana (Reynolds et al., 2015). However,
chlorosis
of the leaves was observed with some gene combinations when MCFA accumulated
in membrane lipids such as PC. The inventors wanted to test whether the
introduction
of a DGAT capable of esterifying MCFA into TAG might increase the MCFA content
and perhaps reduce the chlorosis phenotype.
Gene candidates that might be involved in lipid synthesis pathways were
identified in the Elaeis guineensis (African oil palm) transcriptome (Dussett
et al.,
2013) as described above. The fatty acid profile of the oils from oil palm
(palm oil
and palm kernel oil) (Edem. 2002) suggested that some DGATs from oil palm
might
exhibit preference for MCFA substrates. Sequences for three candidate DGAT1
cDNAs were identified from the E. guineensis transcriptome. Alignment of the
predicted amino acid sequences after translation of the cDNAs revealed that
the
isoforms designated EgDGAT1.2 and EgDGAT1.3 lacked highly conserved C- and
N- terminal motifs (Cao, 2011) which are responsible for the catalytic and
regulatory
activities of DGAT1, respectively (Liu et al., 2012; Xu et al., 2008),
suggesting these
isoforms would be non-functional. The third candidate EgDGAT1.1 had these
conserved motifs and was further tested.
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= A genetic construct with codon optimization for expressing EgDGAT1.1 in
N.
tabacum was synthesized and infiltrated into N. benthamiana in combination
with
genetic constructs to express Arabidopsis thaliana WRI1 and CnLPAAT. The
infiltrations were either with or without a gene for co-expression of a
thioesterase
from Cinnamomum camphora (CcTE), to measure levels of both TAG production and
the incorporation of MCFA into TAG. Five days after infiltration, a strong
chlorosis
phenotype was observed to be associated with several gene combinations,
correlated
in particular with the presence of CcTE. Surprisingly, the chlorosis phenotype
was
alleviated by the addition of the gene encoding EgDGAT1.1 (hereinafter
referred to as
EgDGAT1) mores than with AtDGAT1. It was hypothesized that the alleviation of
the negative chlorosis phenotype was due to the increased capacity of EgDGAT1
to
sequester MCFA into TAG relative to AtDGAT1.
Total lipids were extracted and analysed in order to better understand the
relationship between chlorosis and the particular gene combinations. The total
fatty
acid profile revealed that in the absence of CcTE, the TFA content was similar
in the
presence of either AtDGAT1 or EgDGAT1. In the presence of CcTE, the TFA
content
was significantly greater for treatments including EgDGAT1 relative to
AtDGAT1.
The same correlation was observed for TAG content. Although the TAG content
was
similar for the AtWRI1 + AtDGAT1 and AtWRI1 + EgDGAT1.1 samples, the TAG
content was significantly increased for samples expressing CcTE and EgDGAT1,
compared to samples expressing AtDGAT1. These results suggested that following
CcTE expression, in the presence of AtDGAT1, fatty acid synthesis was
inhibited due
to inefficient assembly of the MCFA into glycerolipids. Conversely, there
appeared to
be no inhibition of fatty acid synthesis following the addition of EgDGAT
highlighted by increases in both the TFA and TAG content, implying improved
incorporation efficiency for MCFAs.
The fatty acid composition of the phospholipid fraction in the infiltrated
leaf
zones was also analysed. Total phospholipids were fractionated by TLC and
prepared
for analysis by the preparation of FAME. Analysis of the fatty acid
composition of the
phospholipids revealed a significant reduction in the accumulation of MCFA,
particularly C14:0 and C16:0, following the expression of the EgDGAT1
construct,
compared to AtDGAT1. This suggested that the reduced accumulation of MCFA into
membrane lipids assisted in reducing the chlorosis phenotype.
Example 14: Reconfiguration of Kennedy Pathway for efficient MCFA
accumulation
Following confirmation of CnGPAT9 activity, its capability to use various
MCFA acyl-CoAs as substrates for TAG assembly was tested. This was done in the
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context of the Kennedy pathway components LPAAT and DGAT1, as well as WRI1
to increase the level of fatty acid synthesis. The fatty acid composition of
TAG and
the TAG content were determined by GC-FID (Figure 13, Tables 13-15). When
combined with co-expression of UcTE, the sequential addition of each
acyltransferase
resulted in both significantly increased total TAG content, and a
significantly
increased accumulation of laurate (C12:0) in the TAG as a percentage of the
total
fatty acid content of the TAG. C12:0 levels were up to 51.6 2.0% in the
presence of
the combined expression of UcTE + AtWRI1 + CnGPAT9 + CnLPAAT +
EgDGATI, at a total TAG content in the leaf tissue of 2.4 0.7%. It was also
observed that this combination was associated with a reduction of the
chlorosis
phenotype, thought by the inventors to be a result of efficient sequestering
of laurate
into TAG, i.e. less inclusion in membrane lipids such as PC. Similar results
were
observed with the co-expression of CcTE. C14:0 accumulated to 40.3 1.2% in
the
presence of the combination of CcTE + AtWRI1 + CnGPAT9 F CnLPAAT. There
was an increase in the TAG content but not significantly compared to CcTE +
CnGPAT9. The greatest TAG production was achieved following the further
addition
of the EgDGAT1, with a total TAG content of 2.8 0.2%. The fatty acid
composition
of TAG was altered following the additional combination with EgDGAT1, with a
significant reduction in C14:0 and a significant increase in C16:0 content,
each as a
percentage of the total fatty acid content of the TAG. This shift in profile
suggested
that EgDGAT1 exhibited a stronger substrate preference for C16:0 compared to
C14:0. Consistent with the observations with UcTE, a significant improvement
in the
chlorotie phenotype was observed following the addition of EgDGAT1. When CnTE2
was used, the sequential addition of the acyltransferases did not result in
any
significant differences in either the fatty acid profile of TAG, or the total
TAG
content. This may have been due to the native acyltransferases' ability to
efficiently
utilise the increased flux of C16:0 acyl-CoA associated with the activity of
CnTE2.
Further investigations into the effects of the sequential addition of
acyltransferases on the utilization of acyl-CoAs for the assembly of MCFA-
enriched
glycerolipids was performed using QQQ-LCMS as described in Example 1, to
reveal
any differences in MCFA assembly and distribution. The integrated analysis
including
DAG, PC and TAG revealed much information about the assembly process of lipids
in the leaf cells. When CnGPAT9 was expressed with UcTE + AtWRI1, it was
observed that CnGPAT9 used C12:0 substrate for assembly, based on the presence
of
PC 30:3 (C12:0 plus C18:3). It was reasoned that the sn-2 position of the PC
was
most likely occupied by C18:3, due to either the esterification of C12:0 to
the sn-1
position via CnGPAT9 or from the absence of CnLPAAT. The presence of some
TAG 42:3 suggested that the native DGATs exhibited some capability of
utilising
CA 2998211 2018-03-16
217
C12:0 for TAG assembly (12:0/18:3/12:0). With the addition of CnLPAAT, a
significant amount of PC 24:0 (di-C12:0) was produced, indicating that C12:0
was
efficiently esterified to both the sn-1 and sn-2 positions of the G3P
backbone.
However, without a strong substrate preference for C12:0, most of the produced
laurate remains sequestered in membrane lipids. However, further addition of
EgDGAT1 increased laurate accumulation. This shift involved the reduction of
MCFAs accumulating in PC and increased production of MCFA-enriched TAG. Most
notable was the shift from PC 24:0 (without EgDGAT1) to the accumulation of
TAG
36:0 (tri-C12:0) (with EgDGAT1), highlighting that laurate was being
efficiently
incorporated into all three position of the G3P backbone in the presence of
EgDGAT1. Significant increases were also observed for other MCFA-enriched TAG
species including TAG 38:0, TAG 40:0 and TAG 42:0. These results confirmed
that
the expression of an appropriate DGAT I was effective for the efficient
incorporation
of the unusual fatty acids of interest (in this instance. C12:0 and other
MCFA) into
TAG. These results highlighted that the expression of the EgDGAT1 in the
enzyme
combination effectively relieved the accumulation of MCFA in PC and promoted
efficient production of MCFA-enriched TAG in plant leaf lipids.
A similar pattern was also observed in the case study involving combinations
including CcTE. When CnGPAT9 was combined with CcTE + AtWRIL it was
observed that CnGPAT9 utilised C14:0 substrate, based on the accumulation of
PC
28:0 (di-C14:0) and PC 30:0 (C14:0 plus C16:0). It appeared that the native
LPAAT
genes were somewhat capable of utilising C14:0-CoA as substrate based on the
presence of PC 28:0, indicating that C14:0 was being esterified at both the sn-
1 and
sn-2 positions of the PC. Similarly, the native DGATs also appeared capable of
utilising C14:0-CoA for TAG assembly, based on the production of TAG 42:0 (tri-
C14:0). However, the subsequent addition of CnLPAAT to the system increased
utilisation of C14:0 acyl-CoA, evident from the significantly increased
abundance of
PC 28:0. which indicated an increased efficiency of esterification to the sn-2
position
of PC. This increased accumulation of MCFA was also correlated with a more
severe
chlorosis phenotype then compared to the CnGPAT9 alone, most likely attributed
to
the increased accumulation in the membrane lipids. The further addition of the
EgDGAT1 to the combination resulted in almost complete absence of MCFA from
PC. This was associated with an increased production of MCFA-enriched TAG
species, particularly TAG 40:0, TAG 42:0, TAG 44:0 and TAG 46:0. all of which
include the incorporation of C14:0.
When CnGPAT9 was combined with CnTE2 + AtWRIE it was observed that
CnGPAT9 also utilised C16:0-CoA as substrate, based on the accumulation of PC
32:0 (di-C16:0). Based on the fatty acid profile of N benthamiana leaves, it
was
CA 2998211 2018-03-16
218
expected that the native LPAATs and DGATs would exhibit substrate preference
for
the incorporation of C16:0 into Oycerolipids, evidenced from the increased
production of C16:0-enriched TAG species, through simply over-expressing a
thioesterase with C16:0 specificity. Although the subsequent additions of the
CnLPAAT and EgDGAT1 did not appear to significantly affect the overall TAG
composition, there was a significant reduction in the total MCFA accumulation
in PC
lipids. Importantly, the addition of the EgDGAT1 to CnTE2 was associated with
a
reduction in the degree of leaf chlorosis, although not as complete as in the
presence
of the other TEs.
It was concluded that a GPAT9 like CnGPAT9 having a preference for MCFA
substrates was an important factor in contributing towards both MCFA
accumulation
and increasing the total production of TAG in plant leaves. In the absence of
a DGAT
having substrate preference for MCFA, the low abundance of MCFA-containing DAG
species suggested that DAG containing the MCFA was efficiently converted to PC
through the activities of either PDCT or CPT (Bates and Browse, 2011; Bates
and
Browse, 2012; Bates et al., 2012). The addition of EgDGAT1 changed the
metabolic
flux of the system, pushing MCFA towards TAG accumulation via the Kennedy
pathway, and thus away from incorporation of the MCFA into membrane lipids
through reducing conversion of DAG to PC.
Table 13. Total leaf fatty acid composition of TAG (% total TAG) of C6:0, C8:0
and
C10:0 fatty acids in Nicotiana benthatniana leaves infiltrated with various
constructs.
Genotype C6:0 C8:0 C10:0
REPLICATE 1
P19 0.000 0.000 4.317
P19+ CincaTE + CnGPAT9 +
AtWRI 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI 0.000 0.000 5.687
P19+ UmbcaTE + CnGPAT9 +
AtWRI 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT 0.000 0.000 0.947
P19+ UmbcaTE + CnGPAT9 +
AtWR1 + CnLPAAT 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT 0.000 0.000 0.000
CA 2998211 2018-03-16
219
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT
EgDGAT 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 1.533
P19+ UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ CincaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ CuplaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 1.643
P19+ UmbcaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
REPLICATE 2
P19 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI 0.000 0.000 4.368
P19+ UmbcaTE + CnGPAT9 +
AtWR1 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT 0.000 0.000 3.523
P19+ UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT ______________ 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+, UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
CA 2998211 2018-03-16
220
P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ CincaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ CuplaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ UmbcaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
REPLICATE 3
P19 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI 0.000 0.000 0.000
P19+ UmbcaTE + CnGPAT9 +
AtWRI 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT 0.000 0.000 0.000
P19+ UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19 UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ CincaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
P19+ CuplaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 0.000 0.000 0.000
CA 2998211 2018-03-16
221
P19+ UmbcaTE + AtGPAT9 +
AtWR1 + CnLPAAT +
EgDGAT 0.000 0.000 0.000
CA 2998211 2018-03-16
222
co Table 14. Total leaf fatty acid composition of TAG (% total TAG) of
12:0, C14:0, C14;1, C15:0, C16:0 and C16:1 fatty acids in Nicotiana
benthamiana leaves infiltrated with various constructs.
co
Genotype C12:0 C14:0 C14:1 C15:0 C16:0 C16:1
1-`
(31
REPLICATE]
P19
3.882 11.116 0.000 1.380 41.258 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI 3.332 , 35.333 0.000
0.226 27.276 0.203
P19+ CuplaTE + CnGPAT9 + '
AtWRI
2.119 10.647 0.000 0.000 47.322 0.000
P19+ UmbcaTE + CnGPAT9 +
AtWRI 32.957 9.794 0.000 0.000
16.217 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI 0.000 17.998 0.000 0.343
56.230 0.578
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT 7.219 41.154 0.000 0.261
24.586 0.334
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT 1.491 7.331 0.000 0.241
57.931 0.315
P19+ UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT 44.742 10.476 0.000 0.000
10.207 0.270
P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT 0.465 14.889 0.000 0.335
56.250 0.481
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT
4.620 30.554 0.000 0.177 37.511 0.475
P19+ CuplaTE + CnGPAT9 + 4.598 5.250 0.000 0.221
51.742 0.320
223
o
co AtWRI + CnLPAAT +
EgDGAT
P19+ UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT +
co
EgDGAT 53.604 7.690 0.000 0.157
11.120 0.094
0
P19+ CocnuTE2 + CnGPAT9
1-` AtWRI + CnLPAAT +
EgDGAT
0.654 14.116 0.000 0.256 53.942 0.306
P19+ CincaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT
4.499 35.151 0.000 0.196 33.202 0.314
P19+ CuplaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 4.943 5.716 _ 0.000 0.262
50.177 -- 0.542
P19+ UmbcaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 49.589 7.781 0.000 0.105
12.284 0.252
REPLICATE 2
P19
6.485 10.998 0.000 0.000 46.160 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI 0.000 0.000 0.000 0.000
0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI
1.758 10.767 0.000 0.000 49.728 0.583
P19+ UmbcaTE + CnGPAT9 +
AtWRI
32.530 10.553 0.000 0.000 15.254 0.544
P19+ CocnuTE2 + CnGPAT9
+ AtWRI 0.628 16.693 0.000 0.327
49.863 -- 0.466
P19+ CincaTE + CnGPAT9 + 3.660 40.701 0.000 0.264
28.736 0.333
224
o
1
co AtWRI + CnLPAAT
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT 2.472 10.374 0.000 0.364
49.195 0.635
P19+ UmbcaTE + CnGPAT9 +
co
AtWRI + CnLPAAT 43 .462 10.775 0.000 0.206
10.328 0.225
0
P19+ CocnuTE2 + CnGPAT9
1-` AtWRI + CnLPAAT 0.000 0.000 0.000 0.000
0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT
4.101 33.380 0.000 0.000 35.431 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 8.061 5.606 0.000 0.000
47.901 0.000
P19+ UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 49.552 6.800 0.000 0.000
12.602 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT +
EgDGAT
0.000 14.374 0.000 0.000 50.723 0.000
P19+ CincaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT
2.758 26.757 0.000 0.000 38.082 0.000
P19+ CuplaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT
2.672 4.771 0.000 0.000 53.725 0.000
P19+ UmbcaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 49.847 6.988 0.000 0.000
11.945 0.000
225
o
co REPLICATE 3
P19 0.000 0.000 0.000 0.000
55.478 0.000
P19+ CincaTE + CnGPAT9 +
0
AtWRI
0.000 32.975 0.000 0.000 29.893 0.000
co
P19+ CuplaTE + CnGPAT9 +
0
AtWRI 0.000 9.743 0.000 0.000
55.084 0.000
1-` P19+ UmbcaTE + CnGPAT9 +
AtWRI 29.807 9.939 0.000 0.000
15.215 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI 0.000 20.098 0.000 ,
0.000 48.646 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT 4.924 38.894 0.000 0.000
22.078 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT 0.000 9.483 0.000 0.000
57.458 0.000
P19+ UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT 46.258 8.809 0.000 0.000
9.487 -- 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT 0.000 ! 18.294 0.000
0.000 56.968 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT
3.909 34.512 0.000 0.000 36.091 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT
0.000 4.605 0.000 0.000 56.818 0.000
P19+ UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 51.506 7.067 0.000 0.000
11.083 0.000
P19+ CocnuTE2 + CnGPAT9
AtWRI + CnLPAAT + 0.000 10.744 0.000 0.000
55.660 0.000
226
o
co EgDGAT
P19+ CincaTE + AtGPAT9 +
AtWRI + CnLPAAT +
0
EgDGAT
3.697 26.670 0.000 0.000 37.159 0.000
co
P19+ CuplaTE + AtGPAT9 +
0
AtWRI + CnLPAAT +
1-` EgDGAT 1.737 4.336 0.000 0.000
54.136 0.000
P19+ UmbcaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT 49.371 6.898 0.000 0.000
10.168 0.000
227
Table 15. Total leaf fatty acid composition of TAG (% total TAG) of C17:0,
C17:1, C18:0, C18:1, C19:0, C18:2, C18:3, C20:0, C20:1, C22:0
1-t
and C24:0 fatty acids in Nicotiana benthamiana leaves infiltrated with various
constructs.
co
Genotype
C17:0 C17:1 C18:0 C18:1 C19:0 C18:2 C18:3 C20:0
C20:1 C22:0 C24:0
0
1-` REPLICATE 1
P19
13.82
0.000 0.000 7.003 3.505 0.000 7.516 7 1.204 1.260 1.303 2.428
P19+ CincaTE + CnGPAT9 +
21.63
AtWRI 0.000
0.381 2.160 1.542 0.363 6.795 6 0.382 0.000 0.208 0.163
P19+ CuplaTE + CnGPAT9 +
20.84
AtWRI 0.000
0.768 3.491 1.051 0.000 7.046 1 0.607 0.000 0.000 0.421
P19+ UmbcaTE + CnGPAT9 +
25.69
AtWRI 0.000
1.251 2.658 1.439 0.000 9.993 0 0.000 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
13.02
+ AtWRI 0.000 0.485 3.864 1.427
0.000 5.547 7 0.503 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
17.21
AtWRI + CnLPAAT 0.000
0.296 1.824 2.156 0.000 4.653 1 0.307 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
14.89
AtWRI + CnLPAAT 0.000
0.242 2.812 5.820 0.616 6.643 2 0.515 0.000 0.203 0.000
P19+ UmbcaTE + CnGPAT9 +
19.32
AtWRI + CnLPAAT 0.000 0.191 1.359
3.790 0.514 8.779 9 0.204 0.000 , 0.140 0.000
P19+ CocnuTE2 + CnGPAT9
16.27
+ AtWRI + CnLPAAT 0.000
0.333 3.431 2.297 0.000 4.517 1 0.552 0.000 0.179 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT +
EgDGAT
0.000 0.244 2.274 6.967 0.414 7.193 8.577 0.560
0.000 0.297 0.137
P19+ CuplaTE + CnGPAT9 +
0.000 0.283 3.698 7.203 0.473 9.780 13.19 0.895
0.000 0.535 0.272
228
o
co AtWRI + CnLPAAT +
8
EgDGAT
P19+ UmbcaTE + CnGPAT9 +
0
AtWRI + CnLPAAT +
co
EgDGAT
0.000 0.237 1.704 6.638 0.460 8.261 8.641 0.521 0.117
0.470 0.286
0
P19+ CocnuTE2 + CnGPAT9
1-` AtWRI + CnLPAAT +
20.24
EgDGAT 0.000 0.457 3.117 1.071 0.324 4.844
8 0.459 0.000 0.205 0.000
P19+ CincaTE + AtGPAT9 +
AtWRI + CnLPAAT +
12.82
EgDGAT 0.000 0.299 2.232 4.203 0.290 5.963
8 0.500 0.000 0.233 _ 0.089
P19+ CuplaTE + AtGPAT9 +
AtWRI + CnLPAAT +
15.84
EgDGAT 0.000 0.321 4.172 5.766 0.479 8.508
5 0.902 0.000 0.501 0.224
P19+ UmbcaTE + AtGPAT9 +
AtWRI + CnLPAAT +
EgDGAT
0.000 0.185 1.873 6.977 0.608 9.240 9.823 0.532 0.095
0.425 0.230
REPLICATE 2
P19
21.10
0.000 0.000 5.724 0.000 0.000 9.527
5 0.000 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
1 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
18.64
AtWRI 0.000 0.450 4.115 0.919 0.000 7.307
6 0.635 0.000 0.440 0.285
P19+ UmbcaTE + CnGPAT9 +
10.35 23.80
AtWRI 0.000 0.432 3.015 2.565 0.000 5
1 0.580 0.000 0.370 0.000
P19+ CocnuTE2 + CnGPAT9
20.59
+ AtWRI 0.000 0.324 3.210
1.170 0.467 5.649 3 0.447 0.000 0.163 0.000
229
o
co P19+ CincaTE + CnGPAT9 +
15.49
AtWRI + CnLPAAT 0.000 0.206 2.042 3.281 0.299 4.527
5 0.334 0.000 0.122 0.000
P19+ CuplaTE + CnGPAT9 +
20.61
0 AtWRI + CnLPAAT 0.000 0.342 3.649 1.568 0.000 6.412
5 0.553 0.000 0.298 0.000
co
P19+ UmbcaTE + CnGPAT9 +
20.20
0
AtWRI + CnLPAAT 0.000 0.197 1.653 3.620 0.431 8.552
5 0.201 0.000 0.145 0.000
1-` P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWRI + CnLPAAT +
11.16
EgDGAT 0.000 1.023 ' 2.522 4.695 0.000 7.688
1 0.000 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT +
11.19 17.10
EgDGAT 0.000 0.000 3.790 5.402 0.000 7
4 0.939 0.000 0.000 0.000
P19+ UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT +
10.57 10.87
EgDGAT 0.000 0.000 1.950 6.434 0.000 2
7 0.615 0.000 0.598 0.000
P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT +
21.58
EgDGAT 0.000 2.364 2.998 1.381 0.000 6.570
9 0.000 0.000 0.000 0.000
P19+ CincaTE + AtGPAT9 +
AtWRI + CnLPAAT +
12.28
EgDGAT 0.000 0.962 2.526 6.126 0.000 9.898
8 0.603 0.000 0.000 0.000
P19+ CuplaTE + AtGPAT9 +
AtWR1 + CnLPAAT +
11.57 14.69
EgDGAT 0.000 1.141 3.660 6.542 0.000 7
3 0.794 0.000 0.424 0.000
P19+ UmbcaTE + AtGPAT9 +
AtWRI + CnLPAAT +
11.99 10.67
EgDGAT 0.000 0.000 1.909 6.636 0.000 8
7 0.000 0.000 0.000 0.000
230
o
co
REPLICATE 3
P19
19.96 24.55
0.000 ' 0.000 , 0.000 0.000 0.000 6
7 0.000 0.000 0.000 0.000
co
P19+ CincaTE + CnGPAT9 +
10.22 20.04
0
AtWRI 0.000 1.600 2.296 2.966 0.000 5
6 0.000 0.000 0.000 0.000
1-` P19+ CuplaTE + CnGPAT9 +
10.71 19.37
AtWRI 0.000 ' 0.000 3.044 2.041 0.000 1
6 0.000 0.000 0.000 0.000
P19+ UmbcaTE + CnGPAT9 +
14.11 24.91
AtWRI 0.000 0.000 2.821 3.186 0.000 3
9 0.000 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
18.75
+ AtWRI 0.000 1.637 2.992
1.264 0.000 6.611 3 0.000 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
22.29
AtWRI + CnLPAAT
0.000 2.465 1.645 1.269 0.000 6.427 8 0.000 0.000 0.000 0.000
P19+ CuplaTE + CnGPAT9 +
18.70
AtWRI + CnLPAAT
0.000 0.000 2.864 3.499 0.000 7.993 2 0.000 0.000 0.000 0.000
P19+ UmbcaTE + CnGPAT9 +
10.69 19.07
AtWRI + CnLPAAT 0.000 0.000 1.356 4.320 0.000 9
0 0.000 0.000 0.000 0.000
P19+ CocnuTE2 + CnGPAT9
15.18
+ AtWRI + CnLPAAT
0.000 0.000 3.467 0.000 0.000 6.091 0 0.000 0.000 0.000 0.000
P19+ CincaTE + CnGPAT9 +
AtWR1 + CnLPAAT +
EgDGAT
0.000 0.875 2.053 5.925 0.000 7.047 9.165 0.422 0.000
0.000 0.000
P19+ CuplaTE + CnGPAT9 +
AtWRI + CnLPAAT +
10.47 15.03
EgDGAT 0.000 1.103 3.685 8.285 0.000 1
4 0.000 0.000 0.000 0.000
P19+ UmbcaTE + CnGPAT9 +
AtWRI + CnLPAAT +
11.36
EgDGAT 0.000 0.631 1.506 6.663 0.000
7 9.290 0.462 0.000 0.425 0.000
231
o
co P19+ CocnuTE2 + CnGPAT9
+ AtWRI + CnLPAAT +
18.18
EgDGAT 0.000 1.705 3.665 2.985 0.000 7.058
2 0.000 0.000 0.000 0.000
0
P19+ CincaTE + AtGPAT9 +
co
AtWRI + CnLPAAT +
10.35
0
EgDGAT 0.000 0.816 2.447 7.987 0.000 9.927
9 0.598 0.000 0.339 0.000
1-` P19+ CuplaTE + AtGPAT9 +
AtWRI + CnLPAAT +
11.96 14.23
EgDGAT 0.000 1.020 3.588 7.767 , 0.000 9
7 0.765 0.000 0.445 0.000
P19+ UmbcaTE + AtGPAT9 +
AtWRI + CnLPAAT +
12.13 10.07
EgDGAT 0.000 0.647 1.538 8.125 0.000 6
0 0.433 0.000 0.377 0.236
232
Discussion
In the seeds of native plants, the incorporation of unusual fatty acids is
almost
exclusively confined to TAG and typically excluded from membrane lipids, most
likely because they interfere with proper membrane functions and are often
deleterious to the plant cells (Millar et al., 2000). A different scenario has
been
observed in transgenic plants that have attempted to modify the oil fatty acid
profiles,
such as increasing the lauric acid content (Knutzon et al., 1999). Although
high levels
of laurate accumulation in plant oils have been achieved in the seeds of
transgenic
canola, there was a significant level of laurate being sequestered in PC
during seed
development (Wiberg et al., 1997). In that work, de novo DAG containing
laurate was
not efficiently converted to TAG by the resident DGAT but was instead
converted to
the membrane lipid PC. The native canola LPCAT lacked the capability to handle
MCFAs (Zhang et al., 2015) so the route to PC could be through PDCT or CPT
activities. Consequently, this inefficient utilization of laurate for TAG
synthesis was
also associated with a negatively correlated penalty in total oil yields
(Knutzon et al..
1999).
Similar to the expression of MCFA in seed oil, the over expression of MCFA
in the leaf cells described here with the co-expression of CnGPAT9 and
CnLPA.AT
identified a metabolic bottleneck through the sequestering of MCFA in PC. The
low
abundance of MCFA-containing DAG species suggested that de novo DAG
containing MCFA was quickly converted to PC through the activities of PDCT or
CPT or both, due to the absence of a DGAT capable of using the MCFA-containing
DAG for TAG assembly. The inventors showed that the addition to the enzyme
combination of a DGAT with a preference for MCFA as substrate, relative to one
or
more CI8 substrates such as oleic acid, LA or ALA, promoted synthesis of MCFA-
enriched TAG and relieved this bottleneck. Endogenous PDAT may also be
involved
in the maintenance of membrane homeostasis, through the removal of unusual
fatty
acids from the membrane lipids and sequestering them into TAG (Fan et al.,
2014;
Fan et al., 2013a and b). This study demonstrated that the expression of the
DGAT
from a species such as E. guineensis (EgDGAT1) was sufficient to restore
membrane
homeostasis by reducing the accumulation of MCFA in PC. The expression of
EgDGAT1 proved that a DGAT with MCFA substrate preference was beneficial for
the efficient assembly of TAG and increased TAG content in the plant cells.
The
reconfigured Kennedy pathway for improving MCFA incorporation into TAG is
expected to benefit seedoil composition and TAG content as well.
CA 2998211 2018-03-16
233
It will be appreciated by persons skilled in the art that numerous variations
and/or modifications may be made to the invention as shown in the specific
embodiments without departing from the spirit or scope of the invention as
broadly
described. The present embodiments are, therefore, to be considered in all
respects as
illustrative and not restrictive.
All publications discussed and/or referenced herein arc incorporated herein in
their entirety.
Any discussion of documents, acts, materials, devices, articles or the like
which has been included in the present specification is solely for the purpose
of
providing a context for the present invention. It is not to be taken as an
admission that
any or all of these matters form part of the prior art base or were common
general
knowledge in the field relevant to the present invention as it existed before
the priority
date of each claim of this application.
CA 2998211 2018-03-16
234
REFERENCES
Adhikari et al. (2016) Plant Physiol 171:179-191.
Alemanno et at. (2008) Planta 227:853-866.
Almeida and Allshire (2005) TRENDS Cell Biol. 15:251-258.
Alonso et al. (2009) Plant Cell 21: 1747-1761.
Alonso et al. (2010) Green Chem. 12:1493-1513.
Alvarez et al. (2000) Theor. App!. Genet. 100:319-327.
Andre at al (2012) Proc. Natl. Acad. Sci. U.S.A. 109:10107-10112.
Arkcoll (1988) Lauric Oil Resources. Economic Botany 42:195-205.
Bartlett et al. (2008) Plant Methods 4:22.
Basiron and Weng (2004) Journal of Oil Palm Research 16.
Bates et al. (2014) PNAS USA 111:1204-1209.
Bates and Browse (2011) Plant J 68:387-399.
Bates and Browse (2012) Frontiers in Plant Science 3:147.
Baud et al. (2007) Plant J. 50:825-838.
Baud and Lepiniec (2010) Progr. Lipid Res. 49: 235-249.
Baumlein etal. (1991) Mol. Gen. Genet. 225:459-467.
Baumlein et al. (1992) Plant J. 2:233-239.
Belide etal. (2013) Plant Cell Tiss. Org. Cult. DOI 10.1007/s11240-013-0295-1.
Ben Saad etal. (2011) Transgenic Res 20: 1003-1018.
Bibikova et al. (2002) Genetics 161:1169-1175.
Bihmidine etal. (2015) BMC Plant Biology 15:186.
Bihmidine et al. (2016) Plant Signaling & Behaviour 11: el 117721.
Bligh and Dyer (1959) Canadian Journal of Biochemistry and Physiology 37:911-
917.
Boutilier etal. (2002) Plant Cell 14:1737-1749.
Bouvier-Nave etal. (2000) European Journal of Biochemistry / FEBS 267:85-96.
Bradford (1976) Anal. Biochem. 72:248-254.
Broothaerts et al. (2005) Nature 433:629-633.
Broun et al. (1998) Plant J. 13:201-210.
Browse et al. (1986) Biochem J 235: 25-31.
Buchanan-Wollaston (1994) Plant Physiol. 105:839-846.
Burgal etal. (2008) Plant Biotechnol J 6:819-831.
Busk etal. (1997) Plant J. 11:1285-1295.
Cai etal. (2015) Plant Cell 27:2616-2636.
Cao (2011) BMC Research Notes 4:249.
Cao et al. (2007) J. Lipid Res. 48:583-591.
Capuano etal. (2007) Biotechnol. Adv. 25:203-206.
CA 2998211 2018-03-16
235
Chapman and Ohlogge (2012) J. Biol. Chem. 287:2288-2294.
Chen et al (2011) Plant Physiol. 155:851-865.
Chen et al. (2016) International Journal of Molecular Sciences 17:507.
Chikwamba et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:11127-11132.
Christensen et al. (1992) Plant Mol Biol 18:675-689.
Christie (1993) Advances in Lipid Methodology-Two, Oily Press, Dundee, pp195-
213.
Chung et al. (2006) BMC Genomics 7:120.
Comai et al. (2004) Plant J 37: 778-786.
Cong et al. (2013) Science 339:819-823.
Corrado and Karali (2009) Biotechnol. Adv. 27:733-743.
Coutu et al. (2007) Transgenic Res. 16:771-781.
Dahlqvist et al. (2000), Proc. Natl. Acad. Sci. U.S.A. 97: 6487-6492.
Damaj et al., (2010) Planta 231:1439-1458.
Dandik and Aksoy (1998) Fuel Process Technol. 57: 81-92.
Dauk et al (2007) Plant Sci. 173:43-49.
Deruyffelaere et al. (2015) Plant Cell Physiol 56:1374-1387.
Dehesh (2001) European Journal of Lipid Science and Technology 103 :688-697.
Durrett et al. (2008) Plant J. 54:593-607.
Dussert et al.. (2013) Plant Physiol 162:1337-1358.
Dyer et al. (2002) Plant Physiol. 130:2027-2038.
Eastmond et al. (2006) Plant Cell 18: 665-675.
Eccleston et al. (1996) Planta 198:46-53.
Eccleston and Ohlrogge (1998) The Plant Cell Online 10:613-621.
Edem (2002) Plant Foods for Human Nutrition 57:319-341.
Ellerstrorn et al. (1996) Plant Mol. Biol. 32:1019-1027.
El Tahchy et al. (2017) FEBS Letters 591:448-456.
Endalew et al. (2011) Biomass and Bioenergy 35:3787-3809.
Fan et al. (2013a) Plant Cell 25: 3506-3518.
Fan et al. (2013b) Plant Journal 76: 930-942.
Fan et al. (2014) Plant Cell 26: 4119-4134.
Fan et al. (2015) Plant Cell 27: 2941-2955.
FAO Animal Production and Health Proceedings (2002) Protein sources for the
animal feed industry, Expert Consultation and Workshop, Bangkok.
Feeney et al. (2012) Plant Physiol 162: 1881-1896.
Finkelstein et al. (1998) Plant Cell 10:1043-1054.
Froissard et al. (2009) FEMS Yeast Res 9:428-438.
CA 2998211 2018-03-16
236
Gan (1995) Molecular characterization and genetic manipulation of plant
senescence.
PhD thesis. University of Wisconsin, Madison.
Gan and Amasino (1995) Science 270:1986-1988.
Gazzarrini et al. (2004) Dev. Cell 7:373-385.
Geurin etal. (2016) Plant Biotech. J 87: 423-441.
Ghosal et al. (2007) Biochimica et Biophysica Acta 1771:1457-1463.
Ghosh et al. (2009) Plant Physiol. 151:869-881.
Gidda et al (2013) Plant Signaling Behay. 8:e27141.
Girijashankar and Swathisree, (2009) Physiol. MA Biol. Plants 15: 287-302.
Gong and Jiang (2011) Biotechnol. Lett. 33:1269-1284.
Gould et al. (1991) Plant Physiol. 95:426-434.
Greenwell et al. (2010) J. R. Soc. Interface 7:703-726.
Guan etal. (2015) Lipids 50:407-416.
Gurel et al. (2009) Plant Cell Rep. 28:429-444.
Gutierrew etal. (2013) BMC Biotechnol. 13: 40.
Hedrich et al. (2015) Curr Opin Plant Biol 25: 63-70.
Henikoff et al. (2004) Plant Physiol. 135:630-636.
Hershey and Stoner (1991) Plant Mol. Biol. 17:679-690.
Hinchee et al. (1988) Biotechnology 6:915-922.
Horn et al. (2007) Euphytica 153:27-34.
Horn et al. (2013). Plant Physiol 162:1926-1936.
Horvath et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:1914-1919.
Hsiao and Tzen (2011) Plant Physiol. Biochem. 49: 77-81.
Hsieh and Huang (2004) Plant Physiol 136:3427-3434.
Huang (1996) Plant Physiol. 110:1055-1061.
Huang and Huang (2016) Plant Physiol. 171: 1867-1878.
Ichihara et al (1988) Biochim. Biophys. Acta 958:125-129.
Ikeda et al. (2006) PI Biotech J. 23: 153-161.
Iwabuchi et al. (2003) J. Biol. Chem. 278:4603-4610.
James et al. (2010) Proc. Natl. Acad. Sci. USA 107:17833-17838.
Jepson et al. (1994) Plant Mol. Biol. 26:1855-1866.
Jing et al. (2011) BMC Biochemistry 12:44.
Jolivet et al. (2014) Plant Physiol. Biochem. 42:501-509.
Jones etal. (1995) Plant Cell 7: 359-371.
Karmakar et al. (2010) Bioresource Technology 101:7201-7210.
Kelly et al. (2011) Plant Physiol. 157: 866-875.
Kelly eta! (2013a) Plant Biotech. J. 11:355-361.
Kelly etal. (2013b) Plant Physiol. 162:1282-1289.
CA 2998211 2018-03-16
237
= Kereszt et al. (2007) Nature Protocols 2:948-952.
Knutzon et al. (1995) Plant Physiol 109:999-1006.
Knutzon etal. (1999) Plant Physiology 120:739-746.
Kim et al. (2014) Biotechnology for Biofuels 7:36.
Kim et al. (2015a) Plant,' 84:1021-1033.
Kim etal. (2015b) Journal of Experimental Botany 66:4251-4265.
Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93:1156-1160.
Kim etal. (2016) Plant Physiol 171: 1951-1964.
Klemens etal. (2013) Plant Physiol 163: 1338-1352.
Koziel et al. (1996) Plant Mol. Biol. 32:393-405.
Kuhn etal. (2009) J. Biol. Chem. 284:34092-102.
Kunst et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:4143-4147.
Kwong et al. (2003) Plant Cell 15:5-18.
Lacroix et al. (2008) Proc. Natl. Acad. Sci.U.S.A. 105: 15429-15434.
Laibach et al. (2015). J. Biotechnol. 201: 15-27.
Lardizabal etal. (2008) Plant Physiol. 148: 89-96.
Laureles etal. (2002) J Agric Food Chem 50:1581-1586.
Lebrun et al. (1987) Nucl. Acids Res. 15:4360.
Laux etal. (1996) Development 122: 87-96.
Lazo et al. (1991) Bio/Technology 9 :963-967.
Lee etal. (1998) Science 280:915-918.
Lee et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2152-2158.
Li et al. (1996) FEBS Lett. 379:117-121.
Li et al. (2006) Phytochemistry 67: 904-915.
Li et al. (2017) Plant Phsyiol 173:2208-2224.
Lin etal. (2005) Plant Physiol. Biochem. 43:770-776.
Linder et al. (2005). FEMS Microbiol. Rev. 29: 877-896.
Liu and Godwin (2012). Plant Cell Reports 31, 999-1007.
Liu etal. (2010) Plant Physiol. Biochem. 48: 9-15.
Liu etal. (2012) Frog Lipid Res 51:350-377.
Liu etal. (2012) J Exp Bot 63: 3727-3740.
Liu et al. (2014) BMC Plant Biol. 14: 73.
Lotan etal. (1998) Cell 93: 1195-1205.
Lu et al. (2009) Proc Natl Acad of Sci USA 106:18837-18842.
Luerssen et al. (1998) Plant J. 15: 755-764.
Lui etal. (2009) J. Agric. Food Chem. 57: 2308-2313.
Ma et al. (2016) Plant Journal doi: 10.1111/tpj.13244.
MacEachran et al. (2010). App!. Environ. Microbiol. 76: 7217-7225.
CA 2998211 2018-03-16
238
Maher and Bressler (2007) Bioresource Technology 98:2351-2368.
Matsuoka et al. (1994) Plant J. 6:311-319.
Matsuoka and Minami (1989) Eur. J. Biochem. 181: 593-598.
McCleary et al. (2013) J AOAC Int 93:221-233.
McCleary et al. (2015) Starch 67:860-883.
McElroy et al. (1990) Plant Cell 2: 163-171.
McKinley et al. (2016) Plant Journal: doi:10.1111/tpj.13269.
Meier et al. (1997) FEBS Lett. 415:91-95.
Millar et al. (2000) Trends in Plant sScience 5:95-101.
Millar and Waterhouse (2005). Funct Integr Genomics 5:129-135.
Miller (1984). Crop Sci 24:1224-1224.
Mojica et al. (2000) Mol Microbiol 36:244-246.
Moreno-Perez (2012) PNAS 109:10107-10112.
Mongrand et al. (1998) Phytochemistry 49:1049-1064.
Moyle and Birch (2013) Theor. Appl. Genet. 126:1775-1782.
Mu etal. (2008) Plant Physiol. 148:1042-1054.
Mudge et al. (2013) Plant Biotechnol. J. 11:502-509.
Murashige and Skoog (1962). Physiol Plant 15:473-497.
Murphy et al. (2012). Protoplasma 249:541-585.
.. Needleman and Wunsch (1970) J. Mol Biol. 45: 443-453.
Nilsson et al. (2012) Physiol. Plantarum 144: 35-47.
Nomura et al. (2000) Plant Mol. Biol. 44: 99-106.
OECD/FAO (2015) OECD-FAO Agricultural Outlook (Edition 2015). Paris:OECD
Publishing.
.. Ohlrogge and Browse (1995) Plant Cell 7: 957-970.
Padidam (2003) Cuff. Opin. Plant Biol. 6:169-77.
Padidam et al. (2003) Transgenic Res. 12:101-9.
Parthibane et al. (2012a) J. Biol. Chem. 287:1946-1965.
Parthibane et al. (2012b) Plant Physiol. 159:95-104.
Pasquinelli et al. (2005). Cuff. Opin. Genet. Develop. 15:200-205.
Perez-Vich etal. (1998) J.A.O.C.S. 75:547-555.
Perrin et al. (2000) Mol. Breed. 6:345-352.
Petrie et al. (2012) PLOS One 7: e35214.
Phillips et al. (2002) Journal of Food Composition and Analysis 12:123-142.
Potenza et al. (2004) In Vitro Cell Dev. Biol. Plant 40:1-22.
Poxleitner et al. (2006) Plant J. 47:917-933.
Prosky et al. (1985) J AOAC Chem 68:677-679.
Pyc et al. (2017) Trends in Plant Sci. 22:596-609.
CA 2998211 2018-03-16
239
=
Qazi etal. (2012) Journal of Plant Physiology 169: 605-613.
Qiu et al. (2001) J. Biol. Chem. 276:31561-3156.
Reynolds et at. (2015) Frontiers in Plant Science 6.
Robson et al. (2004) Plant Biotechnol J 2:101-112.
Rossell and Pritchard (1991) Analysis of Oilseeds, Fats and Fatty Foods.
Elsevier
Ruuska et at. (2002) Plant Cell 14:1191-1206.
Saha et at. (2006) Plant Physiol. 141:1533-1543.
Sanjaya et al. (2011) Plant Biotechnol J 9:874-883.
Santos-Mendoza et al. (2005) FEBS Lett. 579:4666-4670.
Santos-Mendoza et al. (2008) Plant J. 54:608-620.
Schneider etal. (2012) Plant Biol 14: 325-336.
Schnurr et at. (2002) Plant Physiol 129:1700-1709.
Shaw etal. (1959) J Soil Sci 10:316-326.
Shen etal. (2010) Plant Phys. 153: 980-987.
Shen et al. (2014). Biochem. Biophys. Res. Comm. 448: 365-371.
Semwal et al. (2011) Bioresource Technology 102:2151-2161.
Shen et al. (2010) Plant Physiol. 153:980-987.
Shiina et al. (1997) Plant Physiol. 115:477-483.
Shimada and Hara-Nishimura (2010) Biol. Pharm. Bull. 33:360-363.
Shimada et al. (2014) Plant Physiol. 164:105-118.
Shockey et al. (2002) Plant Physiol 129:1710-1722.
Shockey etal. (2016) Plant Physiol 170:163-179.
Singer et al. (2016) Journal of Experimental Botany 67:4627-4638.
Slade and Knauf (2005) Transgenic Res. 14: 109-115.
Smith et al. (2000) Nature 407:319-320.
Somerville et al. (2000) Lipids. In BB Buchanan, W Gruissem, RL Jones, eds,
Biochemisty and Molecular Biology of Plants. American Society of Plant
Physiologists, Rockville, MD, pp 456-527.
Sorokin etal. (2009) Biochemistry Biokhimiia 74:1411-1442.
Srinivasan etal. (2007) Planta 225:341-51.
Stalker et at. 1988 Science 242: 419-423.
Stone et al. (2001) Proc. Natl. Acad. Sci. U.S.A.98: 11806-11811.
Stone et al. (2008) Proc. Natl. Acad. Sci. U.S.A.105: 3151-3156.
Tai etal. (2002). Biosci. Biotechnol. Biochem. 66: 2146-2153.
Tan et al. (2011) Plant Physiol. 156:1577-1588.
Taylor (1997) The Plant Cell 9:1245-1249.
Thillet et al. (1988) J. Biol. Chem 263:12500-12508.
Tingay et al. (1997) Plant J. 11:1369-1376.
CA 2998211 2018-03-16
240
=
Tjellstrom et al. (2013) FEBS Lett 587:936-942.
To etal. (2012) Plant Cell 24:5007-5023.
Ulmasov et al. (1995) Plant Physiol. 108:919-927.
van de Loo etal. (1995) Proc Natl Acad Sci US A. 92:6743-6747.
van Erp etal. (2011) Plant Physiol 155:683-693.
van Erp etal. (2015) Plant Physiol 168:36-46.
Vanhercke et al. (2013) FEBS Letters 587:364-369.
Vanhercke et al. (2014a). Plant Biotech. J. 12:231-239.
Vanhercke et al. (2014b) Biocatalysis and Agricultural Biotechnology 3:75-80.
Vanhercke et al. (2017) Metabolic Engineering 39:237-246.
Vieler etal. (2012) Plant Physiol. 158:1562-1569.
Voinnet et al. (2003) Plant J. 33:949-956.
Voelker et al. (1992) Science 257:72-74.
Voelker etal. (1996) Plant J 9:229-241.
Wang etal. (2002) Plant J 32:831-843.
Wang et al. (2007) Plant J 52: 716-729.
Waterhouse et al. (1998). Proc. Natl. Acad. Sci. U.S.A. 95:13959-13964.
Weissbach and Weissbach, (1989) Methods for Plant Mol Biol, Academic Press.
Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press, San
Diego, Calif., (1988).
Weissman (2001) Molec Cell Biol. 2:169-178.
Wesley etal. (2001) Plant J. 27:581-590.
Wiberg et al. (1997) Planta 203:341-348.
Wiberg et al. (2000) Planta 212:33-40.
Winichayakul et al. (2013) Plant Physiol. 162:626-639.
Wood (2014) EMBO Reports 15:201-202.
Wood etal. (2009) Plant Biotech. J. 7: 914-924.
Wu et al. (2014) In Vitro Cellular and Dev. Biol.-Plant 50:9-18.
Xu et al. (2008) Plant Biotechnol J 6:799-818.
Xu et al (2010) Plant and Cell Physiol. 51:1019-1028.
Xu et al (2005) Plant Cell 17:3094-3110.
Xu et al (2008) Plant Cell 20:2190-2204.
Yamagishi etal. (2005) P1 Physiol 139: 163-173.
Yamasaki et al. (2004) Plant Cell 16 :3448-3459.
Yang et al. (2003) Planta 216:597-603.
Yang et al. (2010) Proc. Natl. Acad. Sci. U.S.A.] 07:12040-12045.
Yeap etal. (2017) Plant Journal 91: 97-113.
Yen etal. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:8512-8517.
CA 2998211 2018-03-16
241
Yen et al. (2005) J. Lipid Res. 46: 1502-1511.
Yokoyama et al. (1994) Mol Gen Genet 244: 15-22.
Yuan et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92 :10639-10643.
Zale etal. (2016) Plant Biotech J. 14: 661-669.
Zhang et al. (2015) PLoS ONE 10, e0144653.
Zheng etal. (2009) P1 Physiol 21: 2563-2577.
Thou et al. (2011) J Biol Chem 286:43644-43650.
Zolman etal. (2001) Plant Physiol. 127:1266-1274.
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this description
contains a sequence listing in electronic form in ASCII text format (file:
84226304
Seq 13-12-2018 v2.txt).
A copy of the sequence listing in electronic form is available from the
Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are reproduced in
the following table.
CA 2998211 2018-12-17
242
SEQUENCE TABLE
<110> Commonwealth Scientific and Industrial Research
Organisation
<120> Plants Producing Modified Levels of Medium Chain Fatty Acids
<130> 524739
<340> Not available
<141> 2018-03-16
<160> 152
<170> PatentIn version 3.5
<210> 1
<211> 520
<212> PRT
<213> Arabidopsis thaliana
<400> 1
Met Ala Ile Leu Asp Ser Ala Gly Val Thr Thr Val Thr Glu Asn Gly
1 5 10 15
Gly Gly Glu Phe Val Asp Leu Asp Arg Leu Arg Arg Arg Lys Ser Arg
20 25 30
Ser Asp Ser Ser Asn Gly Leu Leu Leu Per Gly Ser Asp Asn Asn Ser
35 40 45
Pro Ser Asp Asp Val Gly Ala Pro Ala Asp Val Arg Asp Arg Ile Asp
50 55 60
Ser Val Val Asn Asp Asp Ala Gin Gly Thr Ala Asn Leu Ala Gly Asp
65 70 75 80
Asn Asn Gly Gly Gly Asp Asn Asn Gly Gly Gly Arg Gly Gly Gly Glu
85 90 95
Gly Arg Gly Asn Ala Asp Ala Thr Phe Thr Tyr Arg Pro Ser Val Pro
100 105 110
Ala His Arg Arg Ala Arg Glu Ser Pro Leu Ser Ser Asp Ala Ile Phe
115 120 125
Lys Gin Ser His Ala Gly Leu Phe Asn Leu Cys Val Val Val Leu Ile
130 135 140
Ala Val Asn Ser Arg Leu Ile Ile Glu Asn Leu Met Lys Tyr Gly Trp
145 150 155 160
Leu lie Arg Thr Asp Phe Trp Phe Ser Ser Arg Ser Leu Arg Asp Trp
165 170 175
Pro Leu Phe Met Cys Cys Ile Ser Leu Ser Ile Phe Pro Leu Ala Ala
180 185 190
Phe Thr Val Glu Lys Leu Val Leu Gln Lys Tyr Ile Ser Glu Pro Val
195 270 205
Val Ile Phe Leu His Ile Ile Ile Thr Met Thr Glu Val Leu Tyr Pro
210 215 220
Val Tyr Val Thr Leu Arg Cys Asp Ser Ala Phe Leu Ser Gly Val Thr
225 230 235 240
Leu Met Leu Leu Thr Cys Ile Val Trp Leu Lys Leu Val Ser Tyr Ala
245 250 255
CA 2998211 2018-03-16
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His Thr Ser Tyr Asp Ile Arg Ser Leu Ala Asn Ala Ala Asp Lys Ala
260 265 270
Asn Pro Glu Val Ser Tyr Tyr Val Ser Leu Lys Ser Leu Ala Tyr Phe
275 280 285
Met Val Ala Pro Thr Leu Cys Tyr Gin Pro Ser Tyr Pro Arg Ser Ala
290 295 300
Cys Lie Arg Lys Gly Trp Val Ala Arg Gln Phe Ala Lys Leu Val Ile
305 310 315 320
Phe Thr Gly Phe Met Gly Phe Ile Ile Glu Gin Tyr Ile Asn Pro Ile
325 330 335
Val Arg Asn Ser Lys His Pro Leu Lys Gly Asp Leu Leu Tyr Ala Ile
340 345 350
Glu Arg Val Leu Lys Leu Ser Val Pro Asn Leu Tyr Val Trp Leu Cys
355 360 365
Met Phe Tyr Cys Phe Phe His Leu Trp Leu Asn Ile Leu Ala Glu Leu
370 375 380
Leu Cys Phe Gly Asp Arg Glu Phe Tyr Lys Asp Trp Trp Asn Ala Lys
385 390 395 400
Ser Val Gly Asp Tyr Trp Arg Met Trp Asn Met Pro Val His Lys Trp
405 410 415
Met Val Arg His Ile Tyr Phe Pro Cys Leu Arg Ser Lys Ile Pro Lys
420 425 430
Thr Leu Ala Ile Ile Ile Ala Phe Leu Val Ser Ala Val Phe His Glu
425 440 445
Leu Cys Ile Ala Val Pro Cvs Arg Leu Phe Lys Leu Trp Ala Phe Leu
450 455 460
Gly Ile Met Phe Gin Val Pro Leu Val Phe Ile Thr Asn Tyr Leu Gin
465 470 475 480
Glu Arg Phe Gly Ser Thr Val Gly Asn Met Ile Phe Trp Phe Ile Phe
485 490 495
Cys Ile Phe Giy Gin Pro Met Cys Val Leu Leu Tyr Tyr His Asp Leu
500 505 510
Met Asn Arg Lys Gly Ser Met Ser
515 520
<210> 2
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<400> 2
Tyr Phe Pro
1
<210> 3
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
cA 2998211 2018-03-16
244
<400> 3
His Pro His Giy
1
<210> 4
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<400> 4
Glu Pro His Ser
1
<210> 5
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<220>
<221> X
<222> (2)..(2)
<223> any amino acid
<220>
<221> X
<222> (5)..(5)
<223> any amino acid
<220>
<221> X
<222> (6)..(6)
<223> Lysine (K) or Arginino (R)
<220>
<221> X
<222> (7)..(7)
<223> any amino acid
<220>
<221> X
<222> (9)..(11)
<223> any amino acid
<220>
<221> X
<222> (13)..(15)
<223> any amino acid
CA 2998211 2018-03-16
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<220>
<221> X
<222> (16)..(16)
<223> Leucine (L) or Valine (V)
<220>
<221> X
<222> (19)..(21)
<223> any amino acid
<220>
<221> X
<222> (24)..(24)
<223> Glutamic Acid (E) or Glutamine (Q)
<400> 5
Arg Xaa Gly Phe Xaa Xaa Xaa Ala Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa
1 5 10 15
Val Pro Xaa Xaa Xaa She Gly Xaa
<210> 6
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<220>
<221> X
<222> (3)..(3)
<223> any amino acid
<220>
<221> X
<222> (5)..(7)
<223> any amino acid
<400> 6
She Leu Xaa Leu Xaa Xaa Xaa Asn
1 5
<210> 7
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<400> 7
Gly Asp Leu Val Ile Cys Pro Glu Gly Thr Thr Cys Arg Glu Pro
5 10 15
CA 2998211 2018-03-16
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<210> 8
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<220>
<221> MISC FEATURE
<222> (2).7(2)
<223> any amino acid
<220>
<221> MISC FEATURE
<222> (4).7(4)
<223> any amino acid
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Threonine (T) or Va1ine (V)
<220>
<221> MISC FEATURE
<222> (6).7(6)
<223> Leucine (L) or Valine (V)
<400> 8
Asp Xaa Asp Xaa Xaa Xaa
1 5
<210> 9
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<220>
<221> MISC_FEATURE
<222> (2)..(20)
<223> any amino acid
<220>
<221> MISC_FEATURE
<222> (18)¨(20)
<223> present or absent
<220>
<221> MISC FEATURE
<222> (21)..(21)
<223> Glycine (G) or Serine (S)
<220>
<221> MISC_FEATURE
CA 2998211 2018-12-17
247
=
<222> (22)..(22)
<223> Aspartic Acid (D) or Serine (S)
<220>
<221> MISC_FEATURE
<222> (23)..(25)
<223> any amino acid
<220>
<221> MISC_FEATURE
<222> (26)..(26)
<223> Aspartic Acid (D) or Asparagine (N)
<400> 9
Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25
<210> 10
<211> 393
<212> PRT
<213> Sorghum bicolor
<400> 10
Met Ala Ser Pro Asn Pro Glu Ala Ala Ala Gly Leu Gin Thr Val Ala
1 5 10 15
Val Ala Ala Gly Gly Gly Glu Gly Gly Ser Ser Ser Ser Leu Gly Ala
20 25 30
Val Ala Gly Ala Ala Ala Val Ser Ser Ser Gly Glu Leu Val Pro Arg
35 40 45
Arg Ser Leu Ala Val Arg Lys Glu Arg Val Cys Thr Ala Lys Glu Arg
50 55 60
Ile Ser Arg Met Pro Pro Cys Ala Ala Gly Lys Arg Ser Ser Ile Tyr
65 70 75 80
Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Tyr Glu Ala His Leu
85 90 95
Trp Asp Lys Ser Thr Trp Asn Gin Asn Gin Asn Lys Lys Gly Lys Gin
100 105 110
Gly Ala Tyr Asp Asp Glu Glu Ala Ala Ala Arg Ala Tyr Asp Leu Ala
115 120 125
Ala Leu Lys Tyr Trp Gly Ala Gly Thr Gin Ile Asn Phe Pro Val Ser
130 135 140
Asp Tyr Ala Arg Asp Leu Glu Glu Met Gin Met Ile Ser Lys Glu Asp
145 150 155 160
Tyr Leu Val Ser Leu Arg Arg Gin Leu His Asn Ser Arg Trp Asp Thr
165 170 175
Ser Leu Gly Leu Gly Asn Asp Tyr Met Ser Leu Ser Cys Gly Lys Asp
180 185 190
Ile Met Leu Asp Gly Lys Phe Ala Gly Ser Phe Gly Leu Glu Arg Lys
195 200 205
Ile Asp Leu Thr Asn Tyr Ile Arg Trp Trp Leu Pro Lys Lys Thr Arg
210 215 220
Gin Ser Asp Thr Ser Lys Thr Glu Glu Ile Ala Asp Glu Ile Arg Ala
225 230 235 240
Ile Glu Ser Ser Met Gin Gin Thr Glu Pro Tyr Lys Leu Pro Ser Leu
245 250 255
Gly Leu Gly Ser Pro Ser Lys Pro Ser Ser Val Gly Leu Ser Ala Cys
260 265 270
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Ser Ile Leu Ser Gin Ser Asp Ala Phe Lys Ser Phe Leu Glu Lys Ser
275 280 285
Thr Lys Leu Ser Glu Glu Cys Thr Leu Ser Lys Glu Ile Val Glu Gly
290 295 300
Lys Thr Val Ala Ser Val Pro Ala Thr Gly Tyr Asp Thr Gly Ala Ile
305 310 315 320
Asn Ile Asn Met Asn Glu Leu Leu Val Gin Arg Ser Thr Tyr Ser Met
325 330 335
Ala Pro Val Met Pro Thr Pro Met Lys Thr Thr Trp Ser Pro Ala Asp
340 345 350
Pro Ser Val Asp Pro Leu Phe Trp Ser Asn She Val Leu Pro Ser Ser
355 360 365
Gin Pro Val Thr Met Ala Thr Ile Thr Thr Thr Thr Asn Glu Val Ser
370 375 380
Ser Ser Asp Pro Phe Gin Ser Gin Glu
385 390
<210> 11
<211> 428
<212> PRT
<213> Lupinus angustifolius
<400> 11
Met Ala Ser Ser Ser Ser Asp Pro Gly Lys Ser Glu Ile Gly Gly Gly
1 5 10 15
Ala Ala Glu Thr Ser Glu Ala Ala Ala Val Ala Val Ala Val Thr Asn
20 25 30
Asp Gin Ser Leu Leu Tyr Arg Gly Lou Lys Lys Ala Lys Lys Glu Arg
35 40 45
Gly Cys Thr Ala Lys Glu Arg Ile Ser Lys Met Pro Pro Cys Ala Ala
50 55 60
Gly Lys Arg Ser Ser Ile Tyr Arg Gly Val Thr Arg His Arg Trp Thr
65 70 75 80
Gly Arg Tyr Glu Ala His Leu Arg Asp Lys Ser Thr Trp Asn Gin Asn
85 90 95
Gin Asn Lys Lys Gly Lys Gin Val Tyr Leu Gly Ala Tyr Asp Asp Glu
100 105 110
Glu Ala Ala Ala Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly
115 120 125
Pro Gly Thr Leu Ile Asn Phe Pro Val Thr Asp Tyr Thr Arg Asp Leu
130 135 140
Glu Glu Met Gin Asn Val Ser Arg Glu Glu Tyr Leu Ala Ser Lou Arg
145 150 155 160
Arg Lys Ser Ser Gly She Ser Arg Gly Ile Ser Lys Tyr Arg Ala Leu
165 170 175
Ser Ser Arg Trp Glu Pro Ser Tyr Ser Arg Phe Ala Gly Ser Asp Tyr
180 185 190
She Asn Ser Met His Tyr Gly Ala Gly Asp Asp Ser Ala Ala Glu Ser
195 200 205
Glu Tyr Ala Ser Gly She Cys Ile Glu Arg Lys Ile Asp Leu Thr Gly
210 215 220
His Ile Lys Trp Trp Gly Ser Asn Lys Ser Arg Gin Pro Asp Ala Gly
225 230 235 240
Thr Arg Leu Ser Glu Glu Lys Arg His Gly Phe Ala Gly Asp Ile Cys
245 250 255
CA 2998211 2018-03-16
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Ser Glu Pro Lys Thr Leu Glu Gin Lys Val Gin Pro Thr Giu Pro Tyr
260 265 270
Gin Met Pro Glu Leu Gly Arg Ser His Asn Glu Lys Lys His Arg Ser
275 280 285
Ser Ala Val Ser Ala Leu Ser Ile Leu Ser Gin Ser Ala Ala Tyr Lys
290 295 300
Ser Leu Gin Glu Lys Ala Ser Lys Lys Gin Glu Asn Ser Thr Asp Asn
305 310 315 320
Asp Glu Asn Glu Asn Lys Asn Thr Val Asn Lys Leu Asp His Gly Lys
325 330 335
Ala Val Glu Lys Ser Ser Asn His Asp Gly Gly Ser Asp Arg Val Asp
340 345 350
Ile Giu Ile Gly Thr Thr Gly Ala Leu Ser Leu Gin Arg Asn Ile Tyr
355 360 365
Pro Leu Thr Pro Phe Leu Ser Ala Pro Leu Leu Thr Ala Tyr Asn Thr
370 375 380
Val Asp Pro Ser Leu Val Asp Pro Val Leu Trp Thr Ser Leu Val Pro
385 390 395 400
Met Leu Ser Ala Gly Leu Ser Cys Pro Thr Gin Val Thr Lys Thr Glu
405 410 415
Thr Ser Ser Ser Tyr Thr Ile Phe Gin Pro Glu Gly
420 425
<210> 12
<211> 440
<212> PRT
<213> Ricinus communis
<400> 12
Met Ala Ser Ser Ser Ser Asp Pro Gly Leu Lys Pro Glu Leu Gly Gly
1 5 10 15
Gly Ser Gly Gly Giu Ser Ser Giu Ala Val Ile Ala Asn Asp Gin Leu
20 25 30
Leu Leu Tyr Arg Gin Leu Lys Lys Pro Lys Lys Glu Arg Gly Cys Thr
35 40 45
Ala Lys Glu Arg Ile Ser Lys Met Pro Pro Cys Thr Ala Gly Lys Arg
50 55 60
Ser Ser Ile Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Tyr
65 70 75 80
Chi Ala His Leu Trp Asp Lys Ser Thr Trp Asn Gin Asn Gin Asn Lys
85 90 95
Lys Gly Lys Gin Gly Ala Tyr Asp Asp Glu Glu Ala Ala Ala Arg Ala
100 105 170
Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Pro Gly Thr Leu Ile Asn
115 120 125
Phe Pro Val Thr Asp Tyr Ser Arg Asp Leu Glu Glu Met Gin Asn Val
130 135 140
Ser Arg Glu Glu Tyr Leu Ala Ser Leu Arg Arg Lys Ser Ser Gly Phe
145 150 155 160
Ser Arg Gly Ile Ser Lys Tyr Arg Gly Leu Ser Ser Gin Trp Asp Ser
165 170 175
Ser Phe Gly Arg Met Pro Gly Ser Glu Tyr Phe Ser Ser Ile Asn Tyr
180 185 190
Gly Ala Ala Asp Asp Pro Ala Ala Glu Ser Glu Tyr Val Gly Her Leu
195 200 205
CA 2998211 2018-03-16
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Cys Phe Glu Arg Lys Ile Asp Leu Thr Per Tyr Ile Arg Trp Trp Gly
210 215 220
Phe Asn Lys Thr Arg Glu Ser Val Ser Lys Ser Ser Asp Glu Arg Lys
225 230 235 240
His Gly Tyr Gly Glu Asp Ile Per Glu Leu Lys Ser Ser Glu Trp Ala
245 250 255
Val Gin Ser Thr Glu Pro Tyr Gin Met Pro Arg Leu Gly Met Pro Asp
260 265 270
Asn Gly Lys Lys His Lys Cys Ser Lys Ile Ser Ala Leu Ser Ile Leu
275 280 285
Ser His Ser Ala Ala Tyr Lys Asn Leu Gin Glu Lys Ala Ser Lys Lys
290 295 300
Gin Giu Asn Cys Thr Asp Asn Asp Glu Lys Glu Asn Lys Lys Thr Asn
335 310 315 320
Lys Met Asp Tyr Gly Lys Ala Val Glu Lys Ser Thr Ser His Asp Gly
325 330 335
Ser Asn Glu Arg Leu Gly Ala Ala Leu Gly Met Ser Gly Gly Leu Ser
340 345 350
Leu Gin Arg Asn Ala Tyr Gin Leu Ala Pro Phe Leu Ser A/a Pro Leu
355 360 365
Leu Thr Asn Tyr Asn Ala Ile Asp Pro Leu Val Asp Pro Ile Leu Trp
370 375 380
Thr Ser Leu Val Pro Val Leu Pro Ala Gly Phe Ser Arg Asn Ser Glu
385 390 395 400
Val Gly Met Gly Leu Gin Ile Val Ser Cys His Lys Asp Arg Asp Lys
405 410 415
Phe Asn Leu Tyr Leu Leu Ser Ala Gly Gly Val Ser Thr Phe Leu Leu
420 425 430
Leu Val Val His Trp Arg Phe Cys
435 440
<210> 13
<211> 428
<212> PRT
<213> Lupinus angustifclius
<400> 13
Met Ala Per Ser Ser Ser Asp Pro Gly Lys Ser Glu Ile Gly Gly Gly
1 5 10 15
Ala Ala Glu Thr Per Glu Ala Ala Ala Val Ala Val Ala Val Thr Asn
20 25 30
Asp Gin Ser Leu Leu Tyr Arg Cly Leu Lys Lys Ala Lys Lys Clu Arg
35 40 45
Gly Cys Thr Ala Lys Glu Arg Ile Ser Lys Met Pro Pro Cys Ala Ala
50 55 60
Gly Lys Arg Ser Ser Ile Tyr Arg Gly Val Thr Arg His Arg Trp Thr
65 70 75 80
Gly Arg Tyr Glu Ala His Leu Arg Asp Lys Ser Thr Trp Asn Gin Asn
85 90 95
Gin Asn Lys Lys Gly Lys Gin Val Tyr Leu Gly Ala Tyr Asp Asp Glu
100 105 110
Glu Ala Ala Ala Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Ply
115 120 125
Pro Gly Thr Leu Ile Asn Phe Pro Val Thr Asp Tyr Thr Arg Asp Leu
130 135 140
CA 2998211 2018-03-16
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Glu Glu Met Gin Asn Val Ser Arg Glu Glu Tyr Leu Ala Ser Leu Arg
145 150 155 160
Arg Lys Ser Ser Gly Phe Ser Arg Gly Ile Ser Lys Tyr Arg Ala Leu
165 170 175
Ser Ser Arg Trp Glu Pro Ser Tyr Ser Arg Phe Ala Gly Ser Asp Tyr
180 185 190
Phe Asn Ser Met His Tyr Gly Ala Gly Asp Asp Ser Ala Ala Glu Ser
195 200 205
Glu Tyr Ala Ser Gly Phe Cys Ile Glu Arg Lys Ile Asp Leu Thr Gly
210 215 220
His Ile Lys Trp Trp Gly Ser Asn Lys Ser Arg Gin Pro Asp Ala Gly
225 230 235 240
Thr Arg Leu Ser Glu Glu Lys Arg His Gly Phe Ala Gly Asp Tie Cys
245 250 255
Ser Glu Pro Lys Thr Leu Glu Gin Lys Val Gin Pro Thr Glu Pro Tyr
260 265 270
Gin Met Pro Glu Leu Gly Arg Ser His Asn Glu Lys Lys His Arg Ser
275 280 285
Ser Ala Val Ser Ala Leu Ser Ile Leu Ser Gin Ser Ala Ala Tyr Lys
290 295 300
Ser Leu Gin Glu Lys Ala Ser Lys Lys Gin Glu Asn Ser Thr Asp Asn
305 310 315 320
Asp Glu Asn Glu Asn Lys Asn Thr Val Asn Lys Leu Asp His Giy Lys
325 330 335
Ala Val Glu Lys Ser Ser Asn His Asp Gly Gly Ser Asp Arg Val Asp
34C 345 350
Ile Glu Ile Gly Thr Thr Gly Ala Leu Ser Leu Gin Arg Asn Ile Tyr
355 360 365
Pro Leu Thr Pro Phe Leu Ser Ala Pro Leu Leu Thr Ala Tyr Asn Thr
370 375 380
Val Asp Pro Ser Leu Val Asp Pro Val Leu Trp Thr Ser Leu Val Pro
385 390 395 400
Met Leu Ser Ala Gly Leu Ser Cys Pro Thr Gin Val Thr Lys Thr Glu
405 410 415
Thr Ser Ser Ser Tyr Thr Ile Phe Gin Pro Glu Gly
420 425
<210> 14
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<220>
<221> X
<222> (4)..(4)
<223> Threonine (T) or Serine (S)
<400> 14
Arg Gly Val Xaa Arg His Arg Trp Thr Gly Arg
1 5 10
CA 2998211 2018-03-16
252
<210> 15
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<220>
<221> X
<222> (1)..(1)
<223> Phenylalanine (F) or Tyrosine (Y)
<400> 15
Xaa Giu Ala His Leu Trp Asp Lys
1 5
<210> 16
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<400> 16
Asp Leu Ala Ala Leu Lys Tyr Trp Gly
<210> 17
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> X
<222> (5)..(5)
<223> Serine (S) or Alanine (A)
<220>
<221> X
<222> (8)..(6)
<223> any amino acid
CA 2998211 2018-03-16
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<400> 17
Ser Xaa Gly Phe Xaa Arg Gly Xaa
1 5
<210> 18
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<220>
<221> MISC FEATURE
<222> (3)¨(3)
<223> Histidine (H) or Glutamine (Q)
<220>
<221> MISC FEATURE
<222> (6)¨(6)
<223> Arginine (R) or Lysine (K)
<220>
<221> MISC_FEATURE
<222> (13)..(13)
<223> Arginine (R) or Lysine (K)
<400> 18
His His Xaa Asn Gly Xaa Trp Glu Ala Arg Ile Gly Xaa Val
1 5 10
<210> 19
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> conserved sequence
<220>
<221> MISC FEATURE
<222> (7).7(7)
<223> any amino acid
<400> 19
Gln Glu Glu Ala Ala Ala Xaa Tyr Asp
1 5
CA 2998211 2018-12-17
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<210> 20
<211> 11142
<212> DNA
<213> Artificial Sequence
<220>
<223> TDNA sequence
<400> 20
tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60
aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120
tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa totgctagtg gatctcccag 180
tcacgacgtt gtaaaacgag cgccctagaa tctaattatt ccattcagac taaattagta 240
taagtacttt cttaatcaat aaataataat taataattta ttagtaggag tgattgaatt 300
tataatatat cttttttaat catttaaaga atcttatatc tttaaattga caagagtttt 360
aaatggggag agtattatca tatcacaagt aggattaatg tgttatagtt tcacatgcat 420
tacgataagt tgtgaaagat aacattatta tatataacaa tgacaatcac tagcgatcga 480
gtagtgagag tcgtottatt acactttctt ccttcgatct gtcacatagc ggcggcccga 540
attctcacac aagatagttg caagacactg aagtggtgat agtggtagta gaagaagcag 600
aatcggtaga aaggcaagac aatggagaag atgaagatgg tggagattct cttcccacaa 660
cgcagcaatc aagattttca aggttaaggc actcgtgatt tccatcatcg aacatgaagt 720
cgatgttatc ctcgaaagca agctcgttga agagttctag gtactcaatt gggttctcgt 780
tagcaagott ttgatcggta aggaatgagg agaatccagt atccatcatg cagaagttcc 840
aagcaagttc gttgttatct ccgcacctat ccatttccat gatggtggaa gaatcaatgc 900
agcagttaac aacggcagct tcctcagaat atcccacaat ttcagcctct tgttgctcag 960
cottctattc ctctttttct tcttcctctt gaggtggttc ctcaacgtat tgttgcttaa 100
cctcttccct aggttcctct ttagcttctc tagtctcaac ctcttgctta gcctcaacaa 1080
gaataccctc ttgatggtta gcctggttaa ctgagaatgg gaaaacgccc ttattattaa 1140
gcctgtcgat gtagttggag atatcgaagt tggtaacagc gttagcacct ctgtactcaa 1200
taacagccat atcataagca gctgcagcct cttcttgagt gttgtaaatt ccgaggtaga 1260
ggtacttgtt tccgaaaact cttccaatcc tagcttccca tcttccgtta tgatgatgcc 1320
tagcaactcc cctatactta gaaactcccc tagagaatcc agatgactgc cttctaaggg 1380
aagcaagata ctcttctttg gtcaccctct gcatctcttc aagttctttg gtgtaagtct 1440
cagctgggaa gttaagaatg gtatctgogc cccaatactt aagagcagca agatcatagg 1500
tatgagcagc aacctattca gaatcataag ctccaaggta aacctgcttg cccttcttgt 1560
tttggatgga gttccaagag gacttatccc aaaggtgagc ttcgaatctt ccagtccatc 1620
tatgcctagt aacacctctg tagatagatg accttctggt agaagctgga gaagttaggt 1680
tatgagactt atcgccagat ggagatgact tcttagccct cttagctotc tttggtcttg 1740
gagcttcaga ttgaattggg ctagaggtag tagtagaaga ggacactgaa gaagatggag 1600
aactagagca ggtagaggta gtgagcatct tcttcatgaa ttctgttctt ctttactctt 1860
tgtgtaactg aagtttggtc tagtgatttg gtcatctata tataatgata acaacaatga 1920
gaacaagctt tagagtgatc ggagggtcta ggatacatga gattcaagtg gactaggatc 1980
tacaccgttg gattttgagt gtggatatgt gtgaggttaa ttttacttgg taacggccac 2040
aaaggcctaa ggagaggtgt tgagaccctt atcggcttga accgctggaa taatgccacg 2100
tggaagataa ttccatgaat cttatcgtta tctatgagtg aaattgtgtg atggtggagt 2160
ggtgattgct cattttactt gcctggtgga cttggccctt tccttatggg gaatttatat 2220
tttacttact atagagcttt catacctttt ttttaccttg gatttagtta atatataatg 2280
gtatgattca tgaataaaaa tgggaaattt ttgaatttgt actgctaaat gcataagatt 2340
aggtgaaact gtggaatata tatttttttc atttaaaagc aaaatttgcc ttttactaga 2400
attataaata tagaaaaata tataacattc aaataaaaat gaaaataaga actttcaaaa 2460
aacagaacta tgtttaatgt gtaaagatta gtcgcacatc aagtcatctg ttacaatatg 2520
ttacaacaag tcataagccc aacaaagtta gcacgtctaa ataaactaaa gagtccacga 2580
aaatattaca aatcataagc ccaacaaagt tattgatcaa aaaaaaaaaa cgcccaacaa 2640
agctaaacaa agtccaaaaa aaacttctca agtctccatc ttcctttatg aacattgaaa 2700
actatacaca aaacaagtca gataaatctc tttctgggcc tgtcttccca acctcctaca 2760
tcacttccct atcggattga atgttttact tgtacctttt ccgttgcaat gatattgata 2820
CA 2998211 2018-03-16
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gitatgtztgt gaaaactaat agggttaaca atcgaagtca tggaatatgg atttggtcca 2880
agattttccg agagctttct agtagaaagc ccatcaccag aaatttacta gtaaaataaa 2940
tcaccaatta ggtttottat tatgtgccaa attcaatata attatagagg atatttcaaa 3000
tgaaaacgta tgaatgttat tagtaaatgg tcaggtaaga cattaaaaaa atcctacgtc 3060
agatattcaa ctttaaaaat tcgatcagtg tggaattgta caaaaatttg ggatctacta 3120
tatatatata atgctttaca acacttggat ttttttttgg aggctggaat ttttaatcta 3180
catatttgtt ttggccatgc accaactcat tgtttagtgt aatactttga ttttgtcaaa 3240
tatatgtgtt cgtgtatatt tgtataagaa tttctttgac catatacaca cacacatata 3300
tatatatata tatatattat at,atcatgca cttttaattg aaaaaataat atatatatat 3360
atagtgcatt ttttctaaca accatatatg ttgcgattga tctgcaaaaa tactgctaga 3420
gtaatgaaaa atataatcta ttgctgaaat tatctcagat gttaagattt tcttaaagta 3480
aattctttca aattttagct aaaagtottc taataactaa agaataatac acaatctcga 3540
ccacggaaaa aaaacacata ataaatttgg ggcccctaga atctaattat tctattcaga 3600
ctaaattagt ataagtactt ttttaaccaa taaataataa ttaataattt attagtagga 3660
gtgattgaat ttataatata ttttttttaa tcatttaaag aatcttatat ctttaaattg 3720
acaagagttt taaatgggga gagtgttatc atatcacaag taggattaat gtgttatagt 3780
ttcacatgca ttacgataag ttgtgaaaga taacattatt atatataaca atgacaatca 3840
ctagcgatcg agtagtgaga gtcgtattat tacactttct tccttcgatc tgtcacatgg 3900
cggcggcccg cggccgcttc attactcgag ccaggaggat ggatcgatgc tggtctgaga 3960
ccctgctacc ggttgctgac tgaactgctc ggcacggtcc ttcatttcac gggccttgct 4020
cgccaacttt gtcttggccg actccaacta atccgctccg ggtggatgtt tccccatcag 4080
gtaacggtag atccaggaca gcacagacag agcagcaaca ccaaatcccc cgcttgccag 4140
aaaacccgct cccaacagga agatggtgat gactgcagat cagaaaaact cagattaatc 4200
gacaaattcg atcgcacaaa ctagaaacta acaccagatc tagatagaaa tcacaaatcg 4260
aagagtaatt attcgacaaa actcaaatta tttgaacaaa tcggatgata tctatgaaac 4320
cctaatcgag aattaagatg atatctaacg atcaaaccca gaaaatcgtc ttcgatctaa 4380
gattaacaga atctaaacca aagaacatat acgaaattgg gatcgaacga aaacaaaatc 4440
gaagattttg agagaataag gaacacagaa atttacctgc agggaccagt acaggcgaga 4500
agatcaccag gagaggtgtg gcgattgtca gcgcaatgac cgttccagcc agggtcaacc 4560
cggataacac caacaggcta cctccggcag taaccgcggt cgctgccttt acaacacgct 4620
gagcacgagg ttgcagttgc aagtgggggg cacgtgtttg ttgctgctgc ccgtagtgct 4680
ctgccatggt tttttttaac ggagcaagcg gccgctgttc ttctttactc tttgtgtgac 4740
tgaggtttgg tctagtgott tggtcatcta tatataatga taacaacaat gagaacaagc 4800
tttggagtga toggagggtc taggatacat gagattcaag tggactagga tctacaccgt 4860
tggattttga gtgtggatat gtgtgaggtt aattttactt ggtaacggcc acaaaggcct 4920
aaggagaggt gttgagaccc ttatoggctt gaaccgctgg aataatgcca cgtggaagat 4980
aattccatga atcttatcgt tatctatgag tgaaattgtg tgatagtgga gtqgtgcttg 5040
ctcattttac ttgcctggtg gacttggccc tttcottatg gggaatttat attttactta 5100
ctatagagct ttcatacctt ttttttacct tggatttagt taatatataa tggtatgatt 5160
catgaataaa aatgggaaat ttttgaattt gtactgctaa atgcataaga ttaggtgaaa 5220
ctgtggaata tatatttttt tcatttaaaa gcaaaatttg ccttttacta gaattataaa 5280
tatagaaaaa tatataacat tcaaataaaa atgaaaataa gaactttcaa aaaacagaac 5340
tatgtttaat gtgtaaagat tagtcgcaca tcaagtcatc tgttacaata tgttacaaca 5400
agtcataagc ccaacaaaat tagcacgtct aaataaacta aagagtccac gaaaatatta 5460
caaatcataa gcccaacaaa gttattgatc aaaaaaaaaa aacgcccaac aaagctaaac 5520
aaagtccaaa aaaaacttct caagtctcca tcttccttta tgaacattga aaactataca 5580
caaaacaagt cagataaatc tctttctggg cctgtcttcc caacctccta catcacttcc 5640
ctatcggatt gaatgtttta crtgtacctt ttccgttgca atgatattga tagtatgttt 5700
gtcaaaacta atagggtcaa caatcgaagt catggaatat ggatttggtc caagattttc 5760
cgagagcttt ctagtagaaa gcccatcacc agaaatttac tagtaaaata aatcaccaat 5820
taggtttctt attatgtgcc aaattcaata taattataga ggatatttca aatgaaaacg 5880
tatgaatgtt attagtaaat ggtcaggtaa gacattaaaa aaatcctacg tcagatattc 5940
aactttaaaa attcgatcag tgtggaattg tacaaaaatt tgggatctac tatatatata 6000
taatgcttta caacacttgg attttttttt ggaggctgga atttttaatc tacatatttg 6060
ttttggccat gcaccaactc attgtttagt gtaatacrtt gattttgtca aatatatgtg 6120
ttcgtgtata tttgtataag aatttctttg accatataca cacacacata tatatatata 6180
CA 2998211 2018-03-16
256
tatatatatt atatatcatg cacttttaat tgaaaaaata atatatatat atatagtgca 6240
ttttttctaa caaccatata tgttqcgatt gatctgcaaa aatactgcta gagtaatgaa 6300
aaatataatc tattgctgaa attatctcag atgttaagat tttcttaaag taaattcttt 6360
caaattttag ctaaaagtct tgtaataact aaagaataat acacaatctc gaccacggaa 6420
aaaaaacaca taataaattt cggcgcgccg cgtattggct agagcagctt gccaacatgg 6480
tggagcacga cactctcgtc tactccaaga atatcaaaga tacagtctca qaagaccaaa 6540
aggctattga gacttttcaa caaagggtaa tatcgggaaa cctcctcgga ttccattgcc 6600
cagctatctg tcacttcatc aaaaggacag tagaaaagga aggtggcacc tacaaatgcc 6660
atcattgcga taaaggaaag gctatcgttc aagatgcctc tgccgacagt ggtcccaaag 6720
atggaccccc acccacgagg agca.,,cgtgg aaaaagaaga cgttccaacc acgtcttcaa 6780
agcaagtgga ttgatgtgat aacatggtgg agcacgacac totcgtotac tccaagaata 6840
tcaaagatac agtctcagaa gaccaaaggg ctattgagac ttttcaacaa agggtaatat 6900
cgagaaacct cctcggattc cattgcccag ctatctgtca cttcatcaaa aggacagtag 6960
aaaaggaagg tagcacctac aaatgccatc attgcgataa agaaaaggct atcgttcaag 7020
atgcctctgc cgacagtggt cccaaagatg gaccgccacc cacgaggagc atcgtggaaa 7080
aagaagacgt tccaaccacg tottcaaagc aagtggattg atgtgatatc tccactgacg 7140
taagggatga cgcacaatcc cactatcctt cgcaagacct tcctctatat aaggaagttc 7200
atttcatttg gagaggacac gctgaaatca ccagtctctc tctacaaatc tatctctgcg 7260
atcgcatggc gattttggat tctgctgacg ttactacggt gacggagaac ggtggcggag 7320
agttcgtcga tcttgatagg cttcgtcgac ggaaatcgag atcggattct tctaacggac 7380
ttcttctctc tggttccgat aataattctc cttoggatga tgttggagct cccaccgagg 7440
ttagggatcg gattgattcc gttgttaacg atgacgctca gggaacagcc aatttggccg 7500
gagataataa cggtgatggc gataataacg gtggtggaag aggcggcgga gaaggaagag 7560
gaaacgccga tgctacgttt acgtatcgac cgtoggttcc agctcatcgg agggcgagag 7620
agagtccact tagctccgac acaatcttca aacagagcca tgccggatta ttcaacctct 7680
gtgtagtagt tcttattgct gtaaacagta gactcatcat cgaaaatctt atgaagtatg 7740
gttggttgat cagaacggat ttctggttta gttcaagatc gctgcgagat tggccgcttt 7800
tcatgtattg tatatccctt tcgatctttc ctttggctgc ctttacggtt gagaaattgg 7860
tacttcagaa atacatatca gaacctgttg tcatctttct tcatattatt atcaccatga 7920
cagaggtttt gtatccagtt tacgtcaccc taaggtgtga ttctgctttt ttatcaggtg 7980
tcactttgat gctcctcact tgcattgtgt ggctaaagtt ggtttcttat gctcatacta 8040
gctatgacat aagatcccta gccaatgcag ctgataaggc caatcctgaa gtctcctact 8100
acgttagctt gaagagottg gcatatttca tgatcgctcc cacattgtgt tatcagccaa 8160
gttatccacg ttctgcatgt atacggaagg gttgggtggc tcgtcaattt gcaaaactag 8220
tcatattcac cggattcatg ggatttataa tagaacaata tataaatcct attatcagga 8280
actcaaagca toctttgaaa ggcgatcttc tatatgctat tgaaagagtg ttgaagcttt 8340
cagttccaaa tttatatgtg tggctctgca tgttctactg cttottccac ctttggttaa 8400
acatattggc agagcttctc tgcttcgggg atcgtgaatt ctacaaagat tgatggaatg 8460
caaaaagtgt gagagattac tggagaatgt ggaatatgcc tgttcataaa tggatggttc 8520
gacatatata cttcccgtgc ttgcgcagca agataccaaa gacactcgcc attatcartg 8580
ctttcctagt ctctgcagtc tttcatgagc tatgcatcac agttccttgt cgtctcttca 8640
agctatgggc ttttcttggg attatgtttc aggtgacttt ggtcttcatc acaaactatc 8700
tacaggaaag gtttggctca acggtgggga acatgatctt ctggttcatc ttctgcattt 8760
tcggacaacc gatgtgtgtg cttctttatt accacgacct gatgaaccga aaaggatcga 8820
tgtcatgagc gatcgcgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc 8880
tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat 8940
aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta gagtoccgca 9000
attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc 9060
qcgcgoggtg tcatctatgt tactagatcc ctgcagggcg tattggctag agcagcttgc 9120
caacatggtg gagcacgaca ctctcgtcta ctccaagaat atcaaagata cagtctcaga 9180
agaccaaagg gctattgaga cttttcaaca aagggtaata tcgggaaacc tcctcggatt 9240
ccattgccca gctatctgtc acttcatcaa aaggacagta gaaaaggaag gtggcaccta 9300
caaatgccat cattgcgata aaggaaaggc tatcgttcaa gatgcctctg ccgacagtag 9360
tcccaaagat ggaccoccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac 9420
gtcttcaaag caagtggatt gatgtgataa catggtggag cacgacactc tcgtctactc 9480
caagaatatc aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag 9540
CA 2998211 2018-03-16
9T-0-8TOZ TTZ866Z VD
0801 65pooue-1.1-
4 146-11=22p -48-4-4qeq2eo oeee41q4o5 gooeeqqqqo qbqoae-ego6
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096 b5pbqq4eoP
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006 qebobobbbb
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0D'8 pbbb6go3b eo-
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08Le2eobo4005 epob.640525 6qbae6obbo ob53gqqe6o 4obbeo5.5bb e.64e.E=2bq
OZL Too6bo5o8.6
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099 3330 3E
ep55o6oqoo eq-ebbq5q4B 3E6330336e 0533e3e6e5 obbIlloofre
009 p5ogbooboe
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Obq 647,56go0b6
opeo4obopb pqopooqbuo 4-eopobpobb popqpbogou oo-eoqe-eepo
08f7 eobbo43beo
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2,q4Peoq5 obepqoqi5qq bePqqpq-454 54peoboo4b D22PP211.20 PM-PO:DOD-PO
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OZT e4ao4qqbee
eqpeobb441 epeeeo4.45o 12bq63b3pp e5qoqqo445 ebo-261wgq
09 3303 33D
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TZ <0017>
aouanbas io4oan <zz>
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aouanbas TpToTjTqav <ETz>
kiNCI <ZTZ>
617L91 <ITZ>
TZ <OTZ>
ZbITI oo
4bpo4p000b eobbPoPTeb oqoPo3s.o.4Q ePPopobboq
00ITT obpobboopb
oopoqc62oe epobeoobpo opoobqooqe -4-24-e-eoeoae 36q-44p
Of7OTT pogbp.612plo
161q6epqle qlfil6-12eob oolBoeeeep qqeoegl)poo oo-eope-elaq
08601 qbboebool
b3eboqe5pq oe74.642Loq eoq6q66o0o 6obal-eq4ep eqebbeoee
0Z601 eoBobobele
qepeeo-elp.ep belipboboEq e-eqqq_eoee -44-e-eob000g babpqq-ebaP
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ql2pqegeoq eqap6q2bob ;bob q gbilooq2P6q gebe4goa4
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08901 4344335o-4e
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0Z901 6156gep5ob6
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qbbbqobboo .5157õbg3ebog eoqP6b4oq. gq-aoboobbq ep2pbbqbbq
00801 eoTeqep.boo
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OOT obobElpeogo
66P33bo1-zb qoppboobeo 303b3g3565 bp3-le36ebe eboefe,DE
0801 5-4e86P3b
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OUOT 52POOPOOPb
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09101 6.6qpoqe33q
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00101 bbobbbqq
2g3bqobb-2,0 pbbbePbbBo 5pebq3P3--2.6 q;boE'bo;o6 2,6qobeobob
OtTOT q433g45305
bopboeoobb ;355.453-eq obbobob-eob bpboebepog qoppbqp.281
08001 333b4b033-4
5qopeboo26 e231.64qa1q oqqaboobLE bbbb-eobobp oq6-ao0600q
01001 q5qb335oo5
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0966 55:0ao5
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01786 m24egegogo
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08L6 3g34eqebg0
4p.6-44ebbq15 PP3EPPP3qq 0463P33EPO 3446oebee6 ee-epeaqbo
0ZL6 gEobebbe6o
2000P33000 P.5.61PE,PEPO pobblbeo-e. B336131336 qebppcqq.63
0996 4e3appp5
bpppgebob4 qeo4e3o6Te ep3E,q33e36 54b0ebb-ep ebegbeoeb
0096 bePueoqeog 4o-eoqbqoqp 4obecoobqg 33 6b3
D32.00P2P56 504-PqPPMb
LSZ
9T-E0-8TOZ ITZ866Z 110
ON7V q.-334262-ebe eol.Dlebbue peeppbeobo 5D-87:.qe5eo.6 eD61.2-epeq4-2,
64qqqqq4bb
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peeeobbooq ebo5pq 564q6e6Pae e-ebboqqope
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boeqopeqop
09Zi7 bbqbb46P26 -44oqq.bubPo uqobqbbobb e4baPqbapb 3bebeo6Pq; -ebi5poeeqbb
00217 goppobeobe ob64oppobo 4e44op6opo eb2P46b3op eooqbebya oq63Tegoe2
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6b4Dbeepaq.
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Dbo6.6q.6o6e
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096E B1531 0313 ETe661p3op olq4bobbeo pegEbeeeqls, loebbecebo operTtobb4
006E .65pbpogbpp oqoboebogu -eeeeopogeo beboebqopo Doobooqobb egeopqqm
OBE bobbqp5q45 oboobbpe-22 pgbpovebbp oobbeepeob po36beep23 6e51..blp.opT2
09LE beepbbeobo epqebbb5Po 1P-26P3P33q eqq.66oegpe 16636beeeo go2owbeol
OZLE e4bbobebob 635436b3l. boqbbolobo b4oboloet peogo6o400 .q.o600-4-404
099E 3p-4E6E543e qq43505eop beo5eo4m5.6 656-406booq 4beogb65bq bqqopbbbeg
009E oboeueob42, eobgbbqopp bo,p6gpeo4b 564po6o2oe abbgbbgoob 6obpo6bebo
OPSE bqqbpbeubo 643obepbo6 boqbbpobeb obobbqbepb pebgboboop eopqq.e.65pq
0817E 2563646423 looboo4i.op pepbbqDbEE olob000eb po5e55600e oboao5P011
OZPE leoeboqqb lebboub000 Houboob baeoboo
pq6epbebbb qp4poq4epe
o9es 5aeoe4pepo ebobbgobq4 qbgob4boqb p4T2ppbba6o pEo4,26-4304 Ebeop64q.b3
00CE 3pq.6opep2b eep554boop bb400gb3bo oqqobooeob qp54o5oqbq 6.6plep66-4D
OZE peo56b4gbo booeboogbo polg356bqe 53.156oe6o.66 2pegoo6o5o q.6goob4e-eb
081E beflopEdaeeb Depeq-eq.bb opEbqeeo4e 6oqbbqp566 400ebEepob qq4abebce4
OZIE pq4boeolgo oeporeboobe bobboqegoo Doeobobepb obol2;bebbq qbgbbeobeo
090E bbqbgbogo ebofigeboe bpobbbob4o beboqbobbo peo2qopeog ebe.bqbppb
000E bepoevogOo eopqqq.geoq bbpeoee'ePo bgobobbebo bob000geey ebepoevoho
0f,63 baeooepqq.6 -4 336338 opobboeoe6 pp2e=6-486 pfiebo5ge5o epebboo8bq
088Z 6336p61.4e4 ebaqqbqqoo ;q1obpobqe eebbePoqP6 eobeobee6o epeopppbqo
038Z Elqqboboobe eobepoobbe bobeoq56eo Eveeobbqop ebEcebobboo boopPeeebo
09L3 5eubepooe6 oEbqp4oepb beboboeoeb ogeoppbgeb pq..bep6cb154 qq5.6,20b6ob
OOLZ bebbeoeuto qo4boqbobo qqbabp5b4E, coboobbo4e pobob3335q poob433000
Ot'93 gobb43e2ob 4.bobeoeb35 obeboqe,b2 3obbow4o62 eobpoophEy; pbo4D6bogb
08SZ ooqbeopqlo oe2263600p obobqbob 5000boo
beepboTeop ee6pob-486
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09D'Z leubgpe6gb bbebbeepoo Do4eeo5bbb oqbebqbobb oppobqgpop ob.156epqepb
00P'Z beoqpueoqe bbobbeopPo bbeeopeopb bqoqPq4ebb gEbepqeboo bqq.eogqop6
Of7EZ oop66bop4o 4.65aebe3ob EcebbeebPb aegb-epooeq eebboqqpbp p35bbeo515
0833 o44pgb5gob oqabbbeeob 6544-legbep c615peoJobb e6pobbeoqo go5.6qqqqbq
OZZZ 62-eDqeoboo .4;o4loeboo eobobebbeo beepP.605.6 06412boEpo.6
p6154.6pEpq
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OOTZ e6,66643e4a opb4qq4442 gob-eboqbqp gbepeebeb bEb442qpbb eb156poqebo
OD'OZ lbboogbob2. oggootqlpo e5q-elibbqbp .eoebbobnb-2 obbobeebeb
6.64q=peqq.
0861 erno6,645p eqbepeobb4 25pT2,pbgbgq qole3ee362 Debebb6i.po e6obboe333
0361 4444olbiqo p256E5eeBo pr)beppebbo ebeppqqqq.4 le6qeqbqob ebobobooTe
0981 beuPqqq.poo Doeoebeebe P6b.bqoePP-e 6o5qq.ebbq.b g.eboob6qoq
eboeeTesbq
0081 peq4peggeb 64geTboc6e ggoboobpoe bP44ob-eqp,2 bzeq.pqopo4 5q2,5,634p4
OVLT eppboqepo; 3-2344qp4o5 beoq-eobgbe bbobqpqb43 bcboqeggeb -2Peabqopo5
0891 eepopbqb ep5427,beb bbo oogbob61-
25 pao_5261.6e6 geogo6loge
0391 2ob26,643.56 qeble3663e efq.00e35-2, poqb&ePeoo qqbqpoblo6 eeebbeP6.51
0951 obbqegobqp bqeop6.6ael? 5.563-eub64 bqebqpqope 30e5b.6peq eq_bboobeoe
00gI bEopEqueae eggqe7..eloo eep-52.6qeece b-ebbb2,bbqo bep;pqpqbb
eegobooqo
or7i4b4eebbel2b boegebueue 4bob2,obooe gPeeppLoge bqoeppupeb ;qeebbooeD
08E1 legppbebqp ,obb4p1.=2 Pqe-e-qpet,E, pp6bebe.2 .5q.54opTep .2-11.48-
4,66613
OZEI ggoggobeq; eeqpg7.bqqo gbol_gbebb-a Teeb-1,6eoee bbeeobgeeb eq.q,bbeE4
0931 44p4LB4O4q 4.45qp.opep? -eb-444Deepe e-ee61q4c-es po6oegq;.5 7,-
eqoE.4e6o4
003I bpolobbo4e -epeoqz-46.6q bqq2eqepoo eoTeobpobb qq.bobogoqo b000bboop4
0f7I1 opoupboqoq w000ppobq b6bbbbebo obouabo600 ebq5obobqb q000bobqob
8SZ
259
ttgatctttt ctacggagtc tgacgctcag tggaacgaaa actcacgtta agggattttg 4500
atcatgagat tatcaaaaag gatcttcacc tagatccttt tggatctcct gtggttggca 4560
tgcacataca aatggacgaa cggataaacc ttttcacgcc cttttaaata tccgattatt 4620
ctaataaacg ctcttttctc ttaggtttac ccgccaatat atcctgtcaa acactgatag 4680
tttaaactga aggcgggaaa cgacaatctg ctagtggatc tcccagtcac gacgttgtaa 4740
aacgggcgtc tgcgatcgct gaagttccta tacttttcag agaataggaa cttcggaata 4800
ggaacttccc ataggatcta gtaacataga tgacaccgcg cgcgataatt tatcctagtt 4860
tgcgcgctat attttgtttt ctatcgcgta ttaaatgtat aattgcggga ctctaatcat 4920
aaaaacccat ctcataaata acgtcatgca ttacatgtta attattacat gcttaacgta 4980
attcaacaga aattatatga taatcatcgc aagaccggca acaggattca atattaagaa 5040
actttattgc caaatgtttg aacgatcacg ctagcggata acaatttcac acagggatat 5100
cactagtaaa aagtaccgag ctcctgcagt atcgatgcgg ccacaaaatc gacgaattct 5160
cattagcaga actcaagatg ctgatcctct ggaacgttga acttgagctt gtgttcctcg 5220
aaaagcttgc acaactcttt gatgtaacgc tggtgaagtc tatcaacttc ctctctagaa 5280
ggctgaggag tcattagaac ctcgataggc tttccaacga taatagtgat aggctgtctg 5340
aaaggcatga gtccgaaaga gtattggaaa actccacttc catggaaaag tggaaggctg 5400
attcccataa tattttggag tctgttctgg atccatctaa gccaagttcc aggagtgttc 5460
tcaacctggt tgaagaggtt gttctctccg aatgagaaga taggaacaag aggagcacca 5520
tgcataagag caagtctgat gaatccctta cggttcttca agagaagtct gtaagcacca 5580
ggtctagcat caagagcctc ttgagcacct ccaacgatga taacaagaag gtttccacca 5640
ccctttctgc taaggatgtg atcagcagaa actttctcgc tagacacgag tccaccagac 5700
ataatgtaat ctctgaagaa tggagccctg aaccaaacag taagcatcat aaggtaggat 5760
ctgattccag gqaacaaaga ggtgaatcca gtagactcag tacagaggtt aaggaaagca 5820
ccagcagcaa gaacaccatg aggatggaat ccagcaatgt agttacggct aggatcaagc 5880
tcagcagtct taacgagaga cacagggaag taatccttca tgtacttaca gatggccaat 5940
cttctgaaga attggatagg tctaccacct tgtctaggct tatcccaatc caagtaccac 6000
caggtagcgt aaagaacaga gaaaagccag aacctggtga acaagagtcc aacgaagata 6060
acgatgcaga gttgagcaag agcaaggaat gagaaaaccc actgaagaac agcgaaagtc 6120
tgcaatcttc tctcccaagg aacaagaagt ggagcgaact cgaccatgaa ttcagtcccc 6180
cgtgttctct ccaaatgaaa tgaacttcct tatatagagg aagggtcttg cgaaggatag 6240
tgggattgtg cgtcatccct tacgtcagtg gagatatcac atcaatccac ttgctttgaa 6300
gacgtggttg gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg 6360
ggaccactgt cagcagaggc atcttcaacg atggcctttc ctttatcaca atgatggcat 6420
ttgtaggagc caccttcctt ttccactatc ttcacaataa agtgacagat agctgggcaa 6480
tggaatccga ggaggtttcc ggatattacc ctttgttgaa aaatatcaat tgccatttgg 6540
tcttctgaga ctgtatcttt gatatttttg gagtagacaa gtgtgtcgtg ctccaccatg 6600
ttgacgaaga ttttcttctt gtcattgagt cgtaagagac tctgtatgaa ctgttcgcca 6660
gtctttacgg cgagttctgt taggtcctct atttgaatct ttgactccat gggatccaag 6720
ggccctagaa tctaattatt ctattcagac taaattagta taagtatttt tttaatcaat 6780
aaataataat taataattta ttagtaggag tgattgaatt tataatatat tttttttaat 6840
catttaaaga atcttatatc tttaaattga caagagtttt aaatggggag agtgttatca 6900
tatcacaagt aggattaatg tgttatagtt tcacatgcat tacgataagt tgtgaaagat 6960
aacattatta tatataacaa tgacaatcac tagcgatcga gtagtgagag tcgtcttatt 7020
acactttctt ccttcgatct gtcacatggc ggcggcccga attctcacac aaggtagttg 7080
caagacactg aagtggtggt agtggtagta gaagaagcag aatcggtaga aaggcaagac 7140
aatggagaag atgaagatgg tggagattct cttcccacaa cgcagcaatc aaggttttca 7200
aggttaaggc actcgtgatt tccatcatcg aacatgaagt cgatgttatc ctcgaaagca 7260
agctcgttga agagttctgg gtactcaatt gggttctcgt taacaaggtt ttgatcggta 7320
aggaatgggg agaatccagt atccatcatg cagaagttcc aagcaagttc gttgttatct 7380
ccgcacctat ccatttccat gatggtggaa gaatcaatgc agcagttaac aacggcagct 7440
tcctcagaat atcccacaat ttcagcctct tgttgctcag ccttctattc ctctttttct 7500
tcttcctctt gaggtggttc ctcaacgtat tgttgcttaa cctcttccct aggttcctct 7560
ttagcttctc tagtctcaac ctcttgctta gcctcaacaa gaataccctc ttgatggtta 7620
gcctggttaa ctaggaatgg gaaaacgccc ttcttcttaa gcctgtcgat gtagttggag 7680
atatcgaagt tggtaacagc gttagcacct ctgtactcaa tagcagccat atcataagca 7740
gctgcagcct cttcttgagt gttgtaagtt ccgaggtaga ggtacttgtt tccgaaaact 7800
CA 2998211 2018-03-16
260
cttccaatcc tagattacca tattccgtta tgatgatgcc tagcaactcc cctatactta 7860
gaaactcccc tagagaatcc agatgactgc cttctaaggg aagcaagata ctottotttg 7920
gtcaccctct gcatctcttc aagttctttg gtgtaagtct cagctaggaa gttaagaatg
7980 \
gtatctgggc cccaatactt aagagcagca agatcatagg tatgagcagc agcctcttca 8040
gaatcataag ctccaaggta aacctgcttg ccattattgt tttggatgga gttccaagag 8100
gacttatccc aaaggtgagc ttcgaatctt ccagtccatc tatgcctagt aacacctctg 8160
tagatagatg accttctggt agaagctgga gaagttgggt tatgagactt atcgccagat 8220
ggagatgact tcttagccct cttagctotc tttggtottg gagcttcaga ttgaattggg 8280
ctagaggtag tagtagaaga ggacactgaa gaagatggag aactagagca ggtagaggta 8340
gtgagcctct tcttcatgaa ttcactagtg attaaatttt crttagtgott tgagcatata 8400
acaagcatgg tatatatagg cacgtaaaca agttgagaaa ttttactttg aatttgacat 8460
aaccaataaa agttagtgct gtttattacc tcactcagtt tgcaccgcaa ctgtcgttag 8520
tgatgtttac ctttcctttt tctattattt attagtatta tataatatat atatatatgt 8580
gatgagactt gaaattgttt agcaccgcaa atgtccttct tgaggggagg ttttcttttg 864C
ctgaggttgg ggtgtcacat acaccaccct ctatggactc aacgtccttg ctgaggttta 8700
ccccacacta catgagattt ttctagactc aatactatga tatttctcgc cttatcggaa 8760
ttggttaaac tcagttgaag ttagggtcat atcgataaaa ttgacacatg atcgactctg 8820
atattaaaca gattctctcc ctcgaacctc actcactttc ctttttctat tctttattag 8880
tattatataa tagatccgtt ccaaccattc acgtacataa gaagagaaat attttttttt 8940
aatggactaa catgacaaat aaaacaaaca aaggagtaat gatcactaca acaaattaga 9000
ttatgaggga caaataattt catcatctat aaatcatgtt tcgtcactaa aaattttgtg 9060
tgacgaaaaa gatttcgtca atcagttgtc actaaaaata tacaaagacg atttaatgat 9120
gtttaccttt cottttctat tctttattag tattatataa taaatatatg tgtgatgaga 9180
cttgaaattg tttagcaccg caaatgtcct tgttgaaggg aggttttctt ttgctgaggt 9240
tggggtgtca catacacccc ctctatggac tgaacgtcct ttttgaggtt tattttacac 9300
tgcatgagat ttttctagat tcaacattat gatttctaga ctcaacacta cgatcgtcac 9360
taaagactat tttttatata taaaaaaaat actttgtcct taaatgtata aattagggat 9420
aaatttatta ttataaaaaa ggttaataat tttgtgatta aatctattat tttgtcactg 9480
aaagtgtttg cttttaccga cgacatatat gtcactaaat attatcataa gtagtgacaa 9540
ttacaattgt cacaaaataa aaaaaattat tcatattcaa caaaaaaggg tactacgaca 9600
atacattttt tgtcactgaa agtaatcaag ttgtgataaa ttaatttatt taatgacaaa 9660
aatatttgta tcaaaattca cccatgatca tataataaaa ataactaaaa ttatactaaa 9720
gcataaatga caagaaaatc taactaaaac atatcaaata ttactcctaa acaaagacat 9780
ataagtaaaa atttcttcca aagtatcaat aacgtggtga cacatagctt gcaatcaatc 9840
ttgcrtcaar tttcaccttt tatacctgta aaaagaaaga gaaaataaaa caatgattta 9900
aaaatcgaat toccgaggcc cctagaatct aattattcta ttcagactaa attagtataa 9960
gtattttttt aatcaataaa taataattaa taatttatta gtaggagtga ttgaatttat 10020
aatatatttt ttttaatcat ttaaagaatc ttatatcttt aaattgacaa gagttttaaa 10080
tggggagagt gttatcatat cacaagtagg attaatgtgt tatagtttca catgcattac 10140
gataagttgt gaaagataac attattatat ataacaatga caatcactag cgatcgagta 10200
gtgagagtcg tcttattaca ctttcttcct tcgatctgtc acatggcggc ggcccgcggc 10260
cgcttcatta ctcgagccag gaggatggat cgatgctggt otgagaccct gctaccggtt 10320
gctgactgaa ctgctaggca cggtccttca tttcacgggc cttgctcgcc aactttgtct 10380
tggccgactc caactgatcc gctccgggtg gatgtttccc cgtcaggtaa cggtagatcc 10440
aggacagcac agacagagcg gcaacaccaa atcccccgct tgccagaaaa cccgctccca 10500
acaggaagat ggtgatgact gcagatcaga aaaactcaga ttaatcgaca aattcgatcg 10560
cacaaactag aaactaacac cagatctaga tagaaatcac aaatcgaaga gtaattattc 10620
gacaaaactc aaattatttg aacaaatcgg atgatatcta tgaaacccta atcgagaatt 10680
aagatgatat ctaacgatca aacccagaaa atcgtcttcg atctaagatt aacagaatct 10740
aaaccaaaga acatatacga aattgggatc gaacgaaaac aaaatcgaag attttgagag 10800
aataaggaac acagaaattt acctgcaagg accagtacag gcgagaagat caccaggaaa 10860
ggtgtggcga ttgtcagcgc aatgaccatt ccagccaggg tcaacccgga taacaccaac 10920
aggctacctc cggcagtaac cgcggtcgct gcctttacaa cacgctgagc acgcggttgc 10980
agttgcaagt ggggggcacg tgtttgttgc tactgaccgt agtgctctgc catggaaatt 11040
ttgttggtgc tttgagcata taacaagcat ggtatatata ggcacgtaaa caaottgaga 11100
aattttactt tgagtttgac ataaccaata aaagttagtg ctgtttatta cctcactcag 11160
CA 2998211 2018-03-16
261
tttgcaccgc aactgtcgtt agtgatgttt acctttcctt tttctattat ttattagtat 11220
tatataatat atatatatgt gtgatgagac ttgaaattgt ttagcaccgc aaatgtcctt 11280
cttgagggga ggrtttattt tgctgaggtt agggtgtcac atacaccocc ctctatggac 11340
tcaacgtcct tgctgaggtt taccccacac tacatgagat ttttctagac tcaatactat 11400
gatatttctc gccttatcgg aattagttaa actcaqttga agttagggtc atatcgataa 11460
aattgacaca tgatcgactc tgatattaaa cagattctct ccatcgaacc tcactcactt 11520
tcctttttct attctttatt agtattatat aatagatccg ttccaaccat tcacgtacat 11580
aagaagagag atattttttt ttaatggact aacataacaa ataaaacaaa caaagaagta 11640
atgatcacta caacaaatta gattatgagg gacaaataat ttcatcatct ataaatcatg 11700
tttcgtcact aaaaattttg tgtgacgaaa aagatttcgt caatcagttg tcactaaaaa 11760
tatacaaaga cgatttaatg atgtttacct ttccttttct attctttatt agtattatat 11820
aataaatata tgtgtgatga gacttgaaat tgtttagcac cgcaaatgtc cttgttgaag 11880
ggaggttttc ttttgctgag gttgaggtgt cacatacacc coctctatgg actgaacgtc 11940
ctttttgagg tttattttac actgcatgag atttttctag attcaacatt atgatttcta 12000
gactcaacac tacgatcgtc actaaagact attttttata tataaaaaaa atactttgtc 12060
cttaaatgta taaattaggg ataaatttat tattataaaa aaggttaata attttatgat 12120
taaatctatt attttgtcac tgaaagtgtt tgcttttacc gacgacatat atgtcactaa 12180
atattatcat aagtagtgac aattacaatt gtcacaaaat aaaaaaaatt attcatattc 12240
aacaaaaaaa ggtactacga caatacattt tttgtcactg aaagtaatca agttgtgata 12300
aattaattta tttaatgaca aaaatatttg tatcaaaatt cacccatgat catataataa 12360
aaataactaa aattatacta aagcataaat gacaagaaaa tctaactaaa acatatcaaa 12420
tattactcct aaacaaagac atataagtaa aaatttcttc caaagtatca ataacgtggt 12480
gacacatagc ttgcaatcaa tcttgcttca atzttcacct tttatacctg taaaaagaaa 12540
gagaaaataa aacaatgatt taaaggcgcg ccgcgtattg gctagagcag cttgccaaca 12600
tggtagagca cgacactctc gtctactcca agaatatcaa agatacagtc tcagaagacc 12660
aaagggctat tgagactttt caacaaaggg taatatcggg aaacctcctc ggattccatt 12720
gcccagctat ctgtcacttc atcaaaagga cagtagaaaa ggaaggtggc acctacaaat 12780
gccatcattg cgataaagga aaggctatcg ttcaagatgc ctctgccgac agtggtccca 12840
aagatggacc cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt 12900
caaagcaagt ggattgatgt gataacatgg tggagcacga cactctcgtc tactccaaga 12960
atatcaaaga tacagtctca gaagaccaaa gggctattga gacttttcaa caaagggtaa 13020
tatcgggaaa cctoctogga ttccattgcc cagctatctg tcacttcatc aaaaggacag 13080
tagaaaagga aggtggcacc tacaaatgcc atcattgcga taaaggaaag gctatcgttc 13140
aagatgcctc tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg 13200
aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg 13260
acgtaaggga tgacgcacaa tcccactatc cttcgcaaga ccttcctota tataaggaag 13320
ttcatttcat ttgaagagga cacgctgaaa tcaccagtct ctctctacaa atctatctct 13380
gcgatcgcat ggcgattttg gattctgctg gcgttactac ggtgacggag aacggtggcg 13440
gagagttcgt cgatcttgat aggcttcgtc gacggaaatc gagatcggat tcttctaacg 13500
gacttattct ctctggttcc gatadtaatt ctocttagga tgatgttggd gctcccgccg 13560
acgttaggga tcggattgat tccgttgtta acgatgacgc tcagggaaca gccaatttgg 13620
ccggagataa taacggtggt ggcgataata acgatgatgg aagaggcggc ggagaaggaa 13680
gaggaaacgc cgatgctacg tttacgtatc gaccgtcggt tccagctcat cggagggcga 13740
gagagagtcc acttagctcc gacgcaatct tcaaacagag ccatgccgga ttattcaacc 13800
tctgtgtagt agttcttatt gctgtaaaca gtagactcat catcgaaaat cttatgaagt 13860
atgattgatt gatcagaacg gatttctggt ttaqttcaag atcgctgcga gattggccgc 13920
ttttcatgtg ttgtatatcc ctttcgatct ttcctttggc tgcctttacg gttgagaaat 13980
tggtacttca gaaatacata tcagaacctg ttgtcatctt tcttcatatt attatcacca 14040
tgacagaggt tttgtatcca gtttacgtca ccctaaggtg tgattctgct tttttatcag 14100
gtgtcacttt gatgctcctc acttgcattg tgtggctaaa gttggtttct tatgctcata 14160
ctagctatga cataagatcc ctagccaatg cagctgataa ggccaatcct gaagtotcct 14220
actacgttag cttgaagagc ttggcatatt tcatggtcac tcccacattg tgttatcagc 14280
caagttatcc acgttctgca tgtatacgga agggttgggt ggctcgtcaa tttgcaaaac 14340
tggtcatatt caccggattc ataggattta taatagaaca atatataaat cctattgtca 14400
ggaactcaaa gcatectttg aaaggcgatc ttctatatgc tattgaaaga gtgttgaagc 14460
tttcagttcc aaatttatat gtgtggctct gcatgttcta ctgcttcttc cacctttggt 14520
CA 2998211 2018-03-16
262
taaacatatt ggcagagctt ctotgottcg gagatcgtga attctacaaa gattggtgga 14580
atgcaaaaag tgtgggagat tactggagaa tatggaatat gcctgttcat aaatggatgg :4640
ttcgacatat atacttcccg tgcttgcgca gcaagatacc aaagacactc gccattatca :4700
ttgotttcct agtctctgca gtotttcatg agctatgcat cgcagttcct tgtcgtctct 14760
tcaagctatg ggcttttctt gggattatgt ttcaggtgcc tttggtcttc atcacaaact 14820
atctacagga aaggtttggc tcaacggtgg gaaacatgat cttctggttc atcttctgca 14880
ttttcggaca accgatgtgt atgcttcttt attaccacga cctgatgaac cgaaaaggat 14940
cgatgtcatg agcgatcgcg atcgttcaaa catttggcaa taaagtttct taagattgaa 15000
tcctgttgcc gatcttgcga tgattatcat ataatttctg ttgaattacg t,taagcatgt 15060
aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc 15120
gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt 15180
atcgcgcgcg gtgtcatcta tgttactaga tocctgcagg gcgtattggc aagagcagct 15240
tgccaacatg gtggagcacg acactctcgt ctactccaag aatatcaaag atacagtctc 15300
agaagaccaa aaggcaattg agacttttca acaaagggta atatcgggaa acctcctcgg 15360
attccattgc ccagctatct gtcacttcat caaaaggaca gtagaaaagg aaggtggcac 15420
ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt caagatgcct ctgccgacag 15480
tggtcccaaa gatggacccc cacccacgag gagcaacgtg gaaaaagaag acgttccaac 15540
cacgtcttca aagcaagtgg attgatgtga taacatggtg gagcacgaca ctctcgtcta 15600
ctccaagaat atcaaagata cagtctcaga agaccaaagg gctattgaga cttttcaaca 15660
aaaggtaata acgggaaacc tcctcggatt ccattgccca gctatctgtc acttcatcaa 15720
aaggacagta gaaaaggaag gtggcaccta caaatgccat cattgcgata aaggaaaggc 15780
tatcgttcaa gatgcctctg ccgacagtgg acccaaagat ggacccccac ccacgaggag 15840
catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat 15900
ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc ttcctctata 15960
taaggaagtt catttcattt ggagaggaca cgctgaaatc accagtctct ctctacaaat 16020
ctatctctct cgagatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg 16080
tgaagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg 16140
tgttccggct gtcagcgcag gggaggccgg ttctttttgt caagaccgac ctgtccggtg 16200
ccctgaatga acttcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc 16260
cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg 16320
aaatgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca 16380
tggctgatqc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc 16440
aagcgaaaca tcgcatcgag cgagcacata ctcggatgaa agccggtctt gtcaatcaag 16500
atgatctgga cgaagagcat caggggctcg cgccagccaa actgttcgcc aggctcaagg 16560
cgcgcatgcc cgacggcgag gatctcgtcg tgactcatgg cgatgcctgc ttgccgaata 16620
tcatggzgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcag 16680
accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 16740
gggctgacc 16749
<210> 22
<211> 137
<212> DNA
<213> Artificial Sequence
<220>
<223> linker sequence
<400> 22
atttaaatgc ggccgcgaat tcgtcgattg aggacgtccc tactagacct gctggacctc 60
ctcctgctac ttactacgat tctctcgctg tgcatatggt cagtcatgcc cgggcctgca 120
ggcggccgca tttaaat 137
CA 2998211 2018-03-16
263
<210> 23
<211> 434
<212> DNA
<213> Araificial Sequence
<220>
<223> hpRNAi
<400> 23
gtgagcaatg aaccaagatt tatcaatacc gttacttttg atagcaaaga gggatctcct 60
actcttgtta tggtccacgg atatggtgcc tctcagggtt tcttctttcg gaatttttat 120
gcccttgcga ggcatttcaa agttattgct attgatcagc ttggctgggg tggttcaagc 180
aggcctgaca tcacatgcag aagtacagaa gagactgaag attggtttaa tgattccttt 240
gaggagtggc gcaaagccaa aaaccttagc aactttattt tgcttgggca ctoctttgga 300
gggtatgtcg ctgcaaaata tgctctcaag catccagagc atgttcagca gttgattctg 360
gtaggaccag ctggatttac atcagagact gaacatatgt ccgagcggct tacccagttt 420
agagcaacat ggaa 434
<210> 24
<211> 593
<212> DNA
<213> Artificial Sequence
<220>
<223> hpRNAi
<400> 24 =
actgctgatg ctgtcaggca gtatctatgg ttgtttgagg agcataatgt tcttgaattc 60
ctcgtacttg ctggagatca tctatatcga atggattatg aaaagttcat tcaagcccac 120
agagaaacag atgctgatat tactgttgcc gcactgccaa tggatgaaaa gcgagccact 180
gcatttggtc tcatgaagat tgacgaagaa ggacgcatta ttgaatttgc agagaaaccg 240
aaaggagagc aattgaaagc aatgaaagtg gatacaacca ttttaggtct tgatgatgag 300
agagctaaag agatgccttt tatcgcaagt atgggtatat atgtcattag caaagatgtg 360
atgttaaact tacttcgtga taagttccct ggtgccaatg attttggcag tgaagttatt 420
cctggtgcaa cttcgcttgg gatgagagtg caagcttatt tatatgatgg atactgggaa 480
gatattggta ccatcgaagc tttctacaat gccaatttgg gcattaccaa aaagccagtc 540
ccagatatta gcttctatga ccgatcagct ccaatctaca cccaacctcg ata 593
<210> 25
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> lipase motif
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
CA 2998211 2018-03-16
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<222> (4)..(4)
<223> Xaa can be any naturally occurring amino acid
<400> 25
Gly Xaa Ser Xaa Gly
a 5
<210> 26
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> acyltransferase motif
<220>
<221> X
<222> (2)..(5)
<223> any amino acid
<400> 26
His Xaa Xaa Xaa Xaa Asp
<210> 27
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> probable lipid binding motif
<220>
<221> X
<222> (2)..(4)
<223> any amino acid
<400> 27
Val Xaa Xaa Xaa His Gly Phe
1 5
<210> 28
<211> 584
<212> PRT
<213> Arabidopsis thaliana
<400> 28
Met Asn Ser Met Asn Asn Trp Leu Gly Phe Ser Leu Ser Pro His Asp
1 5 10 15
Gln Asn His His Arg Thr Asp Val Asp Ser Ser Thr Thr Arg Thr Ala
20 25 30
Val Asp Val Ala Gly Gly Tyr Cys Phe Asp Leu Ala Ala Pro Ser Asp
35 40 45
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Glu Ser Ser Ala Val Gin Thr Ser Phe Leu Ser Pro Phe Giy Val Thr
50 55 60
Leu Glu Ala Phe Thr Arg Asp Asn Asn Ser His Ser Arg Asp Trp Asp
65 70 75 80
Ile Asn Gly Gly Ala Cys Asn Thr Leu Thr Asn Asn Glu Gin Asn Gly
85 90 95
Pro Lys Leu Glu Asn Phe Leu Gly Arg Thr Thr Thr Ile Tyr Asn Thr
100 105 110
Asn Glu Thr Val Val Asp Gly Asn Gly Asp Cys Gly Gly Gly Asp Gly
115 120 125
Gly Gly Gly Gly Ser Leu Gly Leu Ser Met Ile Lys Thr Trp Leu Ser
130 135 140
Asn His Ser Val Ala Asn Ala Asn His Sin Asp Asn Gly Asn Gly Ala
145 150 155 160
Arg Gly Leu Ser Leu Ser Met Asn Ser Ser Thr Ser Asp Ser Asn Asn
165 170 175
Tyr Asn Asn Asn Asp Asp Val Val Gin Glu Lys Thr Ile Val Asp Val
180 185 190
Val Ciu Thr Thr Pro Lys Lys Thr Ile Glu Ser Phe Gly Gin Arg Thr
195 200 205
Ser Ile Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Tyr Glu
210 215 220
Ala His Leu Trp Asp Asn Ser Cys Lys Arg Giu Gly Gin Thr Arg Lys
225 230 235 240
Gly Arg Sin Val Tyr Leu Gly Gly Tyr Asp Lys Glu Glu Lys Ala Ala
245 250 255
Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Pro Thr Thr Thr
260 265 270
Thr Asn Phe Pro Leu Ser Glu Tyr Glu Lys Glu Val Glu Glu Met Lys
275 280 285
His Met Thr Arg Gin Glu Tyr Val Ala Ser Leu Arg Arg Lys Ser Ser
290 295 300
Gly Phe Ser Arg Gly Ala Ser Ile Tyr Arg Gly Val Thr Arg His His
305 310 315 320
Gin His Gly Arg Trp Gin Ala Arg Ile Gly Arg Val Ala Gly Asn Lys
325 330 335
Asp Leu Tyr Leu Gly Thr Phe Gly Thr Gin Glu Glu Ala Ala Glu Ala
340 345 350
Tyr Asp Ile Ala Ala Ile Lys Phe Arg Gly Leu Ser Ala Val Thr Asn
355 360 365
Phe Asp Met Asn Arg Tyr Asn Val Lys Ala Ile Leu Glu Ser Pro Ser
370 375 380
Leu Pro Ile Gly Ser Ser Ala Lys Arg Leu Lys Asp Val Asn Asn Pro
385 390 395 400
Val Pro Ala Met Met Ile Ser Asn Asn Val Ser Glu Ser Ala Asn Asn
405 410 415
Val Ser Gly Trp Gin Asn Thr Ala Phe Gin His His Gin Gly Met Asp
420 425 430
Leu Ser Leu Leu Gin Gin Gin Gin Glu Arg Tyr Val Gly Tyr Tyr Asn
435 440 445
Gly Gly Asn Leu Ser Thr Glu Ser Thr Arg Val Cys Phe Lys ,Gin Glu
450 455 460
Glu Glu Gin Gin His Phe Leu Arg Asn Ser Pro Ser His Met Thr Asn
465 470 475 480
Val Asp His His Ser Ser Thr Ser Asp Asp Ser Val Thr Val Cys Gly
485 490 495
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Asn Val Val Ser Tyr Gly Gly Tyr Gin Gly Phe Ala Ile Pro Val Gly
500 505 510
Thr Ser Val Asn Tyr Asp Pro Phe Thr Ala Ala Glu Ile Ala Tyr Asn
515 520 525
Ala Arg Asn His Tyr Tyr Tyr Ala Gin His Gin Gin Gin Gin Gin Ile
530 535 540
Gin Gin Ser Pro Gly Gly Asp Phe Pro Val Ala Ile Ser Asn Asn His
545 550 555 560
Ser Ser Asn Met Tyr Phe His Gly Glu Gly Gly Gly Glu Gly Ala Pro
365 570 575
Thr Phe Ser Val Trp Asn Asp Thr
580
<210> 29
<211> 336
<212> DNA
<213> Artificial Sequence
<220>
<223> inducible promoter
<400> 29
tcgatagtta tgatagttcc cacttgtccg tccgcatcgg catccgcagc tcgggatagt 60
tocgacctaa gattggatgc atgcggaacc gcacgagggc ggggcggaaa ttgacacacc 120
actcctctcc acgcaccgtt caagaggtac gcgtatagag ccgtatagag cagagacgga 180
gcactttctg gtactgtccg cacgggatgt ccgcacggag agccacaaac gagcggggcc 240
ccgtacgtgc tctcctaccc caggatcgca tccccgcata gctgaacatc tatataaaga 300
cccccaaggt tctcagtctc accaacatca tcaacc 336
<210> 30
<211> 2466
<212> DNA
<213> Artificial Sequence
<220>
<223> inducer
<400> 30
atggccgaca ctagaagaag gcagaaccac tcttgtgacc catgccgtaa gggcaagaga 60
agatgtgatg ctccagagaa ccgtaacgag gctaatgaga acggatgggt gtcatgctct 120
aactgcaaga ggtggaacaa ggactgcacc ttcaactgac ttagctccca aaggtctaag 180
gctaagggta ctgctccaag agctaggact aagaaggcta ggactgctac tactacctcc 240
gagccttcta cttccgctgc tactattcca actcccgagt ccgataatca cgatgctcca 300
ccagtgatca actcccacga tgctttgcca tcttggactc agggacttct ttctcaccct 360
ggcgatctct tcgacttctc ccattctgct attccagcta acgctgagga tgctgctaac 420
gtgcaatctg atgctccatt cccatgggat cttgctatcc caogcgattt ctctatagga 480
cagcaacttg agaagcccct ctccccattg tctttccaog ctgttcttct tccaccacac 540
tccccaaaca ctgatgatct cattcgtgag cttgaggaac agactaccga tccagattcc 600
gtgactgaca ctaactccgt tcagcaagtt gctcaggatg gctctctttg gtctgatagg 660
cagtctccac tcctcccaga aaacagtttg tgcatggctt ccgactctac cgctagaaag 720
tatgctaggt ccaccatgac caagaacctc atgaggatct accacgactc catagaaaac 780
gccctttctt gctggcttac tgagcacaac tgcccatact ccgaccagat ttcttacctc 840
ccaccaaagc aaagggctga gtggggacca aattggtcta acaggatgtg cattagggtg 900
tgcaggctog ataaggtgtc aacttctort agaggaaggg ctctctccgc tgaagaagat 960
=
CA 2998211 2018-03-16
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aaggctgctg ctagggcact tcaccttgct attgtagctt tcgottctca gtggactcaa 1020
catgctcaaa ggggagctgg acttaacgtc ccagctgata ttgctgctga cgagcgttct 1080
attaggcgta acgcttggaa tgaggctagg catgcacttc agcacactac tggaatccca 1140
tccttcaggg tgatcttcgc caacatcatc ttcagcctca ctcagtccgt actcgatgat 1200
gatgagcaac atggaatggg agctaggctc gataagcttc tcgagaatga tggtgctcca 1260
gtgttcctcg agactgctaa taggcagctc tacaccttca ggcacaagtt cgctaggatg 1320
cagagaaggg gtaagacttt caataggctt cctggtggat ccgtggcttc tactttcgct 1380
ggaattttcg agactoccac cccctcatct gagtctccac aacttgatcc agtggtggct 1440
tctgaggaac acaggtctac totgtotcto atgttctggc tcgggatcat gttcgacact 1500
ctgtctgctg ctatgtacca gaggccactt gttgtgtccg atgaggactc ccagatctct 1560
tctgcttctc caccaagaag aggtgccgag actcctatta accttgattg ctgggagcca 1620
ccaaggcagg tcccatctaa tcaagagaag tctgatgtgt ggggcgacct gttccttagg 1680
acttctgatt ctztgoccga ccacgagtcc cacactcaaa tttctcaacc agctgctagg 1740
tggccatgca cttatgaaca agctgctgct gctctotcct ctgctactcc tgttaaggtg 1800
ttgctttaca ggcgtgtgac tcagctccag actttgttgt ataggggagc ttctccagot 1860
aggcttgagg ctgctattca gaggactctc tacgtgtaca accactggac tgctaagtac 1920
cagccattca tgcaggattg cgttgccaac catgagattc tcccatccag gatccagtot 1980
tggtacgtga tccttgatgg acactggcac cttgctgcta tgcttttggc tgatgtgctc 2040
gaqtccatcg acagggattc ctactccgat atcaaccaca tcgacctcgt gactaagctc 2100
aggcttgata acgctcttgc tgtgtctgct ctcgctaggt catctcttag aggccaagaa 2160
ctcgatccag gcaaggcttc tccaatgtac aggcacttcc acgactccct tactgaggtt 2220
gcattccttg ttgagccatg gactgtggtg ctcatccact catttgctaa ggctgcttac 2260
atcctoctog attgccttga tcttgatggt cagggaaacg ctctcgctgg ataccttcaa 2340
cttaggcaga actgcaacta ctgcatcagg gctctccagt tccttggccg taagtctgat 2400
atggctgctc tcgtggctaa ggatcttgag aggggactca acggaaaggt cgacagcttc 2460
ctctaa 2466
<210> 31
<211> 208
<212> PRT
<213> Arabidopsis thaliana
<400> 31
Met Thr Ser Ser Val Ile Val Ala Gly Ala Gly Asp Lys Asn Asn Gly
1 5 10 15
Ile Val Val Gin Gin Gin Pro Pro Cys Val Ala Arg Glu Gin Asp Gin
20 25 30
Tyr Met Pro Ile Ala Asn Val Ile Arg Ile Met Arg Lys Thr Leu Pro
35 40 45
Ser His Ala Lys Ile Ser Asp Asp Ala Lys Glu Thr Ile Gin Glu Cys
50 55 60
Val Ser Glu Tyr Ile Ser Phe Val Thr Gly Glu Ala Asn Glu Arg Cys
65 70 75 80
Gin Arg Glu Gin Arg Lys Thr Ile Thr Ala Glu Asp Ile Leu Trp Ala
85 90 95
Met Ser Lys Leu Gly Phe Asp Asn Tyr Val Asp Pro Leu Thr Val Phe
100 105 110
Ile Asn Arg Tyr Arg Glu Ile Glu Thr Asp Arg Gly Ser Ala Leu Arg
115 120 125
Gly Glu Pro Pro Ser Leu Arg Gin Thr Tyr Gly Gly Asn Gly Ile Gly
130 135 140
Phe His Gly Pro Ser His Gly Leu Pro Pro Pro Gly Pro Tyr Gly Tyr
145 150 155 160
Gly Met Leu Asp Gin Ser Met Val Met Gly Gly Gly Arg Tyr Tyr Gin
165 170 175
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Ash Gly Ser Ser Gly Gln Asp Glu Ser Ser Val Gly Gly Gly Ser Ser
180 185 190
Ser Ser Ile Asn Gly Met Pro Ala Phe Asp His Tyr Gly Gln Tyr Lys
195 200 205
<210> 32
<211> 278
<212> PRT
<213> Zea mays
<400> 32
Met Asp Ser Ser Ser Phe Leu Pro Ala Ala Gly Ala Glu Asn Gly Ser
1 5 10 15
Ala Ala Gly Gly Ala Asn Asn Gly Gly Ala Ala Gln Gln His Ala Ala
20 25 30
Pro Ala Ile Arg Glu Gln Asp Arg Leu Met Pro Ile Ala Asn Val Ile
35 40 45
Arg Ile Met Arg Arg Val Leu Pro Ala His Ala Lys Ile Ser Asp Asp
50 55 60
Ala Lys Glu Thr Ile Gln Glu Cys Val Ser Glu Tyr Ile Ser Phe Ile
65 70 75 80
Thr Gly Glu Ala Asn Glu Arg Cys Gln Arg Glu Gln Arg Lys Thr Ile
85 90 95
Thr Ala Glu Asp Val Leu Trp Ala Met Ser Arg Leu Gly Phe Asp Asp
100 105 110
Tyr Val Glu Pro Leu Gly Ala Tyr Leu His Arg Tyr Arg Glu Phe Glu
115 120 125
Gly Asp Ala Arg Gly Val Gly Leu Val Pro Gly Ala Ala Pro Ser Arg
130 135 140
Gly Gly Asp His His Pro His Ser Met Ser Pro Ala Ala Met Leu Lys
145 150 155 160
Ser Arg Gly Pro Val Ser Gly Ala Ala Met Leu Pro His His His His
165 170 175
His His Asp Met Gln Met His Ala Ala Met Tyr Gly Gly Thr Ala Val
180 185 190
Pro Pro Pro Ala Gly Pro Pro His His Gly Gly Phe Leu Met Pro His
195 200 205
Pro Gln Gly Ser Ser His Tyr Leu Pro Tyr Ala Tyr Glu Pro Thr Tyr
210 215 220
Gly Gly Glu His Ala Met Ala Ala Tyr Tyr Gly Gly Ala Ala Tyr Ala
225 230 235 240
Pro Gly Asn Gly Gly Ser Gly Asp Gly Ser Gly Ser Gly Gly Gly Gly
245 250 255
Gly Ser Ala Ser His Thr Pro Gln Gly Ser Gly Gly Leu Glu His Pro
260 265 270
His Pro Phe Ala Tyr Lys
275
<210> 33
<211> 234
<212> PRT
<213> Arabidopsis thaliana
CA 2998211 2018-03-16
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<400> 33
Met Glu Arg Gly Gly Phe His Gly Tyr Arg Lys Leu Ser Val Asn Asn
1 5 10 15
Thr Thr Pro Ser Pro Pro Gly Leu Ala Ala Asn Phe Leu Met Ala Glu
20 25 30
Gly Ser Met Arg Pro Pro Glu Phe Asn Gin Pro Asn Lys Thr Ser Asn
35 40 45
Gly Gly Glu Glu Glu Cys Thr Val Arg Glu Gin Asp Arg Phe Met Pro
50 55 60
Ile Ala Asn Val Ile AiQ Ile Met Arg Arg :le Leu Pro Ala His Ala
65 70 75 80
Lys Ile Ser Asp Asp Ser Lys Glu Thr Ile Gin Glu Cys Val Ser Glu
65 90 95
Tyr Ile Ser Phe Ile Thr Gly Glu Ala Asn Glu Arg Cys Gin Arg Glu
100 105 110
Gin Arg Lys Thr Ile Thr Ala Glu Asp Val Leu Tip Ala Met Ser Lys
115 120 125
Leu Gly Phe Asp Asp Tyr Ile Glu Pro Leu Thr Leu Tyr Leu His Arg
130 135 140
Tyr Arg Glu Leu Glu Gly Glu Arg Gly Val Her Cys Ser Ala Gly Ser
145 150 155 160
Val Ser Met Thr Asn Gly Leu Val Val Lys Arg Pro Asn Gly Thr Met
165 170 175
Thr Glu Tyr Gly Ala Tyr Gly Pro Val Pro Gly Ile His Met Ala Gin
180 . 185 190
Tyr His Tyr Arg His Gin Ash Gly Phe Val Phe Ser Gly Asn Glu Pro
195 200 205
Asn Ser Lys Met Ser Gly Ser Ser Ser Gly Ala Ser Gly Ala Arg Val
210 215 220
Glu Val Phe Pro Thr Gin Gln His Lys Tyr
225 230
<210> 34
<211> 312
<212> PRT
<213> Arabidopsis thaliana
<400> 34
Met Val Asp Glu Asn Val Glu Thr Lys Ala Ser Thr Leu Val Ala Her
1 5 10 15
Val Asp His Gly Phe Gly Ser Gly Ser Gly His Asp His His Gly Leu
20 25 30
Ser Ala Her Val Pro Leu Leu Gly Val Asn Trp Lys Lys Arg Arg Met
35 40 45
Pro Arg Gin Arg Arg Ser Ser Ser Ser Phe Asn Leu Leu Ser Phe Pro
50 55 60
Pro Pro Met Pro Pro Ile Ser His Val Pro Thr Pro Leu Pro Ala Arg
65 70 75 80
Lys Ile Asp Pro Arg Lys Leu Arg Phe Leu Phe Gin Lys Glu Leu Lys
85 90 95
Asn Ser Asp Val Her Her Leu Arg Arg Met Ile Leu Pro Lys Lys Ala
100 105 110
Ala Glu Ala His Leu Pro Ala Leu Glu Cys Lys Glu Gly Ile Pro Ile
115 120 125
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Arg Met Glu Asp Leu Asp Gly Phe His Val Trp Thr Phe Lys Tyr Arg
130 135 140
Tyr Trp Pro Asn Asn Asn Ser Arg Met Tyr Val Leu Glu Asn Thr Gly
145 150 155 160
Asp Phe Val Asn Ala His Gly Leu Gin Leu Gly Asp Phe Ile Met Val
165 170 175
Tyr Gin Asp Leu Tyr Ser Asn Asn Tyr Val Ile Gin Ala Arg Lys Ala
180 185 190
Ser Glu Glu Glu Glu Val Asp Val Ile Asn Leu Glu Glu Asp Asp Val
195 200 205
Tyr Thr Asn Leu Thr Arg Ile Glu Asn Thr Val Val Asn Asp Leu Leu
210 215 220
Leu Gin Asp Phe Asn His His Asn Asn Asn Asn Asn Asn Asn Ser Asn
225 230 235 240
Ser Asn Ser Asn Lys Cys Ser Tyr Tyr Tyr Pro Val Ile Asp Asp Val
245 250 255
Thr Thr Asn Thr Glu Ser Phe Val Tyr Asp Thr Thr Ala Leu Thr Ser
260 265 270
Asn Asp Thr Pro Leu Asp Phe Leu Gly Gly His Thr Thr Thr Thr Asn
275 280 285
Asn Tyr Tyr Ser Lys Phe Gly Thr Phe Asp Gly Leu Gly Ser Val Glu
290 295 300
Asn Ile Ser Leu Asp Asp Phe Tyr
305 310
<210> 35
<211> 321
<212> PRT
<213> Brassica napus
<400> 35
Met Met Ala Asp Glu Asn Val Glu Thr Lys Ala Ser Thr Leu Ile Ala
1 5 10 15
Ser Val Gly His Gin Gly His Gly Phe Gly Ser Gly Ser Gly Gly His
20 25 30
His Gly Leu Ser Ala Ser Val Pro Leu Leu Gly Val Asn Ser Lys Lys
35 40 45
Arg Arg Met Pro Arg Gin Arg Arg Ser Ser Ser Ser Phe Asn Leu Leu
SO 55 60
Ser Leu Pro Pro Pro Met Pro Leu Ser Pro His Val Pro Thr Pro Leu
65 70 75 80
Ser Ala Arg Lys Ile Asp Pro Arg Lys Leu Arg Phe Leu Phe Gin Lys
85 90 95
Glu Leu Lys Asn Ser Asp Val Ser Ser Leu Arg Arg Met Ile Leu Pro
100 105 110
Lys Lys Ala Ala Glu Ala His Leu Pro Ala Leu Glu Cys Lys Glu Gly
115 120 125
Ile Pro Ile Arg Met Glu Asp Leu Asp Gly Leu His Val Trp Thr Phe
130 135 140
Lys Tyr Arg Tyr Trp Pro Asn Asn Asn Ser Arg Met Tyr Val Leu Glu
145 150 155 160
Asn Thr Gly Asp Phe Val Asn Ala His Gly Leu Gln Leu Gly Asp Phe
165 170 175
Ile Met Val Tyr Leu Asp Leu Asp Ser Asn Asn Tyr Val Ile Gin Ala
180 185 190
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Arg Lys Ala Ser Glu Glu G2u Glu Glu Glu Glu Asp Val Thr Ile Ile
195 200 205
Glu Glu Asp Asp Val Tyr Thr Asn Leu Thr Lys Ile Glu Asn Thr Val
210 215 220
Val Asn Asp Leu Leu Ile Gin Asp Phe Asn His His Asn Asp Asn Ser
225 230 235 240
Ser Asn Asn Asn Ser Asn Asn Asn Ile Asn Asn Asn Lys Cys Ser Tyr
245 250 255
Tyr Tyr Pro Val Ile Asp Asp Ile Thr Thr Asn Thr Ala Ser Phe Val
260 265 270
Tyr Asp Thr Thr Thr Leu Thr Ser Asn Asp Ser Pro Leu Asp Phe Leu
275 280 265
Gly Gly His Thr Thr Thr Thr Thr Asn Thr Tyr Tyr Ser Lys Phe Gly
290 295 300
Ser Phe Glu Gly Leu Gly Ser Val Glu Asn Ile Ser Leu Asp Asp Phe
305 310 315 320
Tyr
<210> 36
<211> 314
<212> PRT
<213> Medicage truncatula
<400> 36
Met Met Met Asp Glu Gly Giu Gly Lys Lys Lys Val Val Val Gin Lys
1 5 10 15
Thr Glu Ala Cys Gly Phe Met Ala Gly Val Glu Asp Glu Leu Gly Phe
20 25 30
Val Asn Val Lys Gly Asp Asn Asn Asn Gly Ser Gly Gin Arg Ile His
35 40 45
His Asp His Gly Phe Val Ala Ala Ala Phe Gly Thr Val His Arg Lys
50 55 60
Lys Arg Met Ala Arg Gin Arg Arg Ser Ser Ser Ser Thr Ile Thr Ile
65 70 75 80
His Leu Lys Asn Leu Pro Ser Ser Thr Thr Thr Thr Thr Thr Thr Thr
85 90 95
Thr Ser His Val Pro Ile Ser Pro Ile Pro Pro Leu Phe His Ser Leu
100 105 110
Pro Pro Ala Arg Glu Ile Asp His Arg Arg Leu Arg Phe Leu Phe Gin
115 120 125
Lys Glu Leu Lys Asn Ser Asp Val Ser Ser Leu Arg Arg Met Val Lou
130 135 140
Pro Lys Lys Ala Ala Glu Ala Phe Leu Pro Val Leu Glu Ser Lys Glu
145 150 155 160
Gly Ile Leu Leu Ser Met Asp Asp Leu Asp Gly Leu His Val Trc Ser
165 170 175
Phe Lys Tyr Arg Phe Trp Pro Asn Asn Asn Ser Arg Met Tyr Val Leu
180 185 190
Glu Asn Thr Gly Asp Phe Val Ser Thr His Gly Leu Arg Phe Gly Asp
195 200 205
Ser Ile Met Val Tyr Gln Asp Asn Gin Asn His Asn Tyr Val Ile Gin
210 215 220
Ala Lys Lys Ala Cys Asp Gin Asp Glu Tyr Met Glu Glu Ala Asn Asp
225 230 235 240
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Thr Ile Asn His Ile Phe Val Asp Asp Tyr Glu Val Asn Lys Ser Cys
245 250 255
Phe Asp Val Ala Tyr Pro Ala Met Asn Asp Thr Ser Met Ser Phe Ile
260 265 270
Tyr Asp Thr Thr Ile Ser Asn Asp Ser Pro Leu Asp Phe Leu Sly Gly
275 280 285
Ser Met Thr Asn Tyr Ser Asa Ile Sly Ser Vol Glu Thr Phe Gly Ser
290 295 300
Val Glu Asn Leu Ser Leu Asp Asp Phe Tyr
305 310
<210> 37
<211> 3275
<212> DNA
<213> Arabidopsis thaliana
<400> 37
ggttggctat atggtccaaa trttgatttg caatatgaga ttgcacagag agaacaatct 60
ttcattatga ttaattattg tacaagtaac aaacaccaat ctccgatata ctttggctct 120
ttagcacatt gttatgctag aagttagcgg aaatctatat gttgttaaac gcagcgttta 180
aattgaacag tgtaatttac cttgaaattt taagactaca tactatttag aatttcagat 240
gaaaacatct tgatgtttta gaaatccacg tgggaatagc gtaaaatctt atccaacgaa 300
cttattttgg ttttgttgta tttgtgcaag tcgtcacgct aatcgaaaaa agaaaagaaa 360
aaaagaagcc gtcatgatcg gccatttctc ggccgagtct gagtctgact ctgcgtccgt 420
gtcaccatta tcagatcgag cctgtottat ctcgtr_gcga ttccctatgc aaaaatcttc 480
ttcttttttt tattccccca tttatctctg atctcttctc tcttctcaag taaacctctc 540
tgcttcacgt ctcttctttt cttgtcgatt ttccccagat aatcagttga aaacacaccc 600
aaattcatct tcgaatcaat aatggatata agtaatgagg ctagtgtcga tcccttttcg 660
attggaccat catctatcat ggatcgaacc attgctttca gagtcttgtt ctgtagatca 720
atgtcacagc ttaggcgtga tctotttogg ttcttgttgc attggtttct tagatttaag 780
ctgaccgttt caccgtttgt gtcgtggttt catcctcgga accctcaagg gattttagcg 840
gtagttacaa tcattgcctt tgtgttgaaa cgatacacga atgtgaaaat aaaggcggaa 900
atggcttacc ggaggaagtt ttggaggaat atgatgcgga cggctttgac ttatgaggaa 960
tgggctcatg ctgctaagat gttagagaag gaaacaccaa agatgaatga atctgatctt 1020
tatgatgaag agttggttaa gaacaagctt caggagottc gtcatcgtcg ccaagaaggc 1080
tcacttagag acattatgtt ttgtatgaga gctgatttgg tgaggaatct cggtaatatg 1140
tgtaattcgg agcttcataa agutagactt caggttccta gacatatcaa agagtacatt 1200
gatgaggtgt ctactcagtt gagaatggtt tgtaactctg attcagagga gctttcttta 1260
gaagagaagc sttattttat gcatgaaaca cggcatgcct ttggtagaac ggctttgctt 1320
ttgagtgatg gggcttctct tggtgcgttt catgttggtg tggttaggac tttggttgag 1380
cataagcttt tacctcgaat aattgctggt tctagtgttg aatccatcat ttgtgctgtt 1440
gtggcctcaa ggtcttggcc agaactacag agtttctttg agaattcttt gcattcttta 1500
cagttctttg atcagctcgg aggcgtgttc tcaatagtga aacgggtaat gacacaaggg 1560
gctctacacg atatcagaca gttgcaatgt atgcttagaa acctcacaag caatctcaca 1620
ttccaagaag cttatgacat gacaggaagg attctcggga tcaccatttg ctccccaaga 1680
aagcatgaac ctcctcggtg tcttaactat ttgacttcgc ctcatatggt tatatggagc 1740
gcagtgactg cttcttgtgc ttttcctggt ctctttgaag ctcaaaagct aatggctaaa 1800
gatcgaagtg gagagatcgt accgtatcat ccacctttca atttggatcc agaagtaggc 1860
actaaatcat catctggacg ccggtggaga gatggtagtt tggaggttga tttaccaatg 1920
atgcagctta aagaactgtt caatgtcaat cattttattg tgagccaagc caatcctcac 1980
attgctccat tactgcgtct aaaggattta gttcgagctt atggtggtag attcgcagct 2040
aagctcgcgc atctagtgga gatggaggtc aaacatagat gcaaccaggt attagagctc 2100
gattttcctc tcggtggact cgcaaagctt tttgctcagg agtggaaagg tgatgttaca 2160
gttgtaatgc ctgctactct tgctcagtac tcgaagatta tacaaaatcc gactcatgtc 2220
gagattcaga aagcggctaa ccaaggaaga agatgcactt gggagaagct ctcagccata 2280
CA 2998211 2018-03-16
9T-0-8TOZ TTZ866Z VD
0981 PoPqqp5qee ebeebee5qq oqolq55qDE e34qeepe5p Bepobp4poq oq?-e.66qopq
008T Poo.1.4e66e6 6e4o4epoo5 4oqq6beoqP eobeopq.Bfq. eqe5beeobo Teoboo42ob
OLT PbePubeobq bPbbebpepq. obb-e4bobqq oPooPebqop 4000b4.4&45
4p.2.54eb.5;.o
0891 pob4435e54 qegobgbqqp p2o555eoqp D3beogp43.6 p2e2.565q4o eofq.e0p55p
0Z91 q6bpoqppb 3b4obbeebe og4o8e5gob 42-4gogeoop uPbeael.pbq ebeeo-34qe4
0901 5236351.qoq DE.D36e3Dbq pqm5-1152o2 4-16-125o66p e566 5Ã
oq.abqq.1.2qq.
OOST e2E43.6eql-e 55ebbeq3qo oqq4ebbqqD e-ebb41q1be eo:eelLq55 egeopeepqb
ODI'T eebbls6Ebq D.E.2,peP643 Eq.qo5eeop6 4oboqqa6po 65ebbqeqqo 56.beolboe
OSET eebbeseqoP 664obo oqobequoec qopgpeopbe epobepqbeq eogageoq.
OZET .646,4eeoggp gopp50p2.64 42po6eebTe opoeqqq.e.63 bppp6eggob eb56gebebe
09Z1 5663353o oppoogo52,4 84opebop.26 ee5p4eobbe 41e1.4344e3 oq364e33-44
001 looqq-eopee eDq..66qq;.2b eqebeeeeob 54e6T4e-ebe ep3o6beb1q 11qoeb6qop
OVIT qq.4406q5qo o4i.o6eoeei. beo64.6e6.64 oi.e71.434qbq. epc,poepqeo
epqq4e4DEE
0801 eq.op64e6o4 pobopbub7.6 of5666beeoc qoqq.bqbqbp pEoqb44b44 p44E.bboqbb
OZOI 4opbqeoebq eqq.obebee Dq44eoeb44 qepobeopeq. 4popep5p5i. qq.go6gbbeo
096 b4qpeabbep ge4ebgeDgq. b4-abobeme boe54444pe beE-euggbbq 5googg4oge
006 E.56-46bb-Teb epi.EBT44qq. 4eEe6qopoq. 4-eobbqbE,E,E' 654q4q.q.q.6
ebr66436e6
OD.8 goobbgeo4e beuo.e.eobpq beqp4og:.b4 b4pe4ebogo bbqq.bgbepo qeb6poggge
08L pgebbppoo6 44-44oepp.ge 3.56,pq.15bqq. op-eepeggbq gbgbbb4ble
pq.q.qopq.e6.6
OZL 64qP044o5D, abebbgbppq 364o53o6 eppebe466-4 41.3abge366 ee3e..6264eo
099 bge1.1.42360 goep'ebebee 5;go4poblq. fiebgeftoqq p.63412bq0 qoqbb4PpPP
009 .6qopeoqoEq oq.ugbeaMe Eqqeceqepb beeeeoqop beqoofq.bbe obg.35.6Ebb
OT7g paeoup.52.oe ebooppeeob gbqeq.Peobb ogoopebbuo 4364032155o bobobgeobq
08D' oggo4bo;e3 ebbb2p4o6o Tabbbeboeo obobbooeob bpo4obebob poqbe-eq.P
036 35opqba435 ebbe6oe5oe gpgoce5oo6 oPbobeopbo bo2,6p63oon 5006526p5o
09E 6ebfq.D5qpb b3bp563638 3536654beb 5eboelopeo qoSo600635 oblEbTeae-e,
00E 66355q.315 eEpboobooe qopbooqbec 5-4b8be6o56 5o64a615boo 55.63.5.6063
OD'Z eboobbbbob 40.6gooqo5o 6oqbco15063, oq.boobo4ob qoogebbbob oboupe6oeb
081 oboboopPob qp.6646obbo 5bo5o45000 5gobobo35o 5oo4obobbo 5=66,4335o
OZT oeobbo6qpo bobobbaboo obbobqoq.o 543.6q/565op gabobo4b= 5obo356bi.3
09 bobboe=2.5 33386o3 50-l.q5a5556 5466D36a6.6 abobeDoLog eoebgebbge
80 <00V>
aoTooTq mnqbaos <Etz>
VNG <ZIZ>
I7ZLZ <ITZ>
80 <OTZ>
SLZE pq..6PE
44;.q.eppo e44.poo4eoq. ogoggeqp-eP
OPZE ebeoqq.2. ogog.egeq.2 4q4poqqp-ep
Eq.7..;.Eq4e4q 361-1.egbqqg
081E .61p1518T1.3 po3e3545pq gqbbbeeeoc 44q6e5pJ.64 1011P6eqe1
OZTE beElqbpsoll 26qqEq5q.b.q.6 2.6eqqobae6 eqeqq.brqbel 6baoq.6q.b.63 oe-
ebeeoeqq.
090E lfibooqqebe ebecqqoqqq. bqoPq.4.6Eqo qbabo4o56o ee7leo-el2buP
5epb4epoub
000E perqe.64pbe 2.6,eubcepe bpogegbao4 obp4.6;qpbb qpbpbbpube 55oop4p4eb
0P6Z ogobeoP:..6,4 5pbebeoopq 4beLeo-2-_.'Pe ePoce2bbfq. q2.63-Teop.6.4
2.665443ep
088Z 5p8e5ppppq q53.45oe2g gbgbebb opplbebopp 6p6pEqp36e 3egogq.pqe.6
0Z8Z lb6P261-1.3eq ..6EDeeTeob aeollo-eobe e3pP.615qpq e-eaeu35PqE.
23.6Eoeeeoq.
09LZ e?53quq-e.66 oqq.eoqobbo 52gepogpbe b0006epopq peqqq.6oiob 5q.66qooqop
OOLZ 4q.beo6poo4 p7,.q.ppo33gb eabegbe-zqq. ebbebeoqbb ;.Teobqqpou
bo4e4ebqqo
0P9Z qbebepqabq 2,2,4p.64q2pq 46ceele2;.ob q34-435PoPp bebqe-eq-2..qz
oeb6q5bgoe
08? pbePopbbqq ogq.5e.6446e 5.6q.6c6p6PE) 2opp-e6gbpo ebabeeT:qp.
2.66eD565pq
OZgZ -Tolqp6o-P30 133ee4eeoe bqou3l.6e4D ..ebqe.66-q_pe oq.o.66-eaepc
1peebebefie
09VZ poblqop-2,bo ep.66q4o4po opeebeEte 204435E.ED gq.ebeopoo eEoqq.obbqq.
OOPZ ebboepTeoq oqboqqoqbo epobqp600b pobebebebb obqbep-epbe ec40615-ebbo
Of7EZ Eq.eqpoo-eg ;.344q.o.beq. 5gogge.bqe qgobo544pb 2.6ogeb663b
qOPPPDTPPP
ELZ
274
tctcctcaag gacctggagg agttgctgga acatctacca gaaaccagta tcctcagaga 1920
agtgcacatg agaggagcga caatgaatct gagagtattg atttacactc ttggacaaga 1980
agtggtggcc ctcttatgag gacaacctca gccaataaat tcatcagctt tgttcagaat 2040
cttgagatcg acacagaatc cagaacaatt ccatcgaggg aagacataac tgatcttgtg 2100
acaccaaatg ctggtacctt ggcagctcat gcagtgagta gagaagcaat cgataggagc 2160
ttggacaatt cagctttaga tatccatgat accagtaccc ctagatcgac atttggccct 2220
tcaacaagta ttgtggtttc tgaaggtgac ttgttgcagc ctgaaaagat tgaaaatggt 2280
attttgttta atgttgtaag gagggatact ctgctcgggt ctagtagtgg agttgagtct 2340
caaggatctc ctcgggaacc agatgttgaa acagtacaga cggagtgcct tgatggcgtg 2400
tctacttctg atgatgatga tgacaagaaa ctaaatgcca ttgatgatgg aggaactagt 2460
cccatgagca gaaataatct acaacatcag gggtcctcac tggaagaaaa attataccat 2520
ccctcttcct taaattctga agacgagaca aacacaaaca aaccagaagc tgcatcgatt 2580
tttgatatat gtacagatat gcatccggca tctattagcc tacctgaagg gtcttcagaa 2640
aagacagaac tagaaacaac aaagattcct gatgacaatt caactgttat gaatgatgaa 2700
gttgcctcag gtgctggtaa ctaa 2724
<210> 39
<211> 3470
<212> DNA
<213> Nicotiana benthamiana
<400> 39
gttatctgat ccaaacttct gactttttct attttccgaa tccctatgtt ttttaataaa 60
tccatctctg ccattgcact gatatattca tttattgtta tcaccttctt catttattgg 120
tocctotgtg ttttccatat attgaaggag aaaacattaa ctttatgcga ttttgtagtt 180
tttctggttg attcctacaa ccccttttga cattgatctt gtgggttaca aaaaacattg 240
aatctttatg tcaaaatttg atctttgtat ttcattttaa attgaaattt gatttttggg 300
ggtattaagg attcttttgt cggttgattt tgtgcctttt ttgccaagtt cttgtcggtc 360
tctgagctga atttccataa tttgacaaaa agaaaaggct aaagcagaaa ggttgggagt 420
ttctttcttt gactttcaga aactaaggta ttttctttga tctaattctt gttaatatct 480
ggttcaatct gattccgttg aatcttgtga atagcctttg tttccctatt gtcagaaaat 540
tatttccttt tcactttcct cgactctcag aagttagtac aatctttgtt ctgctaaatc 600
ttgtgaataa cctttagctt agagttttag gtatctgtat attgggttct cttaacattt 660
agcctagaag ccttctctag gattagtacc ccttttcatt gagatggata taagtaatga 720
ggctacaatt gacttctttt ccattggacc tactacgata ttgggtcgaa caatcgcctt 780
tagagtgttg ttctgtaaat caatttcaca attgaagcat cacctatttc atttcttgat 840
atattacttg tacaaattca agaatggttt gtcatactac ttgacaccct tgatctcgtg 900
gttgcaccct cgtaatccac aaggaatatt ggcattggta acgcttctcg ccttcttgtt 960
gaggcgatac acgaatgtaa aaatcaaggc tgagatggcc tataggagga agttttggag 1020
gaatatgatg agatctgcat tgacttatga ggagtgggct catgctgcca agatgctaga 1080
taaagagacc cctaaaatga atgaggcaga tctttatgat gtagaattag ttcgaaataa 1140
actccaagag cttcgacatc gtaggcaaga gggttctatg agggatatca tattctgtat 1200
gagagctgac cttgttagga atcttggtaa tatgtgtaat ccagaacttc acaagggaag 1260
gcttcatgtg cctagactga ttaaggatta tattgatgag gtttcaactc agttgagaat 1320
ggtatgcgac tctgattcgg aggagcttct cttggaagag aagcttgctt tcatgcatga 1380
aacaagacat gcctttggta ggacagcttt gcttttaagt ggaggtgctt ctttaggagc 1440
tttccatatg ggcgtggtga aaacacttgt agaacacaaa ctgatgccac ggataattgc 1500
tggttcaagt gtcggctcga ttatgtgctc catagttgca actcgatctt ggcctgagct 1560
ccagagtttt ttcgaggact cctggcactc tttgcaattt ttcgatcagt ngggtgggat 1620
ttttactatt ttcaggaggg tcatgaccca gggtgctgta catgagatca gacagctgca 1680
ggtgctgtta cgtaatctca cgaataatct tactttccaa gaagcctatg acatgactgg 1740
tagagttctg gggattactg tttgctcgcc taggaaacat gaacctccta gatgcttgaa 1800
ctacttgact tcacctcatg ttgttatatg gagtgccgtt accgcttctt gtgcctttcc 1860
tggtctcttc gaagctcaag aacttatggc aaaggataga agtggagatc ttgttccata 1920
tcacccacca tttcatttgg gtcctgatgc cacttctagt gcatctgctc gtcgttggag 1980
CA 2998211 2018-03-16
275
ggatggtagc ttggaggttg atttgccaat gatacagcta aaggagctct tcaatgtcaa 2040
tcactttatt gtgagccagg cgaatccaca tattgotcca ctgctgagga tcaaagagtt 2100
tgtaagagct tatggaggca actttgctgc caagcttgct caacttacgg aaatggaggt 2160
gaagcacaga tgcaatcagg tattagaact tggttttccc ttgggaggat tagcaaagct 2220
ttttgctcaa gaatgggaag gtaatgtaac tgttgtaatg cctgccactc tagctcagta 2280
ctcaaaaatc atacagaatc cctcgactct ggaactgcaa aaaggagcaa atcaaggaag 2340
aaggtgcact taggaaaaac tctcagccat gaaagcaaac tgtggaattg agcttgcact 2400
tgatgaatgc gttgctatac tgaatcacat gcgtagactg aaaaggagtg ctgagagggc 2460
ggctgctgct tcacatggct tggcaagcac tgtcagagtt aacacttcca gaagaattcc 2520
ttcttggaac tgcattgcac gagagaactc aacaggctcc cttgaagatt ttcttgcgga 2580
tgttgctgct tcacatcatc aaggaggcag tggttcggag gcgcatgtta accgtagttg 2640
gcaaacgoac cggaatgcac atgatggtag tgacagtgag ccggaaaatg tggaccttaa 2700
ttcttggaca agatcgggtg gtcatttgat gagaacaaca tcagctgata agtttattga 2760
ctttgtccag aacttggaaa ttggttcgcg attgaacaaa ggattgacta ttgacctcaa 2820
caatattatt cctcagatgg caagcaggga ccatttctcc ccaagcccaa gggtaacaac 2880
acctgataga agttcagata cagaatttga tcaaagagat tttagttaca gggtccctgc 2940
gagtagttca agcattatgg taagagaagg tgaccttctg caacctgaaa ggactaacag 3000
cggtattatc ttcaatgtag taaggaaagg agacttgacc ccatcgaaca gaagccttga 3060
ttcagaaaat aatagttccg tgcaggatgc agttgctgag tgcgtgcaac ttgaaagtcc 3120
agaaaaggag atggatatta gotcagtatc ggaggatggt gagaatgatg ttgggcaagg 3180
aagtagggta aatgaagttg attgtagtaa aaatcgttca tcaatcggtg atgacaacga 3240
taagcaagtt attgatactt gagagtttag ctttgattat tctacacagg ccattcgaat 3300
tattttttat actcaaatgg agottotttc agagctaaca cactcagaat tggggttgta 3360
aatagtgcaa gtagcaaat.c tgtaataaat gtttagtgta gtcatcaccc ttctactagt 3420
tcaaagtggc tcagttcaat tcaaattcag aacttcgata attcatgttt 3470
<210> 40
<211> 713
<212> DNA
<213> Nicotiana benthamiana
<400> 40
tgtatgagag ctgaccttgt taggaatctt ggtaatatgt gtaatccaga acttcacaag 60
ggaaggcttc atgtgcctag actgattaag gattatattg atgaggtttc aactcagttg 120
agaatggtat gcgactctga ttcggaggag cttctottgg aagagaagct tgctttcatg 180
catgaaacaa gacatgcctt tggtaggaca gctttgcttt taagtggagg tgcttcttta 240
ggagctttcc atgtgggcgt ggtgaaaaca cttatagaac acaaactgat gccacggata 300
attgctggtt caaatgtcgg czcgattatg tgctccatag ttgcaactcg atcttggcct 360
gagctccaga gttttttcga ggactcctgg cactctttgc aatttttcga tcagttgggt 420
gggattttta ctattttcag gagggtcatg acccagggtg ctgtacatga gatcagacag 480
ctgcaggtgc tgttacgtaa tctcacgaat aatcttactt tccaagaagc ctatgacatg 540
actggtagag ttctggggat tactgtttgc tcgcctagga aacatgaacc tcctagatgc 600
ttgaactact tgacttcacc tcatgttgtt atatggagtg ccgttaccgc ttcttgtgcc 660
tttcctggtc tattcgaagc tcaagaactt atggcaaagg atagaagtgg aga 713
<210> 41
<211> 1500
<212> DNA
<213> Arabidopsis thallana
<400> 41
cgaaaaaaga agtagaatat atatatatat atatatatat atatatatat atatatattc 60
gtgtggacat cataaatgcc taaatgataa tagttgattt cgagttttat tttcgttact 120
tccaatcaaa ttctccttgc accatattta tttttttact gtgagaacat atataagtat 180
CA 2998211 2018-03-16
276
atattggaat tacgtatccg agaggttttt gcatatttcg tttatttatt ttcgatatcc 240
acactactgt attattaaaa atttgaaaaa ttcaactagg gcttttcatc ttctctagaa 300
ttattcgttt atttatgtog atgtccacac tattattaaa ataaaacgag aggatatggt 360
tggatcatcc aagtttcgtt tatgactctt tgttcattta caaacgttta gttttccact 420
taagttttga aaagagttaa tttccaatat attcggcaca gtttttcaag tatattcatc 480
tgtttttttt ttttttggtt ggctatatgg tccaaatttt gatttgcaat atgagattgc 540
acagagagaa caatctttca ttatgattaa ttattgtaca agtaacaaac accaatctcc 60C
gatatacttt ggctctttag cacattgtta tgctagaagt tagcggaaat ctatatgttg 660
ttaaacgcag cgtttaaatt gaacagtata atttaccttg aaattttaag actacatgct 720
gtttagaatt tcagatgaaa acatcttgat gttttagaaa tccacgtagg aatagcgtaa 780
aatcttatcc aacgaactta ttttggtttt gttgtatttg tgcaagtcgt cacgctaatc 840
gaaaaaauaa aagaaaaaaa gaagccgtca tgatcggcca tttctcgacc gagtctgagt 900
ctaactctgc gtccgtgtca ccattatcag atcgagcctg tcttatctcg ttgcgattcc 960
ctatgcaaaa atcttcttct tttttttatt cccccattta tctctgatct cttctctctt 1020
ctcaagtaaa cctctctgct tcacgtctct tcttttcttg tcgattttcc ccagataatc 1080
aggtaaataa ggctactttc ttatttgatc tggtggtctt tgtgttgaaa tatctggatt 1140
ttctctgttg atttcaaagt tctctctttt tttttttgtt tactgggtgc tgtgaaaaat 1200
gatcttgtca aagtctcctc ttttcatcga attgaaactc taattagaaa aaagatcata 1260
acttttatta aaaaaatgag tttgctttgc ttaattttgc gaattgotto atagattcat 1320
tgattagcct atttggggta acaaaaaaaa gctgacacgg tttcagattc caaaaataga 1380
tcatgactct gtttcttctc tgcagaggtt ttaataaata tat gcttctt ctcatgagtt 1440
ctcgtttttt ttgtcacctt cgcagttgaa aacacaccca aattcatctt cgaatcaata 1500
<210> 42
<211> 2871
<212> DNA
<213> Artificial Sequence
<220>
<223> Nucleotide sequence of the complement of the pSSU-Oleosin gene in
the T-DNA of pJP3502. In order (complementary sequences):
Glycine max Lectin terminator 348nt, 3' exon 255nt, U3Q10 intron
304nt, 5' exon 213nt, SSU promoter
<400> 42
ggccoctaga atctaattat tctattcaga ctaaattagt ataagtattt ttttaatcaa 60
taaataataa ttaataattt attagtagga gtgattgaat ttataatata ttttttttaa 120
tcatttaaag aatcttatat ctttaaattg acaagagttt taaatgggga gagtgttatc 180
atatcacaag taggattaat gtottatagt ttcacatgca ttacgataag ttgtgaaaga 240
taacattatt atatataaca ataacaatca ctagcgatcg agtagtgaga gtcgtcttat 300
tacactttct tccttcgatc tgtcacatgg cggcggcccg cggccgcttc attactcgag 360
ccaggaggat ggatcgatgc tggtctgaga ccctgctacc ggttgctgac tgaactgctc 420
ggcacggtcc ttcatttcac gggccttcct cgccaacttt gtcttggccg actccaactg 480
atccgctccg ggtggatgtt tccccgtcag gtaacggtag atccaggaca gcacagacag 540
agcggcaaca ccaaatcccc cgcttgccag aaaacccgct cccaacagga agatggtgat 600
gactgcagat cagaaaaact cagattaatc gacaaattcg atcgcacaaa ctagaaacta 660
acaccagatc tagatagaaa tcacaaatcg aagagtaatt attcgacaaa actcaaatta 720
tttgaacaaa tcggatgata tctatgaaac cctaatcgag aattaaqatg atatctaacq 780
atcaaaccca gaaaatcgtc ttcgatctaa gattaacaga atctaaacca aagaacatat 840
acgaaattgg gatcgaacga aaacaaaatc gaagattttg agagaataag gaacacagaa 900
atttacctgc agggaccagt acaggcgaga agatcaccag gagaggtgtg gcgattgtca 960
gcgcaatgac cgttccagcc agggtcaacc cggataacac caacaggcta cctccggcag 1020
taaccgcggt cgctgccttt acaacacgct gagcacgcgg ttgcagttgc aagtgggggg 1080
cacgtgtttg ttgctgctgc ccgtagtgct ctgccatggt tttttttaac ggagcaagcg 1140
gccgctgttc ttctttactc tttgtgtgac tgaggtttgg tctagtgctt tggtcatcta 1200
CA 2998211 2018-03-16
277
tatataatga taacaacaat gagaacaagc tttggagtga tcggagggtc taggatacat 1260
gagattcaag tagactagga tctacaccgt tggattttga gtgtggatat gtgtgaagtt 1320
aattttactt ggtaacggcc acaaaggcct aaggagaggt gttgagaccc ttatcggctt 1380
gaaccgctgg aataatacca cgtggaagat aattccatga atcttatcgt tatctatgag 1440
tgaaattatg tgatggtgga gtggtgcttg ctcattttac ttgcctggtg gacttggccc 1500
tttccttatg gagaatttat attttactta ctatagagat ttcatacctt ttttttacct 1560
tggatttagt taatatataa tggtatgatt catgaataaa aatgggaaat ttttgaattt 1620
gtactgctaa atgcataaga ttaggtgaaa ctgtggaata tatatttttt tcatttaaaa 1680
gcaaaatttg ccttttacta gaattataaa tatagaaaaa tatataacat tcaaataaaa 1740
atgaaaataa gaactttcaa aaaacagaac tatgtataat gtgtaaagat tagtcgcaca 1800
tcaagtcatc tattacaata tgttacaaca agtcataagc ccaacaaagt tagcacatct 1860
aaataaacta aagagtccac gaaaatatta caaatcataa gcccaacaaa gttattgatc 1920
aaaaaaaaaa aacgcccaac aaagctaaac aaagtccaaa aaaaacttct caagtctcca 1980
tattccttta tqaacattga aaactataca caaaacaagt cagataaatc totttctggg 2040
cctgtcaatcc caacctccta catcacttcc ctatcggatt gaatgtttta cttgtacctt 2100
ttccgttgca atgatattga tagtatgttt gtgaaaacta atagggttaa caatcgaagt 2160
catggaatat ggatttggtc caagattttc cgagagcttt ctagtagaaa gcccatcacc 2220
agaaatttac tagtaaaata aatcaccaat taggtttott attatgtgcc aaattcaata 2280
taattataga ggatatttca aatgaaaacg tatgaatgtt attagtaaat ggtcaggtaa 2340
gacattaaaa aaatcctacg tcagatattc aactttaaaa attcgatcag tgtggaattg 2400
tacaaaaatt tgggatctac tatatatata taatgcttta caacacttgg attttttttt 2460
ggaggctgga atttttaatc tacatatttg ttttggccat gcaccaactc attgtttagt 2520
gtaatacttt gattttgtca aatatatgtg ttcgtgtata tttgtataag aatttctttg 2580
accatataca cacacacata tatatatata tatatatatt atatatcatg cacttttaat 2640
tgaaaaaata atatatatat atatagtgca ttttttctaa caaccatata tgttgcgatt 2700
gatctgcaaa aatactgcta gaataatgaa aaatataatc tattgctgaa attatctcag 2760
atgttaagat tttcttaaag taaattcttt caaattttag ctaaaagtct tgtaataact 2820
aaagaataat acacaatctc gaccacggaa aaaaaacaca taataaattt g 2871
<210> 43
<211> 362
<212> PRT
<213> Arabidopsis thaliana
<400> 43
Met Leu Lys Leu Ser Cys Asn Val Thr Asp Ser Lys Leu Gin Arg Ser
1 5 10 15
Leu Leu Phe Phe Set His Ser Tyr Arg Ser Asp Pro Val Asn Phe Ile
20 25 30
Arg Arg Arg Ile Val Ser Cys Ser Gin Thr Lys Lys Thr Gly Leu Val
35 40 45
Pro Leu Arg Ala Val Val Ser Ala Asp Gin Gly Ser Val Val Gin Gly
50 55 60
Leu Ala Thr Leu Ala Asp Gin Leu Arg Leu Gly Ser Leu Thr Glu Asp
65 70 75 80
Gly Leu Ser Tyr Lys Glu Lys Pile Val Val Arg Ser Tyr Glu Val Gly
85 90 95
Ser Asn Lys Thr Ala Thr Val Glu Thr Ile Ala Asn Leu Leu Gin Glu
100 105 110
Val Gly Cys Asn His Ala Gin Ser Val Gly Phe Ser Thr Asp Gly Phe
115 120 125
Ala Thr Thr Thr Thr Met Arg Lys Leu His Leu Ile Trp Val Thr Ala
130 135 140
Arg Met His Ile Glu Ile Tyr Lys Tyr Pro Ala Trp Gly Asp Val Val
145 150 155 160
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Glu Ile Glu Thr Trp Cys Gin Ser Glu Gly Arg Ile Gly Thr Arg Arg
165 170 175
Asp Trp Ile Leu Lys Asp Ser Val Thr Gly Glu Val Thr Gly Arg Ala
180 185 190
Thr Ser Lys Trp Val Met Met Asn Gin Asp Thr Arg Arg Lou Gin Lys
195 200 205
Val Ser Asp Asp Val Arg Asp Glu Tyr Leu Val Phe Cys Pro Gin Glu
210 215 220
Pro Arg Leu Ala Phe Pro Glu Glu Asn Asn Arg Ser Leu Lys Lys Ile
225 230 235 240
Pro Lys Leu Glu Asp Pro Ma Gin Tyr Ser Met Ile Gly Leu Lys Pro
245 250 255
Arg Arg Ala Asp Leu Asp Met Asn Gin His Val Asn Asn Val Thr Tyr
260 265 270
Ile Gly Trp Val Leu Glu Ser Ile Pro Gin Glu Ile Val Asp Thr His
275 280 285
Glu Leu Gin Val Ile Thr Leu Asp Tyr Arg Arg Glu Cys Gin Gln Asp
290 295 300
Asp Val Val Asp Ser Leu Thr Thr Thr Thr Ser Glu Ile Gly Gly Thr
305 310 315 320
Asn Gly Ser Ala Thr Ser Gly Thr Gin Gly His Asn Asp Ser Gin Phe
325 330 335
Leu His Leu Leu Arg Leu Ser Gly Asp Gly Gin Glu Ile Asn Arg Gly
340 345 350
Thr Thr Leu Trp Arg Lys Lys Pro Ser Ser
355 360
<210> 44
<211> 367
<212> PRT
<213> Arabidopsis thaliana
<400> 44
Met Leu Lys Leu Ser Cys Asn Val Thr Asp His Ile His Asn Leu Phe
1 5 10 15
Ser Asn Ser Arg Arg Ile Phe Val Pro Val His Arg Gin Thr Arg Pro
20 25 30
Ile Ser Cys Phe Gin Leu Lys Lys Glu Pro Leu Arg Ala Ile Leu Per
35 40 45
Ala Asp His Gly Asn Ser Ser Val Arg Val Ala Asp Thr Val Per Gly
50 55 60
Thr Ser Pro Ala Asp Arg Leu Arg Phe Gly Arg Leu Met Glu Asp Gly
65 70 75 80
Phe Ser Tyr Lys Glu Lys Phe Ile Val Arg Ser Tyr Glu Val Gly Ile
85 90 95
Asn Lys Thr Ala Thr Ile Glu Thr Ile Ala Asn Leu Leu Gin Glu Val
100 105 110
Ala Cys Asn His Val Gin Asn Val Gly Phe Ser The Asp Gly Phe Ala
115 120 125
Thr Thr Leu Thr Met Arg Lys Lou His Leu Ile Trp Val Thr Ala Arg
130 135 740
Met His Ile Glu Ile Tyr Lys Tyr Pro Ala Trp Ser Asp Val Val Glu
145 . 150 155 160
Ile Glu Thr Trp Cys Gin Ser Glu Gly Arg Ile Gly Thr Arg Arg Asp
165 170 175
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Trp Ile Leu Lys Asp Cys Ala Thr Gly Glu Val Ile Gly Arg Ala Thr
180 185 190
Ser Lys Trp Val Met Met Asn Gin Asp Thr Arg Arg Leu Girl Arg Val
195 200 205
Thr Asp Glu Val Arg Asp Glu Tyr Leu Val Phe Cys Pro Pro Glu Pro
210 215 220
Arg Leu Ala Phe Pro Glu Glu Asn Asn Ser Her Leu Lys Lys Ile Pro
225 230 235 240
Lys Leu Glu Asp Pro Ala Gin Tyr Ser Met Leu Gly Leu Lys Pro Arg
245 250 255
Arg Ala Asp Leu Asp Met Asn Gin His Val Ash Asn Val Thr Tyr Ile
260 265 270
Gly Trp Val Leu Giu Ser Ile Pro Gln Glu Ile Ile Asp Thr His Glu
275 280 285
Leu Lys Val Ile Thr Leu Asp Tyr Arg Arg Glu Cys Gin Gin Asp Asp
290 295 300
Ile Val Asp Ser Leu Thr Thr Ser Glu Thr Pro Asn Giu Val Val Ser
305 310 315 320
Lys Leu Thr Gly Thr Asn Gly Ser Thr Thr Ser Ser Lys Arg Glu His
325 330 335
Asn Glu Ser His Phe Leu His Ile Leu Arg Leu Ser Glu Asn Gly Gin
340 345 350
Glu Ile Asn Arg Gly Arg Thr Gin Trp Arg Lys Lys Ser Ser Arg
355 360 365
<210> 45
<211> 412
<212> PRT
<213> Arabidopsis thaliana
<400> 45
Met Val Ala Thr Ser Ala Thr Ser Ser Phe Phe Pro Val Pro Ser Ser
1 5 10 15
Ser Leu Asp Pro Asn Gly Lys Gly Asn Lys Ile Gly Ser Thr Asn Leu
20 25 30
Ala Gly Leu Asn Ser Ala Pro Asn Ser Gly Arg Met Lys Val Lys Pro
35 40 45
Asn Ala Gin Ala Pro Pro Lys Ile Asn Gly Lys Lys Val Gly Leu Pro
50 55 60
Gly Ser Val Asp Ile Val Arg Thr Asp Thr Glu Thr Ser Ser His Pro
65 70 75 eo
Ala Pro Arg Thr Phe Ile Asn Gin Leu Pro Asp Trp Ser Met Leu Leu
85 90 95
Ala Ala Ile Thr Thr Ile Phe Leu Ala Ala Glu Lys Gin Trp Met Met
100 105 110
Leu Asp Trp Lys Pro Arg Arg Ser Asp Met Leu Val Asp Pro Phe Gly
115 120 125
Ile Gly Arg Ile Val Gin Asp Gly Leu Val Phe Arg Gin Asn Phe Ser
130 135 140
Ile Arg Ser Tyr Glu Ile Gly Ala Asp Arg Ser Ala Ser Ile Glu Thr
145 150 155 160
Val Met Asn His Leu Gin Glu Thr Ala Leu Asn His Val Lys Thr Ala
165 170 175
Gly Leu Leu Gly Asp Gly Phe Gly Ser Thr Pro Glu Met Phe Lys Lys
180 185 190
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Asn Leu Ile Trp Val Val Thr Arg Met Gln Val Val Val Asp Lys Tyr
195 200 205
Pro Thr Trp Gly Asp Val Val Glu Val Asp Thr Trp Val Ser Gln Ser
210 215 220
Gly Lys Asn Gly Met Arg Arg Asp Trp Leu Val Arg Asp Cys Asn Thr
225 230 235 240
Gly Glu Thr Leu Thr Arg Ala Ser Ser Val Trp Val Met Met Asn Lys
245 250 255
Lou Thr Arg Arg Leu Ser Lys Ile Pro Glu Glu Val Arg Gly Glu Ile
260 265 270
Glu Pro Tyr Phe Val Asn Ser Asp Pro Val Leu Ala Glu Asp Ser Arg
275 280 285
Lys Leu Thr Lys Ile Asp Asp Lys Thr Ala Asp Tyr Val Arg Ser Gly
290 295 300
Leu Thr Pro Arg Trp Ser Asp Leu Asp Val Asn Gln His Val Asn Asn
305 310 315 320
Val Lys Tyr Ile Gly Trp Ile Leu Glu Ser Ala Pro Val Gly Ile Met
325 330 335
Glu Arg Gln Lys Leu Lys Ser Met Thr Leu Glu Tyr Arg Arg Glu Cys
340 345 350
Gly Alp Asp Ser Val Leu Gln Ser Leu Thr Ala Val Thr Gly Cys Asp
355 360 365
Ile Gly Asn Leu Ala Thr Ala Gly Asp Val Glu Cys Gln His Leu Leu
370 375 380
Arg Leu Gln Asp Gly Ala Glu Val Val Arg Gly Arg Thr Glu Trp Ser
385 390 395 400
Ser Lys Thr Pro Thr Thr Thr Trp Gly Thr Ala Pro
405 410
<210> 46
<211> 345
<212> PRT
<213> Arabidopsis thaliana
<400> 46
Met Phe Ile Ala Val Glu Val Ser Pro Val Met Glu Asp Ile Thr Arg
1 5 10 15
Gln Ser Lys Lys Thr Ser Val Glu Asn Glu Thr Gly Asp Asp Gln Ser
20 25 30
Ala Thr Ser Val Val Leu Lys Ala Lys Arg Lys Arg Arg Ser Gln Pro
35 40 45
Arg Asp Ala Pro Pro Gln Arg Ser Ser Val His Arg Gly Val Thr Arg
SO 55 60
His Arg Trp Thr Gly Arg Tyr Glu Ala his Leu Trp Asp Lys Asn Ser
65 70 75 80
Trp Asn Glu Thr Gln Thr Lys Lys Gly Arg Gln Val Tyr Leu Gly Ala
85 90 95
Tyr Asp Glu Glu Asp Ala Ala Ala Arg Ala Tyr Asp Leu Ala Ala Leu
100 105 110
Lys Tyr Trp Gly Arg Asp Thr Ile Leu Asn Phe Pro Leu Cys Asn Tyr
115 120 125
Glu Glu Asp Ile Lys Glu Met Glu Ser Gln Ser Lys Glu Glu Tyr Ile
130 135 140
Gly Ser Leu Arg Arg Lys Ser Ser Gly Phe Ser Alp Gly Val Ser Lys
145 150 155 160
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Tyr Arg Gly Val Ala Lys His His His Asn Gly Arg Trp Glu Ala Arg
165 170 175
Ile Gly Arg Val Phe Gly Asn Lys Tyr Leu Tyr Leu Gly Thr Tyr Ala
180 185 190
Thr Gin Glu Glu Ala Ala Ile Ala Tyr Asp Ile Ala Ala Ile Glu Tyr
395 200 205
Arg Gly Leu Asn Ala Val Thr Asn Phe Asp Ile Ser Arg Tyr Leu Lys
210 215 220
Leu Pro Val Pro Glu Asn Pro Ile Asp Thr Ala Asn Asn Lau Leu Glu
225 230 235 240
,Ser Pro His Ser Asp Leu Ser Pro Phe Ile Lys Pro Asn His Glu Ser
245 250 255
Asp Leu Ser Gin Ser Gin Ser Ser Ser Glu Asp Asn Asp Asp Arg Lys
260 265 270
Thr Lys Leu Leu Lys Ser Ser Pro Leu Val Ala Glu Glu Val Ile Gly
275 280 285
Pro Ser Thr Pro Pro Glu Ile Ala Pro Pro Arg Ara Ser Phe Pro Glu
290 295 300
Asp Ile Gin Thr Tyr Phe Gly Cys Gin Asn Ser Gly Lys Lou Thr Ala
305 310 315 320
Glu Glu Asp Asp Val Ile Phe Gly Asp Leu Asp Ser Phe Leu Thr Pro
325 330 335
Asp Phe Tyr Ser Glu Leu Asn Asp Cys
340 345
<210> 47
<211> 303
<212> PRT
<213> Arabidopsis thaliana
<400> 47
Met Ala Lys Val Ser Gly Arg Ser Lys Lys Thr Ile Val Asp Asp Glu
1 5 10 15
Ile Ser Asp Lys Thr Ala Ser Ala Ser Glu Ser Ala Ser Ile Ala Leu
20 25 30
Thr Ser. Lys Arg Lys Arg Lys Ser Pro Pro Arg Asn Ala Pro Leu Gin
35 40 45
Arg Ser Ser Pro Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg
50 55 60
Tyr Glu Ala His Leu Trp Asp Lys Asn Ser Trp Asn Asp Thr Gin Thr
65 70 75 SO
Lys Lys Gly Arg Gin Val Tyr Leu Gly Ala Tyr Asp Glu Glu Glu Ala
85 90 95
Ala Ala Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Arg Asp
100 105 110
Thr Leu Leu Asn Phe Pro Leu Pro Ser Tyr Asp Glu Asp Val Lys Glu
115 120 125
Met Glu Gly Gin Ser Lys Glu Glu Tyr Ile Gly Ser Leu Arg Arg Lys
130 135 140
Ser Ser Gly Phe Ser Arg Gly Val Ser Lys Tyr Arg Gly Val Ala Arg
145 150 155 160
His His His Asn Gly Arg Trp Glu Ala Arg Ile Gly Arg Val Phe Ala
165 170 175
Thr Gin Glu Glu Ala Ala Ile Ala Tyr Asp Ile Ala Ala Ile Glu Tyr
180 185 190
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Arg Gly Leu Asn Ala Val Thr Asn Phe Asp Val Ser Arg Tyr Leu Asn
195 200 205
Pro Asn Ala Ala Ala Asp Lys Ala Asp Ser Asp Ser Lys Pre Ile Arg
210 215 220
Ser Pro Ser Arg Glu Pro Glu Ser Ser Asp Asp Asn Lys Ser Pro Lys
225 230 235 240
Ser Glu Glu Va] Ile Glu Pro Ser Thr Ser Pro Glu Val Ile Pro Thr
245 250 255
Arg Arg Ser Phe Pro Asp Asp Ile Gln Thr Tyr Phe Gly Cys Gln Asp
260 265 270
Ser Gly Lys Leu Ala Thr Glu Glu Asp Val Ile Phe Asp Cys Phe Asn
275 280 285
Ser Tyr Ile Asn Pro Gly Phe Tyr Asn Glu Phe Asp Tyr Gly Pro
290 295 300
<210> 48
<211> 445
<212> PRT
<213> Avena sativa
<400> 48
Met Lys Arg Ser Pro Pro Pro Ala Pro Pro Ala Ala Pro Pro Pro Pro
1 5 10 15
Gln Pro Ser Pro Set Ser Ser Ser Pro Ala Cys Ser Pro Ser Pro Ser
20 25 30
Ser Ser Ser Cys Pro Ser Ser Ser Asp Ser Ser Ser Ile Val Ile Pro
35 40 45
Arg Lys Arg Ala Arg Thr Gln Lys Ala Ala Ser Gly Lys Pro Lys Ala
50 55 60
Lys Ala Ser Ala Lys Arg Pro Lys Lys Asp Ala Ser Arg Ser Ser Lys
65 70 75 80
Glu Thr Asp Ala Asn Gly Ala Ala Ala Ala Ala Gly Lys Arg Ser Ser
85 90 95
Ile Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Phe Glu Ala
100 105 110
His Leu Trp Asp Lys Asn Cys Phe Thr Ser Val Gin Asn Lys Lys Lys
115 120 125
Gly Arg Gln Val Tyr Leu Gly Ala Tyr Asp Thr Glu Asp Ala Ala Ala
130 135 140
Arg Ala Tyr Asp Lou Ala Ala Leu Lys Tyr Trp Gly Ser Glu Thr Ile
145 150 155 760
Leu Asn Phe Ser Val Glu Asp Tyr Ala Lys Glu Met Pro Glu Met Glu
165 170 175
Ala Val Ser Arg Glu Glu Tyr Leu Ala Ala Leu Arg Arg Arg Ser Ser
180 185 190
Gly Phe Ser Arg Gly Vol Ser Lys Tyr Arg Gly Vol Ala Arg His His
195 200 205
His Asn Gly Arg Trp Glu Ala Arg Ile Gly Arg Val Leu Gly Asn Lys
210 215 220
Tyr Leu Tyr Lou Gly Thr Phe Asp Thr Gln Glu Glu Ala Ala Lys Ala
225 230 235 240
Tyr Asp Leu Ala Ala Ile Glu Tyr Arg Gly Ala Asn Ala Val Thr Asn
245 250 255
Phe Asp Ile Ser Cys Tyr Leu Asp Gln Pro Gln Leu Leu Ala Gln Leu
260 265 270
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Gin Gin Gly Pro Gin Val Val Pro Ala Leu Gin Glu Glu Leu Gin His
275 280 285
Asp Val Gin His Asp Leu Gin Asn Aso Asn Ala Val Gin Glu Leu Asn
290 295 300
Ser Gly Glu Val Gin Met Pro Gly Ala Met Asp Glu Pro Ile Ala Leu
305 310 315 ' 320
Asp Asp Ser Thr Glu Cys Ile Asn Thr Pro Phe Glu Phe Asp Phe Ser
325 330 335
Val Glu Glu Asn Leu Trp Ser Pro Cys Met Asp Tyr Glu Leu Asp Ala
340 345 350
Ile Leu Gly Asn Asn Thr Ser Asn Ser Ala Asn Met Asn Glu Trp Phe
355 360 365
Asn Asp Ser Thr Phe Glu Ser Asn Ile Gly Cys Leu Phe Glu Gly Cys
370 375 380
Ser Asn Ile Asp Asp Cys Ser Ser Ser Lys His Cys Ala Asp Leu Ala
385 390 395 400
Ala Phe Asp Phe Phe Lys Glu Gly Asp Asp Asn Asp Phe Ser Asn Met
405 410 415
Glu Met Glu Ile Thr Pro Gin Ala Asn Asp Val Ser Cys Pro Pro Asn
420 425 430
Asp Val Ser Cys Pro Pro Lys Met Ile Thr Val Cys Asn
435 440 445
<210> 49
<211> 420
<212> PRT
<213> Sorghum bicolor
<400> 49
Met Asp Met Glu Arg Ser Gin Gin Gin Lys Ser Pro Thr Glu Ser Pro
1 5 10 15
Pro Pro Pro Ser Pro Ser Ser Ser Ser Ser Ser Val Ser Ala Asp Thr
20 25 30
Val Leu Pro Pro Pro Gly Lys Arg Arg Arg Ala Ala Thr Thr Ala Lys
35 40 45
Ala Lys Ala Gly Ala Lys Pro Lys Arg Ala Arg Lys Asp Ala Ala Ala
50 55 60
Ala Ala Asp Pro Pro Pro Pro Pro Ala Ala Ala Ala Ala Gly Lys Arg
65 70 75 80
Ser Ser Vol Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Phe
85 90 95
Glu Ala His Leu Trp Asp Lys His Cys Leu Ala Ala Leu His Asn Lys
100 105 110
Lys Lys Gly Arg Gln Val Tyr Leu Gly Ala Tyr Asp Ser Glu Glu Ala
115 120 125
Ala Ala Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Pro Glu
130 135 140
Thr Leu Leu Asn Phe Pro Val Glu Asp Tyr Ser Ser Glu Met Pro Glu
145 150 155 160
Met Glu Gly Val Ser Arg Glu Glu Tyr Leu Ala Ser Leu Arg Arg Arg
165 170 175
Ser Ser Gly Phe Ser Arg Gly Vol Ser Lys Tyr Arg Gly Vol Ala Arg
180 185 190
His His His Asn Gly Arg Trp Glu Ala Arg Ile Gly Arg Val Phe Gly
195 200 205
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Asn Lys Tyr Leu Tyr Leu Gly Thr Phe Asp Thr Gin Glu Glu Ala Ala
210 215 220
Lys Ala Tyr Asp Leu Ala Ala Ile Glu Tyr Arg Gly Val Asn Ala Val
225 230 235 240
Thr Asn Phe Asp Ile Ser Cys Tyr Leu Asp His Pro Leu Phe Leu Ala
245 250 255
Gin Leu Gin Gin Glu Pro Gin Val Val Pro Ala Leu Asn Gin Glu Ala
260 265 270
Gin Pro Asp Gin Sex Glu Thr Glu Thr Ile Ala Gin Glu Ser Val Ser
275 280 285
Ser Glu Ala Lys Thr Pro Asp Asp Asn Ala Glu Pro Asp Asp Asn Ala
290 295 300
Glu Pro Asp Asp Ile Ala Glu Pro Leu Ile Thr Val Asp Asp Ser :le
305 310 315 320
Glu Glu Ser Leu Trp Ser Pro Cys Met Asp Tyr Glu Leu Asp Thr Met
325 330 335
Ser Arg Ser Asn Phe Gly Ser Ser Ile Asn Leu Ser Glu Trp Phe Asn
340 345 350
Asp Ala Asp Phe Asp Ser Asn Ile Gly Cys Leu Phe Asp Gly Cys Ser
355 360 365
Ala Val Asp Glu Gly Gly Lys Asp Gly Val Gly Leu Ala Asp Phe Ser
370 375 380
Leu Leu Glu Asp Phe Ser Leu Phe Glu Ala Gly Asp Gly Gin Leu Lys
385 390 395 400
Asp Val Leu Ser Asp Met Glu Glu Gly Ile Gin Pro Pro Thr Met Ile
405 410 415
Ser Val Cys Asn
420
<210> 50
<211> 395
<212> PRT
<213> Zea mays
<400> 50
Met Glu Arg Ser Gin Arg Gin Ser Pro Pro Pro Pro Ser Pro Ser Ser
1 5 10 15
Ser Ser Ser Ser Val Ser Ala Asp Thr Val Leu Val Pro Pro Gly Lys
20 25 30
Arg Arg Arg Ala Ala Thr Ala Lys Ala Gly Ala Glu Pro Asn Lys Arg
35 40 45
Ile Arg Lys Asp Pro Ala Ala Ala Ala Ala Gly Lys Arg Ser Ser Val
50 55 60
Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Phe Glu Ala His
65 70 75 80
Leu Trp Asp Lys His Cys Leu Ala Ala Leu His Asn Lys Lys Lys Gly
85 90 95
Arg Gin Val Tyr Leu Gly Ala Tyr Asp Ser Glu Glu Ala Ala Ala Arg
100 105 110
Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Pro Glu Thr Leu Leu
115 120 125
Asn Phe Pro Val Clu Asp Tyr Ser Ser Glu Met Pro Glu Met Glu Ala
130 135 140
Val Ser Arg Glu Glu Tyr Leu Ala Ser Leu Arg Arg Arg Ser Ser Gly
145 150 155 160
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Phe Ser Arg Gly Val Ser Lys Tyr Arg Gly Val Ala Arg His His His
165 170 175
Asn Gly Arg Trp Glu Ala Arg lie Gly Arg Val Phe Gly Asn Lys Tyr
180 185 190
Leu Tyr Leu Gly Thr Phe Asp Thr Gin Glu Glu Ala Ala Lys Ala Tyr
195 200 205
Asp Leu Ala Ala Ile Glu Tyr Arg Gly Val Asn Ala Val Thr Asn Phe
210 215 220
Asp lie Ser Cys Tyr Leu Asp His Pro Leu Phe Leu Ala Gin Leu Gin
225 230 235 240
Gin Glu Pro Gin Val Val Pro Ala Leu Asn Gin Glu Pro Gin Pro Asp
245 250 255
Gin Ser Glu Thr Gly Thr Thr Glu Gin Glu Pro Glu Ser Ser Glu Ala
260 265 270
Lys Thr Pro Asp Sly Ser Ala Glu Pro Asp Glu Asn Ala Val Pro Asp
275 280 285
Asp Thr Ala Glu Pro Leu Thr Thr Val Asp Asp Ser Ile Glu Glu Ply
290 295 300
Leu Trp Ser Pro Cys Met Asp Tyr Glu Leu Asp Thr Met Ser Arg Pro
305 310 315 320
Asn Phe Gly Ser Ser Ile Asn Leu Ser Glu Trp Phe Ala Asp Ala Asp
325 330 335
Phe Asp Cys Asn Ile Gly Cys Leu Phe Asp Gly Cys Ser Ala Ala Asp
340 345 350
Glu Gly Ser Lys Asp Gly Val Gly Leu Ala Asp Phe Ser Leu Phe Glu
355 360 365
Ala Gly Asp Val. Gin Leu Lys Asp Val Leu Ser Asp Met Glu Glu Gly
370 375 380
Ile Gin Pro Pro Ala Met Ile Ser Val Cys Asn
385 390 395
<210> 51
<211> 430
<212> PRT
<213> Triadica sebifera
<400> 51
Met Ala Ser Ser Ser Ser Asp Pro Val Leu Lys Ala Glu ieu Gly Ser
1 5 10 15
Ser Gly Gly Gly Cys Ser Ser Ply Gly Gly Gly Glu Ser Ser Glu Ala
20 25 30
Val Ile Ala Asn Asp Gin Leu Leu Leu Tyr Arg Gly Leu L)S' Lys Pro
35 40 45
Lys Lys Glu Arg Gly Cys Thr Ala Lys Glu Arg Ile Ser Lys Met Pro
50 55 60
Pro Cys Thr Ala Gly Lys Arg Ser Ser Ile Tyr Arg Gly Val Thr Arg
65 70 75 80
His Arg Trp Thr Gly Arg Tyr Clu Ala His Leu Trp Asp Lys Ser Thr
85 90 95
Trp Asn Gin Asn Gin Asn Lys Lys Gly Lys Gin Val Tyr Leu Gly Ala
100 105 110
Tyr Asp Asp Glu Glu Ala Ala Ala Arg Ala Tyr Asp Leu Ala Ala Leu
115 120 125
Lys Tyr Trp Gly Pro Gly Thr Leu Ile Asn Phe Pro Val Thr Asp Tyr
130 135 140
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Thr Arg Asp Leu Glu Glu Met Gln Asn Met Ser Arg Glu Glu Tyr Leu
145 150 155 160
Ala Ser Leu Arg Arg Lys Ser Ser Gly Phe Ser Arg Gly Ile Ser Lys
165 170 175
Tyr Arg Gly Leu Ser Ser Arg Trp Glu Ser Ser Val Gly Arg Met Pro
180 185 190
Gly Ser Glu Tyr Phe Ser Ser Ile Asn Tyr Val Asp Asp Pro Ala Ala
195 200 205
Glu Ser Glu Tyr Val Gly Ser Leu Cys Phe Glu Arg Lys Ile Asp Leu
210 215 220
Thr Ser Tyr Ile Lys Trp Trp Gly Leu Asn Lys Thr Arg Gin Ala Glu
225 230 235 240
Ser Ile Ser Lys Ser Ala Glu Glu Thr Lys Pro Gly Cys Ala Glu Asp
245 250 255
Ile Gly Gly Glu Leu Lys Thr Thr Glu Trp Ala Ile Gin Pro Thr Glu
260 265 270
Pro Tyr Gin Met Pro Arg Leu Gly Met Pro Val His Val Lys Lys His
275 280 285
Lys Gly Ser Lys Ile Ser Ala Leu Ser Val Leu Ser Gin Ser Ala Ala
290 295 300
?be Lys Ser Leu Gin Glu Lys Ala Ser Lys Lys Gin Glu Asn Ser Thr
305 310 315 320
Asp Asn Asp Glu Asn Glu Asn Lys Asn Thr Asn Thr Asn Lys Ile Asp
325 330 335
Tyr Gly Lys Ala Val Glu Thr Ser Ala Ser His Asp Ser Ser Asn Glu
340 345 350
Arg Pro Val Thr Ala Leu Gly Met Ser Gly Gly Leu Ser Leu Lys Arg
355 360 365
Asn Val Tyr Gin Leu Thr Pro Phe Leu Ser Ala Pro Leu Leu Thr Asn
370 375 380
Tyr Gly Thr Ile Asp Gin Leu Val Asp Pro Ile Leu Trp Ala Ser Leu
385 390 395 400
Val Pro Val Leu Pro Thr Gly Leu Ser Arg Asn Pro Glu Val Thr Lys
405 410 415
Thr Glu Thr Ser Ser Thr Tyr Thr Phe Phe Arg Pro Glu Glu
420 425 430
<210> 52
<211> 1531
<212> DNA
<213> Solanum tuberosum
<400> 52
ttttaaatca ttgttttatt ttctctttct ttttacaggt ataaaaggtg aaaattgaag 60
caagattgat tgcaagctat gtgtcaccac gttattgata ctttggaaga aatttttact 120
tatatgtctt tgtttaggag taatatttga tatgttttag ttagattttc ttgtcattta 180
tgctttagta taattttagt tatttttatt atatgatcat gggtgaattt tgatacaaat 240
atttttgtca ttaaataaat taatttatca caacttgatt actttcagtg acaaaaaatg 300
tattgtcgta gtaccctttt ttgttgaata tgaataattt tttttatttt gtgacaattg 360
taattgtcac tacttatgat aatatttagt gacatatatg tcgtcggtaa aagcaaacac 420
tttcagtgac aaaataatag atttaatcac aaaattatta acctttttta taataataaa 460
tttatcccta atttatacat ttaaggacaa agtatttttt ttatatataa aaaatagtct 540
ttagtgacga tcgtagtgtt gagtctagaa atcataatgt tgaatctaga aaaatctcat 600
gcagtgtaaa ataaacctca aaaaggacgt tcagtccata gagggggtgt atgtgacacc 660
ccaacctcag caaaagaaaa cctcccttca acaaggacat ttgcggtgct aaacaatttc 720
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aagtctcatc acacatatat ttataatata atactaataa agaatagaaa aggaaaggta 780
aacatcatta aatcgtcttt gtatattttt aatgacaact gattgacgaa atctttttcg 840
tcacacaaaa tttttagtga cgaaacatga tttatagatg atgaaattat ttgtacctca 900
taatctaatt tgttgtagtg atcattactc ctttgtttgt tttatttgtc atgttagtcc 960
attaaaaaaa aatatctctc ttcttatgta cgtgaatggt tggaacggat ctattatata 1020
atacaaataa agaataaaaa aaggaaagtg agtgagattc aagggagaga atctgtttaa 1080
tatcagagtc gatcatgtgt caattttatc gatatgaccc taacttcaac taagtataac 1140
caattccgat aaggcgagaa atatcatagt attgagtcta gaaaaatctc atgtagtgtg 1200
gggtaaacct cagcaaggac gttgagtcca tagagggggg tgtatgtgac accocaacct 1260
cagcaaaaga aaaccacccc tcaagaagga catttgaggt gctaaacaat ttcaagtctc 1320
atcacacata tatatatatt atataatact aataaataat agaaaaagga aaggtaaaca 1380
tcactaacga cagttgcggt gcaaactgag acaggtaata aacagcacta acttttattg 1440
gttatgtcaa actcaaagta aaatttctca acttgtttac gtgcctatat ataccatgct 1500
tgttatatgc tcaaagcacc aacaaaattt a 1531
<210> 53
<211> 1970
<212> DNA
<213> Zea mays
<400> 53
ggtaccattt ttcccagaaa taaatgtgga atagctctac aaacaaacgg catgatgctg 60
acacttggat ggcgaccttg caatcccaag aactattgca tacggttgcc agtcgacaaa 120
tatctacgcc atgcatggct acggtcggaa tacaccgtag cggcgggtaa ctcgccgata 180
ccgtccacgt gtcattggat gcccggtcgc tgatacttct ggtcttctgg acatgcacca 240
agacaaacaa gtgattcaac cttaatttaa cataaaataa ataatacgta acatccaact 300
gacgtgttca cctatagaga atattccttc tgattctact ttcagaatga tgccgttgcc 360
gtgtatcgag caagtactct cactcgaagt atcttatctc ccacatccag cacaaaaatc 420
ttctgttcgt ggcaaatctt gtggcggttg aacgaaagaa tgctatataa gtagctatag 480
agaacgtatt atgtgtaaac caaccgttca gtgtaaatcg tgtgtaaata gtcatgttaa 540
ttttttggcg gcaaatcaag tacaaactgt atgcctcgga taaacatgta caaaccacaa 600
cactggccac tagatctata tccaacgttc ataaccatcc atccctctct gctacactct 660
gcaaacaagc acccccatct cgtagcaaca tcttgtctcc gacaagctct cgatgtagtg 720
gaggccotcc accgcaatat cctagtgtat gatgttggag aagcgactcc taaataatgg 760
tgacaagatg ttgctaggtt tgtagccata gcctcaatct aagatcatcc caagccatgg 840
gacctgattc tacgaggcct acaaccaggc atgacacgtc gtctacccac tcttgtgcat 900
catcggtcac ttgatctgac ttggttccta accacttacc ctaggttcca aagocctaag 960
tttctcgtat attgttagtc attcttagtg ggagttttat gtgtatttca ttcctgttaa 1020
atagcatgcc aactaagcaa acataatgat ataatatgca atctaataaa aagatatatg 1080
agtgggtttc ataaaaaagg gagagagttt catgaggagt gaaactctga atacagatac 1140
tgatatgaca gctttaaaag tagtgttatg aaatcatcat tgagaaatgg tattagcact 1200
caatcgattt ctacgctgtc aattgtcatg agcacaattt tcacccaaag aggcacacca 1260
gcaatgtcca cttgtagtgt ccgagacgtt gctccatcgc cgtcgtcttg tttctgtgcg 1320
ctccattcaa tgcggcaagt ggctcaatcc caagcggtcg tcgcctccca gccccagcag 1380
caaaatatct tcccatgcgg ccatgccttg aaaattggaa tagattctct agattcaccg 1440
ccgcatcatc ttcactactt tctcactggc ccaatcagca tctoctLctc cgagctcaat 1500
catgctcagt caagcgtcac caatagcgtc acgattgatt ttqtcactgt ctgcatgcaa 1560
gggtatttta ctacgcaagt gtaaatggaa aatggatcta aacaactgca ctgcaccaat 1620
tttgaaacgc ggaaccgaga gtctgtttgg gttcgtttga aacgcgctga tgtttctcat 1680
tttttaatag atgtagttac ctgatactat ttaagttgga cgatcaaacg acagtgtcaa 1740
gtgtgattaa gaaaagcatc gaaaataaaa tttatcgcca taaaaagtta aaaacagtgg 1800
ataatagtag gacctcataa tagaaaaaat tatcaaacgg aatggagggg cccaacgcag 1860
tatatagcag ccgggtggtg ccggacatcc gacgctcgtg ccagcaggcc attattctcg 1920
cattactacc tcacagaacc cagtaaaata tcgccagtcc cgccgtcgag 1970
CA 2998211 2018-03-16
= 288
<210> 54
<211> 584
<212> DNA
<213> Aeluropus littoralis
<400> 54
occaagottg accgatacac acgctacctg ccaaggctcc ctccatccgc actctgcatc 60
gtogattogg cgtaaacttc cacgtagtac ttgtacgatt ctagctagac ccagtgcgcc 120
caccctaccg ccggcgagcg ggcccccatc tcgcgccagg cttccatgcg ggtccaccgt 180
ggaccagccc tacgccgaac cgagcccatc cctccaccct ttcaccgcca agogggaccc 240
gcgttggacc tttccgcttg gctggccccc accagcgtcc acgcgggcca acggcctcgc 300
gaaatggatc tccacacgac aaaccaaaac gagaagaaaa taaatggaaa ggaaagaaac 360
ggatcgccac gcgttccaga ggcgtccact aaccacccga ttatgcttgc gcagcgtgcg 420
taacctcatc gtggggttaa tccgagtggc cggatcggga aagccacggc ctttataacc 480
catccctgcc ggatcgaacc ggtaccggaa acaaaaacag ggggagaaaa aaagttcttc 540
gcgaggaagg aaaaggaaaa gtcgcgtgcc gtcctcgccc acag 584
<210> 55
<211> 928
<212> DNA
<213> Agrobacterium rhizogenes
<400> 55
ttagcgaaag gatgtcaaaa aaggatgccc ataattggga ggagtggggt aaagcttaaa 60
gttggcccgc tattggattt cgcgaaagcg gcattggcaa acatggagat tgctgcattc 120
aagatacttt ttctattttc tggttaagat gtaaagtatt gccacaatca tattaattac 180
taacattgta tatgtaatat agtgcggaaa ttatctatgc caaaatgatg tattaataat 240
agcaataata atatgtatta atctttttca atcaggaata cgtttaagcg attatcgtgt 300
tgaataaatt attccaaaag gaaatacatg gttttggaga acctgctata gatatatgcc 360
aaatttacac tagtttagtg ggtgcaaaac tattatctct gtttctgagt ttaataaaaa 420
ataaataagc agggcgaata gcagttagcc taagaaggaa tgatggccat gtacgtgctt 480
ttaagagacc ctataataaa ttgccagctg tgttgctttg gtgccgacag gcctaacgtg 540
ggatttagct tgacaaagta gcgoctttcc gcaacataaa taaaggtagg caggtgcgtc 600
ccattattaa aggaaaaagc aaaagctgag attccataga ccacaaacca ccattattgg 660
aggacagaac ctattccctc acgtgggtcg ctacctttaa acctaataag taaaaacaat 720
taaaagcagg caggtgtccc ttctatattc gcacaacgag gcgacgtgga gcatcgacag 760
ccgcatccat taattaataa atttgtggac ctatacctaa ctcaaatatt tttattattt 840
gctccaatac gctaagagct ctggattata aatagtttag atgattcgag ttatgggtac 900
aagcaacctg tttcctactt tgttacca 928
<210> 56
<211> 512
<212> PRI-
<213> Elaeis guineensis
<400> 56
Net Ala Val Ser Lys Asn Pro Glu Thr Leu Ala Pro Asp Gin Glu Pro
1 5 10 15
Ser Lys Glu Ser Asp Leu Arg Arg Arg Pro Ala Ser Ser Pro Ser Ser
20 25 30
Thr Ala Ala Ser Pro Ala Val Pro Asp Ser Ser Ser Arg Thr Ser Ser
35 40 45
Ser Ile Thr Gly Ser Trp Thr Thr Ala Leu Asp Gly Asp Ser Gly Ala
50 55 60
CA 2998211 2018-03-16
289
Gly Ala Val Arg Ile Gly Asp Pro Lys Asp Arg Ile Gly Glu Ala Asn
65 70 75 80
Asp Ile Gly Glu Lys Lys Lys Ala Cys Ser Gly Glu Val Pro Val Gly
85 90 95
Phe Val Asp Arg Pro Ser Ala Pro Val His Val Arg Val Val Glu Ser
100 105 110
Pro Leu Ser Ser Asp Thr Ile Phe Gin Gin Ser His Ala Gly Leu Leu
115 120 125
Asn Leu Cys Val Val Val Leu Ile Ala Val Asn Ser Arg Leu Ile Ile
130 135 140
Glu Asn Leu Met Lys Tyr Gly Leu Leu Ile Gly Ser Gly Phe She Phe
145 150 155 160
Ser Ser Arg Leu Leu Arg Asp Trp Pro Leu Leu Ile Cys Ser Leu Thr
165 170 175
Leu Pro Val Phe Pro Leu Gly Ser Tyr Met Val Glu Lys Leu Ala Tyr
180 185 190
Lys Lys Phe Ile Ser Glu Pro Val Val Val Ser Leu His Val Ile Leu
195 200 205
Ile Ile Ala Thr Ile Met Tyr Pro Val She Val Ile Leu Arg Cys Asp
210 215 220
Ser Pro Ile Leu Ser Gly Ile Asn Leu Met Leu Phe Val Ser Ser Ile
225 230 235 240
Cys Leu Lys Leu Vai Ser Tyr Ala His Ala Asn Tyr Asp Leu Arg Ser
245 250 255
Ser Ser Asn Ser Ile Asp Lys Gly Ile His Lys Ser Gin Gly Val Ser
260 265 270
Phe Lys Ser Leu Val Tyr She Ile Met Ala Pro Thr Leu Cys Tyr Gin
275 280 285
Pro Ser Tyr Pro Arg Thr Thr Cys Ile Arg Lys Gly Trp Val Ile Cys
290 295 300
Gin Leu Val Lys Leu Val Ile Phe Thr Gly Val Met Gly Phe Ile Ile
305 310 315 320
Glu Gin Tyr Ile Asp Pro Ile Ile Lys Asn Ser Gin His Pro Leu Lys
325 330 335
Gly Asn Val Leu Asn Ala Met Glu Arg Val Leu Lys Leu Ser Ile Pro
340 345 350
Thr Leu Tyr Val Trp Leu Cys Val Phe Tyr Cys Thr Phe His Leu Trp
355 360 365
Leu Asn Ile Leu Ala Glu Leu Leu Cys Phe Gly Asp Arg Glu Phe Tyr
370 375 380
Lys Asp Trp Trp Asn Ala Lys Thr Ile Glu Glu Tyr Trp Arg Met Trp
385 390 395 400
Asn Met Pro Val His Lys Trp Met Leu Arg His Val Tyr Leu Pro Cys
405 410 415
Ile Arg Asn Gly Ile Pro Lys Gly Val Ala Met Val Ile Ser Phe She
420 425 430
Ile Ser Ala Ile She His Glu Leu Cys Ile Gly Ile Pro Cys His Ile
435 440 445
Phe Lys Phe Trp Ala Phe Ile Gly Ile Met She Gin Val Pro Leu Val
450 455 460
Ile Leu Thr Lys Tyr Leu Gin Asn Lys Phe Lys Ser Ala Met Val Gly
465 470 475 480
Asn Met Ile She Trp Phe She She Ser Ile Tyr Gly Gin Pro Met Cys
485 490 495
Val Leu Leu Tyr Tyr His Asp Val Met Asn Arg Lys Val Gly Thr Glu
500 505 510
CA 2998211 2018-03-16
290
<210> 57
<211> 74
<212> PRT
<213> Glycine max
<400> 57
Met Ala Asp Ile Asp Arg Ser Phe Asp Asn Asn Val Ser Ala Val Ser
1 5 10 15
Thr Glu Lys Ser Ser Gln Val Ser Asp Val Glu Phe Ser Glu Ala Glu
20 25 30
Glu Ile Leu Ile Ala Met Val Tyr Asn Leu Val Gly Glu Arg Trp Ser
35 40 45
Leu Ile Ala Gly Arg Ile Pro Gly Arg Thr Ala Glu Glu Ile Glu Lys
50 55 60
Tyr Trp Thr Ser Arg Phe Ser Thr Ser Gln
65 70
<210> 58
<211> 146
<212> PRT
<213> Arabidopsis thaliana
<400> 58
Met Gly Ser Leu Gln Met Gln Thr Ser Pro Glu Ser Asp Asn Asp Pro
1 5 10 15
Arg Tyr Ala Thr Val Thr Asp Glu Arg Lys Arg Lys Arg Met Ile Ser
20 25 30
Asn Arg Glu Ser Ala Arg Arg Ser Arg Met Arg Lys Gln Lys Gln Leu
35 40 45
Gly Asp Leu Ile Asn Glu Val Thr Leu Leu Lys Asn Asp Asn Ala Lys
50 55 60
Ile Thr Glu Gln Val Asp Glu Ala Ser Lys Lys Tyr Ile Glu Met Glu
65 70 75 80
Ser Lys Asn Asn Val Leu Arg Ala Gln Ala Ser Glu Leu Thr Asp Arg
85 90 95
Leu Arg Ser Leu Asn Ser Val Leu Glu Met Val Giu Glu Ile Ser Gly
100 105 110
Gln Ala Leu Asp Ile Pro Glu Ile Pro Glu Ser Met Gin Asn Pro Trp
115 120 125
Gin Met Pro Cys Pro Met Gin Pro Ile Arg Ala Ser. Ala Asp Met Phe
130 135 140
Asp Cys
145
<210> 59
<211> 268
<212> PRT
<213> Arabidopsis thaliana
<400> 59
Met Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Ala Asn Ser
1 5 10 15
Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala
20 25 30
CA 2998211 2018-03-16
291
=
Arg Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Val Ile Val Phe
35 40 45
Ser Lys Ser Gly Lys Leu Phe Glu Tyr Ser Ser Thr Gly Met Lys Gln
50 55 60
Thr Leu Ser Arg Tyr Gly Aso His Gin Ser Ser Ser Ala Ser Lys Ala
65 70 75 80
Glu Glu Asp Cys Ala Glu Val Asp Ile Leu Lys Asp Gin Leu Ser Lys
85 90 95
Leu Gin Glu Lys His Leu Gin Leu Gin Gly Lys Gly Leu Asn Pro Leu
100 105 110
Thr Phe Lys Glu Leu Gin Her Leu Glu Gin Gln Leu Tyr His Ala Leu
115 120 125
Ile Thr Val Arg Glu Arg Lys Glu Arg Leu Leu Thr Asn Gin Leu Glu
13C 135 140
Glu Ser Arg Leu Lys Glu Gin Arg Ala Glu Leu Glu Asn Glu Thr Leu
145 150 155 160
Arg Arg Gin Val Gin Glu Leu Arg Ser Phe Leu Pro Ser Phe Thr His
165 170 175
Tyr Val Pro Ser Tyr Ile Lys Cys Phe Ala Ile Asp Pro Lys Asn Ala
180 185 190
Leu Ile Asn His Asp Ser Lys Cys Ser Leu Gin Asn Thr Asp Ser Asp
195 200 203
Thr Thr Leu Gin Leu Gly Leu Pro Gly Glu Ala His Asp Arg Arg Thr
210 215 220
Asn Glu Gly Glu Arg Glu Ser Pro Ser Ser Asp Ser Val Thr Thr Asn
225 230 235 240
Thr Ser Ser Glu Thr Ala Glu Arg Gly Asp Gin Ser Ser Leu Ala Asn
245 250 255
Ser Pro Pro Glu Ala Lys Arg Gin Arg Phe Ser Val
260 265
<210> 60
<211> 437
<212> PRT
<213> Arabidopsis thaliana
<400> 60
Met Glu Phe Glu Ser Val Phe Lys Met His Tyr Pro Tyr Leu Ala Ala
1 5 10 15
Val Ile Tyr Asp Asp Her Her Thr Leu Lys Asp Phe His Pro Ser Leu
20 25 30
Thr Asp Asp Phe Ser Cys Val His Asn Val His His Lys Pro Ser Met
35 40 45
Pro His Thr Tyr Glu Ile Pro Ser Lys Glu Thr Ile Arg Gly Ile Thr
50 55 60
Pro Ser Pro Cys Thr Glu Ala Phe Gly Ala Cys Phe His Gly Thr Ser
65 70 75 80
Asn Asp His Val Phe Phe Gly Met Ala Tyr Thr Thr Pro Pro Thr Ile
85 90 95
Glu Pro Asn Val Ser His Val Ser His Asp Asn Thr Met Trp Glu Asn
100 105 110
Asp Gin Asn Gin Gly Phe Ile Phe Gly Thr Glu Ser Thr Leu Asn Gin
115 120 125
Ala Met Ala Asp Her Asn Gin Phe Asn Met Pro Lys Pro Leu Leu Ser
130 135 140
CA 2998211 2018-03-16
292
Ala Asn Glu Asn Thr Ile Met Asn Arg Arg Gin Asn Asn Gin Val Met
145 150 155 160
Ile Lys Thr Glu Gin Ile Lys Lys Lys Asn Lys Arg Phe Gin Met Arg
165 170 175
Arg Ile Cys Lys Pro Thr Lys Lys Ala Ser Ile Ile Lys Gly Gin Trp
180 185 190
Thr Pro Glu Glu Asp Lys Leu Leu Val Gin Leu Val Asp Leu His Gly
195 200 205
Thr Lys Lys Trp Ser Gin Ile Ala Lys Met Leu Gin Gly Arg Val Gly
210 215 220
Lys Gin Cys Arg Glu Arg Trp His Asn His Leu Arg Pro Asp Ile Lys
225 230 235 240
Lys Asp Gly Trp Thr Glu Glu Glu Asp Ile Ile Leu Ile Lys Ala His
245 250 255
Lys Glu Ile Gly Asn Arg Trp Ala Glu Ile Ala Arg Lys Leu Pro Gly
260 265 270
Arg Thr Glu Asn Thr Ile Lys Asn His Trp Asn Ala Thr Lys Arg Arg
275 280 285
Gin His Ser Arg Arg Thr Lys Gly Lys Asp Glu Ile Ser Leu Ser Leu
290 295 300
Gly Ser Asn Thr Leu Gin Asn Tyr Ile Arg Ser Val Thr Tyr Asn Asp
305 310 315 320
Asp Pro Phe Met Thr Ala Asn Ala Asn Ala Asn Ile Gly Pro Arg Asn
325 330 335
Met Arg Gly Lys Gly Lyn Asn Val Met Val Ala Val Ser Glu Tyr Asp
340 345 350
Glu Gly Glu Cys Lys Tyr Ile Val Asp Gly Val Asn Asn Leu Gly Lou
355 360 365
Glu Asp Gly Arg Ile Lys Met Pro Ser Leu Ala Ala Met Ser Ala Ser
370 375 380
Gly Ser Ala Ser Thr Ser Gly Ser Ala Ser Gly Ser Gly Ser Gly Val
385 390 395 400
Thr Met Glu Ile Asp Glu Pro Met Thr Asp Ser Trp Met Val Met His
405 410 415
Gly Cys Asp Glu Val Met Met Asn Glu Ile Ala Leu Leu Glu Met Ile
420 425 430
Ala His Gly Arg Leu
435
<210> 61
<211> 359
<212> PRT
<213> Arabidopsis thaliana
<400> 61
Met Tyr His Gin Asn Leu Ile Ser Ser Thr Pro Asn Gin Asn Ser Asn
10 15
Pro His Asp Trp Asp Ile Gin Asn Pro Leo Phe Ser Ile His Pro Ser
20 25 30
Ala Clu Ile Pro Ser Lys Tyr Pro Phe Met Gly Ile Thr Ser Cys Pro
35 40 45
Asn Thr Asn Val Phe Glu Glu Phe Gin Tyr Lys Ile Thr Asn Asp Gin
50 55 60
Asn Phe Pro Thr Thr Tyr Asn Thr Pro Phe Pro Val Ile Ser Glu Gly
65 70 75 80
-41
CA 2998211 2018-03-16
= 293
Ile Ser Tyr Asn Met His Asp Val Gin Glu Asn Thr Met Cys Gly Tyr
65 90 95
Thr Ala His Aso Gin Gly Leu Ile Ile Gly Cys His Glu Pro Val Leu
100 105 110
Val His Ala Val Val Glu Ser Gin Gin Phe Asn Val Pro Gin Ser Glu
115 120 125
Asp Ile Asn Leu Val Ser Gin Ser Glu Arg Val Thr Glu Asp Lys Val
130 135 140
Met Phe Lys Thr Asp His Lys Lys Lys Asp Ile Ile Gly Lys Gly Gin
145 150 155 160
Trp Thr Pro Thr Glu Asp Glu Leu Leu Val Arg Met Val Lys Ser Lys
165 170 175
Gly Thr Lys Asn Trp Thr Ser Ile Ala Lys Met Phe Gin Gly Arg Val
180 185 190
Gly Lys Gin Cys Arg Glu Arg Trp Arg Asn His Leu Arg Pro Asn Ile
195 200 205
Lys Lys Asn Asp Trp Ser Glu Glu Glu Asp Gin Ile Leu Ile Glu Val
210 215 220
His Lys Ile Val Gly Asn Lys Trp Thr Glu Ile Ala Lys Arg Leu Pro
225 230 235 240
Gly Arg Ser Glu Asn Ile Val Lys Asn His Trp Asn Ala Thr Lys Arg
245 250 255
Arg Leu His Ser Val Arg Thr Lys Arg Ser Asp Ala Phe Ser Pro Arg
260 265 270
Asn Asn Ala Leu Glu Asn Tyr Ile Arg Ser Ile Thr Ile Asn Asn Asn
275 280 285
Ala Leu Met Asn Arg Glu Val Asp Ser Ile Thr Ala Asn Ser Glu Ile
290 295 300
Asp Ser Thr Arg Cys Glu Asn Ile Val Asp Glu Val Met Asn Leu Asn
305 310 315 320
Leu His Ala Thr Thr Ser Val Tyr Val Pro Glu Gin Ala Val Leu Thr
325 330 335
Trp Gly Tyr Asp Phe Thr Lys Cys Tyr Glu Pro Met Asp Asp Thr Trp
340 345 350
Met Leu Met Asn Gly Trp Asn
355
<210> 62
<211> 386
<212> PRT
<213> Arabidopsis thaliana
<400> 62
Met Ser Lys Arg Pro Pro Pro Asp Pro Val Ala Val Leu Arg Gly His
1 5 10 15
Arg His Ser Val Met Asp Val Ser Phe His Pro Ser Lys Ser Leu Leu
20 25 30
Phe Thr Gly Ser Ala Asp Gly Glu Leu Arg Ile Trp Asp Thr Ile Gin
35 40 45
His Arg Ala Val Ser Ser Ala Trp Ala His Ser Arg Ala Asn Gly Val
50 55 60
Leu Ala Val Ala Ala Ser Pro Trp Leu Gly Glu Asp Lys Ile Ile Ser
65 70 75 80
Gin Gly Arg Asp Gly Thr Val Lys Cys Trp Asp Ile Glu Asp Gly Gly
85 90 95
CA 2998211 2018-03-16
294
Leu Ser Arg Asp Pro Leu Leu Ile Leu Glu Thr Cys Ala Tyr His Phe
100 105 110
Cys Lys Phe Ser Leu Val Lys Lys Pro Lys Asn Ser Leu Gin Glu Ala
115 120 125
Glu Ser His Ser Arg Gly Cys Asp Glu Gin Asp Gly Gly Asp Thr Cys
130 135 140
Asn Val Gin Ile Ala Asp Asp Ser Glu Arg Ser Glu Glu Asp Ser Gly
145 150 155 160
Leu Leu Gin Asp Lys Asp His Ala Glu Gly Thr Thr Phe Val Ala Val
165 170 175
Val Gly Glu Gin Pro Thr Glu Vol Glu Ile Trp Asp Leu Asn Thr Gly
180 185 190
Asp Lys Ile Ile Gin Leu Pro Gin Ser Ser Pro Asp Glu Ser Pro Asn
195 200 205
Ala Ser Thr Lys Gly Arg Gly Met Cys Met Ala Val Gin Leu Phe Cys
210 215 220
Pro Pro Glu Ser Gin Gly Phe Leu His Val Lou Ala Gly Tyr Glu Asp
225 230 235 240
Gly Ser Ile Leu Leu Trp Asp Ile Arg Asn Ala Lys Ile Pro Leu Thr
245 250 255
Ser Val Lys Phe His Ser Glu Pro Val Leu Ser Leu Ser Val Ala Ser
260 265 270
Ser Cys Asp Gly Gly Ile Ser Gly Gly Ala Asp Asp Lys Ile Val Met
275 280 2E5
Tyr Asn Lou Asn His Ser Thr Gly Ser Cys Thr Ile Arg Lys Glu Ile
290 295 300
Thr Leu Glu Arg Pro Gly Val Ser Gly Thr Ser Ile Arg Val Asp Gly
305 310 315 320
Lys Ile Ala Ala Thr Ala Gly Trp Asp His Arg Ile Arg Val Tyr Asn
325 330 335
Tyr Arg Lys Gly Asn Ala Leu Ala Ile Leu Lys Tyr His Arg Ala Thr
340 345 350
Cys Asn Ala Val Ser Tyr Ser Pro Asp Cys Glu Leu Met Ala Ser Ala
355 360 365
Ser Glu Asp Ala Thr Val Ala Leu Trp Lys Leu Tyr Pro Pro His Lys
370 375 380
Ser Leu
385
<210> 63
<211> 292
<212> PRT
<213> Arabidopsis thaliana
<400> 63
Met Glu Pro Pro Gin His Gin His His His His Gin Ala Asp Gln Glu
1 5 10 15
Ser Gly Asn Asn Asn Asn Asn Lys Ser Gly Ser Gly Gly Tyr Thr Cys
20 25 30
Arg Gin Thr Ser Thr Arg Trp Thr Pro Thr Thr Glu Gin Ile Lys Ile
35 40 45
Leu Lys Glu Leu Tyr Tyr Asn Asn Ala Ile Arg Ser Pro Thr Ala Asp
50 55 60
Gin Ile Gin Lys Ile Thr Ala Arg Leu Arg Gin Phe Gly Lys Ile Glu
65 70 75 80
CA 2998211 2018-03-16
295
Gly Lys Asn Val Phe Tyr Trp Phe Gin Asn His Lys Ala Arg Glu Arg
85 9C 95
Gin Lys Lys Arg Phe Asn Gly Thr Asn Met Thr Thr Pro Ser Ser Ser
100 105 110
Pro Asn Ser Val Met Met Ala Ala Asn Asp His Tyr His Pro Leu Leu
115 120 125
His His Hs His Gly Val Pro Met Gin Arg Pro Ala Asn Ser Val Asn
130 135 140
Val Lys Leu Asn Gin Asp His His Leu Tyr His His Asn Lys Pro Tyr
143 150 155 160
Pro Ser Phe Asn Asn Gly Asn Leu Asn His Ala Ser Ser Gly Thr Glu
165 170 175
Cys Gly Val Val Asn Ala Ser Asn Gly Tyr Met Ser Ser His Val Tyr
180 185 190
Gly Ser Met Clu Gin Asp Cys Ser Met Asn Tyr Asn Asn Val Gly Gly
195 200 205
Gly Trp Ala Asn Met Asp His His Tyr Ser Ser Ala Pro Tyr Asn Phe
210 215 220
Phe Asp Arg Ala Lys Pro Leu Phe Gly Leu Glu Giy His Gin Glu Glu
225 230 235 240
Glu Glu Cys Gly Gly Asp Ala Tyr Leu Glu His Arg Arg Thr Leu Pro
245 250 255
Leu Phe Pro Met His Gly Glu Asp His Ile Asn Sly Gly Ser Gly Ala
260 265 270
Ile Trp Lys Tyr Gly Gin Ser Glu Val Arg Pro Cys Ala Ser Leu Glu
275 280 285
Leu Arg Leu Asn
290
<210> 64
<211> 453
<212> PRT
<213> Brassica napus
<400> 64
Met Asp Leu Gly Ser Val Thr Gly Asn Val Asn Gly Ser Pro Ser Leu
1 5 10 15
Lys Glu Leu Arg Glu Ser Lys Gin Asp Arg Ser Glu Phe Asp Gly Glu
20 25 30
Asp Cys Leu Gin Gin Ser Ser Lys Leu Ala Arg Thr Ile Ala Glu Asp
35 40 45
Lys His Leu Pro Ser Ser Tyr Ala Ala Ala Tyr Ser Arg Pro Met Ser
50 55 60
Phe His Gln Gly Ile Pro Leu Ala Arg Ser Ala Ser Leu Leu Ser Ser
65 70 75 80
Asp Ser Arg Arg Gin Glu His Met Leu Ser Phe Ser Asp Lys Pro Glu
85 90 95
Ala Phe Asp Phe Ser Lys Tyr Val Gly Leo Asp Asn Asn Lys Asn Ser
100 105 110
Leu Ser Pro Phe Leu His Gin Leu Pro Pro Pro Tyr Cys Arg Thr Pro
115 120 125
Gly Sly Gly Tyr Gly Ser Gly Gly Met Met Met Ser Met Gin Gly Lys -
130 135 140
Gly Pro Phe Thr Leu Thr Gin Trp Ala Glu Leu Glu Gin Gin Ala Leu
145 150 155 160
CA 2998211 2018-03-16
= 296
Ile Tyr Lys Tyr Ile Thr Ala Asn Val Pro Val Pro Ser Ser Leu Leu
165 170 175
Ile Ser Ile Gin Lys Ser Phe Tyr Pro Tyr Arg Ser Phe Pro Pro Ser
180 185 190
Ser Phe Gly Trp Gly Thr Phe His Leu Gly Phe Ala Gly Gly Lys Met
195 200 205
Asp Pro Glu Pro Gly Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg
210 215 220
Cys Her Lys Asp Ala Val Pro Asp Gin Lys Tyr Cys Glu Arg His Ile
225 230 235 240
Asn Arg Gly Arg His Arg Her Arg Lys Pro Val Glu Val Gin Pro Gly
245 250 255
Gin Thr Ala Ala Ser Lys Ala Ala Ala Val Ala Ser Arg Asn Thr Ala
260 265 270
Ser Gin Ile Pro Asn Asn Arg Val Gin Asn Val Ile Tyr Pro Ser Thr
275 280 285
Val Asn Leu Pro Pro Lys Glu Gin Arg Asn Asn Asn Asn Ser Ser Phe
290 295 300
Gly Phe Gly His Val Thr Ser Pro Ser Leu Leu Thr Ser Ser Tyr Leu
305 310 315 320
Asp Phe Ser Ser Asn Gin Asn Lys Pro Glu Glu Leu Lys Ser Asp Trp
325 330 335
Thr Gin Leu Ser Met Ser Ile Pro Val Ala Ser Ser Ser Pro Ser Ser
340 345 350
Thr Ala Gin Asp Lys Thr Thr Leu Ser Pro Leu Arg Leu Asp Leu Pro
355 360 365
Ile Gin Ser Gin Gin Glu Thr Leu Glu Ala Val Arg Lys Val Asn Thr
370 375 380
Trp Ile Pro Ile Ser Trp Gly Asn Ser Leu Gly Gly Pro Leu Gly Glu
385 390 395 400
Val Leu Asn Ser Thr Thr Ser Ser Pro Thr Leu Gly Ser Ser Pro Thr
405 410 415
Gly Val Leu Gin Lys Ser Thr Phe Cys Ser Leu Ser Asn Ser Ser Ser
420 425 430
Val Thr Ser Pro Val Ala Asp Asn Asn Arg Asn Asn Asn Val Asp Tyr
435 440 445
Phe His Tyr Thr Thr
450
<210> 65
<211> 461
<212> PRT
<213> Brassica napus
<400> 65
Met Asp Leu Gly Ser Val Thr Gly Asn Val Asn Gly Ser Pro Gly Leu
1 5 10 15
Lys Glu Leu Arg Gly Ser Lys Gin Asp Arg Ser Gly Phe Asp Gly Glu
20 25 30
Asp Cys Leu Gin Gin Ser Ser Lys Leu Ala Arg Thr Ile Ala Glu Asp
35 40 45
Lys His Leu Pro Ser Ser Tyr Ala Ala Tyr Ser Arg Pro Met Ser Phe
50 55 60
His Gin Gly Ile Pro Leu Thr Arg Ser Ala Ser Leu Leu Ser Ser Asp
65 70 75 80
CA 2998211 2018-03-16
297
Ser Arc? Arg Gln Glu His Met Leu Ser Phe Ser Asp Lys Pro Giu Ala
85 90 95
Phe Asp Phe Ser Lys Tyr Val Gly Leu Asp Asn Asn Lys Asn Ser Leu
100 105 110
Ser Pro Phe Leu His Gln Leu Pro Pro Pro Tyr Cys Arg Ser Ser Gly
115 120 125
Gly Gly Tyr Gly Ser Gly Gly Met Met Met Ser Met Gln Gly Lys Gly
130 135 14C
Pro Phe Thr Leu Thr Gln Trp Ala Glu Leu Glu Gln Gln Ala Leu Ile
145 150 155 160
Tyr Lys Tyr Ile Thr Ala Asn Val Pro Val Pro Ser Ser Leu Leu lie
165 170 175
Ser lie Gln Lys Ser Phe Tyr Pro Tyr Arg Ser Phe Pro Pro Ser Ser
180 185 190
Phe Gly Trp Gly Thr Phe His Leu Gly Phe Ala Gly Gly Lys Met Asp
195 200 . 205
Pro Glu Pro Gly Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg Cys
210 215 220
Ser Lys Asp Ala Val Pro Glu Gln Lys Tyr Cys Glu Arg His Ile Asn
225 230 235 240
Arg Gly Arg His Arg Ser Al-C1 Lys Pro Vol Glu Val Gln Pro Gly Gln
245 250 255
Thr Ala Ala Ser Lys Ala Val Ala Ser Arg Asp Thr Ala Ser Gln Ile
260 - 265 270
Pro Ser Asn Arg Val Gln Asn Val Ile Tyr Pro Ser Asn Val Asn Leu
275 280 285
Gin Pro Lys Glu Gln Arg Asn Asn Asp Asn Ser Pro Phe Gly Phe Gly
290 295 300
His Val Thr Ser Ser Ser Leu Leu Thr Ser Ser Tyr Leu Asp Phe Ser
305 310 315 320
Ser Asn Gln Glu Lys Pro Ser Gly Asn His His Asn Gln Ser Ser Trp
325 330 335
Pro Glu Glu Leu Lys Ser Asp Trp Thr Gln Leu Ser Met Ser Ile Pro
340 345 350
Vol Ala Ser Ser Ser Pro Ser Ser Thr Ala Gln Asp Lys Thr Ala Leu
355 360 365
Ser Pro Leu Arg Leu Asp Leu Pro Ile Gln Ser Gln Gln Glu Thr Leu
370 375 380
Glu Ser Ala Arg Lys Val Asn Thr Trp Ile Pro Ile Ser Trp Gly Asn
385 390 395 400
Ser Leu Gly Gly Pro Leu Gly Glu Vai Leu Asn Ser Thr Thr Ser Ser
405 410 415
Pro Thr Leu Gly Ser Ser Pro Thr Gly Val Leu Gin Lys Ser Thr Phe
420 425 430
Cys Ser Leu Ser Asn Ser Ser Ser Val Thr Ser Pro Ile Ala Asp Asn
435 440 445
Asn Arg Asn Asn Asn Val Asp Tyr Phe His Tyr Thr Thr
450 455 460
<210> 66
<211> 409
<212> PRT
<213> Arabidopsis thaliana
CA 2998211 2018-03-16
298
<400> 66
Met Glu Ala Arg Pro Val His Arg Ser Gly Ser Arc, Asp Leu Thr Arg
1 5 10 15
Thr Ser Ser Ile Pro Ser Thr Gin Lys Pro Ser Pro Val Glu Asp Ser
20 25 30
Phe Met Arg Ser Asp Asn Asn Ser Gin Leu Met Ser Arg Pro Leu Gly
35 40 45
Gin Thr Tyr His Leu Leu Ser Ser Ser Asn Gly Gly Ala Val Gly His
50 55 60
Ile Cys Ser Ser Ser Ser Ser Gly Phe Ala Thr Asn Leu His Tyr Ser
65 70 75 80
Thr Met Val Ser His Glu Lys Gin Gin His Tyr Thr Gly Per Ser Ser
85 90 95
Asn Asn Ala Val Gin Thr Pro Ser Asn Asn Asp Ser Ala Trp Cys His
100 105 110
Asp Ser Leu Pro Gly Gly Phe Leu Asp Phe His Glu Thr Asn Pro Ala
115 120 125
Ile Gin Asn Asn Cys Gin Ile Glu Asp Gly Gly Ile Ala Ala Ala Phe
130 135 140
Asp Asp Ile Gin Lys Arg Ser Asp Trp His Glu Trp Ala Asp His Leu
145 150 155 160
Ile Thr Asp Asp Asp Pro Leu Met Ser Thr Asn Trp Asn Asp Leu Leu
165 170 175
Leu Glu Thr Asn Ser Asn Ser Asp Ser Lys Asp Gin Lys Thr Leu Gin
180 185 190
Ile Pro Gin Pro Gin Ile Val Gin Gin Gln Pro Ser Pro Ser Val Glu
195 200 205
Leu Arg Pro Val Ser Thr Thr Ser Ser Asn Ser Asn Asn Gly Thr Gly
210 215 220
Lys Ala Arg Met Arg Trp Thr Pro Glu Leu His Glu Ala Phe Val Glu
225 230 235 240
Ala Val Asn Ser Leu Gly Gly Ser Glu Arg Ala Thr Pro Lys Gly Val
245 250 255
Leu Lys Ile Met Lys Val Glu Gly Leu Thr Ile Tyr His Val Lys Ser
260 265 270
His Leu Gin Lys Tyr Arg Thr Ala Arg Tyr Arg Pro Glu Pro Ser Glu
275 280 285
Thr Gly Ser Pro Glu Arg Lys Leu Thr Pro Leu Glu His :le Thr Ser
290 295 300
Leu Asp Leu Lys Gly Gly Ile Gly Ile Thr Glu Ala Leu Arg Leu Gin
305 310 315 320
Met Glu Val Gin Lys Gin Leu His Glu Gin Leu Glu Tie Gin Arg Asn
325 330 335
Leu Gin Leu Arg Ile Glu Giu Gin Gly Lys Tyr Leu Gin Met Met Phe
340 345 350
Glu Lys Gin Asn Ser Gly Leu Thr Lys Gly Thr Ala Ser Thr Ser Asp
355 360 365
Ser Ala Ala Lys Ser Glu Gin Glu Asp Lys Lys Thr Ala Asp Ser Lys
370 375 380
Giu Val Pro Glu Glu Glu Thr Arg Lys Cys Glu Glu Leu Glu Ser Pro
385 390 395 400
Gin Pro Lys Arg Pro Lys Ile Asp Asn
405
CA 2998211 2018-03-16
299
<210> 67
<211> 1173
<212> DNA
<213> Sapium sebiferum L.
<400> 67
tgccaatagc cagccaataa aacatctaca cgttttcaca cgacttttca tcagagccgt 60
tgtttttctc atctcactcc gtgccttcat ottcatcotc ttctcctcto tctatgtcto 120
tatatgtata gaagcgttag atgtcttgcg ttgttaacca attcattttt cgctttctgc 180
ttottctaat attataagaa agtttgattc ttcttcttgt caatotttgt tcgcggcttt 240
taacgatatc cgctaaagga aatttgaaat ttcaattatg gccgatggaa acgtcaattc 300
gcaagaacag atggctaagc aggaggaaca gaggctgaag tatttggagt ttgtacaagt 360
ggctccaata catgctgtgg tgaccttcac aaacctctat gtttatgcca aaaacaagtc 420
gggtccattg aagcccggtg ttgagactgt tgaaggtacg gtcaagagtg tggttggacc 480
tgtttatggc aagttccatg atgttcccat tgaggttctc aagtttgtcg atcgcaagat 540
tgatcaatct gtaagcagcc tagacagccg tgtgcctcca gttgtgaagc agttatcggc 600
ccaagcattt tcagtggctc gcgaagcccc agtggctgct cgtgctgtgg cttctgaagt 660
gcagactgct ggagtgaagg aaactgcatc tgggttggca agaactctgt acttcaaata 720
tgaacccaag gccaaggagc tatacaccaa gtatgaacca aaagcggaag agtgtgctgc 780
ctctgcctgg cgtaagctca atcaactccc agtottccct catgtagctc aggttgttat 840
gccaacagca gcttattgtt ctgaaaagta caaccaggca gtacttacca ccgctgagaa 900
aggatacaga gtgtcctott atttgccttt tgtgcccact gagagaattg ctaagttgtt 960
taggaatgag gcacctgaat ctacccottt cotttccaat tgagcaagat gctgataaat 1020
gattcacaat ggacatgtgg acagaataaa aatctttgga tattatatgg tactgtgtat 1080
ttcaaggttc aagattactc tctacaatgt gtgaattttt gtttcagatg acttaattct 2740
tgttcattca ttatatatat atatatatat ate 1173
<210> 68
<211> 241
<212> PRT
<213> Sapium sebiferum L.
<400> 68
Met Ala Asp Gly Asn Val Asn Ser Gin Glu Gin Net Ala Lys Gin Glu
1 5 10 15
Glu Gin Arg Leu Lys Tyr Leta Glu Phe Val Gin Val Ala Ala Ile His
20 25 30
Ala Val Val Thr Phe Thr Asn Leu Tyr Val Tyr Ala Lys Asn Lys Ser
35 40 45
Gly Pro Leu Lys Pro Gly Val Glu Thr Val Glu Gly Thr Val Lys Ser
50 55 60
Val Val Gly Pro Val Tyr Gly Lys Phe His Asp Val Pro Ile Glu Val
65 70 75 BO
Leu Lys Phe Val Asp Arg Lys Ile Asp Gin Ser Val Ser Ser Leu Asp
85 90 95
Ser Arg Val Pro Pro Val Val Lys Gin Leu Ser Ala Gin Ala Phe Ser
100 105 110
Val Ala Arg Clu Ala Pro Val Ala Ala Arg Ala Val Ala Ser Glu Val
115 120 125
Gin Thr Ala Gly Val Lys Glu Thr Ala Ser Gly Len Ala Arg Thr Leu
130 135 140
Tyr Phe Lys Tyr Glu Pro Lys Ala Lys Glu Leu Tyr Thr Lys Tyr Glu
145 150 155 160
Pro Lys Ala Glu Gin Cys Ala Ala Ser Ala Trp Arg Lys Leu Asn Gin
165 170 175
CA 2998211 2018-03-16
300
Leu Pro Val Phe Pro His Val Ala Gin Val Val Met Pro Thr Ala Ala
180 185 190
Tyr Cys Ser Glu Lys Tyr Asn Gin Ala Val Leu Thr Thr Ala Glu Lys
195 200 205
Gly Tyr Arg Val Ser Ser Tyr Leu Pro Phe Val Pro Thr Glu Arg Ile
210 215 220
Ala Lys Leu Phe Arg Asn Giu Ala Pro Glu Ser Thr Pro Phe Leu Ser
225 230 235 240
Asn
<210> 69
<211> 1252
<212> DNA
<213> Sapium sebiferam L.
<400> 69
ctacttttcc ctagcattag tattctaggc cccactctgt agattcctcc agctgcctga 60
tctaattttt tatcaactct tgaccgttcg atcatcccaa cggctcagat tcactagtac 120
ttttctcaca ccgtatctcc gattctccat gactccatcg atataaatcg cagtgatcat 180
caactgaatt ctcgaaattg cgattacaag ctgctataag aagcgaaaag aaacgctgag 240
aaacaggatc cgttcctcct ccatcgcttt ttactcctta caagatggag accgagaaga 300
agattcctga attgaagcac ttagggttcg tgaggatggc tgctattcag tcactgattt 360
gcgtctcgaa tctctacgat tacgcgaagc ataactcagg acctttgaga tccactgttg 420
gaaccgtgga gggtgccgta accaccgtag taggtccagt ttaccagaaa ttcaaagacc 480
ttcctgatga tattattgta tatgttgata agaaggtgga tgaaggaaca cacaagtttg 540
ataagcatgc tccacctatt gctaagaagg ctgcgagcca agcccatagt ttgtttcata 600
tagccttgga gaaggtcgaa aaactcgtgc aggaggctcg tgcaggagga cctcgtgotg 660
ctctgcattt tgtggctaca gagtcgaagc acttggcgtt gacccaatct gtgaagctgt 720
atagtaaact taatcagttc cctgtcattc acactgttac agatgtaacc cttcccacag 780
ctactcactg gtcagataag tataaccata ccattatgga cctgacccgg aagggttata 840
cgatctttgg ttatttgcct ttgattccta ttgatgacat atctaagaca tttaaacaaa 900
gtaaagcaga ggagaaagaa aatgcaacta cgcataaatc tgattcatcg gattccgact 960
aaacggttgc catcatgtct aatgggtctg gtttgrtaag tatagtggtt tgcgaaaatg 1020
ttctagggtt tatgagcctg ctcgaaagat gctgagaaat ggaaatctgt actatttagg 1080
agtttttccg tactataata atgagtatga atgatttgta aattctgcct tgtgctttct 1140
cgacaagtat atcatgattc tattttttac tactacttac tggactactg aattgtctca 1200
taattgtocc tagtgtctaa ttaaatatca cctccaaaat attattgaaa as 1252
<210> 70
<211> 225
<212> PRT
<213> Sapium sebiferum L.
<400> 70
Met Glu Thr Glu Lys Lys Ile Pro Glu Leu Lys His Leu Gly Phe Val
1 5 10 15
Arg Met Ala Ala Ile Gin Ser Leu Ile Cys Val Ser Asn Leu Tyr Asp
20 25 30
Tyr Ala Lys His Asn Ser Gly Pro Leu Arg Ser Thr Val Gly Thr Val
35 40 45
Glu Gly Ala Val Thr Thr Val Val Gly Pro Val Tyr Gin Lys Phe Lys
50 55 60
Asp Leu Pro Asp Asp Leu Leu Val Tyr Val Asp Lys Lys Val Asp Glu
65 70 75 80
CA 2998211 2018-03-16
301
Gly Thr His Lys Phe Asp Lys His Ala Pro Pro Ile Ala Lys Lys Ala
25 90 95
Ala Ser Gln Ala His Ser Leu Phe His Ile Ala Leu Glu Lys Val Glu
100 105 110
Lys Leu Val Gin Glu Ala Rig Ala Gly Gly Pro Arg Ala Ala Leu His
115 120 125
Phe Val Ala Thr Glu Ser Lys His Leu Ala Leu Thr Gln Ser Val Lys
130 135 140
Leu Tyr Ser Lys Leu Asn Gin Phe Pro Val 71e His Thr Val Thr Asp
145 150 155 160
Val Thr Leu Pro Thr Ala Thr His Trp Ser Asp Lys Tyr Asn His Thr
165 170 175
Leu Met Asp Leu Thr Arg Lys Gly Tyr Thr Ile Phe Gly Tyr Leu Pro
180 185 190
Leu Val Pro lie Asp Asp Ile Ser Lys Thr Phe Lys Gin Ser Lys Ala
195 200 205
Glu Glu Lys Glu Asn Ala Thr Thr His Lys Ser Asp Ser Ser Asp Ser
210 215 220
Asp
225
<210> 71
<211> 938
<212> DNA
<213> Sapium sebiferum L.
<400> 71
gagtattcac actctggcct gattgggttt gctataaagg gcgatcgttg caacgctcca 60
tattgtctac ttggttttgt ttcaaatctc atcattttgt aaatttgcga cagtgtagcg 120
ttttctagga aaaaggttgc taaaggaaag tagttatcaa accgcagaaa tggcggaatc 180
cgaacttaat caacacacag atatggttca agatgatgat aaaaaactca agtatctaga 240
ttttgtacaa gtggccgcga tctatgttgt gatttatttc tctagtatct atgaatatgc 300
taaggaaaac tccggtccac taaaaccagg ggtocaagcc gttgagtgta ccgtcaaaac 360
tgtaataagt ccggtttacg agaagtttcg cgacgtacct tttgaactcc ttaaattcgt 420
cgatcgtaaa gttgacaact ctctaggcga gttggacagg cacgtgccgt cgctggtgaa 480
gcaggcatca agccaagctc gagctgtggc tagtgaaatt caacatgctg gattggtaga 540
cgcaactaag aacattgcga agacgatgta tacaaagtat aaactgacgg cttggcagct 600
ctactgcaaa tacaagccgg tggctaagcg ttacgoggtg tcgacctggc gctcattgaa 660
ccagcttcct ctgtttcctc aagcggctca gattgcaatc ccaactgctg cttcgtggtc 720
tgagaaatac aataagatgg ttcgttacac gaaagataga ggatatccag cggcggtgta 780
totgccattg atctoggttg agaggattgc caaggtgttc aatgaagact taaacgggcc 840
caccgtccct accaatggat catccgccgc agcacaatag ttttcatttt atgtatttat 900
gtcagattga agacgctccg gagattttga aaacctga 938
<210> 72
<211> 194
<212> PRT
<213> Sapium sebiferum L.
<400> 72
Met Ala Glu Ser Glu Leu Asn Gin His Thr Asp Met Val Gin Asp Asp
1 5 10 15
CA 2998211 2018-03-16
302
Asp Lys Lys Leu Lys Tyr Leu Asp Phe Val Gln Val Ala Ala Ile Tyr
20 25 30
Val Val Val Cys Phe Ser Ser Ile Tyr Glu Tyr Ala Lys Glu Asn Ser
35 40 45
Gly Pro Leu Lys Pro Gly Val Gin Ala Val Glu Cys Thr Val Lys Thr
50 55 60
Val Ile Ser Pro Val Tyr Glu Lys Phe Arg Asp Val Pro Phe Glu Leu
65 70 75 80
Leu Lys Phe Val Asp Arg Lys Val Asp Asn Ser Leu Gly Glu Leu Asp
85 90 95
Ara His Val Pro Ser Leu Val Lys Gln Ala Ser Ser Gln Ala Arg Ala
10C 105 110
Val Ala Ser Glu Ile Gln his Ala Gly Leu Val Asp Ala Thr Lys Asn
115 120 125
Ile Ala Lys Thr Met Tyr Thr Lys Tyr Glu Leu Thr Ala Trp Gln Leu
130 135 140
Tyr Cys Lys Tyr Lys Pro Vol Ala Lys Arg Tyr Ala Val Ser Thr Trp
145 150 155 160
Arg Ser Leu Asn Gln Leu Pro Leu Phe Pro Gln Ala Ala Gln Ile Ala
165 170 175
Ile Pro Thr Ala Ala Ser Trp Ser Glu Lys Tyr Asn Lys Met Val Arg
180 185 190
Tyr Thr
<210> 73
<211> 2526
<212> DNA
<213> Sorghum bicolor
<400> 73
atggacgagt ccggggaagc gagcgtcggc tccttcagga tcggcccgtc gacgotgctg 60
ggccgcgggg tggcgctccg cgtgcttctc ttcagctcgc tgtggcgcct gcgggcgcgc 120
gcgtacgccg ccatctcgcg cgtgcgcagc gcggtgctgc cggtggcggc gtcctggctt 180
cacctcagga acacccacgg cgtcctcctc atggtcgtcc tcttcgccct ctccctgagg 240
aagctctccg gcgcgoggtc gcgggcggcg ctcgcgcgcc ggcgcaggca gtacgagaag 300
gccatgctgc atgccgggac gtacgaggtc tgggcccgcg ccgccaatgt gctcgacaag 360
atgtctgatc aggtccatga ggcggatttc tatgacgagg agctgatcag gaacaggctt 420
gaggacctcc ggaggcggag ggaggacgga tcgctgcggg acgtggtgtt ctgtatgcgc 480
ggcgatcttg ttaggaactt ggggaacatg tgcaatcctg aacttcacaa gggcaggcta 540
gaggttccta agcttataaa ggaatagatt gaagaggttt ctattcaact aagaatggtg 600
tgcgaatctg acactgatga gttgctattg ggagagaagc ttgcctttqt tcaggagacc 660
aggcatgcct ttgggaggac agccctactc ttaagtgggg gtgcttcact ggggtctttc 720
catgtaggtg tagtgaaaac attggttgag cataagcttc tgcctcggat tatagcagga 780
tcaagcgttg gttccattat at_gttcgatt gttgcl.-accc ggacatggcc tgagattgag 840
agcttcttca cagactcatt acagacctta cagttctttg ataggatggg tggaattttt 900
gcagtgatga ggcaagtcac cactcatgqt gcactgcatg acattagcca gatgcaaagg 960
cttctgaggg atctcacaag taacttaaca tttcaaoagg cttatgacat gactggccgt 1020
gtccttggga tcaccgtttg ctctcctaga aaaaatgagc caccccgctg cctcaactat 1080
ctgacgtcgc cgcacgttgt tatttggagt gctgtaactg cctcttgtgc atttcctggg 1140
ctctttgaag ctcaggaact gatggcgaag gatagattcg gcaacatagt tcccttccat 1200
gcaccctttg ccacagatcc tgaacaaggt cctogagcat caaagcgccg gtggagagat 1260
gggagcctgg aaatggattt gcccatgatg agactcaagg agttgtttaa tgtaaaccat 1320
ttcattgtga gccaaactaa tcctcacatt totccoctoc tccgaatgaa agagcttgtt 1380
agagtctatg gagggcgctt tgctggaaag cttgctcgtc ttgctgagat ggaggttaag 1440
tatcgatgta accaaatcct agagattggt cttccaatgg gaggacttgc aaaattgttt 1500
CA 2998211 2018-03-16
9T-0-8TOZ TTZ866Z VD
0081 pobbbeogrq 35logoqobu P2P556-1.7,DP 36q2.63.65e4 bb5eo.45-eeo 5oobeeP5-
eo
06L1 llobe54o44 elb3lloo2.e EBE'ouqeoqe BepboTeqb eoqo614peo epo6eoo5qe
0891 qq6pleeppE, g6le6156p-e bbbggeopeo qaErn.qbqqb e2;obuqPe6 bpbbeqq2po
0Z91 qqqebbqqoE abbqoqqbee p4ppoq;bb-e qeobeeqqbP ebb4ebebqo 5.4.43eebqob
09ST -44obep2obq obmcbeob bebboegap5 5b2oqeD4P5 Pbbpepqoef, eb4oeqqpoo
00ST qp_64qopoq poqeepobee poft-eqbei.e oqqopoqePp qbopepq;eq qpp.bbee.61;
()DDT pe3bee5gP3 p3eqqq2.5a6 eePbPq4bp 3606p525 54a63b5peo eqobqpboeb
08E1 q36e6pee66 26.63515564 ;52,434geop q354eoql1 opq.6PoPPe Sebboggebe
OZET ..e.beepoobb 3ef4:45e6be opo6bp57,41 qqoeb6Doq qqq064.64o3 lqobqceeqb
093I eobgbp66qq. qeogolq151.e. o4poroqeoe bqqqeqoeb goobquboeo poob,abgpo
003I peeebepoq pqqbqbq54o eqqbqgbo43 qq56boobbq a2b4e3eb4e qeobbebepo
OP1I qq1-4.E.5qqq. epobeepeqg 3Teep6p_61-4 bpoboe61PD5 qqaeob5elq eb4eoqq6
0801 b3beb54eob Debq4qqee5 eepplqleq8 qooqq.pqpf) 551655qe5e posblqqoqg
OZOI pppblqooqq. eob6qbe5be 5E5egoepbu
pobbquo4b6 oepeo3begb
096 44p4obqbgb qpeqeeobob bqqbpbeeol e5be3q4qe; metbQ433eq ogqobEe4eo
006 bebeqb5q4o oe2ee54.544 bqbb5-454eo oqq4o3D6bq qqP0143.646 bebbqbeeqo
0D8 6-43eqopob6 D4p6e185q1 qoobleoebe EDESe6TeDb qe-2_1qED-657. oeeE'bebeeb
08L 1.1opooeqq6 eboebboqqe 51D1gr.7081 qq56-4ePeeE, qoPpo;oeqo qeqbbebbr,b
OZL qu'oe-45E'66 eeoqPooepe Egoob4562o 15-4obbe6qqb e-eoeco4ob-e, bobq
099 bquoe-eqbbq gooepbbeog o5qp4pbo3b abobqeobqo 443q50.42oe Mbpogobcq
009 obbbebbe34 bootiDcPobb eogobefq.bo oqobeeDePo 6-1.6q5D4ob2 5.62.6oeboeq
ol3pEopfio ebooeoqbob ooboboo6oE 5e56606-ebo lob4ebbo6o 6po6oeo5ob
08D 56abpb5Eto eloorowbo boobobobqu Eq?oeebbob bqpqqbaeob opbooeqopb
OZD op;beabg66 bobobobobq bbbboobobo pbbbbboboo bbobobqobq obqoboboqb
09 opbobqp4bo oboqobqooq ebbbbboboe opP.oeca5ob 3poeob4356 gopbbobbob
00 Eq.otoobobb abootoboBq pOobooboofi ogoo5331235 50.6q065D6o agobogobb
OPZ 35-4D1.3o4D.6 qba63543bo bogo835360 5B6606bobb oepolflopob bol.rbcboq
081 bobb6bi5T6o bobobbeboe epaeoleoq6 DPbbleopob 3pboo.46336 Droobqooqo
OZT opapqopobq ft000bbqbb obboboobbr. eoogeboo2,0 bqopobb000 bb;apftgoo
09 poqbobooqq eqoqqopopq o5opeopoo6 epDobeoob obqbbeobqo obob3oobqe
VL <00V>
tunATqsae lunoTqTay <ETD.
<Z1Z>
660 <TIZ>
171. <OTZ>
9ZSZ beq4eb
OZSZ 425gobeoq4 opeupTeobo qq4begbqqg gobqp5q4pq >f)qq.opp-4D-2 Dq2pobppoq
09D3 oebqobqoeq ebbppoPeqp bepbqp6qop obtqoqb6be oqoqPooppe E;bqqoeeub
OODZ bobqqepee ebgaeoLqop 4obe-efoob Eqqr-pb-lpee -4536eoebqe eueol.obbeo
ODEZ bqqoablpbe 61212226-45-44 epe-eqobqo oqegHoebq ob..;Pobeee bqoa6voblo
0833 bqggebebb,2 eb4ogq-15o boquobeE'pe L'oploeqoqq_ 35BEoqEmoo 5e5popeoe
OZZZ oqbweopbo eqeobbeboo e5ebqoqqae erbeoeobqu beTbEcecoqo .qopqq.bqbe
0913 epqobbgeep e-ebopepaeo oqpb42ePoo oqqoaegoge P5Poqbcqqq. -
4".6b2obqo.e.
0010 q6bqbbi5bE, qbqoe-4.6qo p5bP4,52-eqg e-ebboe3Pbq le62533e .pqpooTeoll
Ot'OZ qbpoq=qqb qeop61o513 qeDbeoebbe Eqe2loloob 551.6bgEebb eooEf)84Doq
0861 42-eq.lopebq popeeebeo qe-a60,LebeE, qbepbboebq eoqcqqeob oeboqqbgeo
0061 peoqoqqqp:. opooftoupb Boqbqqbqqo gob;obqbbe eobeobaboo eepqqaegob
0981 qqbe.-Dbeoqa Tebebeubbo qoqogoTeeb eoqqoqqepb ebeboqbpog yobqofreabq
0081 eogpooeqbb beobepooqo .22-2-eopebo qq5qegoeeq pq4poqbbefe. 06-4-
4T364D6
OD=LT bebeopEbpb 2Logeobepb PPPP_Pq3bbP Eboeeeoeop eeeqqq.CDE.
0891 leffyl.geob74. qoeebaleo 5bloeeeob ebeoTepobq oqoqobepbe 5.65geoeqbq
0091 bbpobocbbe eopueop5qo 55TeE'Pooqo 6pbbobgege oepooqe-ebv 3q4pqqebep
0901 Eqqq-eq6eoq obegb-eoepo bboabqeqqb Ege00046q bqt,bbebbb goebbeowb
EOE
304
aactgcgcta ttgagcttgc aatagatgaa tgagttgocc tcctgaacca catgcgtagg 1860
caaaagagaa gtgcagaaag agcagctgct tcacaaggat atggtgctac aattagactc 1920
tgtccaacta gaaggattcc atcatggaat ctcatagcaa gagaaaattc aactggtact 1980
ctcgatgagg aaatgctcac aaatcccact gttacgagcc atcaagcagt tggagggact 2040
gctgggccat ctaacagaaa tcaccatctc caacatagta tgcatgatag cagtgacagt 2100
gaatctgaga gtatagactt gaactcatgg acgagaagtg gtggccctct catgagaaca 2160
gcctcagcta ataaattcat cagotttatt cagaaccttg agattgacac agaattcaga 2220
acaatttcac caagggggag cgaaggtgat attgttacac cgaatagtaa cttatttgct 2280
ggtcacccaa ttggtagaga gccagttgat aaccatccag ggcctgctac tcctggtagg 2340
acctcaggca attcaggttg cgatcctcat gatactcctg ttcctaggtc tccatttggt 2400
cattccacaa gtatcatggt ccctgaaggt gacttgctgc agccggaaaa gattgagaat 2460
ggtattttat tcaatgttat gagaagggat gctottgtag cgactactag cggagttgaa 2520
cctcatggat cttcacagaa agcagatgtg gaaactgtac cgaccgagtg cctttatggt 2580
gottoggatg acgacgacga caacgtggaa ctgaatgctg atcatgaagc attatctgac 2640
cctggagatc agagatcctc agttgcagga aacctagatc cgtccacttc catggatagt 2700
caagCtgatg aaacaagtac tactcgatca gaagctccat ctctctttaa tatctgtgtg 2760
gagattcctc cagcaaccat gatcagagaa aatagtcggc ccgacgagcc ttattcagac 2820
ataagactga agattgtaaa gacagaatgc cctgatgaga attcagctgc tgggaacgat 2880
gaagttggct cagttcctgc caataaagaa tcttcctatt gttctcagac agctgaaaat 2940
agacaggagc atcaagttga tatgggatct gtgaactcct gtagtgtttc agtttcagaa 3000
gatgataggc atgtcagcct catttcgaac gagaaaccag ttactacttc cagtggcgga 3060
gcggagagta tgacatctgg aagaaatgaa gctgactag 3099
<210> 75
<211> 2198
<212> DNA
<213> Artificial Sequence
<220>
<223> S. bicolor SDP1 hpRNAi fragment
<400> 75
gcggcggcgt ggctgcaccc gcgcgacaac acgcgcggga tcctgctcgc cgtctgcgcc 60
gtcgcgctgg gtgcagtccg cctaccgccg caagttctgg cggaacatga tgcgcgccgc 120
gctcacctac gaggagtggg cgcacgcggc geggatgatt ggagtgcagt aacagcttcc 180
tgtgcttttc ctggactttt tgaggcccac catctaggag gattccatcc tggaatctca 240
tagCaagaga aaattcaact ggttctctat gtgcaatcct gaacttcaca aggacaggct 300
agaggttcct aagcttataa aggaatacat tgaagaggtt tctattcaac taagaatggt 360
gtgcgaatct gacactgatg agttgctatt gggagagaag cttgcctttg ttcaggagac 420
caggcatgcc tttgggagga cagccctact cttaagtggg ggtgcttcac tggagtcttt 480
ccatgtaggt gtagtgaaaa cattggttga gcataagctt ctgcctcgga ttatagcagg 540
atcaagaagg gtggacccag cattottgta caaagtggtc tcgaggaatt cggtacccca 600
gcttggtaag gaaataatta ttttcttttt tccttttagt ataaaatagt taagtgatgt 660
taattagtat gattataata atatagttgt taaaattgtg aaaaaataat ttataaatat 720
attgtttaCa taaacaacat agtaatgtaa aaaaatatga caagtgatgt gtaagacgaa 760
gaagataaaa gttgagagta agtatattat ttttaatgaa tttgatcgaa catgtaagat 840
gatatactag cattaatatt tgttttaatc ataatagtaa ttctagctgg tttgatgaat 900
taaatatcaa tgataaaata ctatagtaaa aataagaata aataaattaa aataatattt 960
ttttatgatt aatagtttat tatataatta aatatctata ccattactaa atattttagt 1020
ttaaaagtta ataaaLattt tgttagaaat tccaatctgc ttgtaattta tcaataaaca 1080
aaatattaaa taacaagcta aagtaacaaa taatatcaaa ctaatagaaa cagtaatcta 1140
atgtaacaaa acataatcta atgctaatat aacaaagcgc aagatctatc attttatata 1200
gtattatttt caatcaacat tcttattaat ttctaaataa tacttgtagt tttattaact 1260
tctaaatgga ttgactatta attaaatgaa ttagtcgaac atgaataaac aaggtaacat 1320
gatagatcat gtcattgtgt tatcattgat cttacatttg gattgattac agttgggaag 1380
CA 2998211 2018-03-16
305
ctaggttcga aatcgataag cttgcgctgc aattatcatc atcatcatag acacacgaaa 1440
taaagtaatc agattatcag ttaaagctat gtaatatttg cgccataacc aatcaattaa 1500
aaaatagatc agtttaaaga aagatcaaag ctcaaaaaaa taaaaagaga aaagggtcct 1560
aaccaagaaa atgaaggaga aaaactauaa atttacctgc acaagcttgg atcctctaga 1620
ccactttata caagaaagct gggtccaccc ttcttgatcc tgctataatc cgaggcagaa 1680
gcttatgctc aaccaatgtt ttcactacac ctacatggaa agaccccagt gaagcacccc 1740
cacttaagag cagggctgtc ctcccaaagg catgcctggt ctcctgaaca aaggcaagct 1800
tctctcccaa cagcaactca tcagtgtcag attcgcacac cattcttagt tgaatagaaa 186C
cctcttcaat gtattccttt ataagcttag gaacctctag cctgccattg tgaagttcag 1920
gattgcacat agagaaccag ttgaattttc tattgctatg agattccagg atggaatcct 1980
cctagatggt gagcctcaaa aagtccagga aaagcacagg aagctgttac tgcactccaa 2040
gcatccgcgc cgcgtgcgcc cactcctcgt aggtgagcgc ggcgcgcatc atattccgcc 2100
agaacttgcg gcggtaggcg gactgcaccc agcgcgacgg cgcagacggc gagcaggatc 2160
ccgcgcgtgt tatcgcgogg gtgcagccac gccgccgc 2198
<210> 76
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 76
ttttaacgat atccgctaaa gg 22
<210> 77
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 77
aatgaatgaa caagaattaa gtc 23
<210> 78
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 78
cttttctcac accgtatctc cg 22
<210> 79
<211> 25
<212> DNA
<213> Artificial Sequence
CA 2998211 2018-03-16
306
<220>
<223> Oligonucleotide primer
<400> 79
agcatgatat acttgtcgag aaagc 25
<210> 80
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 80
gcgacagtgt agcgtttt 18
<210> 81
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Oliggnucleotide primer
<400> 81
atacataaaa tgaaaactat tgtgc 25
<210> 82
<211> 2631
<212> DNA
<213> Saccharum hybrid
<400> 82
ctgcgacagc tagaggcgcc accgcgtcct agcttcctcc aacttctcgt cggagatccc 60
ttcagggatg cccaatgcca ccgcccctaa gtcaacctgc gggagctgga gcttcgccag 120
ggtcagagct gcggcagcac cctggtagac cgcattcctg atgacccgcg gggtgcgctc 180
catgaagaag tgcattcgcc caaccaagtc gagtgggtcg cctggagggg gcggggaagc 240
aaaacgttgc atgcacctag cgccctggca gcgagctcct gtagtatcac ctgcgtcgcc 300
tccagctcat gctcgcaagc ctccagggcg gcccggcagt gctccaacac tttcgcctcc 360
tcctacagct ccttccacat gcagtcgtgc tccgcacgca ccttctccac ctttttactc 420
ttttctttct cttttcttgg cocatotttg gtattttcac aaatgtcccc ctacaaatga 480
taaatcacca aaactcatgg agottgctag ttataaactc taattctaag tttggtgttt 540
atttgagtgg attttctgtg aaagttggtg gttagaaata ggagttaagg accgccaaca 600
agatccccca cacttagccc tttgctcatc ctcgagtaaa gttcaaggac taaggtggaa 660
catctcctca aatggtacga tgcctgcata taagttattc caagcctcac ctatacatgt 720
gaactttgaa gtgtctacca cgccatcttg ggtggttgag aaatggaaca gatcagaatc 780
cagtcatctt tacctctctt gcttagataa cttgggtttt tgtaaggttt tcaaatttaa 840
aacatagtct tgctcctcaa atgattctct catatagctc aatgtgtatg gtttctcacc 900
aaggcaatgt tttgcctctt ttcatcctac ttctaatatt tcttttgtgg agcttagggt 960
agggaatgaa aaggaagcat acttgcatr_g catatgttac taagtcaaaa accaaatctg 1020
aggagaagca agtcatacaa tctgatcaag atgtgcaagt gtgtggatat gtggattaag 1080
ataactcctg tttattcatg ctctcctcct taataaactt tagagggcat ggcaatcttt 1140
CA 2998211 2018-03-16
307
ccatgggcct tcatgagctc atcgtatgtc taagcatgga gctcatcatt tatataagca 1200
tggtgatacc aaaattactc cttttaagca tgtttatatt taggaggacg ttttacctgt 1260
tgaggtaaat ctgaacgcta ataaatcggc taagcaaaat aatttatcac ctgttgattc 1320
taacaatttg atgatggaca atattgatga ggtgactgac aaatgattga aggctttaaa 1380
agagattgag aaggataaat ctacaataaa aatgtaaaga agaaagcatt caaagtgtga 1440
gatctggtgt ggaagactat tttgcctott gggggtaaaa gacaacaagt ttagtaagtg 1500
gcctcaaaat tgggagggcc catgcaagat tgttaaagaa attgttttgg attgacggag 1560
gcatttcaag gtgatcatct acctagagct ctcaatggga ggagctcgaa gacatattac 1620
ccatgtgtaa ggcaagatgt ttagctagta actgactgat agagtaaacg atcaccaatg 1680
aggcaagaca tattacctaa cgccaggctg gtttttgcaa gtacgagtag gatatagaga 1740
ttctcgtgcg agttgtaaac gatctccaaa ggggcaagac atcctaccct atatatagtg 1800
aaggggcagt agctgattga gaatcaatca atcaagcaca atataattta ttaatttttt 1860
atacaaaccc aatttttttc cttttccaac cctaattata gtattccttt tgcctctagg 1920
acaaattgac gtgttccggg tatcctgctg aataaagaac aaccctaggt gcacctgtcc 1980
cgatagagtc ccacctgggt aggcattcaa agggattcgt gtatttcctg caaaaaagcg 2040
attaagctgg cttctaaaac tggctaggcc ggattctgtg gccttcacta ccaggtgatt 2100
ttcaagtgat ccgtgcattc tagcactttg ctatgtaaCc caaacttaag tcgacaacta 2160
taaatatgct acttgcagga tgttatcacg acacaactcc taatctacgg aagcctaagt 2220
ttagatttgc tcggagacaa gcaattgtgg ccagtcacta tagctacgtc agagggtagt 2280
aggagcagtt gcgtcgttgg attgaaaaca ggtggatcgt atcagatatt, atgcattcac 2340
atggacagta aatgtggtac agtaacttcg caaacaataa aatctgtcac aatttattag 2400
tgcactoctc tgacgtaaat acttctacgt cagaggattt gattccgagg gccgctgcac 2460
ccatcactaa tgacggtctt tacccatcat catggaccat tgttcacatc catgctatca 2520
ctgtcgtcct gtccatgcac tgCagccctc tataaatact ggcatccctc coccgttcac 2580
agatcacaca acacaagcaa gaaataaacg gtagctgcca taactagtac a 2631
<210> 83
<211> 2907
<212> DNA
<213> Saccharum hybrid
<400> 83
gcataggcat tgtaaaagcg gtatgcctct tcttcagtgc agaatttcat accaacctta 60
ggtatcctgt cttccataga attttctacc tgagtaggat cggtotgatt ggaattgtag 120
cgggtttcat gcaaaataag ttagaaatcg tqcaaacttg caatggaggt taaatttgaa 180
atatatttgc atagacaaaa caaatataga ttatgaatgg taatccaata tgacttgcat 240
tttctaactc tattgctact gtgccagatg aagaatgttg atctgaagaa gttttgtgag 300
aatgtgacaa caacgggagg tcatatcaag attctgggta cccgcggaga atcggcctcc 360
atgtagttag cctcgtcagg catgggggga attggctgag atgcccccat gtagtcgtca 420
ggcatggaga gtactggctg agatgccatt gttgtgtaga tcgagagaaa cgagaagaat 480
gctagtctaa taataccctt ccgtatgcta accaactatt ataattggca ccatttttca 540
catgctagcg ccttttgcct gctttattta attcaattgg gtccgataag catgtgaacg 600
tgggagacgg ttccgtcgga cggctccgtt ttcttgtagc gtacggcgtg gacggagaaa 660
aggtgagggc ctatctctaa aggggaacga atggatggtg gacacatgtg gggagacacc 720
gaagggacat gccgaggagg cacacaagct tcagcaggcg tctccagact ctcagaagaa 780
gaagaagctc acggcacggt tgcggctggt tattgctgtc gctgtctcgt ggtgcacgtt 840
tctgtgatca cgctgaaatc gaccggccgg cggaccaaca ggaggtcagc tcggccactc 900
cgtctccgag cgcatgagtg caccgttcgt ccgcggttcc ttttctcgtg gtgccgtgca 960
cgcctctgcg ttcaccggca ccctgaaacc aatcagaacg ttccctttac aggggaaagg 1020
gacaagtctg ataacctctc tgtttccatc gtcctctaac cgcgaagagc ggacgcacaa 1080
gacttagagt ctatttgttc gaaatttttt actctcacaa aagctagctt ttatagacgg 1140
gcataaaagc tatcatgtcg accggcacgt ttaatattta acttatacca tatgaatatc 1200
atgtcgaact atgaggatga tacttttctg aacgtgattg cgtgagttat taaattgtac 1260
ttttagttgt ttgagcatga aggtctgaac tatgaattaa tgatgtattg tggcttgtga 1320
gctactccgc tctacattta gttggtatca taaatattat tatattatca tataaatttg 1380
CA 2998211 2018-03-16
308
atcaacttga gatgctttga ctcttcaaga ttcatggaat gacttatcat ttggggtagg 1440
gagtaggttt ctaaggccag tctcagtggg gtttcatcag agtttcatgg acattaaata 1500
agctgatgtg acaccgtatt gatgaagaga gagatgataa gagtttcatg cgagtagaga 1560
gagtttcatg gggatgaaac tcttcttcac tgtttccaaa atatagatgc attggtaaga 1620
gggccatgaa atcactagtg acactaacct aagatgagat tgactctagc actatgtttc 1680
aaaatctgca tgcatgcatg ctttgaatat tgtaacctca cattaactcc cctcacacat 1740
gcatgcaaac gggcggtgca cgcaaaagaa ttgagtgaag atgcacatga aaaataagta 1800
aaatgctttg gcttcatcac ccgqcttaaa tgatcgacag aaaaacacgt cggtagtcaa 1860
gattgtgact aacaaactgg ggttcacatg taaaacacgt tcatgcctta gaaacggcct 1920
ggagggatta gatacaactt caattatatc ttagggcccc tccaatattg tcagctctaa 1980
actagtttta tgtgtcacgg tggaggagag ggaggctaaa aatataatct tgagctaacg 2040
tgaagagaag agctattttt ttttgctccc caatacatga tagatacaat atgagagaaa 2100
aaatatatga ataaagaaca ctttacatgc cagccataca atatgagatt tcatctaaga 2160
accaacacca gactcgtact gttgaaggtg tcctagttgg agtggtcgat cttttagttg 2220
ttagtagtga aagacctagt ttagtgctct tttcttgtct aggtttatgt tgtgttttgg 2280
ctgccaagtg ttgaacaact caaggtaagg tcccatctaa ttctaaaatg atgccaaata 2340
aagatagatt acaaagttaa acgacggaaa aactctaaaa taggatggaa agttttatag 2400
agtaataatt ggtatgaagt ggcgaagtcg accacaacca aacataaaga gttaaatgca 2460
tggtaggctc ttgatcttgt ctggaggtgc cacttaggtc cacaaactct caaattgcat 2520
ttttgacacc ctaatgttat tcaagtgtgc cacttagatc tacaaactct caaaatgcat 2580
ttctgatacc ctagtgttgt tcaagtgtgt cacttaggca agaaaagtta gataaatttg 2640
ataagctatg ggaccaaatt aatttatgta tgcatgctcg aactagttga taatgatgga 2700
ccccataata gacactagtt catgggctgg tttccttgta tagtactagc tagtataact 2760
ttttcaagta gtagctacta ctttagctta tactccacat attacaatca aatagaattc 2820
ggaagtacta taaacgagag cctataaatg gagacgtttt acatcatgag gctataacaa 2880
cttgagcaaa aacagaagcc gtgcgcc 2907
<210> 84
<211> 1141
<212> DNA
<213> Saccharam hybrid
<400> 84
actatagqqc acgcgtqgtc qacqgccogg qctggtctgg ttttggcctc ttttagttac 60
taaattgcca aaaagagtga ctaaaaagtg actaaactga tttagtcctc tagtcaaggg 120
actaaaccag ctaaaagaca tccgctgccc ctcattaatg cacagaagga gagagagagg 180
gagagggagg acattttggt ctttatatag tagctttaat ggactttagt acctagatcc 240
aaaccggtag tgactaaagt ttagtcattg aactgaactt taatccaggg acatggaacc 300
aaacatgccc ttaacratttt tttattctaa tacctattac attcacttgt ctcacaaagt 360
ggcaagtcat ttgccaccct cactaccagt ggcgactggt taaatatcct catgtttggt 420
tttttttagt aaccaaatac tgcaagctat tgggaaaaaa ggcaaaaaat tatctccttg 480
cttatagttg tataatccat gatccggcaa atgtttgtta cggagatcct gaatcctctg 540
acgtagagtt taatcaattt tagctcaaga ataatacact ataaagtgga tatgacaatc 600
accgtagtac ttatttatct tgtagtagta tactgaattc gacctgcaat tatgataaag 660
gcatcagaaa ctagagtact ttctagaatc tttagtcagt ttctgtaaga tgaacgtgac 720
taagaaactt atactgttgc aatcctctga cattctctga ttgaaactcg gtttccaaaa 780
atcatatgtt actaaacaaa acatatctaa ccaaatacta tgtgataatg tagatttata 840
tgctgtgtac aaaaagtgac gtcaagaata gtagtggcag agactcaaaa gatacctgcg 900
gattctgaat accacaacca taaaaaacag gatgatgtta tacttgtccc cttccatgat 960
acaggactgt atagtaattt cccaaacagc ccataataca ttctgcaccc tttattaaac 1020
ctotactagc tacaacatct tactccatct tgtctagttg gacaagttct ctotttcttg 1080
gctgactcca acttactaca ccgcaacttc ttgtgccctt gttccaacca tcacaattga 1140
1141
CA 2998211 2018-03-16
309
<210> 85
<211> 4438
<212> DNA
<213> Saccharum hybrid
<400> 85
aaatacaaac gtagactctg acatacacgc acgtagactc tgacatacac gcataaacga 60
acgaagaatg atattattta tgttttgagt gggaatattt ggtactgcta tgattcacgt 120
gtgtaaggaa ggattcaaaa agaaaggatg cgtttagttc gcgaaaattt ttgactttta 180
ccactatagc actttcgttt gtatttgtta attagtgacc aatcatggac taattagact 240
caaaagatcc gtctcgtggt tttaaaccaa actgtgtaat taattttttt tatctatatt 300
taatgctcca aatatgagtc aaatattcga tataacgaag aatcttgaaa atttttagga 360
actaaacatg gccaaagtgt tgtcccgact gagaaacttt ggaagcagaa taaaggctca 420
aaggaacatt aaaaagaaga ggatgatata taatcaaaag tgacgacaaa gaagtgtgta 480
cgacccactc gagattgacg aaggacagct tcattgttct tttgtgtatt actgaatatg 540
taataatctt gtatagattg gtttttaaaa tacagtggca aattaaagac gatatcactt 600
acaaagacat ggacaatgtg gaggggccaa aagttatata aacgacacqc cgaatoggtg 660
ataaacacca catgcctccc ataaagacgg tgaatcaatc tttgatataa tgggtatccg 720
tttgaggcgg catttatact tgatctagta aaattacaag gagaggaaaa gaagtttaag 780
agaatgataa agataatgaa aaaaatcgga ggaaaaagaa catgaacaaa gcaagaggag 840
atagccgtgc acacaaaata gagataattt cctattagaa ctatgaaaac ttcctcatact 900
ttCtgcaaca ctgatttgag tttttattct ctatctagca tttcagtcca tcttgatgtc 960
aagtgacatg taaaaagacg tattgccccc attgctattt taaattgtct ccacacttga 1020
caacaattta atgagttgtt aaaatattat gtgtatttat ggccaaatat acattttagt 1080
tatgagattt tcatgaagtc aataagatgc taaaaataat ataaagttgt caatgattgt 1140
cggaagcccc aatatgtgac taaaatgctg ctaaaagttt atagcatttt ttaaaaaatc 1200
taaacaaatt gaaaaaagaa atccaaacta gaaattgtag aacttatcga aaactataag 1260
ttttatataa aaggcgactt tatctaacac cacacaagaa agatgtactt ttactaagaa 1320
gacaagtctt agtatgtgat taatatgcta ctgaaaattt atattatttt taagcatttt 1380
aataacctca aatggaaaca tacaaaacta agttgcagat catatcaaga gcaataattt 1440
ttatataaaa tgtatatzta aataacacca tacaagaaag atatatgatt ttttctaaga 1500
cgacaaagct ttgtatgcaa tttaatatgt tgctaaaaaa tcatattatt ttttttatca 1560
tcttaacgtc ctcaaataaa aaaaaatcag actagttggt atagacctca tcgaggctac 1620
aatttttata aaaactcaac ttcatccggt gttgtataaa aatgatataa tttttcctag 1680
atagagcgtt gccataagtg tattttggtc aagaaatata tgtatactta ttaatgaaat 2740
cctaacaaaa tatactttaa aatctgacgg aaatgttgga taggaaagaa aagcttaaat 1800
caatgctaaa tagggaagtt ttcatcatag ttataatgag tgatttctcc acaaaatatg 1860
atgtaccaca tgttaaatat tactcgcgca caaataatca gagcatatta ctatcatagc 1920
gtggtcgtgg ccatggccta gacttggttg tggacgtctc acttcaccaa ttgatagaaa 1980
aaaaacattt ataagaaaga aaagatacaa aaaccatcac acgcgacaac atgacttgcc 2040
gaaacacaaa accaaaaccc aaactcgaga agatgctttc gagaaaaagc ctgaaaagaa 2100
aaaaaatttg cacgtaaaat caaattcgga cggcgaagag ggcaaacgag acagacaact 2160
gggtccactt gctgataaaa aagagagaga ggagggcaga cttgccggcq ggcaccactc 2220
agactgtctc caacaatact gacgcaaaca gaagacgcat tggatgcaat gcgttgcgct 2280
gtggcaaaaa attaggtacc tatttctagt gtattccaac agagaacgca aaagaagatg 2340
ccgtactgcg ccatgcattc atgtgggacc ggggaggatg cgggcaacag cagtttgcac 2400
gacccattgg ccggagcatg cgacgtatat ttgcgttgcg cctcgcttcc tacgcaaaat 2460
gtgtcgttgg tatgactacc ctattggaga gcgttttott ctgctaaagt aacgtggagc 2520
acgcatttgc gtaggctgtt ggagatagtc tcaccacgcg gtgaccggac caggccaatt 2580
cccgagccca aaaagaaaaa agcacacaca cagagacaca cgctctcgct ctcgcctccc 2640
tgacgctgga tttaagcaga gcagggagca gaggtgcaac cgcccaccac gatctcccct 2700
cccgcacgcc ccgcgggcag acccagccaa ggcaaggcag ccgcgaaccg gagcacgccg 2760
gCCggtgtcg cctcccgcgc cggcggcctg ctgctcgctc gocctcgott ccgcattgga 2820
tcacgcggcg gttggcgact tggtggtgtc tgctgctggt gattgcgcct agccggccga 2880
cgaggagagg gtgaggcgct gctottcgct tctctcccca ctgctcccct cagcggtttc 2940
tctctccctg ttatgcgtgg aggagccctg cccccgcgga acggaagcct ccgccggatc 3000
CA 2998211 2018-03-16
310
tctgttacgc cgcggttact gcctcgccct ggatttgaac ttgtttcgta attttccctt 3060
gctgcgcttc tcgatttcgg ggaggggttc tgccggcagc totgccgcto cacctgactt 3120
ggggaccttt ctatgttccg cgacaggagc attgatgatc tgcttgtctc ttgagttttt 3180
ttttcgtgcg atgcatcgag cgcgtgggga cacgatcacg cctgatgggc ggtagtccgc 3240
gatccgcatt tctgaatccc ggcgcctagc cgaggtgcct cggtgcttcc tggttgcott 3300
gctgctattc ccttcttcgg atccgctctc gtacggctgg cacggtggtt gcggccttag 3360
aatttcgtgg cggcggtttg gttggattgg tgatgctgct ccgtccgcat ttatgaagga 3420
atgttotoca aacttttaag ctgctcgtgt actaggagta ttgaattgcc tgttccttgc 3480
cgctatagga ggccctgggc cagcctaccc cgctttgggt tgtgattggt gatttccggc 3540
agctgttatt gtttcatgat tcgtgtgggg aaaaaaagtt tttttggttc acgagtggtt 3600
tctggtgcat gttttgacaa gttttctatg atgctggtac tgtctttacc cctgctagag 3660
tagtttggtg gtgcgttttc ctattaggtg ggaatttaat cactttccca ctttatcgta 3720
tctctactat ggtaaccatc ttttggcaat tttgattggt atagtcatgt ttaagataag 3780
cttttqaatt caatgatctt gccgttcatt agctagcact taattttgta gagctgcttg 3840
gatcaccaaa gtgccgctca atcttattca agtgcctatg atatatggga ttctgatgga 3900
actcttagca gtcgtgtcct taggcagtcg gcaccttgat aaggttccaa gagttcaatc 3960
ttacggaaga aatagtgagc ttgatctgag ttcagatcgg ttgtcttcac acttcacgat 4020
taattaccac gtttttaagg tgtgcattct cacttcttta cttccatcgt caatcttctt 4080
aactggttgg gttggaggtg tggtcatgca cccaaccaca taggttgagt cctcttcaac 4140
tcgaatttag gtgcctattt ttttcttaat aaaaaaggcc acctgattct ccttggttgg 4200
tcacattttt ttcttaataa aaaaaogcca cctcaatgtt tctcctttta gcttgagcac 4260
tttttctgga tctcctcttt cttcttaatt ctgatccaag tgtcatcagc gttatattta 4320
tttgaacctg cttgcttttg taagcctgat cagtttgcaa aagttactag aacaatttaa 4380
ccatctgtgc ttgttatttc tgcaggcatc aagtttctaa caatttgaag tacctaaa 4438
<210> 86
<211> 145
<212> PRT
<213> Sesamum indicum
<400> 86
Net Ala Glu His Tyr Gly Gin Gin Gin Gin Thr Arg Ala Pro His Leu
10 15
Gin Leu Gin Pro Arg Ala Gin Arg Val Val Lys Ala Ala Thr Ala Vol
20 25 30
Thr Ala Gly Gly Ser Leu Leu Val Leu Ser Gly Leu Thr Leu Ala Gly
35 40 45
Thr Val Ile Ala Leu Thr Ile Ala Thr Pro Leu Leu Val Ile Phe Ser
50 55 60
Pro Val Leu Val Pro Ala Val Ile Thr Ile Phe Leu Leu Gly Ala Gly
65 70 75 80
Phe Leu Ala Ser Gly Gly Phe Gly Val Ala Ala Leu Ser Val Leu Ser
85 90 95
Trp Ile Tyr Arg Tyr Leu Thr Gly Lys His Pro Pro Gly Ala Asp Gin
100 105 110
Leu Glu Ser Ala Lys Thr Lys Leu Ala Ser Lys Ala Arg Glu Met Lys
115 120 125
Asp Arg Ala Glu Gin Phe Per Gin Gin Pro Vol Ala Gly Ser Gln Thr
130 135 140
Ser
145
<210> 87
<211> 382
CA 2998211 2018-03-16
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<212> PRT -
<213> Ginnamomum camphora
<400> 87
Met Ala Thr Thr Ser Leu Ala Ser Ala Phe Cys Ser Met Lys Ala Val
1 5 10 15
Met Leu Ala Arg Asp Gly Arg Gly Met Lys Pro Arg Ser Ser Asp Leu
20 25 30
Gln Leu Arg Ala Gly Asn Ala Gln Thr Ser Leu Lys Met Ile Asn Gly
35 40 45
Thr Lys Phe Ser Tyr Thr Glu Ser Leu Lys Lys Leu Pro Asp Trp Ser
50 55 60
Met Leu Phe Ala Val Ile Thr Thr Ile Phe Ser Ala Ala Glu Lys Gln
65 70 75 80
Trp Thr Asn Leu Glu Trp Lys Pro Lys Pro Asn Pro Pro Gln Leu 1,eu
85 90 95
Asp Asp His Phe Gly Pro His Gly Leu Val Phe Arg Arg Thr Phe Ala
100 105 110
Ile Arg Ser Tyr Glu Val Gly Pro Asp Arg Ser Thr Ser :le Val Ala
115 120 125
Val Met Asn His Leu Gln Glu Ala Ala Leu Asn His Ala Lys Ser Val
130 135 140
Gly Ile Leu Gly Asp Gly Phe Gly Thr Thr Leu Glu Met Ser Lys Arg
145 150 155 160
Asp Leu Ile Trp Val Val Lys Arg Thr His Val Ala Val Glu Arg Tyr
165 170 175
Pro Ala Trp Gly Asp Thr Val Glu Val Glu Cys Trp Val Gly Ala Ser
180 185 190
Gly Asn Asn Gly Arg Arg His Asp Phe Leu Val Arg Asp Cys Lys Thr
195 200 205
Gly Glu Ile Leu Thr Arg Cys Thr Ser Leu Ser Val Met Met Asn Thr
210 215 220
Arg Thr Arg Arg Leu Ser Lys Ile Pro Glu Glu Val Arg Gly Glu Ile
225 230 235 240
Gly Pro Ala Phe Ile Asp Asn Val Ala Val Lys Asp Glu Glu Ile Lys
245 250 255
Lys Pro Gln Lys Leu Asn Asp Ser Thr Ala Asp Tyr Ile Gln Gly Gly
260 265 270
Lou Thr Pro Arg Trp Asn Asp Leu Asp lie Asn Gln His Val Asn Asn
275 280 285
Ile Lys Tyr Val Asp Trp Ile Leu Glu Thr Val Pro Asp Ser Ile Phe
290 295 300
Glu Ser His His Ile Ser Ser Phe Thr Ile Glu Tyr Arg Arg Glu Cys
305 310 315 320
Thr Met Asp Ser Val Leu Gln Ser Leu Thr Thr Val Ser Gly Gly Ser
325 330 335
Ser Glu Ala Gly Leu Val Cys Glu His Leu Lou Gln Lou Glu Gly Gly
340 345 350
Ser Glu Val Leu Arg Ala Lys Thr Glu Trp Arg Pro Lys Leu Thr Asp
355 360 365
Ser Phe Arg Gly Ile Ser Val Ile Pro Ala Glu Ser Ser Val
370 375 380
<210> 88
<211> 417
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<212> PRT
<213> Cocos nucifera
<400> 88
Met Val Ala Ser Val Ala Ala Ser Ala Phe Phe Pro Thr Pro Ser Phe
1 5 10 15
Ser Ser Thr Ala Ser Ala Lys Ala Ser Lys Thr Ile Gly Glu Gly Ser
20 25 30
Glu Ser Leu Asp Val Arg Gly lie Val Ala Lys Pro Thr Ser Ser Ser
35 40 45
Ala Ala Met Gin Gly Lys Val Lys Ala Gln Ala Val Pro Lys Ile Asn
50 55 60
Gly Thr Lys Val Gly Leu Lys Thr Glu Ser Gln Lys Ala Glu Glu Asp
65 70 75 80
Ala Ala Pro Ser Ser Ala Pro Arg Thr Phe Tyr Asn Gln Leu Pro Asp
85 90 95
Trp Ser Val Leu Leu Ala Ala Va] Thr Thr Ile Phe Leu Ala Ala Glu
100 105 110
Lys Gln Trp Thr Leu Leu Asp Trp Lys Pro Arg Arg Pro Asp Met Leu
115 120 125
Thr Asp Ala She Ser Leu Gly Lys Ile Val Gln Asp Gly Leu Ile Phe
130 135 140
Arg Gln Asn Phe Ser Ile Arg Ser Tyr Glu Ile Gly Ala Asp Arg Thr
145 150 155 160
Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr Ala Leu Asn
165 170 175
His Val Arg Asn Ala Gly Leu Leu Gly Asp Gly Phe Gly Ala Thr Pro
180 185 190
Glu Met Ser Lys Arg Asn Leu Ile Trp Val Val Thr Lys Met Gin Val
195 200 205
Leu Val Glu His Tyr Pro Ser Trp Gly Asp Val Val Glu Val Asp Thr
210 215 220
Trp Val Gly Ala Ser Gly Lys Asn Gly Met Arg Arg Asp Trp His Val
225 230 235 240
Arg Asp Tyr Arg Thr Gly Gln Thr Ile Leu Arg Ala Thr Ser Val Trp
245 250 255
Val Me-J. Met Asn Lys His Thr Arg Lys Leu Ser Lys Met Pro Glu Glu
260 265 270
Val Arg Ala Glu Ile Gly Pro Tyr Phe Val Glu His Ala Ala Ile Val
275 280 285
Asp Glu Asp Ser Arg Lys Leu Pro Lys Leu Asp Asp Asp Thr Ala Asp
290 295 300
Tyr Ile Lys Trp Gly Leu Thr Pro Arg Trp Ser Asp Leu Asp Val Asn
305 310 315 320
Gln His Val Asn Asn Val Lys Tyr Ile Gly Trp lie Leu Glu Ser Ala
325 330 335
Pro Ile Ser Ile Leu Glu Asn His Glu Leu Ala Ser Met Thr Leu Glu
340 345 350
Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Gln Ser Leu Thr Ala
355 360 365
Ile Ser Asn Asp Cys Thr Gly Gly Leu Pro Glu Ala Ser Ile Glu Cys
370 375 380
Gln His Leu Leu Gln Leu Glu Cys Gly Ala Glu Ile Val Arg Gly Arg
385 390 395 400
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Thr Gln Trp Arg Pro Arg Arg Ala Ser Gly Pro Thr Ser Ala Gly Ser
405 410 415
Ala
<210> 89
<211> 423
<212> PRT
<213> Cocos nucifera
<400> 89
Met Val Ala Ser Ile Ala Ala Ser Ala Phe Phe Pro Thr Pro Ser Ser
1 5 10 15
Ser Ser Ser Ala Ala Ser Ala Lys Ala Her Lys Thr Ile Gly Glu Gly
20 25 30
Pro Gly Ser Leu Asp Val Arg Gly Ile Val Ala Lys Pro Thr Ser Ser
35 40 45
Ser Ala Ala Met Gln Glu Lys Val Lys Val Gln Pro Val Pro Lvs Ile
50 55 60
Asn Gly Ala Lys Val Gly Leu Lys Val Glu Thr Gln Lys Ala Asp Glu
65 70 73 80
Glu Ser Ser Pro Ser Ser Ala Pro Arg Thr Phe Tyr Asn Gln Leu Pro
85 90 95
Asp Trp Ser Val Leu Leu Ala Ala Val Thr Thr Ile Phe Leu Ala Ala
100 105 110
Glu Lys Gln Trp Thr Leu Leu Asp Trp Lys Pro Arg Arg Pro Asp Met
115 120 125
Leu. Ala Asp Ala Phe Gly Leu Gly Lys Ile Val Gln Asp Gly Leu Val
130 135 140
Phe Lys Gln Asn Phe Ser Ile Arg Ser Tyr Glu Ile Gly Ala Asp Arg
145 150 155 160
Thr Ala Ser Ile Glu Thr Leu Met Asn His Leu Gln Glu Thr Ala Leu
165 170 175
Asn His Val Lys Ser Ala Gly Leu Met Gly Asp Gly Phe Gly Ala Thr
18C 185 190
Pro Glu Met Ser Lys Arg Asn Leu Ile Trp Val Val Thr Lys Met Arg
195 200 205
Val Leu Ile Glu Arg Tyr Pro Ser Trp Gly Asp Val Val Glu Val Asp
210 215 220
Thr Trp Val Gly Pro Thr Gly Lys Asn Gly Met Arg Arg Asp Trp His
225 230 235 240
Val Arg Asp His Arg Ser Gly Gln Thr Ile Leu Arg Ala Thr Ser Val
245 250 255
Trp Val Met Met Asn Lys Asn Thr Arg Lys Leu Ser Lys Val Pro Glu
260 265 270
Glu Val Arg Ala Glu Ile Gly Pro Tyr Phe Val Glu Arg Ala Ala Ile
275 280 285
Val Asp Glu Asp Ser Arg Lys Leu Pro Lys Leu Asp Glu Asp Thr Thr
290 295 300
Asp Tyr Ile Lys Lys Gly Leu Thr Pro Arg Trp Gly Asp Leu Asp Val
305 310 315 320
Asn Gin His Val Asn Asn Val Lys Tyr Ile Gly Trp Ile Leu Glu Ser
325 330 335
Ala Pro Ile Ser Ile Leu Glu Asn His Glu Leu Ala Ser Met Ser Leu
340 345 350
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Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Val Leu Gin Ser Leu Thr
355 360 365
Ala Val Ser Asn Asp Leu Thr Asp Gly Leu Val Glu Ser Gly Ile Glu
370 375 380
Cys Gin His Leu Leu Gin Leu Glu Cys Gly Thr Glu Leu Val Lys Gly
385 390 395 400
Arg Thr Glu Trp Arg Pro Lys His Ser Pro Ala Leu Gly Asn Met Gly
405 410 415
Pro Thr Pro Gly Gly Ser Ala
420
<210> 90
<211> 414
<212> PRT
<213> Cocos nucifera
<400> 90
Met Val Ala Ser Val Ala Ala Ser Ser Ser Phe Phe Pro Val Pro Ser
1 5 10 15
Ser Ser Ser Ser Ala Ser Ala Lys Ala Ser Arg Gly Ile Pro Asp Gly
20 25 30
Leu Asp Val Arg Gly Ile Val Ala Lys Pro Ala Ser Ser Ser Gly Trp
35 40 45
Met Gin Ala Lys Ala Ser Ala Arg Ala Ile Pro Lys Ile Asp Asp Thr
50 55 60
Lys Val Gly Leu Arg Thr Asp Val Glu Glu Asp Ala Ala Ser. Thr Ala
65 70 75 80
Arg Arg Thr Ser Tyr Asn Gin Leu Pro Asp Trp Ser Met Leu Leu Ala
85 90 95
Ala Ile Arg Thr Ile Phe Ser Ala Ala Glu Lys Gin Trp Thr Leu Leu
100 105 110
Asp Set Lys Lys Arg Gly Ala Asp Ala Val Ala Asp Ala Ser Gly Val
115 120 125
Gly Lys Met Val Lys Asn Gly Leu Val Tyr Arg Gin Asn Phe Ser Ile
130 135 140
Arg Ser Tyr Glu Ile Gly Val Asp Lys Arg Ala Ser Val Glu Ala Leu
145 150 155 160
Met Asn His Phe Gin Glu Thr Ser Leu Asn His Cys Lys Cys Ile Gly
165 170 175
Leu Met His Gly Gly Phe Gly Cys Thr Pro Glu Met Thr Arg Arg Asn
180 185 190
Leu Ile Trp Val Val Ala Lys Met Leu Val His Val Glu Arg Tyr Pro
195 200 205
Trp Trp Gly Asp Val Val Gin Ile Asn Thr Trp Ile Ser Ser Ser Gly
210 215 220
Lys Asn Gly Met Gly Arg Asp Trp His Val His Asp Cys Gln Thr Gly
225 230 235 240
Leu Pro Ile Met Arg Gly Thr Ser Val Trp Val Met Met Asp Lys His
245 250 255
Thr Arg Arg Leu Ser Lys Leu Pro Glu Glu Val Arg Ala Glu Ile Thr
260 265 270
Pro Phe Phe Ser Glu Arg Asp Ala Val Leu Asp Asp Asn Gly Arg Lys
275 280 285
Leu Pro Lys Phe Asp Asp Asp Ser Ala Ala His Val Arg Arg Gly Leu
290 295 300
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Thr Pro Arg Trp His Asp Phe Asp Val Asn Gin His Val Asn Asn Val
305 310 315 320
Lys Tyr Vai Gly Trp Ile Leu Glu Ser Val Pro Val Trp Met Leu Asp
325 330 335
Gly Tyr Glu Val Ala Thr Met Ser Leu Glu Tyr Arg Arg Glu Cys Arg
340 345 350
Met Asp Ser Val Val Gin Ser Leu Thr Ala Val Ser Ser Asp His Ala
355 360 365
Asp Gly Ser Pro Ile Val Cys Gin His Leu Leu Arg Leu Glu Asp Gly
370 375 380
Thr Glu Ile Val Arg Gly Gin Thr Glu Tip Arg Pro Lys Gin Gin Ala
385 390 395 400
Arg Asp Leu Gly Asn Met Gly Leu His Pro Thr Glu Ser Lys
405 410
<210> 91
<211> 419
<212> PRT
<213> Cuphea lanceolate
<400> 91
Met Val Ala Ala Ala Ala Thr Ser Ala Phe Phe Pro Val Pro Ala Pro
10 15
Gly Thr Ser Pro Lys Pro Gly Lys Ser Gly Asn Trp Pro Ser Ser Leu
20 25 30
Ser Pro Thr Phe Lys Pro Lys Ser Ile Pro Asn Ala Gly Phe Gin Val
35 40 45
Lys Ala Asn Ala Ser Ala His Pro Lys Ala Asn Gly Ser Ala Val Asn
50 55 60
Leu Lys Ser Gly Ser Leu Asn Thr Gin Glu Asp Thr Ser Ser Ser Pro
65 70 75 80
Pro Pro Arg Ala Phe Leu Ash Gin Leu Pro Asp Trp Ser Met Leu Leu
85 90 95
Thr Ala Ile Thr Thr Val Phe Val Ala Ala Glu Lys Gin Trp Thr Met
100 105 110
Leu Asp Arg Lys Ser Lys Arg Pro Asp Met Leu Val Asp Ser Val Gly
115 120 125
Leu Lys Ser Ile Val Arg Asp Gly Leu Val Ser Arg Gin Ser Phe Leu
130 135 140
lie Arg Ser Tyr Glu Ile Gly Ala Asp Arg Thr Ala Ser Ile Glu Thr
45 150 155 160
Leu Met Asn His Leu Gin Glu Thr Ser Ile Asn His Cys Lys Ser Leu
165 170 175
Gly Leu Leu Asn Asp Gly Phe Gly Arg Thr Pro Gly Met Cys Lys Asn
180 185 190
Asp Leu Ile Trp Val Leu Thr Lys Met Gin Ile Met Val Asn Arc Tyr
195 200 205
Pro Thr Trp Gly Asp Thr Val Glu lie Asn Thr Trp Phe Ser Gin Ser
210 215 220
Gly Lys Ile Gly Met Ala Ser Asp Trp Leu Ile Ser Asp Cys Asn Thr
225 230 235 240
Gly Glu Ile Leu Ile Arg Ala Thr Ser Val Trp Ala Met Met Asn Gin
245 250 255
Lys Thr Arg Arg Phe Ser Arg Leu Pro Tyr Glu Val Arg Gin Glu Leu
260 265 270
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Thr Pro His Phe Val Asp Ser Pro His Val Ile Glu Asp Asn Asp Gin
275 280 285
Lys Leu His Lys Phe Asp Val Lys Thr Gly Asp Ser Ile Arg Lys Gly
290 295 300
Leu Thr Pro Arg Trp Asn Asp Leu Asp vial Asn Gin His Val Ser Asn
305 310 315 320
Val Lys Tyr Ile Gly Trp Ile Leu Glu Ser Met Pro Ile Glu Val Leu
325 330 335
Glu Thr Gin Glu Leu Cys Ser Leu Thr Val Glu Tyr Arg Arg Glu Cys
340 345 350
Gly Met Asp Ser Val Leu Glu Ser Val Thr Ala Val Asp Pro Ser Glu
355 360 365
Asn Gly Gly Arg Ser Gin Tyr Lys His Leu Leu Arg Leu Glu Asp Gly
370 375 380
Thr Asp Ile Val Lys Ser Arg Thr Glu Trp Arg Pro Lys Asn Ala Gly
385 390 395 400
Thr Asn Gly Ala Ile Ser Thr Ser Thr Ala Lys Thr Ser Asn Gly Asn
405 410 415
Ser Ala Ser
<210> 92
<211> 419
<212> PRT
<213> Cuphea viscosissima
<400> 92
Met Val Ala Ala Ala Ala Thr Ser Ala Phe Phe Pro Val Pro Ala Pro
10 15
Gly Thr Ser Pro Lys Pro Gly Lys Ser Gly Asn Trp Pro Ser Ser Leu
20 25 30
Ser Pro Thr Phe Lys Pro Lys Ser Ile Pro Asn Gly Gly Phe Gin Val
35 40 45
Lys Ala Asn Ala Ser Ala His Pro Lys Ala Asn Gly Ser Ala Val Asn
50 55 60
Leu Lys Ser Gly Ser Leu Ash Thr Gin Glu Asp Thr Ser Ser Ser Pro
65 7C 75 80
Pro Pro Arg Ala Phe Leu Asn Gin Leu Pro Asp Trp Ser Met Leu Leu
85 90 95
Thr Ala Ile Thr Thr Val Phe Val Ala Ala Glu Lys Gin Trp Thr Met
100 105 110
Leu Asp Arg Lys Ser Lys Arg Pro Asp Met Leu Val Asp Ser Val Gly
115 120 125
Leu Lys Ser Ile Val Arg Asp Gly Leu Val Ser Arg His Ser Phe Ser
130 135 140
Ile Arg Ser Tyr Glu Ile Gly Ala Asp Arg Thr Ala Ser Ile Glu Thr
145 150 155 160
Leu Met Asn His Leu Gin Glu Thr Thr Ile Asn His Cys Lys Ser Leu
165 170 175
Gly Leu His Asn Asp Gly Phe Gly Arg Thr Pro Gly Met Cys Lys Asn
180 185 190
Asp Leu Ile Trp Val Leu Thr Lys Met Gin Ile Met Val Asn Arg Tyr
195 200 205
Pro Thr Trp Gly Asp Thr Val Glu Ile Asn Thr Trp Phe Ser Gin Ser
210 215 220
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317
Gly Lys lie Gly Met Ala Ser Asp Trp Leu Ile Ser Asp Cys Asn Thr
225 230 235 240
Gly Glu Ile Leu Ile Arg Ala Thr Ser Val Trp Ala Met Met Asn Gin
245 250 255
Lys Thr Arg Arg Phe Ser Arg Leu Pro Tyr Glu Val Arg Gin Glu Leu
260 265 270
Thr Pro His Phe Val Asp Ser Pro His Val Ile Glu Asp Asn Asp Gin
275 280 285
Lys Leu Arg Lys Phe Asp Val Lys Thr Gly Asp Ser Ile Arg Lys Gay
290 295 300
Leu Thr Pro Arg Trp Asn Asp Leu Asp Val Asn Gin His Val Ser Asn
305 310 315 320
Val Lys Tyr Ile Gly Trp Ile Leu Glu Ser Met Pro Ile Glu Val Lou
325 330 335
Giu Thr Gin Glu Leu Cys Ser Leu Thr Val Glu Tyr Arg Arg Glu Cys
340 345 350
Gly Met Asp Ser Val Leu Glu Ser Val Thr Ala Val Asp Pro Ser Glu
355 360 365
Asn Gly Gly Arg Ser Gin Tyr Lys His Leu Leu Arg Leu Glu Asp Gly
370 375 380
Thr Asp Ile Val Lys Ser Arg Thr Glu Trp Arg Pro Lys Asn Ala Gly
385 390 395 400
Thr Asn Gly Ala Ile Ser Thr Ser Thr Ala Lys Thr Ser Asn Gly Asn
405 410 415
Ser Val Ser
<210> 93
<211> 382
<212> PRT
<213> Umbellularia californica
<400> 93
Met Ala Thr Thr Ser Leu Ala Ser Ala Phe Cys Ser Met Lys Ala Val
1 5 10 15
Met Leu Ala Arg Asp Gly Arg Gly Met Lys Pro Arg Ser Ser Asp Leu
20 25 30
Gin Leu Arg Ala Gly Asn Ala Pro Thr Ser Leu Lys Met Ile Asn Gly
35 40 45
Thr Lys Phe Ser Tyr Thr Glu Ser Leu Lys Arg Leu Pro Asp Trp Ser
50 55 60
Met Leu Phe Ala Val Ile Thr Thr Ile Phe Her Ala Ala Glu Lys Gin
65 70 75 BO
Trp Thr Asn Leu Glu Trp Lys Pro Lys Pro Lys Leu Pro Gin Leu Leu
85 90 95
Asp Asp His Phe Gly Leu His Gly Leu Val Phe Arg Arg Thr Phe Ala
100 105 110
Ile Arg Ser Tyr Glu Val Gly Pro Asp Arg Ser Thr Ser Ile Leu Ala
115 120 125
Val Met Asn His Met Gin Glu Ala Thr Leu Asn His Ala Lys Ser Val
130 135 140
Gly Ile Leu Gly Asp Gly Phe Gly Thr Thr Leu Glu Met Ser Lys Arg
145 150 155 160
Asp Leo Met Trp Val Val Arg Arg Thr His Val Ala Val Glu Arg Tyr
165 170 175
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Pro Thr Trp Gly Asp Thr Val Glu Val Glu Cys Trp Ile Gly Ala Ser
180 185 190
Gly Asn Asn Gly Met Arg Arg Asp Phe Leu Val Arg Asp Cys Lys Thr
195 200 205
Gly Glu Ile Leu Thr Arg Cys Thr Ser Leu Ser Val Leu Met Asn Thr
210 215 220
Arg Thr Arg Arg Leu Ser Thr Ile Pro Asp Glu Val Arg Gly Glu Ile
225 230 235 240
Gly Pro Ala Phe Ile Asp Asn Val Ala Val Lys Asp Asp Glu Ile Lys
245 250 255
Lys Leo Gin Lys Leu Asn Asp Ser Thr Ala Asp Tyr Ile Gin Gly Gly
260 265 270
Leu Thr Pro Arg Trp Asn Asp Leu Asp Val Asn Gin His Val Asn Asn
275 280 285
Leu Lys Tyr Val Ala Trp Val Phe Glu Thr Val Pro Asp Ser Ile Phe
290 295 300
Glu Ser His His Ile Ser Ser Phe Thr Leu Glu Tyr Arg Ara Glu Cys
305 310 315 320
Thr Arg Asp Ser Val Leu Arg Ser Leu Thr Thr Val Ser Gly Sly Ser
325 330 335
Ser Glu Ala Gly Leu Val Cys Asp His Leu Leu Gin Leu Glu Gly Gly
340 345 250
Ser Glu Val Leu Arg Ala Arg Thr Glu Trp Arg Pro Lys Leu Thr Asp
355 360 365
Ser Phe Arg Gly Ile Ser Val Ile Pro Ala Glu Pro Arg Val
370 375 380
<210> 94
<211> 308
<212> PRT
<213> Cocos nucifera
<400> 94
Met Asp Ala Ser Gly Ala Ser Ser Phe Leu Arg Gly Arg Cys Leu Clu
1 5 10 15
Ser Cys Phe Lys Ala Ser Phe Gly Met Ser Gin Pro Lys Asp Ala Ala
20 25 30
Gly Gin Pro Ser Arg Arg Pro Ala Asp Ala Asp Asp Phe Val Asp Asp
35 40 45
Asp Arg Trp Ile Thr Val Ile Leu Ser Val Val Arg Ile Ala Ala Cys
50 55 60
Phe Leu Ser Met Met Val Thr Thr Ile Val Trp Asn Met Ile Met Leu
65 70 75 80
Ile Leu Leu Pro Trp Pro Tyr Ala Arg Ile Arg Gin Gly Asn Leu Tyr
85 90 95
Gly His Val Thr Gly Arg Met Leu Met Trp Ile Leu Gly Asn Pro Ile
100 105 110
Thr Ile Glu Gly Ser Glu Phe Ser Asn Thr Arg Ala Ile Tyr Ile Cys
115 120 125
Asn His Ala Ser Leu Val Asp Ile Phe Leu Ile Met Trp Leu Ile Pro
130 135 140
Lys Gly Thr Val Thr Ile Ala Lys Lys Glu Ile Ile Trp Tyr Pro Leu
145 150 155 160
Phe Gly Gin Leu Tyr Val Leu Ala Asn His Gin Arg Ile Asp Arg Ser
165 170 175
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Asn Pro Ser Ala Ala Ile Glu Ser Ile Lys Glu Val Ala Arc Ala Val
180 185 190
Val Lys Lys Asn Lou Ser Leu Ile Ile Phe Pro Glu Gly Thr Arg Ser
195 200 2C5
Lys Thr Gly Arg Leu Leu Pro Phe Lys Lys Gly Phe Ile His Ile Ala
210 215 220
Leu Gln Thr Arg Leu Pro Ile Val Pro Met Val Leu Thr Gly Thr His
225 230 235 240
Leu Ala Trp Arg iys Asn Ser Leu Arg Val Arg Pro Ala Pro Ile Thr
245 250 253
Val Lys Tyr Phe Ser Pro He Lys Thr Asp Asp Trp Glu Glu Glu Lys
260 265 270
Ile Asn His Tyr Vai Glu Met Ile His Ala Leu Tyr Vai Asp His Leu
. 275 280 285
Pro Glu Ser Gin Lys Pro Leu Vai Ser Lys Gly Arg Asp Ala Ser Gly
290 295 300
Arg Ser Asn Ser
305
<210> 95
<211> 356
<212> PRT
<213> Arabidopsis thaliana
<400> 95
Met Asp Val Ala Ser Ala Arg Ser Ile Ser Ser His Pro Ser Tyr Tyr
1 5 10 15
Gly Lys Pro Ile Cys Ser Per Gin Ser Ser Leu Ile Arg Ile Ser Arg
20 25 30
Asp Lys Val Cys Cys Phe Gly Arg Ile Ser Asn Gly Met Thr Ser Phe
35 40 45
Thr Thr Ser Leu His Ala Val Pro Ser Glu Lys Phe Met Gly Glu Thr
50 55 60
Arg Arg Thr Gly Ile Gin Trp Ser Asn Arg Ser Leu Arg His Asp Pro
65 70 75 80
Tyr Arg Phe Leu Asp Lys Lys Ser Pro Arg Ser Ser Gin Leu Ala Arg
85 90 95
Asp Ile Thr Val Arg Ala Asp Leu Ser Gly Ala Ala Thr Pro Asp Ser
100 105 110
Ser Phe Pro Glu Pro Glu Ile Lys Leu Ser Ser Arg Leu Arg Gly Ile
115 120 125
Phe Phe Cys Val Val Ala Gly Ile Ser Ala Thr Phe Leu Ile Val Leu
130 135 140
Met Ile Ile Gly His Pro Phe Val Leu Leu Phe Asp Pro Tyr Arg Arg
145 150 155 160
Lys Phe His His Phe Ile Ala Lys Leu Trp Ala Ser Ile Ser Ile Tyr
165 170 175
Pro Phe Tyr Lys Ile Asn Ile Glu Gly Leu Glu Asn Leu Pro Ser Ser
180 185 190
Asp Thr Pro Ala Val Tyr Val Ser Asn His Gin Ser Phe Leu Asp Ile
195 200 205
Tyr Thr Leu Leu Ser Leu Gly Lys Ser Phe Lys Phe Ile Ser Lys Thr
210 215 220
Gly Ile Phe Val Ile Pro Ile Ile Gly Trp Ala Met Ser Met Met Gly
225 230 235 240
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Val Val Pro Leu Lys Arg Met Asp Pro Arg Ser Gin Val Asp Cys Leu
245 250 255
Lys Arg Cys Met Glu Leu Leu Lys Lys Gly Ala Ser Val Phe Phe Phe
260 265 270
Pro Glu Gly Thr Arg Ser Lys Asp Gly Arg Leu Gly Ser Phe Lys Lys
275 280 285
Gly Ala Phe Thr Val Ala Ala Lys Thr Gly Val Ala Val Val Pro Ile
290 295 300
Thr Leu Met Gly Thr Gly Lys Ile Met Pro Thr Gly Ser Glu Gly Ile
305 310 315 320
Leu Asn His Gly Asn Val Ara Val Ile Ile His Lys Pro Ile His Gly
325 330 335
Ser Lys Ala Asp Val Leu Cys Asn Glu Ala Ara Ser Lys Ile Ala Glu
340 345 350
Ser Met Asp Leu
355
<210> 96
<211> 1539
<212> DNA
<213> Artificial Sequence
<220>
<223> Codon optimised sequence
<400> 96
atggctgtgt ccaagaaccc agagactctc gctccagatc aagagccatc caaagagtct 60
gatcttaggc gtaggccagc ttcctctcca tcttctactg ctgcttctcc agctgtgcca 120
gattcctcat ctaagacttc cagttccatc actggctctt ggactactgc tctcgatggt 180
gattctggtg ctgatactgt taggatcgga gatccaaagg ataggatcgg cgaggctaac 240
gatatcggcg aaaagaaaaa ggcttgctcc ggtgaggttc cagtgggatt tgttgataga 300
ccatctgcto cagtgcacgt gagagttgtt gagtctcctc tctcctccga tacaatcttc 360
cagcagtctc acgctggact cctcaatctt tgcgtggtgg tgcttatcgc tgtgaactcc 420
aggctcatta tcgagaacct tatgaagtac ggcctcctca tcggctccgg atttttcttc 480
tcatctcgtt tgctcaggga ttggcctctc cttatctgct ctottactct cccagtgttc 540
ccactcgcat cctacatggt tgagaagctc gcttacaaga agttcatctc cgagccagtg 600
gtggtgtctc ttcacgtgat cctcatcatt gctactatca tgtaccctgt 4ttcgtgatt 660
ctcaggtgcg attccccaat cctctccgga atcaacctca tgattttcgt gtoctccatc 720
tgcctcaagc tcgtttctta cgctcacgct aactacgatc tcaggtcctc ctccaactcc 780
atcgataagg gaatccacaa gtcccagggc gtgtccttca agtctctcgt gtactttatc 840
atcgctccaa cactctgcta ccagccatct tacccaagga ctacttgcat taggaagggc 900
tgggttatct gccagcttgt gaagctcgtg atcttcactg gtgtgatggg cttcatcatc 960
gagcagtaca tcgatccaat catcaagaac tcccagcacc cactcaaggg aaacgtgttg 1020
aacgctatgg aaagggtgct caagctctcc atcccaacac tttacgtgtg gctctgcgtg 1080
ttctactgca ctttccacct ctagctcaat atcctcgctg agcttctttg cttoggcgat 1140
cgtgagttct acaaggattg gtggaacact aagadtatcg aagagtactg gcgtatgtgg 1200
aacatgccag tgcacaagtg gatgcttagg cacgtttacc tcccatgcat ccgtaacggt 1260
attccaaagg gtgtggctat ggtgatctcc ttcttcatct ctgctatctt ccacgacttg 1320
tgcatcggaa tcccatgcca catcttcaag ttctgggctt tcatcagcat catgttccag 1380
gtgccactcg ttatcctcac taagtacctc cagaacaagt tcaagtccgc tatggtaggc 1440
aacatgattt tctggttctt tttctcaatc tacggccagc caatgtgcgt gctcctttac 1500
taccacgatg tgatgaatag gaaggtgggc actgagtaa 1539
CA 2998211 2018-03-16
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<210> 97
<211> 371
<212> PRT
<213> Cocos nucifera
<400> 97
Met Val Glu Leu Arg Ser Ser Ser Ser Glu Met Asp Leu Asp Arg Pro
1 5 10 15
Asn Ile Glu Glu Tyr Leu Thr Thr Asp Ser Ile Gin Glu Ser Pro Lys
20 25 30
Lys Leu His Leu Arg Asp Leu Leu Asp Ile Ser Pro Thr Leu Thr Glu
35 40 45
Ala Thr Gly Ala Ile Val Asp Asp Ser Phe Thr Arg Cys Phe Lys Ser
50 SS 60
Asn Pro Pro Glu Pro Trp Ass Trp Asn Val Tyr Leu Phe Pro Leu Trp
65 70 75 80
Cys Leu Gly Val Ile Ile Arg Tyr Gly Ile Leu Phe Pro Leu Arg Val
85 90 95
Ala Ile Leu Thr Ala Gly Trp Leu Val Phe Phe Ala Ala Phe Ile Pro
100 105 110
Val His Phe Leu Leu Thr Ala His Asn Lys Trp Arg Arg Lys Ile Glu
115 120 125
Arg Lys Leu Val Glu Met Ile Cys Ser Val Phe Val Ala Ser Trp Thr
130 135 140
Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser Met Arg Pro Gln Gin
145 150 155 160
Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe Ile Ile Leu Glu
165 170 175
Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys His Pro Gly Trp Val
180 185 190
Gly Phe Ile Gin Lys Thr Ile Leu Glu Gly Val Gly Cys Ile Trp Phe
195 200 205
Asn Arg Thr Glu Ser Lys Asp Arg Glu Val Val Ala Arg Lys Leu Arg
210 215 220
Glu His Ile His Gly Ala Asp Asn Asn Pro Leu Leu Ile Phe Pro Glu
225 230 235 240
Gly Thr Cys Val Asn Asn His Tyr Thr Val Met Phe Lys Lys Gly Ala
245 250 255
Phe Glu Leu Gly Cys Ala Val Cys Pro Val Ala Ile Lys Tyr Asn Lys
260 265 270
Ile Phe Val Asp Ala Phe Trp Asn Ser Lys Lys Gin Ser Phe Thr Met
275 280 285
His Leu Phe His Leu Met Thr Ser Trp Ala Val Val Cys Asp Val Trp
290 295 300
Tyr Leu Glu Pro Gin Tyr Ile Arg Pro Gly Glu Thr Pro Ile Glu Phe
305 310 315 320
Ala Glu Arg Val Arg Asp Met Ile Ser Val Arg Ala Gly Leu Lys Lys
325 330 335
Val Pro Trp Asp Gly Tyr Leu Lys Tyr Phe Arg Pro Ser Pro Lys Leu
340 345 350
Thr Glu Arg Lys Gin Gin Ile Phe Ala Glu Ser Val Leu Gin Arg Leu
355 360 365
Glu Glu Lys
370
CA 2998211 2018-03-16
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<210> 98
<211> 376
<212> PRT
<213> Arabidopsis thaliana
<400> 98
Met Ser Ser Thr Ala Gly Arg Leu Val Thr Ser Lys Ser Glu Leu Asp
1 5 10 15
Len Asp His Pro Asn Ile Giu Asp Tyr Leu Pro Ser Gly Ser Ser Ile
20 25 30
Asn Glu Pro Arg Gly Lys Leu Ser Leu Arg Asp Len Leu Asp Ile Ser
35 40 45
Pro Thr Leu Thr Glu Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr
50 55 60
Arg Cys Phe Lys Ser Asn Pro Pro Glu Pro Trp Asn Trp Asn Ile Tyr
65 70 75 80
Len Phe Pro Leu Tyr Cys Phe Gly Val Val Val Arg Tyr Cys Ile Leu
85 90 95
Phe Pro Leu Arg Cys Phe Thr Leu Ala Phe Gly Trp Ile Ile Phe 1,eu
100 105 110
Ser Leu Phe Ile Pro Val Asn Ala Leu Leu Lys Gly Gin Asp Arg Leu
115 120 125
Arg Lys Lys Ile Glu Arg Val Leu Val Glu Met Ile Cys Ser Phe Phe
130 135 140
Val Ala Ser Trp Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser
145 150 155 160
Ile Arg Pro Lys Gin Val Tyr Val Ala Asn His Thr Ser Met Ile Asp
165 170 175
Phe Ile Val Leu Glu Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys
180 les 190
His Pro Gly Trp Val Gly Leu Leu Gin Ser Thr Ile Leu Glu Ser Val
195 200 205
Gly Cys Ile Trp Phe Asn Arg Ser Glu Ala Lys Asp Arg Glu Ile Val
210 215 220
Ala Lys Lys Len Arg Asp His Val Gin Gly Ala Asp Ser Asn Pro Leu
225 230 235 240
Leu Ile Phe Pro Glu Gly Thr Cys Val Asn Asn Asn Tyr Thr Val Met
245 250 255
Phe Lys Lys Gly Ala Phe Glu Leu Asp Cys Thr Val Cys Pro Ile Ala
260 265 270
Ile Lys Tyr Asn Lys Ile Phe Val Asp Ala Phe Trp Asn Ser Arg Lys
275 280 285
Gin Ser Phe Thr Met His Leu Leu Gin Leu Met Thr Ser Trp Ala Val
290 295 300
Val Cys Glu Val Trp Tyr Leu Glu Pro Gin Thr Ile Arg Pro Gly Glu
305 310 315 320
Thr Gly Ile Glu Phe Ala Glu Arg Val Arg Asp Met Ile Ser Leu Arg
325 330 335
Ala Gly Leu Lys Lys Val Pro Trp Asp Gly Tyr Leu Lys Tyr Ser Arg
340 345 350
Pro Ser Pro Lys His Ser Glu Arg Lys Gin Gin Ser Phe Ala Glu Ser
355 360 365
Ile Len Ala Arg Len Glu Glu Lys
370 375
CA 2998211 2018-03-16
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<210> 99
<211> 371
<212> PRT
<213> Eiaeis guineensis
<400> 99
Met Val Glu Leu Arg Ser Ser Ser Ser Slu Met Asp Leu Asp Arg Pro
1 5 10 15
Asn Ile Glu Glu Tyr Leu Pro Pro Thr Pro Ser Lys Asn Pro Pro Lys
20 25 30
Lys Leu His Leu Arg Asp Leu Leu Asp Ile Ser Pro Thr Leu Thr Glu
35 40 45
Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr Arg Cys Phe Lys Ser
50 55 60
Ash Pro Pro Glu Pro Trp Asn Trp Asn Val Tyr Leu Phe Pro Leu Trp
65 70 75 80
Cys Leu Gly Val Ile Ile Arg Tyr Gly Ile Leu Phe Pro Leu Arg Val
85 90 95
Ala Ile Leu Thr Ala Gly Trp Leu Val Phe Phe Ala Ala Phe Ile Pro
100 105 110
Val His Phe Leu Leu Thr Ala His Asn Lys Trp Arg Arg Lys Ile Glu
115 120 125
Arg Lys Leu Val Glu Met Ile Cys Ser Val Phe Val Ala Ser Trp Thr
130 135 140
Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser Met Arg Pro Gin Gln
145 150 155 160
Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe Ile Ile Leu Glu
165 170 175
Gln Met Thr Ala Phe Ala Val Ile Met Gln Lys His Pro Gly Trp Val
180 185 190
Gly Phe Ile Gln Lys Thr lie Leu Glu Gly Val Gly Cys Ile Trp Phe
195 200 205
Asn Arg Thr Glu Ser Lys Asp Arg Glu Val Val Ala Arg Lys Leu Arg
210 215 220
Glu His Ile His Gly Ala Asp Asn Asn Pro Leu Leu Ile Phe Pro Glu
22E 230 235 240
Gly Thr Cys Val Asn Asn His Tyr Thr Val Met Phe Lys Lys Gly Ala
245 250 255
Phe Glu Leu Gly Cys Ala Val Cys Pro Val Ala Ile Lys Tyr Asn Lys
260 265 270
Ile Phe Val Asp Ala Phe Trp Asn Ser Lys Lys Gln Ser Phe Thr Met
275 280 285
His Leu Phe His Leu Met Thr Ser Trp Ala Val Val Cys Asp Val Trp
290 295 300
Tyr Leu Glu Pro Gln Tyr Ile Arg Pro Gly Glu Thr Pro Ile Glu Phe
305 310 315 320
Ala Glu Arg Val Arg Asp Met Ile Ser Tie Arg Ala Gly Leu Lys Lys
325 330 335
Val Pro Trp Asp Gly Tyr Leu Lys Tyr Phe Arg Pro Ser Pro Lys Leu
340 345 350
Thr Glu Arg Lys Gln Gln Ile Phe Ala Glu Ser Val Leu Gln Arg Leu
355 360 365
Glu Glu Lys
370
CA 2998211 2018-03-16
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<210> 100
<211> 371
<212> PRT
<213> Phoenix dactylifera
<400> 100
Met Val Gly Leu Arg Ser Ser Ser Ser alu Met Asp Leu Asp Arg Pro
1 5 10 15
Asn Ile Glu Glu Tyr Leu Thr Thr Asp Ser Ile Glu Glu Ser Pro Lys
20 25 30
Lys Leu His Leu Arg Asp Leu Leu Asp Tie Ser Pro Thr Leu Thr Glu
35 40 45
Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr Arg Cys Phe Lys Ser
50 55 60
Asn Pro Pro Glu Pro Trp Asn Trp Asn Val Tyr Leu Phe Pro Leu Trp
65 70 75 80
Cys Leu Gly Val Ile Ile Arg Tyr Gly Ile Leu Phe Pro Leu Arg Val
85 90 95
Ala Val Leu Thr Ala Gly Trp Leu Val Phe Phe Ala Ala Phe Ile Pro
10C 105 110
Ala His Phe Leu Leu Thr Ala His Asn Lys Trp Arg Arg Lys Ile Glu
115 120 125
Arg Lys Leu Val Glu Met Ile Cys Ser Val Phe Val Ala Ser Trp Thr
130 135 140
Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser Met Arg Pro Gin Gin
145 150 155 160
Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe Ile Ile Leu Glu
165 170 175
Gin Meo Thr Ala Phe Ala Val Ile Met Gin Lys His Pro Gly Trp Val
180 185 190
Gly Phe Ile Gin Lys Thr Ile Leu Glu Gly Val Gly Cys Ile Trp Phe
195 200 205
Asn Arg Thr Glu Ser Lys Asp Arg Glu Val Val Ala Arg Lys Leu Arg
210 215 220
Glu His Ile Gin Gly Ala Asp Asn Asn Pro Leu Leu Ile Phe Pro Glu
225 230 235 240
Gly Thr Cys Val Asn Asn His Tyr Thr Val Met Phe Lys Lys Gly Ala
245 250 255
Phe Glu Lau Gly Cys Ala Val Cys Pro Val Ala Ile Lys Tyr Asn Lys
260 265 270
Ile Phe Val Asp Ala Phe Trp An Ser Lys Lys Gin Ser Phe Thr Met
275 280 285
His Leu Phe His Leu Met Thr Ser Trp Ala Val Val Cys Asp Val Trp
290 295 300
Tyr Leu Glu Pro Gin Tyr Ile Arg Pro Gly Glu Thr Pro Ile Glu Phe
305 310 315 320
Ala Glu Arg Val Arg Asp Met Ile Ser Val Arg Ala Gly Leu Arg Lys
325 330 335
Val Pro Trp Asp Gly Tyr Leu Lys Tyr Phe Arg Pro Ser Pro Lys Leu
340 345 350
Thr Glu Arg Lys Gin Gin Ile Phe Ala Glu Ser Val Leu Gin Arg Leu
355 360 365
Glu Glu Lys
370
CA 2998211 2018-03-16
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<210> 101
<211> 371
<212> PRT
<213> Musa acuminata
<400> 101
Met Ala Gly Leu Ala Thr Sec Her Thr Glu Met Asp Leu Asp Arg Pro
1 5 10 15
Asn Ile Asp Glu Tyr Leu Thr Val Glu Ser Ile Arg Glu Ala Pro Lys
20 25 30
Lys Leu His Leu Arg Asp Leu Leu Asp Ile Ser Pro Thr Leu Lys Glu
35 40 45
Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr Arg Cys Phe Lys Ser
50 55 60
Asn Pro Ser Glu Pro Trp Asn Trp Asn Ile Tyr Leu Phe Pro Leu Trp
65 70 75 80
Cys Leu Gly Val Val Ile Arg Tyr Gly Ile Leu Phe Pro Phe Arg Vol
85 90 95
Ile Ile Leu Val Ala Gly Trp Ile Val Phe Phe Ala Ala Phe Ser Leu
100 105 110
Val His Phe Leu Leu Gly Glu His Asn Lys Trp Lys Arg Glu Ile Glu
115 120 125
Arg Lys Leu Val Glu Met Ile Cys Ser Val Phe Val Ala Ser Trp Thr
130 135 140
Ala Val Ile Lys Tyr His Gly Pro Arg Pro Ser Met Arg Pro Gin Gin
145 150 155 160
Val Phe Val Ala Asn His Thr Her Met Ile Asp Phe Ile Ile Leu Glu
165 170 175
Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys His Pro Gly Trp Vol
180 185 190
Gly Phe Ile Gin Lys Ile Ile Val Glu Ser Leu Gly Cys Ile Trp Phe
195 200 205
Asn Arg Thr Glu Ala Lys Asp Arg Glu Ile Val Ala Arg Lys Leu Arg
210 215 220
Glu His Ile Gin Gly Val Asp Asn Asn Pro Leu Leu Ile Phe Pro Glu
225 230 235 240
Gly Thr Cys Val Asn Asn His Tyr Thr Val Met Phe Lys Lys Gly Ala
245 250 255
Phe Glu Leu Gly Cys Ala Val Cys Pro Val Ala Ile Lys Tyr Asn Lys
260 265 270
Ile Phe Val Asp Ala Phe Trp Asn Ser Lys Lys Gin Ser Phe Thr Met
275 280 285
His Leu Val Gin Leu Met Thr Ser Trp Ala Vol Val Cys Asp Val Trp
290 295 300
Tyr Leu Glu Pro Gln Tyr Ile Arg Pro Gly Glu Thr Pro Ile Glu Phe
305 310 315 320
Ala Glu Arg Val Gin Asp Met Ile Ser Val Arg Ala Gly Leu Lys Lys
325 330 335
Val Pro Trp Asp Gly Tyr Leu Lys Tyr Phe Arg Pro Ser Pro Lys Leu
340 345 350
Ile Glu Arg Lys Gin Gin lie Phe Ala Glu Ser Val Leu Gin Arg Leu
355 360 365
Glu Glu Lys
370
CA 2998211 2018-03-16
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<210> 102
<211> 372
<212> PRT
<213> Ananas comosus
<400> 102
Met Ala Glu Ala Leu Gly Ser Ser Ser Ala Glu Met Asp Leu Asp Arg
1 5 10 15
Pro Asn Leu Glu Glu Tyr Leu Pro Thr Asp Ser Ile Gln Asp Ser Pro
20 25 30
Lys Asn Leu His Leu Arg Asp Leu Leu Asp Ile Ser Pro Thr Leu Thr
35 40 45
Glu Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr Arg Cys Phe Lys
50 55 60
Ser Asn Pro Pro Glu Pro Trp Asn Trp Asn Ile Tyr Leu Phe Pro Leu
65 70 75 80
Trp Cys Leu Gly Val Val Val Arg Tyr Gay Ile Leu Phe Pro Leu Arg
85 90 95
Val Ala Val Leu Ala Ile Gly Trp Ile Val Phe Phe Ser Ala Phe Phe
100 105 110
Pro Val His Phe Leu Leu Lys Gly Tyr Pro Lys Trp Arg Arg Lys Leu
115 120 125
Glu Arg Lys Leu Val Glu Met Met Cys Ser Val Phe Val Ala Ser Trp
130 135 140
Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser Thr Arg Pro His
145 150 155 160
Gln Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe Ile Ile Leu
165 170 175
Glu Gln Met Thr Ala Phe Ala Val Ile Met Gln Lys His Pro Gly Trp
180 185 190
Val Gly Phe Ile Gln Lys Thr Ile Leu Glu Ser Val Gly Cys Ile Trp
195 200 205
Phe Asn Arg Thr Glu Ser Lys Asp Arg Gly Val Val Gly Arg Lys Leu
210 215 220
Arg Glu His Val Gln Gly Val Asp Asn Asn Pro Leu Leu Ile Phe Pro
225 230 235 240
Glu Gly Thr Cys Val Asn Asn His Tyr Thr Val Met Phe Lys Lys Gly
245 250 255
Ala Phe Glu Leu Gly Cys Ala Val Cys Pro Ile Ala Ile Lys Tyr Asn
260 265 270
Lys Ile Phe Val Asp Ala Phe Trp Asn Ser Lys Lys Gln Ser Phe Thr
275 280 285
Met His Leu Val Arg Leu Met Thr Ser Trp Ala Val Val Cys Asp Val
290 295 300
Trp Tyr Leu Glu Pro Gln Tyr Leu Arg Pro Gly Glu Thr Pro Ile Glu
305 310 315 320
Phe Ala Glu Arg Val Arg Asp Met Ile Ser Ala Arg Ala Gly Leu Lys
325 330 335
Lys Val Pro Trp Asp Gly Tyr Leu Lys Tyr Phe Arg Pro Ser Pro Lys
340 345 350
His Thr Glu Arg Lys Gln Gin Ile Phe Ala Glu Ser Ile Leu Arg Arg
355 360 365
Leu Glu Arg Lys
370
CA 2998211 2018-03-16
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<210> 103
<211> 370
<212> PRT
<213> Asparagus officinalis
<400> 103
Met Ala Gly Leu Glu Ser Ser Ser Ala Gly Ile Asp Val Asp Pro Pro
10 15
Asn Ile Glu Asp Tyr Leu Thr Ser Asp Ala Leu His Gln Pro His Lys
20 25 30
Lys Leu Gln Leu Lys Asp Leu Leu Asp Ile Ser Pro Thr Leu Thr Glu
35 40 45
Ala Ala Gly Ala Ile Vol Asp Asp Ser Phe Thr Arg Cys Phe Lys Her
50 55 60
Asn Pro Pro Glu Pro Trp Asn Trp Asn Val Tyr Leu Phe Pro Leu Trp
65 70 75 80
Cys Leu Gly Val Val Vol Arg Tyr Gly Ile Leu Phe Pro Leu Arg Val
85 90 95
Met Thr Leu Ala Ala Gly Trp Ile Vol Phe Phe Ser Ala Phe Leu Pro
100 105 110
Val His Tyr Leu Met Lys Gly Gln Asn Lys Trp Lys Ash Asn Ile Glu
115 120 125
Arg Lys Leu Val Glu Met Ile Cys Ser Val Phe Vol Ala Ser Trp Thr
130 135 140
Gly Val Val Arg Tyr His Gly Pro Arg Pro Ser Met Arg Pro Gln Gln
145 150 155 160
Vol Phe Val Ala Asn His Thr Ser Met Ile Asp Phe Ile Ile Leu Glu
165 170 175
Gln Met Ala Ala Phe Ala Val Ile Met Gln Lys His Pro Gly Trp Val
180 185 190
Gly Phe Leu Gln Thr Thr Ile Leu Glu Ser Ile Gly Ser Ile Trp Phe
195 200 205
Asn Arg Thr Glu Ala Lys Asp Arg Glu Val Vol Ala Arg Lys Leu Arg
210 215 220
Glu His Thr Glu Gly Asp Asn Asn Pro Leu Leu Ile Phe Pro Glu Gly
225 230 235 240
Thr Cys Val Asn Asn Asp Tyr Thr Val Met Phe Lys Lys Gly Ala Phe
245 250 255
Glu Leu Gly Cys Ala Vol Cys Pro Val Ala Ile Lys Tyr Asn Lys Ile
260 265 270
Phe Val Asp Ala Phe Trp Asn Ser Lys Lys Gln Ser Phe Thr Met His
275 280 285
Leu Met Arg Leu Met Thr Her Trp Ala Val Vol Cys Asp Vol Trp Tyr
290 295 300
Leu Glu Pro Gln Tyr Leu Lys Pro Gly Glu Thr Ser Ile Glu Phe Ala
305 310 315 320
Glu Arg Val Arg Asp Met Ile Ser Vol Arg Ala Gly Leu Arg Lys Val
325 330 335
Pro Trp Asp Gly Tyr Leu Lys Tyr Phe Arg Pro Ser Pro Lys Leu Thr
340 345 350
Glu Arg Lys Gln Gln Ile Phe Ala Glu Ser Val Leu Arg Arg Leu Glu
355 360 365
Glu Lys
370
CA 2998211 2018-03-16
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<210> 104
<211> 370
<212> PRT
<213> Oryza brachyaneha
<400> 104
Met Ala Ser Ser Ser Val Ala Gly Asp Ile Glu Leu Asp Arg Pro Asn
1 5 10 15
Leu Glu Asp Tyr leu Pro Pro Asp Ser Leu Pro Gln Glu Ser Pro Gly
20 25 30
Aso Leu His Leu Arg Asp Leu -ieu Asp Ile Ser Pro Val Leu Thr Glu
35 4C 45
Ala Ala Gly Ala Ala Val Asp Asp Ser Phe Thr Rig Cys Phe Lys Ser
50 55 60
Asn Ser Pro Glu Pro Trp Asn Trp Asn lie Tyr Leu Phe Pro Leu Trp
65 70 75 80
Cys Leu Gly Val Val Ile Arg Tyr Gly Ile Leu Phe Pro Leu Arg Gly
85 90 95
Leu Thr Leu Leu Val Gly Trp Ile Ala Phe Phe Ala Ala Phe Phe Ser
100 105 110
Val His Phe Leu Phe Lys Gly Gin Lys Met Arg Ser Lys Ile Giu Arg
115 120 125
Lys Leu Val Glu Met Met Cys Ser Val Phe Val Ala Ser Trp Thr Gly
130 135 140
Val Ile Lys Tyr His Gly Pro Arg Pro Ser Thr Arg Pro His Gin Val
145 150 155 160
Phe Val Ala Asn His Thr Ser Met Ile Asp Phe Ile Ile Leu Glu Gin
165 170 175
Met Thr Ala Phe Ala Val Ile Met Gin Lys His Pro Gly Trp Val Gly
180 195 190
Phe Ile Gin Lys Thr Ile Leu Glu Ser Val Gly Cys Ile Trp Phe Asn
195 200 205
Arg Asn Asp Leu Lys Asp Arg Glu Val Val Ala Lys Lys Leu Arg Asp
210 215 220
His Val Gin His Pro Asp Asn Asn Pro Leu Leu Ile Phe Pro Glu Gly
225 230 235 240
Thr Cys Val Asn Asn Gin Tyr Thr Val Met Phe Lys Lys Gly Ala Phe
245 250 255
Glu Leu Gly Cys Ala Val Cys Pro Ile Ala Ile Lys Tyr Asn Lys Ile
260 265 270
Phe Val Asp Ala Phe Trp Asn Ser Lys Lys Gin Ser Phe Thr Met His
275 280 285
Leu Val Arg Leu Met Thr Ser Trp Ala Val Val Cys Asp Val Trp Tyr
290 295 300
Leta Glu Pro Gin Tyr Leu Lys Glu Gly Glu Thr Ala Ile Gin Phe Ala
305 310 315 320
Glu Arg Val Arg Asp Met Ile Ala Ala Arg Ala Gly Leu Lys Lys Val
325 330 335
Pro Trp Asp Gly Tyr Leu Lys His Asn Arg Pro Ser Pro Lys His Thr
340 345 350
Glu Glu Lys Gin Arg Ile Phe Ala Asp Ser Val Leu Gin Arg Leu Glu
355 360 365
Glu Ser
370
CA 2998211 2018-03-16
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<210> 105
<211> 370
<212> PRT
<213> Oryza sativa
<400> 105
Met Ala Thr Ser Ser Val Ala Gly Asp Ile Glu Leu Asp Arg Pro Asn
1 5 10 15
Leu Glu Asp Tyr Leu Pro Ser Asp Ser Len Pro Gin Glu Phe Pro Arg
20 25 30
Asn Leu His Leu Arg Asp Leu Leu Asp Iie Ser Pro Vol Leu Thr Glu
35 40 45
Ala Ala Gly Ala Ile Vol Asp Asp Ser Phe Thr Arg Cys Phe Lys Ser
50 55 60
Asn Ser Pro Glu Pro Trp Asn Trp Asn Tie Tyr Leu Phe Pro Leu Trp
65 70 75 80
Cys Leu Gly Val Val Ile Arg Tyr Gly Iie Leu Phe Pro Leu Arg Gly
85 90 95
Leu Thr Leu Leu Val Gly Trp Leu Ala Phe Phe Ala Ala Phe Phe Pro
100 105 110
Val His Phe Leu Leu Lys Gly Gin Lys Met Arg Ser Lys Ile Glu Arg
115 120 125
Lys Leu Val Glu Met Met Cys Ser Vol Phe Val Ala Ser Trp Thr Gly
130 135 140
Val Ile Lys Tyr His Gly Pro Arg Pro Ser Thr Arg Pro His Gin Val
145 150 355 160
Phe Val Ala Asn His Thr Ser Met :le Asp Phe Ile Ile Leu Glu Gin
165 170 175
Met Thr Ala Phe Ala Val Ile Met Gin Lys His Pro Gly Trp Val Gly
180 185 190
Phe Ile Gin Lys Thr Ile Leu Glu Ser Val Gly Cys Ile Trp Phe Asn
195 200 205
Arg Asn Asp Leu Lys Asp Arg Glu Val Val Ala Lys Lys Leu Arg Asp
210 215 220
His Val Gin His Pro Asp Ser Asn Pro Leu Leu Ile Phe Pro Glu Gly
225 230 235 240
Thr Cys Val Asn Asn Gin Tyr Thr Val Met Phe Lys Lys Gly Ala Phe
245 250 255
Glu Leu Gly Cys Ala Val Cys Pro Ile Ala Ile Lys Tyr Asn Lys Ile
260 265 270
Phe Val Asp Ala Phe Trp Asn Ser Lys Lys Gin Ser Phe Thr Met His
275 280 285
Leu Val Arg Leu Met Thr Ser Trp Ala Vol Val Cys Asp Vol Trp Tyr
290 295 300
Leu Glu Pro Gin Tyr Leu Arg Asp Gly Gin Thr Ala Ile Glu Phe Ala
305 310 315 320
Glu Arg Vol Arg Asp Met Ile Ala Ala Arg Ala Gly Leu Lys Lys Val
325 330 335
Pro Trp Asp Gly Tyr Leu Lys His Asn Arg Pro Ser Pro Lys His Thr
340 345 350
Glu Glu Lys Gin Arg Ile Phe Ala Asp Ser Vol Leu Arg Arg Leu Glu
355 360 365
Glu Ser
370
CA 2998211 2018-03-16
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<210> 106
<211> 363
<212> PRT
<213> Nelumbo nucifera
<400> 106
Met Asp Leu Asp Arg Pro Asn Ile Glu Glu Tyr Leu Pro Ser Glu Ala
1 5 10 15
Ile Gin Glu Ser Asn Glu Lys Leu His Leu Arg Asp Leu Leu Asp Ile
20 25 30
Ser Pro Thr Leu Thr Glu Ala Ala Gly Ala Ile Val Asp Asp Ser Phe
35 40 45
=
Thr Arg Cys Phe Lys Ser Asn Pro Ser Glu Pro Trp Asn Trp Asn Val
50 55 60
Tyr Leu Phe Pro Leu Trp Cys Phe Gly Val Val Val Arg Tyr Gly Ile
65 70 75, 80
Leu Phe Pro Val Arg Val Leu Val Leu Thr lie Gly Trp Ile Ile Phe
85 90 95
Leu Ser Ser Phe Ile Pro Ala His Phe Leu Leu Arg Ser His Asp Lys
100 105 110
Trp Arg Lys Lys Ile Glu,Arg Tyr Leu Val Glu Leu Ile Cys Ser Phe
115 120 125
Phe Val Ala Ser Trp Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro
130 135 140
Ser Met Arg Pro Lys Gin Val Phe Val Ala Asn His Thr Ser Met Ile
145 150 155 160
Asp Phe Ile Val Leu Glu Gin Met Thr Ala Phe Ala Val Ile Met Gin
165 170 175
Lys His Pro Gly Trp Val Gly Leu Leu Gin Ser Thr Ile Leu Glu Ser
180 185 190
Val Gly Cys Ile Trp Phe Asn Arg Ala Glu Ala Lys Asp Arg Giu Ile
195 200 205
Val Ala Arg Lys Leu Arg Asp His Ile Gin Gly Val Asp Asn Asn Pro
210 215 220
Leu Leu Ile Phe Pro Glu Gly Thr Cys Val Asn Asn His Tyr Thr Val
225 230 235 240
Met Phe Lys Lys Gly Ala Phe Glu Leu Gly Cys Thr Val Cys Pro Ile
245 250 255
Ala Ile Lys Tyr Asn Lys lie Phe Val Asp Ala Phe Trp Asn Ser Lys
260 265 270
Lys Gin Ser Phe Thr Met His Leu Leu His Leu Met Thr Ser Trp Ala
275 280 285
Val Val Cys Asp Val Trp Tyr Leu Gin Pro Gin Asn Ile Arg Pro Gly
290 295 300
Glu Thr Pro Ile Glu Phe Ala Glu Arg Val Arg Asp Ile Ile Ser Val
305 310 315 320
Arg Ala Gly Leu Lys Lys Val Pro Trp Asp Gly Tyr Leu Lys Tyr Ser
325 330 335
Arg Pro Ser Pro Lys His Arg Glu Arg Lys Gin Gin Arg Phe Val Glu
340 345 350
Ser Val Lou Gin Arg Leu Glu Lys Lys Giy Lys
355 360
<210> 107
<211> 376
CA 2998211 2018-03-16
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<212> PRT
<213> Vitis vinifera
<400> 107
Met Ala Asn Ala Pro Asp Asn Lys Leu Thr Ser Ser Ser Ser Glu Leu
1 5 10 15
Asp Leu Asp Arg Pro Asn Leu Glu Asp Tyr Leu Pro Ser Gly Ser Met
20 25 30
Gin Glu Pro Arg Sly Lys Leu Arg Leu Arg Asp Leu Leu Asp Ile Ser
3" 40 45
Pro Thr Leu Thr Glu Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr
50 55 60
Arg Cys Phe Lys Ser Asn Pro Pro Glu Pro Trp Ash Trp Asn Val Tyr
65 70 75 80
Leu Phe Pro Leu Trp Cys Leu Gly Val Val Ile Arg Tyr Gly Ile Leu
85 90 95
Phe Pro Thr Arg Val Leu Val Leu Thr Leu Gly Trp Ile Ile Phe Leu
100 105 110
Ser Ser Phe lie Pro Val His Phe Leu Leu Lys Gly Asn Asp Lys Leu
115 120 125
Arg Lys Lys Leu Glu Arg Cys Leu Val Glu 1,eu Ile Cys Ser Phe Phe
130 135 140
Val Ala Ser Trp Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser
145 150 155 160
Arg Arg Pro Gin Sin Val Phe Val Ala Asn His Thr Ser Met Ile Asp
165 170 175
Phe Ile Val Leu Glu Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys
180 185 190
His Pro Gly Trp Val Gly Leu Leu Gin Ser Thr Ile Leu Glu Ser Val
195 200 205
Gly Cys Ile Trp Phe Asn Arg Thr Glu Ala Lys Asp Arg Glu Ile Val
210 215 220
Ala Arg Lys Leu Arg Asp His Val Gin Gly Ala Asp Asn Asn Pro Leu
225 230 235 240
Leu Ile Phe Pro Glu Gly Thr Cys Val Asn Asn His Tyr Thr Val Met
245 250 255
Phe Lys Lys Gly Ala Phe Glu Leu Gly Cys Thr Val Cys Pro Ile Ala
260 265 270
Ile Lys Tyr Asn Lys Ile Phe Val Asp Ala Phe Trp Asn Ser Lys Lys
275 280 285
Gin Ser Phe Thr Met His Leu Leu Gin Leu Met Thr Ser Trp Ala Val
290 295 300
Val Cys Asp Val Trp Tyr Leu Glu Pro Gin Thr Leu Lys Pro Gly Glu
305 310 315 320
Thr Pro Ile Glu Phe Ala Glu Arg Val Arg Asp Ile Ile Ser Leu Arg
325 330 335
Ala Gly Leu Lys Lys Val Pro Trp Asp Gly Tyr Leu Lys Tyr Ser Arg
340 345 350
Pro Ser Pro Lys His Arg Glu Gin Lys Gin Gin Ser Phe Ala Asp Ser
355 360 365
Val Leu Arg Arg Leu Glu Glu Lys
370 375
<210> 108
<211> 373
CA 2998211 2018-03-16
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<212> PRT
<213> Nicotiana tomentosiformis
<400> 108
Met Asn Met Asn Lys Leu Lys Thr Ser Ser Ser Glu Leu Asp Leu Asp
1 5 10 15
Arg Pro Asn Leu Glu Asp Tyr Leu Pro Thr Gly Ser Ile Pro Glu Pro
20 25 30
His Gly Lys Leu Arg Leu Arg Asp Leu Ile Asp Ile Ser Pro Thr Leu
35 40 45
Thr Glu Ala Ala Gly Ala Ile VaL Asp Asp Ser Phe Thr Arg Cys Phe
50 55 60
Lys Ser Asn Pro Pro Glu Pro Trp Asn Trp Asn Ile Tyr Leu Phe Pro
65 70 75 80
Leu Trp Cys Leu Gly Val Val Val Arg Tyr Gly Ile Leu Phe Pro Ile
85 90 95
Are Val :le Val Leu Thr Ile Gly Trp Ile :le Phe Leu Ser Cys Tyr
100 105 110
Ile Pro Val His Phe Leu Leu Lys Gly His Asp Lys Phe Arg Lys Lys
115 120 125
Leu Glu Arg Cys Leu Val Glu Leu Ile Cys Ser Phe Phe Val Ala Ser
130 135 140
Trp Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser Ile Arg Pro
145 150 155 160
Lys Gin Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe Ile Val
165 170 175
Leu Glu Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys His Pro Gly
180 185 190
Trp Val Gly Leu Leu Gin Ser Thr Ile Lou Glu Gly Val Gly Cys Ile
195 200 205
Trp Phe Asn Arg Ser Glu Ala Lys Asp Arg Glu Ile Val Ala Arg Lys
210 215 220
Leu Arg Gin His Vol Glu Gly Ala Asp Asn Asn Pro Leu Leu Ile Phe
225 230 235 240
Pro Glu Gly Thr Cys Val Asn Asn His Tyr Thr Val Met Phe Lys Lys
245 250 255
Gly Ala Phe Glu Leu Gly Cys Thr Val Cys Pro Val Ala Ile Lys Tyr
26C 265 270
Asn Lys Ile Phe Val Asp Ala She Trp Asn Ser Arg Lys Gin Ser Phe
275 280 285
Thr Met His Leu Leu Gin Leu Met Thr Ser Trp Ala Val Vol Cys Asp
290 295 300
Val Trp Tyr Leu Glu Pro Gin Asn Ile Arg Pro Gly Glu Thr Pro Ile
305 310 315 320
Glu She Ala Glu Arg Val Arg Asp Ile Ile Ser Ala Arg Ala Gly Leu
325 330 335
Lys Lys Val Pro Trp Asp Gly Tyr Leu Lys Tyr Ser Arg Pro Ser Pro
340 345 350
Lys His Arg Glu Arg Lys Gin Gin Ser She Ala Glu Ser Val Leu Arg
355 360 365
Arg Leu Glu Glu Lys
370
<210> 109
<211> 375
CA 2998211 2018-03-16
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=
<212> SRI
<213> Jatropha curcas
<400> 109
Met Ala Thr Pro Gly Lys Leu Lys Thr Ser Ser Ser Glu Leu Asp Leu
1 5 10 15
Asp Arg Pro Asn Ile Glu Asp Tyr Leu Pro Ser Gly Val Ser Ile din
20 25 30
Glu Pro Arg Gly Lys Leu Arg Leo Arg Asp Leu Leu Asp Ile Ser Pro
35 40 45
Thr Leu Thr Glu Ala Ala Gly Ala Ile Val Asp Asp Thr She Thr Arg
50 55 60
Cys Phe Lys Ser Asn Pro Pro Glu Pro Trp Asn Trp Asn Ile Tyr Leu
65 70 75 80
Phe Pro Leu Trp Cys Cys Gly Val Val Cys Arg Tyr Gly Ile Leu Phe
85 90 95
Pro Ile Arg Val Leu Val Leu Thr Ile Gly Trp Ile Ile Phe Leu Ser
100 105 110
Cys Tyr Ile Pro Val His Phe Leu Leu Lys Gly His Asp Lys Leu Arg
115 120 125
Lys Lys Leu Glu Arg Cys Leu Val Glu Leu Ile Cys Ser Phe She Val
130 135 140
Ala Ser Trp Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser Ile
145 150 155 160
Arg Pro Lys Gin Val She Val Ala Asn His Thr Ser Met Ile Asp Phe
165 170 175
Ile Ile Leu Glu G2n Met Thr Ala Phe Ala Val Ile Met Gin Lys His
180 185 190
Pro Gly Trp Val Gly Leu Leu Gin Ser Thr Ile Leu Glu Ser Val Gly
195 200 205
Cys Ile Trp Phe Asn Arg Ser Glu Ala Lys Asp Arg Glu Ile Val Thr
210 215 220
Lys Lys Leu Arg Asp His Val Gin Gly Ala Asp Asn Asn Pro Leu Leu
225 230 235 240
Ile Phe Pro Glu Gly Thr Cys Val Asn Asn His Tyr Thr Val Met Phe
243 250 255
Lys Lys Gly Ala Phe Glu Leu Gly Cys Thr Val Cys Pro Ile Ala Ile
260 265 270
Lys Tyr Asn Lys Ile Phe Val Asp Ala Phe Trp Asn Ser Arg Lys Gin
275 280 285
Ser She Thr Thr His Leu Leu Gin Leu Met Thr Ser Trp Ala Val Val
290 296 300
Cys Asp Val Trp Tyr Leo Glu Pro Gin Asn Leu Lys Pro Gly Glu Thr
305 310 315 320
Pro Ile Glu Phe Ala Glu Arg Val Arg Asp Ile Ile Ser Val Arg Ala
325 330 335
Gly Leo Lys Lys Val Pro Trp Asp Gly Tyr Leu Lys Tyr Ser Arg Pro
340 345 350
Ser Pro Lys His Arg Glu Arg Lys Gin Gin Ser Phe Ala Glu Ser Val
355 360 365
Leu Gin Arg Leu Glu Glu Lys
370 375
<210> 110
<211> 376
CA 2998211 2018-03-16
=
334
<212> PRT
<213> Glycine max
<400> 110
Met Asn Asn Ser Gly Thr Pro Lys Ser Ser Ser Ser Glu Leu Asp Leu
1 5 10 15
Asp Arg Pro Asn Ile Glu Asp Tyr Leu Pro Ser Gly Ser Thr Ile Gin
20 25 30
Gin GiAa Pro His Gly Lys Leu Phe Leu His Asp Leu Leu Asn Ile Ser
35 40 45
Pro Thr Leu Ser Glu Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr
50 55 60
Arg Cys Phe Lys Ser Asn Pro Pro Glu Pro Trp Asn Trp Asn Val Tyr
65 70 75 80
Leu Phe Pro Leu Trp Cys Phe Gly Val Val Ile Arg Tyr Leu Ile Leu
85 90 95
She Pro Ile Arg Val Ile Gly Leu Thr Ile Gly Trp Ile :le Phe Leu
100 105 110
Ser Ser Phe Ile Pro Val His Phe Leu Leu Lys Gly His Asp Lys Leu
115 120 125
Arg Arg Ser Ile Glu Arg Ser Leu Val Glu Met Met Cys Ser Phe Phe
130 135 140
Val Ala Ser Trp Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser
145 150 155 160
Arg Arg Pro Lys Gin Val Phe Val Ala Asn His Thr Ser Met Ile Asp
165 170 175
She Ile Ile Leu Glu Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys
180 185 190
His Pro Gly Trp Val Gly Leu Leu Gin Ser Thr Ile Leu Glu Ser Leu
195 200 205
Gly Cys Ile Trp Phe Asn Arg Thr Glu Ala Lys Asp Arg Glu Tie Val
210 215 220
Ala Arg Lys Leu Arg Asp His Val Gin Gly Ala Asp Asn Asn Pro Leu
225 230 235 240
Leu Ile Phe Pro Clu Gly Thr Cys Val Asn Asn His Tyr Thr Val Met
245 250 255
Phe Lys Lys Gly Ala Phe Glu Leu Gly Cys Thr Val Cys Pro Val Ala
260 265 270
Ile Lys Tyr Asn Lys Ile Phe Val Asp Ala Phe Trp Asn Ser Arg Lys
275 280 285
Gin Ser Phe Thr Met His Leu Leu Gin Leu Met Thr Ser Trp Ala Val
290 295 300
Val Cys Asp Val Trp Tyr Leu Glu Pro Gin Asn Leu Lys Pro Gly Glu
305 310 315 320
Thr Pro Ile Glu She Ala Glu Arg Val Arg Asp Ile Ile Ser Val Arg
325 330 335
Ala Gly Leu Lys Lys Val Pro Trp Asp Gly Tyr Leu Lys Tyr Ser Arg
340 345 350
Pro Ser Pro Lys His Arg Glu Arg Lys Gin Gin Asn Phe Ala Glu Ser
355 360 365
Val Leu Arg Arg Trp Glu Glu Lys
370 375
<210> 111
<211> 371
CA 2998211 2018-03-16
335
4
<212> PRT
<213> Sesamum indicum
<400> 111
Met Ser Lys Leu Asn Thr Ser Ser Ser Glu Leu Asp Phe Asp Arg Pro
1 5 10 15
Asn Ile Glu Asp Tyr Leu Pro Ser Gly Ser Ile Gin Glu Pro His Gly
20 25 30
Lys Leu Arg Leu Arg Asp Leu Leu Asp Ile Ser Pro Thr Leu Thr Glu
35 40 45
Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr Arg Cys Phe Lys Ser
50 55 60
Asn Pro Pro Glu Pro Trp Asn Trp Asn Ile Tyr Leu Phe Pro Leu Trp
65 70 75 80
Cys Leu Gly Val Val lie Arg Tyr Gly Leu Leu Phe Pro Leu Arg Val
85 90 95
Ile Val Leu Thr Ile Gly Trp Ile :le Phe Leu Ser Cys Tyr Phe Pro
100 105 110
Val His Phe Leu Leu Arg Gly His Asp Lys Leu Arg Lys Arg Leu Glu
115 120 125
Arg Sly Leu Val Glu Leu Ile Cys Ser Phe Phe Val Ala Ser Trp Thr
130 135 140
Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser Met Arg Pro Lys Gin
145 150 155 160
Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe Ile Val Leu Glu
165 170 175
Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys His Pro Gly Trp Val
180 185 190
Gly Leu Leu Gin Ser Thr Ile Leu Glu Ser Leu Gly Cys Ile Trp Phe
195 200 205
Asn Arg Ser Glu Ser Lys Asp Arg Glu Ile Vai Ala Lys Lys Leu Arg
210 215 220
Glu His Val His Asp Ala Asp Asn Asn Pro Leu Leu Ile Phe Pro Glu
225 230 235 240
Gly Thr Cys Val Asn Asn His Tyr Thr Val Met Phe Lys Lys Gly Ala
245 250 255
Phe Glu Leu Gly Cys Thr Val Cys Pro Ile Ala Ile Lys Tyr Asn Lys
260 265 270
Ile Phe Val Asp Ala Phe Trp Asn Ser Arg Lys Gin Ser Phe Thr Thr
275 280 285
His Leu Lou Gin Leu Met Thr Ser Trp Ala Val Val Cys Asp Val Trp
290 295 300
Tyr Leu Glu Pro Gin Asn Leu Lys Pro Gly Glu Thr Pro Ile Glu Phe
305 310 315 320
Ala Glu Arg Val Arg Asp Ile Ile Ser Val Arg Ala Gly Lela Arg Lys
325 330 335
Val Pro Trp Asp Gly Tyr Leu Lys Tyr Ser Arg Pro Ser Pro Lys His
340 345 350
Arg Glu Arg Lys Gin Gin Ser Phe Ala Giu Ser Ile Lou Arg Arg Leu
355 360 365
Glu Glu Lys
370
<210> 112
<211> 364
CA 2998211 2018-03-16
a
336
<212> PRT
<213> Brachypodium distachyon
<400> 112
Met Ala Ser Ser Leu Asp Ala Pro Asn Leo Asp Asp Tyr Leu Pro Thr
1 5 10 15
Asp Ser Leu Pro Gin Glu Pro Pro Arg Ser Leu Asn Leu Arg Asp Leu
20 25 30
Leu Asp Ile Ser Pro Val Leu Thr Glu Ala Ala Gly Ala Ile Val Asp
35 40 45
Asp Ser Phe Thr Arg Cys Phe Lys Ser Asn Ser Pro Glu Pro Trp Asn
50 55 60
Trp Asn Ile Tyr Leu Phe Pro Leu Trp Cys Phe Gly Val Val Val Arg
65 70 75 80
Tyr Gly Leu Leu Phe Pro Leu Arg Val Leu Thr Leu Gly Leu Gly Trp
85 90 95
Met Val Phe Phe Ala Ala Phe Phe Pro Val His Phe Leu Leu Lys Gly
100 105 110
Gin Asn Lys Leu Arg Ser Lys Ile Glu Arg Lys Leu Vol Clu Met Met
115 120 125
Cys Ser Val Phe Val Ala Ser Trp Thr Gly Val Ile Lys Tyr His Gly
130 135 140
Pro Arg Pro Ser Ser Arg Pro Tyr- Gin Val Phe Val Ala Asn His Thr
145 150 155 160
Ser Met Ile Asp Phe Ile Ile Leu Glu Gin Met Thr Ala Phe Ala Val
165 170 173
Ile Met Gin Lys His Pro Gly Trp Val Gly Phe Ile Gin Lys Thr Ile
180 185 190
Leu Glu Ser Val Gly Cys Ile Trp Phe Asn Arg Asn Asp Leu Lys Asp
195 200 205
Arg Glu Val Val Gly Arg Lys Leu Arg Asp His Val Gin Arg Pro Asp
210 215 220
Asn Asn Pro Leu Leu Ile Phe Pro Glu Gly Thr Cys Val Asn Asn Gin
225 230 235 240
Tyr Thr Val Met Phe Lys Lys Gly Ala Phe Glu Leu Gly Cys Ala Vol
245 250 255
Cys Pro Ile Ala Ile Lys Tyr Asn Lys Ile Phe Val Asp Ala Phe Trp
260 265 270
Asn Ser Lys Lys Gin Ser Phe Thr Met His Leu Gly Arg Leu Met Thr
275 280 285
Ser Trp Ala Val Val Cys Asp Val Trp Phe Leu Glu Pro Gin Tyr Leu
290 295 300
Arg Glu Gly Glu Thr Ser Ile Ala Phe Thr Glu Arg Val Arg Asp Met
305 310 315 320
Ile Ala Ala Arg Ala Gly Leu Lys Lys Val Leu Trp Asp Gly Tyr Leu
325 330 335
Lys His Asn Arg Pro Ser Pro Lys His Thr Glu Glu Lys Gin Arg Ile
340 345 350
Phe Ala Glu Ser Val Leu Lys Arg Leu Giu Glu Ser
355 360
<210> 113
<211> 371
<212> PRT
<213> Setaria italica
CA 2998211 2018-03-16
ft
337
4
<400> 113
Met Ala Her Ser Ser Val Ala Ala Asp Met Glu Leu Asp Arg Pro Asn
1 5 10 15
Leu Glu Asp Tyr Leu Pro Pro Asp Ser Leu Pro Gin Glu Ala Pro Arg
20 25 30
Asn Leu His Leu Arg Asp Leu Leu Asp Ile Ser Pro Val Leu Thr Glu
35 40 45
Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr Arg Cys Phe Lys Ser
50 55 60
Asn Ser Pro Glu Pro Trp Asn Trp Asn Ile Tyr Leu Phe Pro Leu Trp
65 70 75 80
Cys Leu Gly Val Val Ile Arc Tyr Gly Ile Leu Phe Pro Leu Arg Ser
85 90 95
Leu Thr Leu Ala Ile Gly Trp Leu Ala Phe Phe Ala Ala Phe Phe Pro
100 105 110
Val His Phe Leo Leu Lys Gly Gin Asp Lys Leu Arg Ser Lys Ile Glu
115 120 125
Arg Lys Leu Val Glu Met Met Cys Ser Val Phe Val Ala Ser Trp Thr
130 135 140
Gly Val Ile Lys Tyr His Gly Pro Arg Pro Ser Thr Arg Pro His Gin
145 150 155 160
Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe Ile Ile Leu Glu
165 170 175
Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys Kis Pro Gly Trp Val
180 185 190
Gly Phe Ile Gin Lys Thr Ile Leu Glu Ser Val Gly Cys Ile Trp Phe
195 200 205
Asn Arg Asn Asp Leu Arg Asp Arg Glu Val Thr Ala Arg Lys Leu Arg
210 215 220
Asp His Val Gin Gin Pro Asp Lys Asn Pro Leu Leu Ile Phe Pro Glu
225 230 235 240
Gly Thr Cys Val Asn Asn Gln Tyr Thr Val Met Phe Lys Lys Gly Ala
245 250 255
Phe Glu Leu Gly Cys Ala Val Cys Pro Ile Ala Ile Lys Tyr Asn Lys
260 265 270
Ile Phe Val Asp Ala Phe Trp Asn Ser Lys Lys Gin Ser Phe Thr Met
275 280 285
His Leu Val Arg Leu Met Thr Ser Trp Ala Val Val Cys Asp Val Trp
290 295 300
Tyr Leu Pro Pro Gin Tyr Leu Arg Glu Gly Glu Thr Ala Tie Ala Phe
305 310 315 320
Ala Glu Arg Val Arg Asp Met Ile Ala Ala Arg Ala Gly Leu Lys Lys
325 330 335
Val Pro Trp Asp Gly Tyr Leu Lys His Asn Arg Pro Ser Pro Lys His
340 345 350
Thr Glu Glu Lys Gln Arg Ile Phe Ala Glu Ser Val Leu Met Arg Leu
355 360 365
Glu Glu Lys
370
<210> 114
<211> 376
<212> PRT
<213> Cicer arietinum
CA 2998211 2018-03-16
338
<400> 114
Met Asn Ser Thr Gly Thr Leu Lys Ser Ser Ser Ser Glu Leu Asp Leu
1 5 10 15
Asp Arg Pro Asn Ile Glu Asp Tyr Leu Pro Ser Gly Ala Ala Ile Gln
20 25 30
Gin Glu Pro Arg Gly Lys Leu Arg Leu His Asp Leu -Leu Asp Ile Ser
35 40 45
Pro Thr Leu Ser Glu Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr
50 55 60
Arg Cys Phe Lys Ser Asn Pro Pro Glu Pro Trp Asn Trp Asn Ile Tyr
65 70 75 80
Leu Phe Pro Leu Trp Cys Phe Gly Val Val Val Arg Tyr Leu Ile Leu
85 90 95
Phe Pro Thr Arg Vol Leu Gly Leu Thr Leu Gly Trp Ile Ile Phe Leu
100 105 110
.Ser Ala Phe Ile Pro Val His Leu Leu Leu Lys Gly His Asp Lys Leu
115 120 125
Arg Arg Asn Ile Glu Arg Ser Leu Val Glu Met Met Cys Gly Phe Phe
130 135 140
Val Ala Ser Trp Thr Gly Val Val Lys Tyr His Gly Pro Lys Pro Ser
145 150 155 160
Arc Arg Pro Lys Gln Vol Phe Vol Ala Asn His Thr Ser Met Ile Asp
165 170 175
Phe Ile :le Leu Glu Gln Met Thr Ala Phe Ala Val Ile Met Gln Lys
180 185 190
His Pro Gly Trp Val Gly Leu Leu Gln Ser Thr Ile Leu Glu Ser Vol
195 200 205
Gly Cys Ile Trp Phe Asn Arg Thr Glu Ala Lys Asp Arg Glu Ile Val
210 215 220
Ala Arg Lys Leu Arg Glu His Val Gln Gly Ala Asp Asn Asn Pro Leu
225 230 235 240
Leu Ile Phe Pro Glu Gly Thr Cys Vol Asn Asn His Tyr Thr Val Met
245 250 255
Phe Lys Lys Gly Ala Phe Glu Leu Gly Cys Thr Val Cys Pro Val Ala
260 265 270
Ile Lys Tyr Asn Lys Ile Phe Val Asp Ala Phe Trp Asn Ser Arg Lys
275 280 285
Gln Ser Phe Thr Lys His Leu Leu Gin Leu Met Thr Ser Trp Ala Val
290 295 300
Vol Cys Asp Val Trp Tyr Leu Glu Pro Gln Asn Leu Lys Pro Gly Glu
305 310 315 320
Thr Pro lie Glu Phe Ala Glu Arg Vol Arg Asp Ile Tie Ser His Arg
325 330 335
Ala Gly Leu Lys Lys Vol Pro Trp Asp Gly Tyr Leu Lys Tyr Ser Arg
340 345 350
Pro Ser Pro Lys His Arg Glu Arg Lys Gln Gln Asn Phe Ala Giu Ser
355 360 365
Vol Leu Arg Arg Leu Glu Glu Lys
370 375
<210> 115
<211> 371
<212> PRT
<213> Zea mays
CA 2998211 2018-03-16
339
<400> 115
Met Ala Ser Ser Ser Val Ala Ala Asp Met Glu Leu Asp Arg Pro Asn
10 15
Leu Glu Asp Tyr Leu Pro Pro Asp Ser Leu Pro Gln Glu Ala Pro Arg
20 25 3C
Asn Leu His Leu Arg Asp Leu Leu Asp Ile Ser Pro Val Leu Thr Glu
35 40 45
Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr Arg Cys Phe Lys Ser
50 55 60
Asn Ser Pro Glu Pro Trp Asn Trp Asn Ile Tyr Leu Phe Pro Leu Trp
65 70 75 80
Cys Phe Gly Val Val Ile Arc Tyr Gly Leu Leu Phe Pro Leu Arg Ser
85 90 95
Leu Thr Leu Ala Ile Gly Trp Leu Ala Phe Phe Ala Ala Phe Phe Pro
100 105 110
Val His Phe Leu Leo Lys Gly Gln Asp Lys Leu Arg Asn Lys Ile Glu
115 120 125
Arg Lys Leu Val Giu Met Met Cys Ser Val Phe Val Ala Ser Trp Thr
130 135 140 ,
Gly Val Ile Lys Tyr His Gly Pro Arg Pro Ser Thr Arg Pro His Gln
145 150 155 160
Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe Ile Ile Leu Glu
165 170 175
Gln Met Thr Ala Phe Ala Val Ile Met Gln Lys His Pro Gly Trp Val
180 185 190
Gly Phe Ile Gln Lys Thr Ile Leu Glu Ser Val Gly Cys Ile Trp Phe
195 200 205
Asn Arg Asn Asp Leo Arg Asp Arg Glu Val Thr Ala Arg Lys Leu Arg
210 215 220
Asp His Val Gln his Pro Asp Lys Asn Pro Leu Leu Ile Phe Pro Glu
225 230 235 240
Gly Thr Cys Val Asn Asn Gln Tyr Thr Val Met Phe Lys Lys Gly Ala
245 250 255
Phe Glu Leu Gly Cys Ala Val Cys Pro Ile Ala Ile Lys Tyr Asn Lys
260 265 270
:le Phe Val Asp Ala Phe Top Asn Ser Lys Lys Gln Ser Phe Thr Met.
275 280 285
His Leu Val Arg Leu Met Thr Ser Trp Ala Val Val Cys Asp Val Trp
290 295 300
Tyr Leu Glu Pro Gln Tyr Leu Arg Glu Gly Glu Thr Ala Ile Ala Phe
305 310 325 320
Ala Glu Arg Val Arg Asp Met Ile Ala Ala Arg Ala Gly Leu Lys Lys
325 330 335
Vol Pro Trp Asp Gly Tyr Leo Lys His Asn Arg Pro Ser Pro Lys His
340 345 350
Thr Glu Glu Lys Gln Arg Ile Phe Ala Glu Ser Val Leu Arg Arg Leu
355 360 365
Glu Glu Lys
370
<210> 316
<211> 378
<212> PRT
<213> Gossypium hirsutum
CA 2998211 2018-03-16
340
<400> 216
Met Asn Ser Ser Glu Gly Lys Leu Lys Ser Ser Ser Ser Glu Leu Asp
1 5 10 15
Leu Asp Arg Pro Asn :le Glu Asp Tyr Leu Pro Ser Gly Ser Ser Ile
20 25 30
GlE Glu Pro His Gly Lys Leu Arg Leu Arg Asp Leu Leu Asp Ile Ser
35 40 45
Pro Ala Leu Thr Glu Ala Ala Gly Ala Ire Val Asp Asp Ser Phe Thr
50 55 60
Arg Cys Phe Lys Set Asn Pro Pro Glu Pro Trp Asn Trp Asn Val Tyr
65 70 75 80
Leu Phe Pro Leu Trp Cys Cys Gly Val Val The Arg Tyr Leu Ile Leu
85 90 95
Phe Pro Met Arg Ala Leu Ile Leu Thr Ile Gly Trp Ile Ile Phe Leu
100 105 110
Ser Cys Phe Ile Pro Val His Phe Leu Leu Lys Gly Asn Asp Asn Leu
125 120 125
Arg Lys Lys Met Glu Arg Ala Leu Val Glu Leu Ile Cys Ser Phe Phe
130 135 140
Val Ala Ser Trp Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser
145 150 155 160
Met Arg Pro Lys Gin Val Phe Val Ala Asn His Thr Ser Met Ile Asp
165 170 175
Phe Ile Ile Leu Glu Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys
180 185 190
His Pro Gly Trp Val Gly Leu Leu Gin Ser Thr Ile Leu Glu Ser Val
195 200 205
Gly Cys Ile Trp Phe Ash Arg Ser Glu Ala Lys Asp Arg Glu Ile Val
210 215 220
Thr Arg Lys Leu Arg Glu His Set Gln Gly Ala Asp Asn Asn Pro Leu
225 230 235 240
Leu Ile Phe Pro Glu Gly Thr Cys Val Asn Asn Gin Tyr Ser Val Met
245 250 255
Phe Lys Lys Gly Ala Phe Glu Leu Gly Cys Thr Val Cys Pro Ile Ala
260 265 270
Ile Lys Tyr Asn Lys Ile Phe Val Asp Ala Phe Trp Asn Ser Arg Lys
275 280 285
Gin Ser Phe Thr Met His Leu Leu Gin Leu Met Thr Ser Trp Ala Val
290 295 300
Val Cys Asp Val Trp Tyr Leu Glu Pro Gin Asn Leu Arg Pro Gly Glu
305 310 315 320
Thr Pro Ile Glu Phe Ala Glu Arg Ile Arg Asp Ile Ile Ser Val Arg
325 330 335
Ala Gly Leu Lys Lys Val Pro Trp Asp Gly Tyr Leu Lys Tyr Her Arg
340 345 350
Pro Ser Pro Lys His Arg Glu Arg Lys Gin Gin Ser Phe Ala Glu Set
355 360 365
Val Leu Arg Gly Leu Glu Leu Glu Glu Lys
370 375
<210> 117
<211> 375
<212> PRT
<213> Eucalyptus grandis
CA 2998211 2018-03-16
341
<400> 117
Met Ala Ser Pro Arg Lys Leu Pro Thr Ser Ser Per Glu Leu Asp Leu
1 5 10 15
Asp Arg Leu Asn Ile Glu Asp Tyr Leu Pro Ser Gly Ser Ser Ile His
20 25 30
Glu Pro Pro Gly Pin Leu Arg Leu Arg Asp Leu Leu Asp Ile Thr Pro
35 40 45
Thr Leu Thr Glu Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr Arg
50 55 60
Cys Phe Lys Ser Asn Ser Gin Glu Pro Trp Asn Trp Asn Val Tyr Leu
65 70 75 80
Phe Pro Leu Trp Cys Phe Gly Val Val Val Arg Tyr Leu Ile Leu Phe
85 90 95
Pro Ala Arg Val Leu Val Leu Thr Ile Gly Trp Ile Ile Phe Leu Ser
100 105 110
Ser Phe Ala Ile Val His Phe Met Leu Lys Ala His Asp Ala Leu Arg
115 120 125
Arg Lys Leu Glu Arg Leu Leu Val Glu Leu Ile Cys Ser Phe Phe Val
130 135 140
Ala Ser Trp Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser Ile
145 150 155 160
Arg Pro Lys Gin Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe
165 170 175
Ile Ile Leu Glu Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys His
180 185 190
Pro Gly Trp Val Gly Leu Leu Gin Ser Thr Ile Leu Glu Ser Val Gly
195 200 205
Cys Ile Trp Phe Asn Arg Ser Glu Ala Lys Asp Arg Glu Ile Val Ala
210 215 220
Arg Lys Leu Arg Asp His Val Leu Gly Thr Asp Asn Asn Pro Leu Leu
225 230 235 240
Ile Phe Pro Glu Gly Thr Cys Val Asn Asn His Tyr Thr Val Met Phe
245 250 255
Lys Lys Gly Ala Phe Glu Leu Gly Cys Thr Val Cys Pro Ile Ala Ile
260 265 270
Lys Tyr Asn Lys Ile Phe Val Asp Ala Phe Trp Asn Ser Arg Lys Gin
275 280 285
Ser Phe Thr Met His Leu Leu Gin Leu Met Thr Ser Trp Ala Val Val
290 295 300
Cys Asp Val Trp Tyr Leu Glu Pro Gin Thr Leu Lys Pro Gly Glu Thr
305 310 315 320
Pro Ile Glu Phe Ala Glu Arg Val Arg Asp Ile Ile Ser Val Arg Ala
325 330 335
Gly Leu Lys Lys Val Pro Trp Asp Gly Tyr Leu Lys Tyr Ser Arg Pro
340 345 350
Ser Pro Lys His Arg Glu Gly Lys Gin Arg Ser Phe Ala Glu Trp Val
355 360 365
Leu Gin Arg Leu Glu Glu Arg
370 375
<210> 118
<211> 375
<212> PRT
<213> Cucumis sativus
CA 2998211 2018-03-16
342
<400> 118
Met Ser Gly Ala Ala Leu Leu Lys Ser Ser Ala Ser Glu Leu Asp Leu
1 5 10 15
Asp Arg Pro Asn Ile Glu Asp Tyr Leu Pro Ser Gly Ser Ser Ile Gin
20 25 30
Gin Pro Thr Ala Lys Leu Arg Leu Arg Asp Leu Leu Asp Ile Ser Pro
35 40 45
Thr Leu Thr Glu Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr Arg
50 55 60
Cys Phe Lys Ser Asn Pro Pro Clu Pro Trp Asn Trp Asn :le Tyr Leu
65 70 75 80
Phe Pro Leu Trp Cys Cys Gly Val Val lie Arg Tyr Leu Phe Leu Phe
85 90 95
Pro Ala Arg Val Leu Ile Leu Thr Ile Gly Trp Ile Ile Phe Leu Ser
100 105 110
Thr Phe Ile Pro Val Asn Leu Leu Leu Lys Gly His Pro Lys Leu Arg
115 120 125
Ala Lys Leu Glu Arg Phe Leu Val Glu Leu Ile Cys Ser Phe Phe Val
130 135 140
Ala Ser Trp Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser Ile
145 150 155 160
Arg Pro Lys Gin Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe
165 170 175
Ile Val Leu Glu Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys His
180 185 190
Pro Gly Trp Val Gly Leu Leu Gin Ser Thr Ile Leu Clu Ser Ile Gly
195 200 205
Cys Ile Trp Phe Asn Arg Thr Glu Leu Lys Asp Arg Glu Ile Val Ala
210 215 220
Lys Lys Leu Asn Asp His Val Gin Gly Ala Asp Asn Asn Pro Leu Leu
225 230 235 240
Ile Phe Pro Glu Gly Thr Cys Val Asn Asn His Tyr Ser Val Met Phe
245 250 255
Lys Lys Gly Ala Phe Glu Leu Gly Cys Ser Val Cys Pro Ile Ala Ile
260 265 270
Lys Tyr- Asn Lys Ile Phe Val Asp Ala Phe Trp Asn Ser Arg Lys Gin
275 280 285
Ser Phe Thr Met His Leu Leu Gin Leu Met Thr Ser Trp Ala Val Val
290 295 300
Cys Asp Val Trp Tyr Leu Glu Pro Gin Val Leu Lys Pro Gly Giu Thr
305 310 315 320
Pro Ile Glu Phe Ala Glu Arg Val Arg Asp Ile Ile Cys Ala Arg Ala
325 330 335
Gly Leu Lys Lys Val Pro Trp Asp Gly Tyr Leu Lys His Ser Arg Pro
340 345 350
Ser Pro Lys Tyr Arg Glu Ara Lys Gln Gin Ser Phe Ala Clu Ser Val
355 360 365
Leu Gin Leu Leu Asp Asn Lys
370 375
<210> 119
<211> 375
<212> PRT
<213> Gossypium arboreum
CA 2998211 2018-03-16
343
<400> 119
Met Ser Gly Ala Ala Leu Leu Lys Ser Ser Ala Ser Glu Leu Asp Leu
1 5 10 15
Asp Arg Pro Asn Ile Glu Asp Tyr Leu Pro Ser Gly Ser Ser Ile Gin
20 25 30
Gin Pro Thr Ala Lys Leu Arg Leu Arg Asp Leu Leu Asp Ile Ser Pro
35 40 45
Thr Leu Thr Glu Ala Ala Gly Ala Ile Val Asp Asp Ser Phe Thr Arg
50 55 60
Cys Phe Lys Ser Asn Pro Pro Glu Pro Trp Asn Trp Asn :le Tyr Leu
65 70 75 80
Phe Pro Leu Top Cys Cys Gly Val Val Ile Arg Tyr Leu Phe Leu Phe
25 90 95
Pro Ala Arg Val Leu Ile Leu Thr Ile Gly Trp Ile Ile Phe Leu Ser
100 105 110
Thr Phe Ile Pro Val Asn Leu Leu Leu Lys Gly His Pro Lys Leu Arg
115 120 125
Ala Lys Leu Glu Arg Phe Leu Val Glu Leu Ile Cys Ser Phe Phe Val
130 135 140
Ala Ser Trp Thr Gly Val Val Lys Tyr His Gly Pro Arg Pro Ser Tie
145 150 155 160
Arg Pro Lys Gin Val Phe Val Ala Asn His Thr Ser Met Ile Asp Phe
165 170 175
Ile Val Leu Glu Gin Met Thr Ala Phe Ala Val Ile Met Gin Lys His
186 185 190
Pro Gly Trp Val Gly Leu Leu Gin Ser Thr Ile Leu Glu Ser Tie Gly
195 200 205
Cys Ile Trp Phe Asn Arg Thr Glu Leu Lys Asp Arg Glu Ile Val Ala
210 215 220
Lys Lys Leu Asn Asp His Val Gin Gly Ala Asp Asn Asn Pro Leu Leu
225 230 235 240
Ile Phe Pro Glu Gly Thr Cys Val Asn Asn His Tyr Ser Val Met Phe
245 250 255
Lys Lys Gly Ala Phe Glu Leu Gly Cys Ser Val Cys Pro Ile Ala Ile
260 265 270
Lys Tyr Asn Lys Ile Phe Val Asp Ala Phe Trp Asn Ser Arg Lys Gin
275 280 285
Ser Phe Thr Met His Leu Leu Gin Leu Met Thr Ser Trp Ala Val Val
290 295 300
Cys Asp Val Trp Tyr Lou Glu Pro Cln Vol Leu Lys Pro Gly Glu Thr
305 310 315 320
Pro Ile Glu Phe Ala Glu Arg Val Arg Asp Ile Ile Cys Ala Arg Ala
325 330 335
Gly Leu Lys Lys Val Pro Trp Asp Gly Tyr Lou Lys His Ser Arg Pro
340 345 350
Ser Pro Lys Tyr Arg Glu Arg Lys Gin Gin Ser Phe Ala Glu Ser Val
355 360 365
Leu Gin Leu Leu Asp Asn Lys
370 375
<210> 120
<211> 1116
<212> DNA
<213> Cocos nucifera
CA 2998211 2018-03-16
344
<400> 120
atagttgagc tgaggtcatc gagctcggaa atggatctgg accgccccaa catcgaggag 60
tacctcacca cggactccat ccaagaatcc cccaagaagc tccacctaag ggacttgctc 120
gacattactc ccacgctgac ggaggccacc ggcgccatcg ttgatgattc cttcactcgc 180
tgctttaaat cgaatcctcc agagccctgg aattggaatg tctatttatt tcccttatgg 240
Lgcttgggag agatLattag atatggaatt ctttttcccc taagagttgc aatcttgaca 300
gcaggttggc aagtgttatt tgcagccttc attcctgtac atttcttatt gacagcacat 360
aataagtgga ggcgtaaaat agagaggaag ttggttgaga tgatatgcag tgtotttgtt 420
gcttcatgga caggggtagt caagtatcat gggcctcgtc ctagcatacg ccctcagcag 480
gtatttgttg ccaaccacac ttccatgatt gatttcatca tactagaaca gatgacagca 540
tttgctgtta taatgcaaaa gcatcctgga tgggttggat ttattcaaaa gaccatattg 600
gaaggtgttg gttgtatttg gttcaaccgt acagaatcaa aggatcgtga aattgtggca 660
cgaaagttaa gagaacatat tcatggagct gacaacaacc ctcttctgat atttccagaa 720
ggaacttgtg ttaacaacca ttacactgtc atgttcaaga agggtgcttt tgaacttggt 780
tgtgctgttt gccoggtagc aataaagtac aacaaaattt ttgtggatgc cttctggaac 840
agtaagaagc aatatttaac aatgcatttg tttcacctta tgacatcatg ggctgttgtt 900
tgcgatgttt ggtacctgga gactcagtac ataagacctg gagagacgcc cattgaattt 960
gctgaaaggg ttagagacat gatatctgtt cgagctggtc tcaaaaaagt cccgtaggat 1020
ggatatttga agtacttccg ccccagtcct aagctaacag agaggaagca gcagatcttt 1080
goggagtogg tcttgcagag gttggaggaa aaataa 1116
<210> 121
<211> 1131
<212> DNA
<213> Arabidopsis thaliana
<400> 121
atgagcagta cggcagggag gctcgtgact tcaaaatccg agcttgacct cgatcaccct 60
aacatcgaag attacctacc ttctggttct tccatcaatg aacctcgcgg caagctcagc 120
ctgcgtgatt tgctagacat cactccaaca ctcactgaag ctgctggtgc cattgttgat 180
gactcgttca caaaatgttt caaatcaaat octccagaac cttggaactg gaataattac 240
ttattcccac tatactgctt tgggattatt gttagatact gtatcctctt tccattgagg 300
tgcttcactt tagcttttgg gtggattatt ttcctttcat tgtttatccc tataaatgcg 360
ttgctgaaag gtcaagatag gttgaggaaa aagatagaga gggtattggt ggaaatgatt 420
tgcagctttt ttgtcgcctc atggaccgga gttgtcaaat atcacgggcc acgtcctagc 480
atccgtccta agcaggtcta tgttgccaac catacttcaa tgattgattt catcgaattg 540
gagcagatga ccgcatttgc tgttataatg cagaagcatc ctggttgggt tggtcatctg 600
caaagcacaa tattagagag tgtgggatgt atctggttca atcgttcaga ggcaaaggat 660
cgtgaaattg tagcaaaaaa gttaagggac catgtccaag gagctgacag taatcctctt 720
ctcatatttc ccgaagggac atgtgtaaat aataattaca cagtgatgtt taagaagggt 780
gcttttgaat tggactgcac tgtttgtcca atagcaatta aatacaacaa gatttttgtt 840
gacgccttct ggaatagcag aaaacaatca tttactatgc acttgctgca actcatgaca 900
tcatgggctg ttgtatgtga agtgtggtac ttgaaaccac aaaccataag gcccggtgaa 960
acaggaattg aatttgcaga gagggtcaga gacatgatat ctattagggc gggtctcaaa 1020
aaggtccctt gggatggata cttgaagtat tcgagaccaa gccccaagca tagtgaacgc 1080
aagcaacaga gtttcgcaga gtcgatcctg gctagattgg aagagaagtg a 1131
<210> 122
<211> 1116
<212> DNA
<213> Elaeis guineensis
<400> 122
atagttgagc tgaggtcatc gagctoggaa atggatctgg accgccccaa catcgaggag 60
CA 2998211 2018-03-16
345
tacctoccac cgactccatc caagaatccc cccaagaagc tccacctgag ggacttgctc 120
gacatttctc ccacgctgac ggaggccgcc ggcgccatcg ttgatgattc cattcactcgc 180
tgctttaaat cgaatcctcc agagccctgg aattgaaatg tctatttatt tcccttatgg 240
tgcttgggag tgattattag atacggaatt ctttttcccc taagagttgc aatcttgaca 300
gcagggtggc tagtattctt tgcagccttt attcctgtac atttottgtt gacagcacat 360
aataagtgga ggcgtaaaat agagaggaag ttggttgaga tgatatgcag tgtatttatt 420
gcttcatgga cagggatggt caagtatcat gggcctcgtc ctagcatgcg ccotcaggag 480
gtatttgttg ccaaccacac ttccatgatt gatttcatca tactagagca gatgacagca 540
tttgctgtta taatgcaaaa gcatcctgga taggtaggat ttattcaaaa gactattttg 600
gaaggtgttg gttgtatttg gttcaaccgt acagaatcaa aggatcgtga agttgtggca 660
cgaaagttaa gagaacatat tcacggagct gacaacaacc ctcttctgat atttccagaa 720
ggaacctgcg tcaacaacca ttacactgtc atgttcaaga agggtgcttt tgaacttggt 780
tgtactgttt gcccaatcgc aataaagtac aacaaaattt ttgtggatgc cttctggaac 840
agtaagaagc aatcttttac aatgcatttg tttcacctta tgacatcgtg ggctgttgtt 900
tgcgatgttt ggtacctgga gcctcagtac ataagacctg gagagacgcc tattgaattt 960
gctgaaaggg ttagggacat gatttccatt cgagctggtc tcaaaaaggt gccgtgggat 1020
ggatatttga aatacttccg ccccagtoct aagctcactg aaagaaagca gcagatattt 1080
gcagagtcag tcttgcagcg gttgaaggaa aaataa 1116
<210> 123
<211> 1057
<212> DNA
<213> Phoenix dactylifera
<400> 123
atggttggac tgaggtcatc gagctcggag atggatcttg accggccgaa cattgaggag 60
tacctcacca ccgactccat cgaagaatcc cccaagaagc tccacttgaa ggacttgctc 120
gacatttctc ccacgctgac ggagaccgct ggtgctatcg ttgatgattc tttcactcgg 180
tgctttaaat cgaatcctcc agaaccctgg aattggaatg tctatttatt tcccttatgg 240
tgcttgggag tgattattag atacggaatt ctttttcccc taagagttgc agtcttgaca 300
gcagggtggc tagtattctt tgcagccttt attcctgcac atttcctgtt gacagctcat 360
aataagtgga ggcgtaaaat agagaggaag ttggttgaga tgatatgcaa tgtatttgtt 420
gcttcatgga caggggtggt caagtatcat gggcctcgtc ctagcatgcg ccaccagcag 480
gtatttgtta ccaaccacac ttcaatgatt gatttcatca tactagagca gatgacagca 540
tttgctgtta tcatgcaaaa gcatcctgga tgggtaggat ttattcagaa gactattttg 600
gaaggtgttg gttgtatttg gttcaaccgt acagaatcaa aggatcgtga agttgtggca 660
cgaaagttaa gagaacatat tcaaggagct gacaacaacc ctcttctgat atttccagaa 720
ggaacctgcg ttaacaacca ttacactgtc atattcaaga agagtgottt tgaacttggt 780
tgtgctgttt gcccagttgc aataaagtac aacaaaaftt ttctggatgc tttctggaac 840
agtaagaagc aatcttttac gatgcatttg tttcacctta tgacatcatg ggctgttgtt 900
tgtgatgttt ggtatctgga gcctcagtac ataagacctg gagagacgcc cattgaattt 960
gctgaaaggg ttagagacat gatttctgtt cgagctggtc tcagaaaggt cccatgggat 1020
ggatatttga aatacttccg cccgagtoct aagctaa 1057
<210> 124
<211> 1116
<212> DNA
<213> Musa acuminata
<400> 124
atggctgggt tggctacctc gagcacggag atggatctcg accgccccaa catcgacgag 60
tacctcaccg tggagtcgat ccgggaggcc cccaagaagc tccacctgag ggacctcctc 120
gacatttctc ctactctcaa agaagctgcc ggcaccatcg tggacgactc cttcactcgt 180
tgctttaagt cgaatccttc agaaccctgg aattggaata totatttott ccatttatgg 240
CA 2998211 2018-03-16
346
tgcttgggag tagttattag atatgggatt ctttttccat tcagagttat aatcttggtt 300
gcaggataga tagtatatctt tgcagccttt tcactggtgc atttcctttt aggagaacat 360
aataagtgga aacgtgaaat agagaggaaa ctggttaaga tgatatgcag cgtatttgtt 420
gattcataga cggcagtgat taaataccat ggacctcgtc ccagcatgcg ccctcaacag 480
gtcttcgttg ccaaccacac ttctatgatt gatttcatca tcttagaaca gatgacagca 540
tttgctgtca ttatgcaaaa gcatcctagt tgggttggat ttatccaaaa gatcatcgta 600
gaaagtttag gttgtatatg gttcaaccgt acagaggcta aggaccgtga aattattgcat 660
agaaagttga gagaacacat tcaaggaatt gacaacaacc ctcttctgat atttcctgag 720
ggaacttqcg ttaacaacca ttatactgtt atgttcaaga agggtgcttt taaacttggt 780
tgtgatgttt gtcctgtaac aatcaagtac aacaagattt ttgtggatgc tttctggaac 840
agcaaaaagc aatctttcac gatgcactta gtacagctta tgacatcatg goctgttgtt 900
tgtgatgttt ggtacctgga gcctcagtat ataaggcctg gagagactcc tattgaattt 960
gctgaaaagg ttcaagacat gatctctgtt cgagctggtc tcaaaaaggt cccatgggat 1020
ggctatctaa agtacttccg coccagtocc aagctcatag agcgcaagca gcagatcttt 1080
gcggagtcag tcttacagcg attggaggag aaatga 1116
<210> 125
<211> 1119
<212> DNA
<213> Ananas comosus
<400> 125
atggctgaag ctctgggctc gtcgagcgcg gagatggatc tcgaccgtcc caacctcgag 60
gagtacctcc ccaccgactc catccaagac tccaccaaaa acctccacct gagggacctg 120
ctcgatatct cccccacgct caccgaggcc gcgggcgcca tcgttgatga ctcattcact 180
cgctgcttta aatcaaatcc tccagaacca tggaattgga atatatattt gttccctcta 240
tggtgcctcg gagtcgttgt aagatatggg attctttttc cactcagagt tgcagtcttg 300
gcgatagggt ggatagtatt tttttctgcc ttcttccctg tacatttctt attgaaaggg 360
tatcccaagt ggaggcgcaa actagagaga aaattggttg agatgatgtg cagtgtattt 420
gttgcttcat ggactggagt cgtaaaatat catggaccac gcccaagcac gcgccctcat 480
caggtatttg ttgctaatca cacctccatg atcgatttca tcattttaga acaaatgact 540
gcatttgctg ttatcatgca aaaacatcct ggatgggttg gatttattca gaagaccatc 600
ttagaaagtg taggatgtat ttggttcaac cgaacggagt ctaaggatcg cggagttgtc 660
gggcggaagc taagagaaca tgttcaagga gtagacaaca accctcttct gatatttcca 720
gaaggaacct gcgtaaacaa tcactacact gtcatgttta agaagggtgc ttttgagctt 780
ggatgtgctg tttgcccaat agcaatcaaa tacaacaaaa tttttgtgga tgccttctgg 840
aacagtaaga agcaatcrtt taccatgcat ctggtccgcc tcatgacgtc gtgggctgtt 900
gtctgtgatg tgtggtactt ggagcctcag tacctgagac ctggggagac gccaattgaa 960
tctgctgaaa gggttagaga catgatttct gctcgagctg gtctaaagaa ggttccatgg 1020
gatgggtatc tgaagtactt tcgtcctagc cccaagcata cacaacggaa gcaocagatc 1080
tttgcagagt caatcttgcg gcggttggag aggaaatga 1119
<210> 126
<211> 1113
<212> DNA
<213> Asparagus officinalis
<400> 126
atggcggggc tggagtcctc gagcgcaggg atcgacgtcg accctccaaa tattgaagac 60
tatctcacat ccgatgccct ccatcaacct cataagaagc ttcaattgaa ggatttactc 120
gatatttctc ctacactaac tgaggctgca ggagcaattg ttgatgactc atttacacga 180
tgtttcaagt caaatcctcc cgaaccctgg aattggaatg tctacctatt tcccttgtgg 240
tgcttgggag tgattgttcg atatgggatc ctttttccct tgagagttat gactctggca 300
gctggatgga ttgtgttctt ttcagccttt cttcctgttc attatctaat gaaagggcag 360
CA 2998211 2018-03-16
347
aacaaatgga aaaataatat agagagaaaa ttggtggaoa tgatatgtag gtttttgtt 420
gcttcttgga ctggtgttat caggtatcac ggacctcgtc ctagcatgcg ccatcaacag 480
gratttgtgg cgaatcatac ttcgatgatt gatttcatca ttttagagca gatggctgca 540
tttgctgtaa tcatgcagaa gcatcctgga tgggttggtt tccttcagac gacaattttg 600
gaaagcatag gttctatttg gttcaatcgt accgaggcca agaatcgcga agttgtagca 660
agaaagttaa gagaaca-Lac tgaaggggac aacaatcctt tactaatatt r_ccggaagga 720
acttgtgtga acaatgacta cactgttatg ttcaaaaaag gcacatttga actaggatgt 780
gctgtttgtc ctgtagccat caagtacaat aaaattttcg tgaacgcctt ctggaacagc 840
aagaagcaat cttttacgat gcatctgatg cgccttataa catcatgggc tgtggtatgt 900
gatgtttggt atcttgaacc acagtatctg aaaccgggag agacttctat tgaattcgct 960
gaaagggtca gggatatgat ttcggtccga gctggtctca gaaaggtccc gtgggatgga 1020
tatttgaagt acttccgccc aagtcctaag cttacagagc gcaagcagca aatatttgcg 1080
gaatcagtcc tacggcggct ggaagaaaag tga 1113
<210> 127
<211> 1113
<212> DNA
<213> Oryza brachyantha
<400> 127
atggcgtcct catcggtggc gggggacatc gagctggacc ggccgaacct ggaggattac 60
ctcccgcccg actcgctgcc gcaggaatcc cccgggaatc tccatctgcg cgatctgctt 120
gacatctcgc cggtgctcac tgaagcggcg ggggcggccg tcaatgattc attcacacgt 180
tgctttaagt ccaattctcc agagccatgg aattggaaca tttatttatt cccactatgg 240
tgcttgggag tagtgataag atatggaata ctattaccac taaggggctt aactcttcta 300
gttggatgga tagctttctt cgctgccttt ttctctgtgc atttcttatt taaagggcaa 360
aagatgagaa gtaaaataga gagaaaactg gttgaaatga tgtgcagtgt ttttgttgct 420
tcttggactg gagtgatcaa gtatcatgga cctcgcccaa gcacacgacc tcatcaggta 480
tttgttgcaa accacacatc gatgatagac ttcattattc tggagcagat gacagcattt 540
gctgtcatta tgcaaaagca tcctggatgg gttggattta ttcagaagac tatattggaa 600
agtgtgggtt gcatctggtt caatcgtaac gatctcaagg atcgtgaagt agtagcaaaa 660
aagttacgag atcatgttca acatccagac aacaatcctc tcctaatttt ccctgaagga 720
acttgtgtta acaaccagta cactgtcatg ttcaagaagg gtgcttttga gcttggctgt 780
gctgtatacc caatagctat caaatacaat aaaatatttg ttgatgcctt ctggaatagt 840
aagaagcaat cttttacaat gcacttgatt cggcttatga catcatgggc agttgtgtgt 900
gatgtatggt acttggagcc gcaatatcta aaggagggag aaacagcaat tcaatttgct 960
gaaagagtaa gagacatgat agctgctaga gctggtctta agaaggttcc atgggacgga 1020
tatctgaaac acaaccgccc tagccccaaa cacactgaag agaagcagcg catctttgct 1080
gattctgtgt tgcagagact ggaggaaagc taa 1113
<210> 128
<211> 1113
<212> DNA
<213> Oryza sativa
<400> 128
atggcgacct cgtcggtggc gggggacatc gagctggacc ggccgaacct ggaggactac 60
ctcccatccg actcgctgcc gcaggagttc cccaggaatc tccatctacg caatctgctg 120
gacatctcgc cgatgatcac tgaagaggcg ggcgccatcg tcgatgattc attcacacgt 180
tgctttaagt caaattctcc agagccatgg aattggaaca tttatttatt cccattgtgg 240
tgcttgggag tagtgataag atacggaata ctattcccgc tgaggggcct aactattcta 300
gttggatggt tagcattott tgctgccttt tttcctgtac atttcttatt gaaaggtcaa 360
aagatgagaa gtaaaataga gagaaagctg gttgaaatga tgtgcagtgt ttttgttgct 420
tcttggactg gagtgatcaa gtatcatggg cctcgcccaa gcacacggcc tcatcaggta 480
CA 2998211 2018-03-16
348
tttgttgcaa accatacatc gatgatagat ttcattattc tggagcagat gacagcattt 540
gctgtcatta tgcaaaagca tcctggatgg gttggattta ttcagaagac tatcttggaa 600
agtgttggtt gcatctgatt taatcgcaat gatctcaagg atcgtgaagt ggttgcaaaa 660
aagttacgag atcatgttca acatccagac agcaatcctc tcctgatttt ccctgaagga 720
acttgtgtta acaaccagta cactgtcatg ttcaagaagg gtgcttttga gcttggctgt 780
gctgtatgcc caatagctat caaatacaat aaaatatttg ttgatgcctt ctggaatagt 840
aagaagcaat cgtttacaat gcacttggtt aggcttatga catcatgggc agttgtgtgt 900
gatgtatggt acttggagcc tcagtatctg agggatggag aaacagcaat tgaatttgct 960
gaaagaqtaa gagacatgat agctgctaga gctggtotta agaaggttcc gtgggacggg 1020
tatctgaaac acaaccgccc tagtcccaaa cacactgaag agaaggagcg catctttgct 1080
gactctgtgt tacggagact ggaggaaagc taa 1113
<210> 129
<211> 1092
<212> DNA
<213> Nelumbc nucifera
<400> 129
atggacttgg atcgaccaaa catagaggaa tatttacctt cagaagccat tcaagagtct 60
aacgagaagc ttcacttgcg tgatttgctc gacatttcgc ctactctaac coaggctgct 120
ggtgccattg ttgatgattc tttcactcgt tgtttcaagt caaatccgtc agaaccttgg 180
aattggaatg tatatttatt tccactttgg tgctttggag tggtggtaag atatggcatt 240
ctttttcctg ttagagttct agtgttaaca attgggtgga taatattcct ttcatccttc 300
attcctgcac atttcctatt gagaagtcat gataagtgga ggaagaagat agagagatat 360
ctagtggagt taatatgcag cttctttgtt gcatcatgga ctggggttgt caaatatcat 420
gggccacggc caagcatgcg acccaagcag gtttttgtgg ccaatcatac ttccatgata 480
gattttattg utttagaaca gatgactgca tttgctgtaa ttatgcagaa gcatcctgga 540
tgggttgggc ttttgcaaag cactattttg gagagtgtag gttgtatctg gttcaatcgt 600
gcagaagcaa aggaccgtga aattgtagca agaaagttaa gagaccacat tcaaggggtt 660
gacaacaatc ctcttcttat atttccagaa ggaacatgtg taaataacca ctatacagtc 720
atcrttcaaga agggtgcatt tgaacttgga tgcactgttt gtccaatagc aatcaagtac 780
aataaaattt ttgttgatgc cttctggaat agtaagaagc aatcttttac catgcactta 840
ctgcacctta tgacttcatg ggctgttgtt tgtgatgttt ggtatttgga gccgcaaaat 900
attagacctg gagagacacc catagaattt gcagagaggg tacgagacat aatttctgtt 960
cgaggaggtc ttaaaaaggt tccatqggat ggatatttga aatattctcg tcctagcccc 1020
aaacacagag aaagaaagca acaaaggttt gtagagtcgg tattgcagcg cttggagaaa 1080
aagggaaaat ga 1092
<210> 130
<211> 1131
<212> DNA
<213> Vitis vinifera
<400> 130
atggccaacg ctcccgataa taagctcact tcctcaagct ccgagctcga cttggatcgc 60
cccaatctcg aagactacct tccctccgga tccatgcaag aacctcgcgg caagcttcgc 120
ctgcgtgatt tattggacat ttcgccgacc ctaaccgagg ctgctggggc cattgttgac 180
gactcatttca cacgatgttt caagtcgaac cctccggagc cttggaactg gaatgtgtat 240
ttatttcctc tttggtgttt gggagtggta attcgatatg gaattttatt tcccacaagg 300
gttctagtac tcacactggg gtggataata ttcctttcat cctttattcc agtacatttt 360
ctattgaagg gaaacgataa gttgaggaaa aagttggaga gatgtctagt ggagttaatt 420
tgcagcttct ttgttgcatc atggactgga gttgtcaagt accatgggcc acggcctagc 480
aggaggcctc agcaggtttt tgttgccaat catacttcca tgattgattt tatcgtttta 540
gaacagatga ctgcatttgc agttattatg cagaagcatc ctggctgggt tggattqctg 600
CA 2998211 2018-03-16
349
caaagtacca ttttggagag tgtaggatgt atctggttca atcgtacaga agcaaaggac 660
cgtgaaattg ttgctaggaa gctaagggat catgttcaag gggctgacaa caaccctatt 720
ctcatattcc cagaaggaac ttgtgtgaat aaccactaca ctgtcatgtt caagaagggc 780
gcattcgaac ttggctgcac tgtttgccct attgcaataa agtacaataa gattttcgtt 840
gatgatttct ggaacagtaa gaagcaatcc tt':,acaatgc atcttctgca gcttatgaca 900
tcctgggctg -ccgtttgtga tatttgatac ttagagcccc aaacattgaa gccaggagag 960
acacccattg aatttgcaaa gaaggtcagg gacataattt ctattcgagc tggtttgaaa 1020
aaggttcctt gggatggata tttgaagtac tctcgcccta gcccaaagca tagagagcag 1080
aagcagcaga gctttgctga ttcagtatta cggcgcctgg aagagaagtg a 1131
<210> 131
<211> 1122
<212> DNA
<213> Nicotiana tomentosiformis
<400> 132
atgaatatga ataagctaaa aacatcaagc tccgaattag acttggatcg acccaatctc 60
gaagattatc ttccaactgg atccatccca gaaccccatg gcaagottcg cctgcgtgat 120
ttaattgata tttctcccac cctaactgaa gctgctggtg ccattgttga cgattctttc 180
accagatgct tcaagtcaaa tccaccagag ccttggaact ggaacattta t_tEtgttccct 240
ttatggtgct tgggggttgt tgttagatat gggattcttt tccctataag agttattgtc 300
ttgacaatag gatggataat attcctotct tgctatatcc cgatgcattt cctgctgaaa 360
ggacacgata agttcaggaa aaagcttgag agatgtctgg tggagctgat atgcagtttc 420
tttgttgcat cttagactgg ggttgtcaaa taccatggtc cacggcctag catacgacct 480
aagcaggttt ttgtggcgaa tcacacgtca atgatagatt ttattgtcct agagcagatg 540
actgcatttg cagtgatcat gcagaagcat cctggatagg ttggactact gcagagtacc 600
attttagaag gtgttggatg tatctggttc aaccgctcag aagccaagga tcgtgaaatt 660
gtagcacgaa agttgaggca acatgttgaa ggggccgata acaaccctct tcttatattc 720
cccgagggaa cttgcgtaaa taaccactac actgtcatgt tcaaaaaggg agcatttgaa 780
ctcggttgca ctgtttgtcc tgttgcaatc aagtacaaca aaatttttgt tgacgccttt 840
tggaatagta gaaaacaatc cttcacaatg cacctcttgc agctcatgac atcttgggct 900
gttgtctgtg atgtttggta cctggagcct cagaacataa gacctgggga gactccaatc 960
gagtttgcag agagggtgag ggacatcatt tctgctagag caagtcttaa aaaggttact 1020
tgggatggat atttaaaata ctctcgtcct agocccaagc atcgagagag gaagcaacag 1080
agttttgcag aatcagtgct gcgtcgcctg gaagagaagt ag 1122
<210> 132
<211> 1128
<212> DNA
<213> Jatropha curcas
<400> 132
atggctactc caggtaagct aaagacctca agctctgaat tggacttgga tcgacccaat 60
atcgaagact accttccttc tggagtctct attcaagaac ctcgtggcaa gcttcgtctg 120
cgtgatttgc ttgacatttc gccgacccta acggaggctg ctggtgccat tgttgatgac 180
acctttacaa ggtgtttcaa gtcaaatcct ccagaaccat ggaattggaa catatatcta 240
tttccccttt ggtgctgagg tgtggtgtgt cgatatggga ttttgtttcc catcagggtt 300
ctagtactga caatagggtg gataattttc ctttcatgct acattcctgt gcatttccta 360
cttaaagaac atgacaagtt gagaaaaaag cttgagagat gtttggtgga gttaatttgc 420
agottotttg tggcatcatg gaccggagtt gtcaagtacc atggtccacg gcctagcatc 480
cgacctaaac aggtttttgt ggccaatcat acctccatga ttgattttat catcttggaa 540
cagatgactg catttgctgt tattatgcag aagcatcctg gatgggttgg actactgcaa 600
agcactatat tagagagtgt cggatgtatc tggttcaatc gttcagaggc aaaggatcgt 660
gaaattgtaa caaaaaagtt aaaggatcat gtacaggggg ctgacaataa cactottctc 720
CA 2998211 2018-03-16
350
atatttcctg aaggaacttg tgtaaataae cactatactg taatattcaa gaagggtgca 180
ttcgaactgg gatgtactgt ttgtccaatt gcaatcaaat acaacaaaat ttttgttgat 840
gctttttgga acagccggaa gcagtcattt acaacgcatt tgctgcaact catgacttcc 900
tgggctgttg tttgtgatgt atggtacttg gagccacaaa atctgaaacc tggagagaca 960
cccattgagt ttgctgagag ggtcagggac ataatatctg tacgagcagg tctcaaaaag 1020
gttccttggg atggatatct aaagtattct cgccctagcc caaagcatag agagcgaaag 1080
caacaaagct ttgctgagtc agtgctgcag cgactggagg agaaatga 1128
<210> 133
<211> 1132
<212> DNA
<213> Glycine max
<400> 133
atgaataact cagggacacc caagtcttca agttctgaat tggatcttga tcgacccaac 60
attgaagatt acctcccttc agggtccacc attcaacaag aacctcatgg aaagcttttc 120
Ctgcatgatt tgcecaatat ttctcctact ttgtctgagg ctgcaggtgc tattgtagat 180
gactcattca caagatgctt caagtcaaat cctccagaac catggaattg gaatgtttat 240
ttgtttcctt tgtggtgttt tggagttgtg attcgatact tgattctgtt cccaatcagg 300
gttatagggt taacaatagg atggataata tttctttcat ccttcattcc ggtgcacttc 360
ctattgaaag gacatgacaa gttaaggaga agtattoaga ggtctttggt agagatgatg 420
tgcagtttct ttgttgcatc ttggactggg gttgttaagt atcatogacc caggcctagc 480
aggagaccaa agcaggtttt tgtagccaac catacttcca tgattgattt cattatctta 540
gaacaoatga ctgcttttgc tgttattatg cagaagcatC ctggatgggt tggattattg 600
cagagtacca ttttggagag tctaggatgC atctggttca accgtacaga ggcaaaggat 660
cgggaaatag tagcaaggaa attgagggat catgtccagg gagctgataa caacccoctt 720
ctcatatttc ctgaaggaac ttgtgtaaat aatcactata cagtcatgtt caagaaoggt 780
gcatttgaac ttggctgcac agtttgccca gttgcaatca agtacaataa gatttttgta 840
qatgcttttt ggaatagtcg aaagcaatca ttcactatgc atctgttgca gctaatgacg 900
tcttgggcag ttgtttgtga tgtttggtac ttggagccac aaaatctgaa gccaggagag 960
acgcctattg agttcgcaga gagggtgaga gacataatct cagttcgtgc tggccttaaa 1020
aaggttcctt gggatggata tctgaagtat tctcgtccta gcccaaagca tagagaaagg 1080
aagcaacaga actttgctga gtcagtgctg cggcgatggg aggaaaagtg a 1131
<210> 134
<211> 1116
<212> DNA
<213> Sesamum indicum
<400> 134
atgagtaagc ttaacacatc cagctccgaa ttggattttg atcgccccaa catcgaggac 60
tatctcccat ccggatccat tcaagagcct cacggcaaac tccgcctgcg tgatttgctc 120
gatatttcac caactctcac tgaggccgct ggtgcaattg ttgatgactc tttcaccaga 180
tgcttcaagt caaatcctcc agaaccctgg aactggaaca tatacttgttt tectttatgg 240
tgcttgggag tggtcatcag atatggcctt cttttcccat taagggtaat agtgttgaca 300
ataggatgga ttatatttct atcatgctat tttcctgtgc atttcctgtt aagagggcac 360
gacaaattga ggaaaagatt agagagaggt ctagtggagt tgatttgcag tttcttcgtt 420
gcatcatgga caggggttgt caagtatcat ggtccacggc cgtccatgcg acctaagcag 480
gtttttgtgg cgaatcacac atccatgatt gatttcattg ttttggaaca aatgactgct 540
tttgcagtga ttatgcagaa gcatcctggg tgggttggat tattgcagag cacaattttg 600
gaaagtctag gatgtatctg gttcaaccgc tcagagtcca aggatcgtga aattgttgca 660
aaaaagctaa gggaacatgt ccatgatgct gataacaatc ctcttcttat attcccggaa 720
ggaacttgtg tgaataacca ttacactgtt atgtttaaga agggtgcatt tgaacttggc 780
tgcactgtct gtccaatagc aatcaagtac aacaagatat ttgttgatgc cttctggaat 840
CA 2998211 2018-03-16
351
agccgaaagc aatccttcac tacacacttg ttgcagctta tgacatcctg ggctgttgtt 900
tgtgacgttt ggaacctaga gcctcaaaat ctgaaacctg gggaaacacc cattgaattt 960
gcagagaggg tgagggacat tatttctgtt cgggccggcc tcagaaaggt gccttgggat 1020
ggatatttga agtactctcg gcctagtccg aagcatcgtg aacgcaagca acaaagcttt 1080
gcagagtcaa ttctccgtcg cttggaagag aaatag 1116
<210> 135
<211> 1095
<212> DNA
<213> Brachypodium distachyon
<400> 135
atggcgtcgt cgctcgacgc gccgaacctt gatgattacc tccccacgga ctcgctcccg 60
caggaacccc ccaggagcct caatctgcgc gatctgctgg acatctcgcc agtgctcact 120
gaagcggcgg gcgccatcgt ggatgattcg ttcacacgct gctttaagtc aaattctcca 160
gagccatgga actggaacat ttatttgttc ccgttatggt gcttcggagt agtcgtaaga 240
tacgaactac tgtttccact cagggtatta acgcttggat taggatggat ggtattcttt 300
gctgccttzt ttcccgtgca tttcctattg aaagggcaaa ataaactgag aagtaaaata 360
gagagaaagc tcgttgaaat gatgtgcagt gtttttgttg cttcttggac tggagtaatc 420
aagtaccaag gaccacgccc aagctcacgg ccttatcagg tatttgttgc aaaccataca 480
tcaatgatag atttcattat tcatggaggag atgacagcat ttgctgtcat tatgcaaaag 540
catcctggat gggttggatt tattcagaag actattttgg aaagtgtggg ttgcatctgg 600
tttaatcgaa atgatcttaa ggaacgtgaa gtagttggca gaaagttacg tgatcatgtt 660
caacgtccag acaacaaccc tctcttgatt ttcccagaag gaacttgtgt taacaaccag 720
tacactgtaa tgatcaagaa gggtgotttt gagcttgggt gtgctgtatg tccgatagct 780
atcaagtata ataaaatatt tgttgatgcc ttctgaaata gtaaaaagca atctttcaca 840
atgcacttgg gtcggcttat gacatcatga gctgtagtgt gtgatattta, gtacttggaa 900
cctcaatatc tcaggaaagg agagacatcg attgcattta ctgaaagagt aagggacatg 960
atagctgctc gagccagtct taagaaggtt ctgtgggatg ggtatctgaa gcataaccgt 1020
cctagcccca aacacactga ggagaagcag cgcatatttg cagaatcggt gttgaagaga 1080
ctagaggaaa gctaa 1095
<210> 136
<211> 1116
<212> DNA
<213> Setaria italica
<400> 136
atggcgagct cctcggtggc ggcggacatg gagctggacc gccccaatct ggaggactac 60
ctcccgcccg actcgctccc gcaggaggcg ccccggaatc tccatctgcg cgatttgctg 120
gacatctcgc cagtgctcac cgaggcagca ggcgccatcg tcgatgactc cttcacgcgt 180
tgctttaagt caaattctcc agagccatgg aattggaaca tatatctgtt ccccttatgg 240
tgcttgggag tagtaataag atatggaata ctcttcccac tgaggtcctt aacgcttgca 300
ataggatggt tagcattttt tgctgccttt tttcctgtcc atttcctatt gaaagggcaa 360
gacaagttga gaagtaaaat tgagaggaag ttggttgaaa tgatgtgcag tgtttttgtt 420
gcttcatgga ctggagtgat caagtatcat ggaccacgcc caagcacacg acctcatcag 480
gtattcgttg caaaccatac atcaatgata gatttcatta ttctggagca aatgacagca 540
tttgctgtca tcatgcagaa gcatcctgga tgggttggat ttattcagaa gactatcttg 600
gaaagtgtcg gttgcatctg gtttaatcgt aatgatcttc gggatcgtga agttacggca 660
cggaagttac gtgatcatgt tcaacaacca gacaaaaatc ctctcttgat ttttccggaa 720
ggaacttgtg ttaacaacca gtacacggtc atgttcaaga agggtgcctt tgagcttggc 780
tgcgctgtct gtccaatagc tatcaagtac aataaaatat ttgttgatgc cttttggaac 840
agtaagaagc aatcttttac aatgcacttg gtccggctga tgacatcatg ggctgttgtg 900
tgtgatgttt ggtacttacc tccacaatat ctgagggagg gagagacggc aattgcattt 960
CA 2998211 2018-03-16
352
gctgagagag taagggacat gatagccgct agagctggac taaaaaaggt tccgtgggat 1020
ggctatctga aacacaaccg tcctagtccc aaacacactg aagagaaaca acgcatattt 1080
gccgaatcta tcctgatgag actgaaggag aaatga 1116
<210> 137
<211> 1131
<212> DNA
<213> Cicer arietinum
<400> 137
atgaatagca ctgaaacact taagtcttca agttctgagt tggatcttga tcgacccaac 60
attgaggatt atctccattc aggaaccgcc attcaacaag aacctcgcgg caagcttcac 120
cagcatgact tgottgatat ttctcctaca ctatctgagg cagctggtgc tattgtagat 180
gactcattca caagatgttt caagtcaaat cctccagaac catggaattg gaatatatat 240
trgtttcctt tgtggtgttt tggagttgtt gttcgatatt tgatactgtt ccctacaagg 300
gttcttgggt taacattagg aaggataata tttctttctg ctttcattcc agtgcacctc 360
ctattgaaag gacatgacaa gatgaggaga aaaattgaga ggtctttagt agagatgatg 420
tgaggtatct ttgttgcatc ttggactggg gttgtcaagt accatgggcc aaagcccagc 480
aggcgaccaa aacaggtatt tgttgccaac cacacttcca tgattgattt cattatctta 540
gaacagatga ctgcttttgc tgttattatg cagaagcatc ctggatgggt tggattgttg 600
caaagcacca ttttggagag tgtaggatgt atctggttca atcgcacaga ggcaaaggat 660
cgagaaattg tggcaagaaa attgagggaa catatccagg gagctgacaa caatcctctt 720
ctcatatttc cagagggaac ttgtgtaaat aatcactaca cagtcatatt taagaagggt 780
gcatttgaac ttggctgcac agtttgccct gttacaatca aatacaacaa aatttttgtc 840
gatgcatttt ggaatagtcg aaagcaatca ttcactaaac atctgttgca gctaaagaca 900
tcatgggctg ttgtttgtga tgtttggtac ttggagccac aaaacctaaa gccaggagag 960
acaccaattg agtttgccga aagggtgaga gacataatct cacatcgtgc tggtcttaaa 1020
aaggttcctt gggatggata tctgaagtat tcgcgaccta gcccaaaaca tagagaaaga 1080
aaacaacaga actttgctaa gtoggtgctg cggcgtttgg aagaaaaata a 1131
<210> 138
<211> 1116
<212> DNA
<213> Zea mays
<400> 138
atggcgagct cgtctgtggc ggcggacatg gagctggacc gccccaacct ggaggactac 60
ctcccgcccg actcgctccc gcaggaggcg cccaggaatc tccatctgcg cgatctactt 120
gacatctcgc cggtgctaac cgaggcagcg ggtgccatag tcgatgattc attcacacgc 180
tgctttaagt cgaattctcc agaaccatgg aactggaaca tatatttgtt ccctttatgg 240
tgcttcggtg tagtaattcg atatggatta ctcttcccac tgaggtcctt aacgcttgca 300
ataggatggt tagcattttt tgctgccttt ttccccgtgc atttcctatt gaaaggtcaa 360
gacaagttga gaaataaaat tgagaggaag atggttgaaa tgatgtgcag tatttttatt 420
gcttcatgga ctggagtgat caagtaccat ggaccacgcc caagcacacg acctcatcag 480
gtatttgttg caaaccatac atcaatgata gatttcatta ttctggagca aatgacagca 540
tttgctgtca tcatgcagaa gcatcctgga tgggttggat ttattcagaa gactatcttg 600
aaaagtgtgg gttgcatctg gtttaaccgt aatgatctcc gggatcgtga agttacggca 660
cggaagttgc gtgatcatgt tcaacatcca gacaaaaacc ctctcttgat tttcccagaa 720
ggaacttgtg ttaacaacca gtatacggtc atgttcaaga agggtgcctt tgagcttggg 780
tgtgctgtct gtccaatagc tatcaaatac aataaaatat ttgttgatgc cttttggaac 840
agtaaaaagc aatcttttac gatgcacttg gtccggttga tgacatcatg ggctgttgtg 900
tgtgatgttt ggtacttgga gcctcaatat ctgagggagg gagagactgc aattgCgttt 960
gctgagagag taagggacat gatagcagct agagctgatc ttaagaaggt cccgtgggat 1020
CA 2998211 2018-03-16
353
ggctatctga aacacaaccg ccctagaccc aaacacaccg aagagaagca acgcatattc 1080
gccgaatctg tottgaggag actagaggag aaatga 1116
<210> 139
<211> 1137
<212> DNA
<213> Gossypium hirsutum
<400> 139
atgaacagta gtgaagggaa gttgaaatca tcgagttccg aattggattt ggatcgaccc 60
aacatcgaag attatctocc tactggatct tccattcaag aaccacatgg caagcttcgc 120
ctgcgggatt tgattgatat ttctcccgct ttaactgaag ctactggtgc tattgttgat 180
gattctttca cacggtgttt taagtcgaat cceccggaac cgtggaactg gaatgtgtat 240
cattttcctc tctgatattg tggtgtggta tttcggtact tgattttgtt ccctatgagg 300
gctttaattt tgacaatagg atggataata tttctgtcat gcttcattcc tgtgcacttt 360
catctcaaaa ggaacgaaaa cttgoggaaa aagatggaga gggcgttggt ggagctaatc 420
tgcagcttct ttgttgcatc ctggactgga gtagttaagt accatggacc gcggcctagc 480
atgcggccca agcaggtgtt tgtggccaat catacttcta tgattgattt catcatatta 540
gaacagatga ctgcatttgc tgtcattatg cagaagcacc ctggataggt tggactgcta 600
cagagcacta ttttagagag tgtagggtgt atttggttta accgttcaga ggccaaagat 660
cgtgaaatta taacaaggaa gttaagggag catagtcagg gagctgacaa taaccctott 720
ctcatatttc ccgaagggac atgtgtaaac aatcaataca gcgttatatt caagaagggt 780
gcattcgaac ttggttgcac tgtttgcccg attgcaataa agtacaataa aatttttgtt 840
gatgcctttt ggaatagccg gaagcagtcc tttacaatgc atttattgca gcttatgaca 900
tcctgggcta ttgtttgcga tgtttggtac ctagagcccc aaaatctaag gcctggagaa 960
acacccatcg agtttgcaga gaggatcaga gacataatct ctattcgagc aggtcttaaa 1020
aaggttccat gggacggata tttgaagtat tctcgcccga gccctaagca tagagagcga 1080
aaacaacaaa gttttgccga atctattatt cgaggactgg aactggaaga aaaatga 1137
<210> 140
<211> 1128
<212> DNA
<213> Eucalyptus grandis
<400> 140
atggcgagcc ccaggaagct gccgacctcg agctqcgagc tggacctgga tcgcctcaac 60
atcgaggatt acctccattc cggatcctcc atccacgagc ccccaggcca gctccgcctg 120
cgcgatttgc ttgatatcac gccgactctg accgaggccg ccggtgctat cgtcgatgac 180
tcgttcacgc ggtgcttcaa gtcgaattcg caggaaccgt ggaactggaa cgtgtacctc 240
ttcccgctgt ggtgcttcgg ggtggtggtt cggtacttga toctattccc ggcaagggtt 300
ttagtgttga caattggatg gataatattc ctctcatcat ttgccattgt tcactttatg 360
cttaaagcac atgatacact gagaaggaag ctggagaggt tgctggtgga gttaatttgc 420
agcttctttg ttgcttcatg gactggtgtc gtcaaatacc atgggccacg gcctagcatt 480
cggcctaaac aagtttttgt tgccaaccac acttccatga ttgatttcat catcttagag 540
caaatgactg ccttcgctgt tattatgcaa aagcatcctg gatgggttgg actactgcaa 600
agcactattt tggagagtgt aggatgcatc tggtttaatc gttctgaggc caaagatcgt 660
gaaattgtgg caagaaagtt gagagatcac gtactgggaa ctgataacaa tcctcttctc 720
atatttcctg aagggacttg tgtgaacaat cactatactg tcatattcaa aaagggtgca 780
tttgagcttg ggtgcactgt ttgccctatc gcaatcaagt acaataagat cttcgtggat 840
gccttttgga acagcaggaa acaatctttc acaatgcatc tactgcaact tatgacatct 900
tgggctgttg tttgtaacgt ctggtacttg gaaccccaaa ccttgaaacc tgatgaaacq 960
ccaattgaat ttgcagagag ggtccgtgac atcatatctg ttcgagctgg tttgaagaag 1020
gttccttgag atggatatct gaagtactct cgccctagcc ccaagcatag agaagggaag 1080
caacgaagct ttgctgagtg ggtgctgcag cgacttgagg agaggtga 1128
CA 2998211 2018-03-16
354
<210> 141
<211> 1128
<212> DNA
<213> Cucumis sat vus
<400> 141
atgagtggtg ctgctattct caaatcctcc gcctctaaat tggacttaga tcgacccaat 60
atcgaagatt acttgccttc cggatcctct atccaacaac ccactgccaa gottcgcctt 120
cgtgatttgc tcgatatttc gccgaccctt accgaggctg ctggtgctat tgttgatgat 180
tcgtttacaa ggtgtttcaa atcaaaccca ccagagccat agaattggaa tatttatttg 240
ttccctttgt ggtgctatgg agtggtgatt cggtatttgt ttctottocc ggcaagggtt 300
ctcatattga cgaLaggatg gataattttc ctttcaacgt tcattccagt gaatctcctt 360
ctgaaagagc atcctaaact gagagctaag ttagagaggt ttttggtgga gttgatttgc 420
agcttctttg ttgcatcttg gactggagtt gttaagtatc atgggccacg gcctagcatc 480
agaccaaaac aggttttcgt ggccaaccac acttccatga ttgatttcat agtcttagag 540
caaatgactg catttgctgt tattatgcaa aaacatcctg ggtgggttgg actgttgcaa 600
agcactatat tggagagtat aggatgtata tggttcaacc gtacagagtt gaaggaccgt 660
gaaattgtag caaagaagtt aaatgaccac gttcaagggg ctgacaacaa tcctcttctt 720
atatttcctg aaggaacttg tgtaaataac cactactctg ttatgttcaa gaagggtgca 780
tttgaacttg gatgctotgt ttgcccaatt gcaatcaaat acaataaaat tttcgttgat 840
gctttttgga acagcaggaa gcagtcgttc actatgcatc tgctgcagct catgacct_ct 900
tgggctgttg tttgtgatgt ttggtacctg gagccccaag ttttgaagcc tggagaaaca 960
cccattgagt ttgcagaaag ggtcagggac ataatatgtg ctcgagcagg tcttaagaag 1020
gttccatggg atggatattt gaagcactcc cgtccgagcc caaaataccg agaacgtaaa 1080
caacaaagct tcgoggagtc agtgctgcag ctattggaca ataagtga 1128
<210> 142
<211> 1137
<212> DNA
<213> Gossypium arboreum
<400> 142
atgaacagta gtgaagggaa gttgaaatca tcgagttccg aattggattt ggatcgaccc 60
aacatcgaag attatctccc ttctggatct tccattcaag aaccacatgg caagcttcgc 120
ctgagggatt tgattgatat ttctcccgct ttaactgaag ctgctggtgc tattgttgat 180
gattcattca cacggtgttt taagtcgaat cccccggaac cgtggaactg gaatgtgtat 240
ctgtttcctc tctggtgttg tggtgtggta tttcggtact tgattttatt ccctatgagg 300
gctttagttt tgacaatagg atggataata tttctgtcat gcttcattcc tgtgcacttt 360
cttctcaaag ggaacgataa cttgcggaaa aagatggaga gggcgttggt ggagctaatc 420
tgtagcttct ttgttgcgto ctggactgga gttgttaagt accatggacc acggcctagc 480
atgcggccca agcaggtgtt tgtggccaat catacttcta tgattgattt catcatatta 540
gaacagatga ctgcatttgc tgtcattatg cagaagcacc ctggatgggt tggactgcta 600
cagagcacta ttttagagag tgtagggtgt atttggttta accgttcaga ggccaaagat 660
cgtgaaattg taacaaggaa gttaagggag catagtcagg gagctgacaa taaccctctt 720
ctcatatttc ccgaagggac atgtgtaaac aatcaataca gcgttatgtt caagaagggt 780
gcattcgaac ttggttgcac tgtttgcccg attgcaataa agtacaataa aatttttgtt 840
gatgcctttt ggaatagccg gaagcagtcc tttacaatgc atttattaca gctaatgaca 900
tcctgggctg ttgtttgcga tgtttggtac ctagagcccc aaaatctaag gcctggagaa 960
acacccatcg agtttgcaaa gaggatcaga gacataatct ctgttcgagc aggtcttaaa 1020
aaggttccat gggacggata tttgaagtat tctcgcccga gccctaagca tagagagcga 1080
aaacaacaaa gttttgccga atctgttctt cggggactgg aactggaaga aaaatga 1137
<210> 143
<211> 215
CA 2998211 2018-03-16
355
<212> PRT
<213> Elaeis guineensis
<400> 143
Met Pro Asp Ser Asp Asn Glu Ser Gly Gly Gin Asn Asn Ser His Asn
1 5 10 15
Asn Asn Val Gly Glu Tyr Ser Ser Ser Arg Glu Gin Asp Arg Phe Leu
20 25 30
Pro Ile Ala Asn Val Ser Arg Ile Met Lys Lys Ala Len Pro Ala Asn
35 40 45
Ala Lys Ile Ser Lys Asp Ala Lys Glu Thr Val Gin Glu Cys Val Ser
50 55 60
Glu Phe Ile Ser Phe lie Thr Gly Glu Ala Ala Asp Lys Cys Gin Arg
65 70 75 80
Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr
85 90 95
Thr Leu Gly Phe Glu Asp Tyr Val Asp Pro Leu Lys Val Tyr Leu His
100 105 110
Arg Phe Arg Glu Met Glu Gly Asp Lys Cys Ser Ala Gly Ala Ser Ala
115 120 125
Ser Her Gin Pro Gin His Lys Asp Gly Gly Asp Gly Gly Gly Gly Gly
130 135 140
Gly Gly Gly Ala Pro Ser Met Gly Asn Asn Val Val Gly Leu Gly Gly
145 150 155 160
Gly Gly Gly Gly Ala Gly Gly Met Met Met Met Met Gly Gin Gin Met
165 170 175
Tyr Ala Th/ Pro Pro Ser Tyr His His His Met Ser Thr Met Ser Gly
180 185 190
Lys Ser Ser Met Gly Gly Gly Ser Ser Ala Ser Ser Ser Ser Pro Gly
195 200 205
Phe Gly Arg Gin Gly Arg Val
2.70 215
<210> 144
<211> 133
<212> PRT
<213> Elaeis guineensis
<400> 144
Met Glu Pro Glu Asn Pro Glu Leu Asn Leu Asp Leu Ala Leu Gin Pro
1 5 10 15
Ser Her Pro Pro Glu Pro Ala Arg Val Phe Ser Cys Asn Tyr Cys Gin
20 25 30
Lys Lys Phe Tyr Ser Ser Gin Ala Leu Gly Gly His Gin Asn Ala His
35 40 45
Lys Leu Glu Arg Ser Leu Ala Lys Arg Ser Trp Glu Leu Ala Thr Ala
50 55 60
Leu Arg Pro His Ala Gly Ser Thr Ile Gly Gin His Thr Ser Thr Val
65 70 75 80
Val Leu Val Glu Arg Gin Arg Glu Glu Cys Cys Tyr Asn Gly Val Gly
85 90 95
Leu Ala Thr Arg Gly Arg Glu Ala Ser Arg Ala Ser Ile Arg Leu Gly
100 105 110
CA 2998211 2018-03-16
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Ser Arg Lys Glu Ser Asp Asp Lys Arg Glu Leu Ala Asp Gly Ile Asp
115 120 125
Leu Ser Leu Arg Leu
130
<210> 145
<211> 190
<212> PRT
<213> Arabidopsis thaliana
<400> 145
Met Gly Asp Ser Asp Arg Asp Ser Gly Gly Gly Gin Asn Gly Asn Asn
1 5 10 15
Gin Asn Gly Gin Ser Ser Leu Ser Pro Arg Glu Gin Asp Arg Phe Leu
20 25 30
Pro Ile Ala Asn Val Ser Arg Ile Met Lys Lys Ala Leu Pro Ala Asn
35 40 45
Ala Lys Ile Ser Lys Asp Ala Lys Glu Thr Met Gin Glu Cys Val Ser
50 55 60
Glu She Ile Ser She Val Thr Gly Glu Ala Ser Asp Lys Cys Gin Lys
65 70 75 80
Glu Lys Arg Lys Thr Ile Asn Gly Asp Asp Leu Leu Trp Ala Met Thr
85 90 95
Thr Leu Gly Phe Glu Asp Tyr Val Glu Pro Leu Lys Val Tyr Leo Gin
100 105 110
Arg Phe Arg Glu Ile Glu Gly Glu Arg Thr Gly Leu Gly Arg Pro Gin
115 120 125
Thr Gly Gly Glu Val Gly Glu His Gin Arg Asp Ala Val Gly Asp Gly
130 135 140
Gly Gly Phe Tyr Gly Gly Gly Gly Gly Met Gin Tyr His Gin His His
145 150 155 160
Gin Phe Leu His Gin Gin Asn His Met Tyr Gly Ala Thr Gly Gly Gly
165 170 175
Ser Asp Ser Gly Gly Gly Ala Ala Ser Gly Arg Thr Arg Thr
180 185 190
<210> 146
<211> 161
<212> PRT
<213> Arabidopsis thaliana
<400> 146
Met Ala Asp Ser Asp Asn Asp Ser Gly Gly His Lys Asp Gly Gly Asn
1 5 10 15
Ala Ser Thr Arg Glu Gin Asp Arg Phe Leo Pro Ile Ala Asn Val Ser
20 25 30
Arg Ile Met Lys Lys Ala Leu Pro Ala Asn Ala Lys Ile Ser Lys Asp
35 40 45
Ala Lys Glu Thr Val Gin Glu Cys Val Ser Glu She Ile Ser She Ile
50 55 60
Thr Gly Glu Ala Ser Asp Lys Cys Gin Arg Glu Lys Arg Lys Thr Ile
65 70 75 80
Asn Gly Asp Asp Leu Leu Trp Ala Met Thr Thr Leu Gly Phe Glu Asp
85 90 95
CA 2998211 2018-03-16
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Tyr Val Glu Pro Leu Lys Val Tyr Leu Gln Lys Tyr Arg Glu Val Glu
100 105 110
Gly Glu Lys Thr Thr Thr Ala Gly Arg Gln Gly Asp Lys Glu Gly Gly
115 120 125
Gly Gly Gly Gly Gly Ala Gly Ser Gly Ser Gly Gly Ala Pro Met Tyr
130 135 140
Gly Gly Gly Met Val Thr Thr Met Gly His Gln Phe Ser His His Phe
145 150 155 160
Ser
<210> 147
<211> 150
<212> PRT
<213> Arabidopsis thaliana
<400> 147
Met Asp Tyr Gln Pro Asn Thr Ser Leu Arg Leu Ser Leu Pro Ser Tyr
10 15
Lys Asn His Gln Leu Asn Leu Glu Leu Val Leu Glu Pro Ser Ser Met
20 25 30
Ser Ser Ser Ser Ser Ser Ser Thr Asn Ser Ser Ser Cys Leu Glu Gln
35 40 45
Pro Arg Val Phe Ser Cys Asn Tyr Cys Gin Arg Lys Phe Tyr Ser Ser
50 55 60
Gln Ala Leu Gly Gly His Gln Asn Ala His Lys Leu Glu Arg Thr Leu
65 70 75 80
Ala Lys Lys Ser Aug Glu Leu Phe Arg Ser Ser Asn Thr Val Asp Ser
85 90 95
Asp Gln Pro Tyr Pro Phe Ser Gly Arg Phe Glu Leu Tyr Gly Arg Gly
100 105 110
Tyr Gin Gly Phe Leu Glu Ser Gly Gly Ser Arg Asp Phe Ser Ala Arg
115 120 125
Arg Val Pro Glu Ser Gly Leu Asp Gln Asp Gln Glu Lys Ser His Leu
130 135 14C
Asp Leu Ser Leu Arg Leu
145 150
<210> 148
<211> 399
<212> PRT
<213> Elaeis guineensis
<400> 148
Met Ala Ser Ala Ser Glu Ser Arg Asn Val Thr Ser Glu Glu Thr Glu
1 5 10 15
Val Thr Ser Glu Arg Arg Pro Glu Glu Gly Lys Glu Glu Arg Glu Leu
20 25 30
Gly Leu Glu Phe Pro Lou Met Arg Gln Ser Ser Ile Tyr Ser Leu Thr
35 40 45
Leu Asp Glu Ile Gln Asn Thr Val Cys Glu Pro Gly Lys Ser Phe Gly
50 55 60
Ser Met Asn Met Asp Glu Phe Leu Thr Asn Ile Trp Asn Val Glu Glu
65 70 75 80
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Gly Gin Ile Ala Ser Ala Asn Ala Gin Asn Gin Gin His Ile Gly Gly
85 90 95
Gly Gly Pro Pro Ala Ala Pro Pro Leu Gln Arg Gin Gly Per Ile Ala
100 105 110
Val Pro Ala Pro Leu Cys Arg Lys Thr Val Asp Glu Val Trp Ser Asp
115 120 115
Ile His Arg Gly Gin Asn Ala Arg Arg Gin Asn Val Asp Arg Pro Pro
130 135 140
Pro Pro Ser Gin Gin Gin Glu Ser Asn Cys Ala Ala Pro Arg Lys Pro
145 150 155 160
Thr Phe Gly Glu Ile Thr Leu Glu Asp Phe Leu Val Lys Ala Gly Val
165 170 175
Val Arg Glu Gly Tyr Gin Pro Gly Ser Ala Pro Ser Ala His Ala Pro
180 185 190
Val Pro Pro Ala Thr Gin Tyr Gly Met Pro Ala Gly Tyr Gin Met Val
195 200 205
Gly Thr Glu Gly Ala Pro Val Phe Gly His Val Val Gly Val Gin Ala
210 215 220
Tyr Gly Asp His Gin Val Thr Ala Ala Asn Ala Met Tyr Pro Val Val
225 230 235 240
Gly Asp Gly Gly Gly Pro Gly Tyr Ala Val Gly Asn Gly Phe Gly Gly
245 250 255
Arg Val Gly Asn Gly Tyr Gly Ala Val Ala Ala Val Gly Gly Ser Pro
260 265 270
Ala Ser Pro Gly Ser Ser Glu Gly Val Gly Gly Gly Gin Val Glu Asn
275 280 285
Ser Gly Ala Ala Glu Gly Gly Gly Gly Gly Lys Gly Gly Arg Lys Arg
290 295 300
Pro Leu Asp Gly Thr Val Glu Lys Val Val Glu Arg Arg Gin Arg Arg
305 310 315 320
Met Ile Lys Asn Arg Glu Ser Ala Ala Arg Ser Arg Ala Arg Lys Gin
325 330 335
Ala Tyr Thr Val Glu Leu Glu Ala Glu Leu Asn Gin Leu Lys Glu Glu
340 345 350
Asn Ala Arg Leu Lys Glu Ala Glu Lys Lys Met Leu Ala Leu Lys Lys
355 360 .365
Gin Leu Leu Met Gin Ala Met Ala Glu Arg Ala Arg Val Asn Ala Gin
370 375 380
Lys Thr Ile Leu Thr Met Arg Arg Cys Asn Ser Ser Lys Trp
385 390 395
<210> 149
<211> 272
<212> PRT
<213> Elaeis guineensis
<400> 149
Met Glu Gin Ser Thr Gin Pro Ser His Pro Val Met Gly Ile Val Thr
1 5 10 15
Gly Ala Ala Gin Ile Ala Tyr Ala Ala Pro Thr Tyr Gin Ser Ala Ala
20 25 30
Met Val Thr Gly Ala Pro Ala Val Ile Gly Ala Ile Pro Ser Pro Ala
35 40 45
Gin Pro Thr Ser Thr Phe Pro Thr Ser Pro Ala Gin Leu Thr Ser Gin
50 55 60
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His Gin Leu Ala Tyr Gin Gin Val Arg Gin Phe His His Gin Gin Gin
65 70 75 80
Gin Gin Gin Gin Gin Gin Leu Gin Thr Phe Trp Ala Asn Gin Met Leu
85 90 95
Glu Ile Glu His Ala Thr Asp Phe Lys Asn His Ser Leu Pro Leu Ala
100 105 110
Arg Ile Lys Lys Ile Met Lys Ala Asp Glu Asp Val Arg Met Ile Ser
115 120 125
Ala Glu Ala Pro Vai Ile Phe Ala Lys Ala Cys Glu Met Phe Ile Leo
130 135 14C
Glu Leu Thr Leu Arg Ser Trp Ile His Thr Glu Glu Asn Lys Arg Arg
145 150 155 160
Thr Leu Gin Lys Asn Asp Ile Ala Ala Ala Ile Thr Arg Thr Asp Ile
165 170 175
Phe Asp Phe Leu Val Asp Ile Val Pro Arg Asp Glu Leu Lys Glu Glu
180 185 190
Gly Ile Gly Ile Ala Arg Ala Ala Leu Pro Thr Met Gly Ala Pro Ala
195 200 205
Asp Ser Gly Pro Tyr Tyr Tyr Val Pro Ala Gin His Gin Leu Ala Gly
210 215 220
Pro Gly Met Ile Met Gly Lys Pro Val Asp Gin Ala Thr Thr Ala Ala
225 230 235 240
Met Tyr Thr Ala Gin Pro Pro His Pro Val Ala Tyr Met Trp Gin Gin
245 250 255
Pro Gin Gin Gin Gin Ala Gin Gin Gin Gin Gin Met Pro Asp Ser Gly
260 265 270
<210> 150
<211> 352
<212> PRT
<213> Elaeis guineensis
<400> 150
Met Fro Leu Asp Asn Ala Asn Ala Phe Asp Thr Gin His Phe Ser Asn
1 5 10 15
Lys Asp Ser Glu His Ser Ser Val Thr Ser Val His Ser Ala Ser Asn
20 25 30
Cys Val Asp Asn Phe Pro Ser Leu Trp Lys Gin Ser Gly Ser His She
35 40 45
Pro Gin Ser Thr Tyr Phe Lys Asn Phe Cys Met Asn Met Gly Phe Leu
50 55 60
Ala Gin Pro Asp Asn Gin Met Lys Gin Leo Gly Gly Gin Met Pro Asp
65 70 75 BO
Gin Asp Ser Ser Ser Ser Gin Ser Thr Gly Gin Ser His Gin Glu Val
85 90 95
Ser Gly Thr Ser Glu Gly Asn Leu His Glu Gin Ser Ile Ser Ala Gin
100 105 110
Ala Gly Asn Asp Lys Thr Cys Gly Lys Gln Val Glu Gly His Val Asn
115 120 125
Ser Val Leu Phe Leu Gly Thr Pro Glu Ala Ala Phe Val Ser Pro Arg
130 135 140
Leu Asp Tyr Gly Gin Ser She Ala Cys Val Pro Tyr Thr Tyr Ala Asp
145 150 155 160
Pro Ser Phe Gly Gly Val Leu Ala Ala Tyr Gly Ser Pro Ala Ile Ile
165 170 175
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His Pro Gin Met Val Gly Val Pro Pro Ser Ser Arg Val Pro Leu Pro
180 185 190
Leu Glu Pro Ala Ala Glu Glu Pro Ile Tyr Val Asn Ala Lys Gin Tyr
195 200 205
Arg Ala Ile Leu Arg Arg Arg Gin Leu Arg Ala Lys Leu Glu Ala Gin
210 215 220
Asn Lys Leu Ile Lys Ala Arg Lys Pro Tyr Leu His Glu Ser Arg His
225 230 235 240
Leu His Ala Met Lys Arg Ala Arg Gly Ser Gly Gly Arg Phe Leu Asn
245 250 255
Thr Lys Gin Leu Glu Gin Gin Gin Gin Arg Pro Leu Leu Pro Pro Pro
260 265 270
Pro Ser Val Ser Thr Gly Leu Gly Asn Leu Ser Ala Ser Asn Leu His
275 280 285
Phe Glu Asn Gly Pro Ser Gly Ser Ser Ala Ala Pro Thr Ser Ser Ala
290 295 300
Asp Val Val Arg Val Ser Thr Ser Gly Gly Met Leu Glu Gin Gin Gly
305 310 315 320
His Leu Ser Phe Leu Ser Ala Asp Phe His Ser His Val Arg Ser Thr
325 330 335
Gin Gly Gly Gly Asp Ser Gly Ser Gin Pro Arg Ile Thr Ile Met Arg
340 345 350
<210> 151
<211> 300
<212> PRT
<213> Glycine max
<400> 151
Met Gin Gin Ile His Ser Met Pro Gly Gly Arg Phe Phe Ser Gly Ser
1 5 10 15
Gly Ser Ala Asp Arg Arg Leu Arg Pro His His Gin Asn Gin Gin Ala
20 25 30
Leu Lys Cys Pro Arg Cys Asp Ser Leu Asn Thr Lys Phe Cys Tyr Tyr
35 40 45
Asn Asn Tyr Asn Leu Ser Gin Pro Arg His Phe Cys Lys Asn Cys Arg
50 55 60
Arg Tyr Trp Thr Lys Gly Gly Val Leu Arg Asn Val Pro Val Gly Gly
65 70 75 BO
Gly Cys Arg Lys Ser Lys Arg Ser Ser Lys Pro Asn Lys Ile Thr Pro
85 90 95
Ser Glu Thr Ala Ser Pro Pro Pro Pro Pro His Pro Asp His Asn Asn
100 ]05 110
Asn Ser Asn Ser His Ser Ser Ser Glu Ser Ser Ser Leu Thr Ala Ala
115 120 125
Val Ala Thr Thr Thr Glu Ala Val Ser Ala Pro Glu Thr Leu Asn Ser
130 135 140
Asp Ser Asn Asn Asn Asn Asn Met Gin Glu Ser Lys Leu Leu Ile Pro
145 150 155 160
Ala Leu Glu Thr Asn Asn Pro Leu Glu Gin Gly Thr Gly Asp Cys Gly
165 170 175
Gly Ile Phe Ser Giu Ile Gly Pro Phe The Ser Leu Ile Thr Thr Thr
180 185 190
Thr Ser Thr Asn Glu Pro Leu Gly Ser Gly Phe Gly Phe Gly Asn Ser
195 200 205
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Thr Leu Pro Asp Ala Ser Ser Phe Gin Trp His Tyr Gin Lys Val Ser
210 215 220
Ser Asn Asn Glu Glu Leu Lys Leu Pro Glu Asn Ser Phe Leu Asp His
225 230 235 240
Thr Val Asp Leu Ser Gly Met His Ser Lys Thr Ser His Gly Gly Gly
245 250 255
Phe Gly Ser Leu Asp Trp Gin Gly Gly Ala Asp Gin Gly Leu Phe Asp
260 265 270
Leu Pro Asn Thr Val Asp His Ala Tyr Trp Ser His Thr His Trp Ser
275 280 285
Asp His Asp Asn Ser Ser Ser Leu Phe His Leu Pro
290 295 300
<210> 152
<211> 351
<212> PRT
<213> Giycine max
<400> 152
Met Ser Ser Val Phe Ser Glu His Lys Elie Gin Leu Gin Pro Ser His
1 5 10 15
Gin Leu Leu Ser Leu Lys Lys Ser Leu Gly Asp Ile Asp Ile Pro Val
20 25 30
Pro Pro Arg Lys Leu Leu Thr Arg Arg Ser Ala Ala Val His Asp Gly
35 40 45
Ser Gly Asp Ile Tyr Leu Pro His Ser Gly Ser Thr Asp Ser Ser Thr
50 55 60
Asp Asp Asp Ser Asp Gly Asp Pro Tyr Ala Ser Asp Gin Phe Arg Met
65 70 75 80
Phe Glu Phe Lys Val Arg Arg Cys Ser Arg Ser Arg Ser His Asp Trp
85 90 95
Thr Asp Cys Pro Phe Val His Pro Gly Glu Lys Ala Arg Arg Arg Asp
100 105 110
Pro Arg Arg Phe Tyr Tyr Ser Gly Thr Val Cys Pro Glu Phe Arg Arg
115 120 125
Gly Gin Cys Asp Arg Gly Asp Ala Cys Glu Phe Ser His Gly Val Phe
130 135 140
Glu Cys Trp Leu His Pro Ser Arg Tyr Arg Thr Giu Ala Cys Lys Asp
145 250 155 160
Gly Lys Asn Cys Lys Arg Lys Val Cys Phe Phe Ala His Thr Pro Arg
165 170 175
Gin Leu Arg Val Phe His Ser Asn Asp Asn Ser Asn Lys Lys Lys Cys
180 185 190
Thr Asp Ile Ser Pro His Asn Asn Asn Asn Cys Cys Leu Val Cys His
195 200 205
Cys Ser Asn Ser Thr Arg Ser Pro Thr Ser Thr Leu Phe Gly Met Ser
210 215 220
His Phe Ser Pro Pro Leu Ser Pro Pro Ser Pro Ser Ser Pro Ser Met
225 230 235 240
Phe Glu Thr Asn Asn His His His Gly Val Val Lys Tyr Asn Lys Asp
245 250 255
Val Phe Ser Glu Leu Val Cys Ser Met Glu Gly Leu Asn Phe Asp Glu
260 265 270
Ala Ser Ser Leu Leu Ser Ala Ala Ser Lys Pro His His His Asn Asn
275 280 285
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Leu Ser Ser Trp Leu Asp Val Ser Lys Asp His Asn Gin Lys Gin Phe
290 295 300
Asn Thr Leu Asn Ser Pro Thr Ile Thr Ala Cys Gly Ser Phe Ser Asn
305 310 315 320
Asn Gly Asn Gly Gly Phe Leu Arg Ala Glu Asn Gly Vai Val Val Asp
325 330 335
Asp Val Ile Ala Pro Asp Leu Ala Trp Val Asn Glu Leu Leu Met
340 345 350
CA 2998211 2018-03-16
