Note: Descriptions are shown in the official language in which they were submitted.
CA 02205657 1997-05-20
WO 97/12~47 PCT/US96/16078
PI.aWT ST13:aROYI.--ACP '~-H ~ QI ~ ~ AND E~ ~vS TO
A-~ ST~R~ ;u ~ IN PI.aNT SEED OI~S
INq!Rb~u~: ~ lU~
Field of the Invention
This invention relates to the application of genetic
engineering techniques to plants. More specifically, the
invention relates to plant acyl-ACP thioesterase sequences
having substantial activity on C18: 0-ACP and methods for the
use of such sequences to increase 18:0 levels in plant seed
oils.
BACKGROUND
Fatty acids are organic acids having a hydrocarbon chain
of from about 4 to 24 carbons. Many different kinds of fatty
acids are known which differ from each other in chain length,
and in the presence, number and position of double bonds. In
cells, fatty acids typically exist in covalently bound forms,
the carboxyl portion being referred to as a fatty acyl group.
The chain length and degree of saturation of these molecules
is often depicted by the formula CX:Y, where "X~ indicates
2~ number of carbons and "Y" indicates number of double bands.
Fatty acyl groups are major components of many lipids,
and their long, non-polar hydrocarbon chain is responsible for
the water-insoluble nature of these lipid molecules. The type
of covalent linkage of the fatty acyl group to other factors
can vary. For example, in biosynthetic reactions they may be
covalently bound via a thioester linkage to an acyl carrier
protein (ACP) or to CoenzymeA (CoA), dep~n~1ng on the
particular enzymatic reaction. In waxes, fatty acyl groups
are linked to fatty alcohols via an ester linkage, and
triacylglycerols have three fatty acyl groups linked to a
glycerol molecule via an ester linkage.
The production of fatty acids in plants begins in the
plastid with the reaction between acetyl-CoA and malonyl-ACP to
produce butyryl-ACP catalyzed by the enzyme, ~-ketoacyl-ACP
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WO97/12047 PCT~S96/16078
synthase III. Elongation of acetyl-ACP to 16- and 18- carbon
fatty acids involves the cyclical action of the following
sequence of reactions: condensation with a two-carbon unit from
malonyl-ACP to form a ~-ketoacyl-ACP ($-ketoacyl-ACP synthase),
reduction of the keto-function to an alcohol (~-ketoacyl-ACP
reductase), dehydration to form an enoyl-ACP (~-hydroxyacyl-ACP
dehydrase), and finally reduction of the enoyl-ACP to form the
elongated saturated acyl-ACP (enoyl-ACP reductase). ~-ketoacyl-
ACP synthase I, catalyzes elongation up to palmitoyl-ACP
(C16:0), whereas ~-ketoacyl-ACP synthase II catalyzes the final
elongation to stearoyl-ACP (C18:0). The longest chain fatty
acids produced by the FAS are typically 18 carbons long. A
further fatty acid biochemical step occurring in the plastid is
the desaturation of stearoyl-ACP (C18:0) to form oleoyl-ACP
(C18:1) in a reaction catalyzed by a ~-9 desaturase, also often
referred to as a "stearoyl-ACP desaturase~ because of its high
activity toward stearate the 18 carbon acyl-ACP.
Carbon-chain elongation in the plastids can be t~rm;n~ted
by transfer of the acyl group to glycerol 3-phosphate, with the
resulting glycerolipid retained in the plastidial,
"prokaryotic~', lipid biosynthesis pathway. Alternatively,
specific thioesterases can intercept the prokaryotic pathway by
hydrolyzing the newly produced acyl-ACPs into free fatty acids
and ACP.
Subsequently, the free fatty acids are converted to fatty
acyl-CoA's in the plastid envelope and exported to the
cytoplasm. There, they are incorporated into the ''eukaryoticl'
lipid biosynthesis pathway in the endoplasmic reticulum which
is responsible for the formation of phospholipids,
triglycerides and other neutral lipids. Following transport
of ~atty acyl CoA's to the endoplasmic reticulum, subsequent
sequential steps for triglyceride production can occur. For
example, polyunsaturated fatty acyl groups such as linoleoyl
and ~-linolenoyl, are produced as the result of sequential
desaturation o oleoyl acyl groups by the action of membrane-
bound enzymes. Triglycerides are formed by action of the 1-,
2-, and 3- acyl-ACP transferase enzymes glycerol-3-phosphate
acyltransferase, lysophosphatidic acid acyltransferase and
diacylglycerol acyltransferase. The fatty acid composition of
,
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WO 97/12047 PCT/US96/16078
a plant cell is a reflection of the ~ree fa~ty acid pool and
the ~atty acids (fatty acyl groups) incorporated into
triglycerides as a result of the acyltransferase activities.
The fatty acid composition of an oil determines its
physical and chemical properties, and thus its uses. Plants,
especially plant species which synthesize large amounts o~
oils in plant seeds, are an important source of oils both for
edible and industrial uses. Various combinations of fatty
acids in the different positions in the triglyceride will
alter the properties of triglyceride. For example, if the
fatty acyl groups are mostly saturated fatty acids, then the
triglyceride will be solid at room temperature. In general,
however, vegetable oils tend to be mixtures of di~ferent
triglycerides. The triglyceride oil properties are therefore
a result of the combination of triglycerides which make up the
oil, which are in turn influenced by their respective fatty
acid compositions.
For example, cocoa-butter has certain desirable qualities
(mouth feel, sharp melting point, etc.) which are a function
of its triglyceride composition. ~ocoa-butter contains
a~oximately 24.4% palmitate (16:0), 3g.5~ stearate (18:0),
39.1~ oleate (18:1) and 2% linoleate (18:2). Thus, in cocoa
butter, palmitate-oleate-stearate (POS) comprises almost 50%
of triglyceride composition, with stearate-oleate-stearate
(SOS) and palmitate-oleate-palmitate (POP) comprising the
ma~or portion of the hAl~nce at 39% and 16%, respectively, of
the triglyceride composition. Other novel oils compositions
of interest might include trierucin (three erucic) or a
triglyceride with medium chain fatty acids in each position of
the triglyceride molecule.
Of particular interest also are triglyceride molecules in
which stearate is esterified at the sn-1 and sn-3 positions of
a triglyceride molecule and oleate is at sn-2. Vegetable oils
rich in such SOS (Stearate-Oleate-Stearate) molecules share
certain desirable qualities with cocoa butter yet have a
degree of additional hardness when blended with chocolate-
based fats. SOS - cont~; n; ng vegetable oils are currently
extracted from relatively expensive oilseeds from certain
trees grown in tropical areas such as Sal, Shea, and Illipe
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WO97/12047 PCT~S96/16078
trees from India, Africa, and Indonesia respectively. Cheaper
and more conveniently grown sources for SOS -type vegetable
oils are desirable.
In addition, vegetable oils rich in stearate fatty acid
content tend to be solid at room temperature. Such vegetable
fats can be used directly in shortenings, margarine and other
food "spread" products, obviating the need for chemical
hydrogenation. Hydrogenation is a process to convert
unsaturated ~atty acids in liquid oils to a saturated form
which in turn converts the oil into a solid fat useful in
margarine and shortening applications. The cost and any other
factors associated with chemical hydrogenation, such as the
production of trans fatty acids, can be avoided if the
vegetable oil is engineered to be stearate rich in the plant
seed.
Moreover, some plant tissues use 18 carbon fatty acids as
precursors to make other compounds. These include saturated
long chain fatty acids longer than 18 carbons in length.
Since very little stearate typically accumulates in common
oilseed crops, it may be necessary to increase stearate
accumulation if one wants to increase production of compounds
which depend upon supply of stearate fatty acids for
synthesis.
Thus, a variety of plant oils modifications are desired,
including alternative crop sources for certain oils products
and/or means to provide novel fatty acid compositions for
plant seed.
n~R~ u,~ ûF THE ~ K~.~
Figure 1. An amino acid sequence alignment of
representative Class I (FatA) and Class II (FatB)
thioesterases is provided. UcFatBl (SEQ ID NO:l) is a
California bay C12 thioesterase. CcFatBl (SEQ ID NO:2) is a
c~m~h~r C14 thioesterase. CpFatBl (SEQ ID NO:3) is a Cuphea
palustris C8 and C10 thioesterase. CpFatB2 (SEQ ID NO:4) is a
Cuphea palustris C14 thioesterase. GarmFatAl (SEQ ID NO:5) is
a mangosteen 18:1 thioesterase which also has considerable
activity on C18:0 acyl-ACP substrates. BrFatAl (SEQ ID NO:6)
is an 18:1 thioesterase from Brassica rapa (aka Brassica
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WO97/12047 PCT~S96/16078
campestris). Amino acid se~uences which are identical in all
of the represented thioesterases are indicated by bold
,~h~ln~.
Figure 2. The nucleic acid sequence and translated amino
acid sequence of a mangosteen Class I acyl-ACP thioesterase
cDNA clone, GARM FatAl (SEQ ID NO:7), are provided.
Figure 3. The nucleic acid sequence and translated amino
acid sequence of a mangosteen Class I acyl-ACP thioesterase
cDNA clone, GARM FatA2 (SEQ ID NO:8), are provided.
Figure ~ Data from fatty acid composition (weight
percent) analyses of T2 mature pooled seed from Brassica
plants transformed with napin/mangosteen TE construct pCGN5266
are provided.
Figure 5. Fatty acid composition data (weight percent)
from analyses of T2 half seeds from events 5266-LP004-2 and
5266-SP30021-29 are provided.
Figure 6 The nucleic acid sequence and translated amino
acid sequence of a Brassica napus BNDll stearoyl-ACP
desaturase cDNA clone are provided.
Figure 7. The nucleic acid sequence and translated amino
acid sequence of a Brassica napus BND9 stearoyl-ACP desaturase
cDNA clone are provided.
s~n~M~Y OF THE lNV~ 1 l~N
This invention relates to plant thioesterases,
specifically plant acyl-ACP thioesterases having substantial
activity on 18:0-ACP substrates such that the Cl8:0 content in
a target plant seed oil can be dramatically increased upon
expression of the plant acyl-ACP thioesterase in the seeds of
the target plant.
DNA constructs useful for the expression in a plant seed
cell of a plant acyl-ACP thioesterase having substantial
activity on 18:0-ACP substrates are described herein. ~uch
constructs will contain a DNA sequence encoding the plant
acyl-ACP thioesterase under the control of regulatory elements
capable of preferentially directing the expression of the
plant acyl-ACP thioesterase in seed tissue, as compared with
other plant tissues. At least one element of the DNA
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WO97/12047 PCT~S96/16078
construct will be heterologous to at least one other of the
elements, or when found in a plant cell, at least one element
will be heterologous to the plant cell. Of particular
interest for use in the constructs of the present invention is
a Class I acyl-ACP thioesterase, Garm FatAl, obtained from
mangosteen ( Garcinia mangifera) .
In yet a different embodiment, host plant cells
cont~;n;ng a first DNA construct capable of expressing a plant
acyl-ACP thioesterase having substantial activity on 18:0-ACP
substrates and a second DNA construct capable of expressing an
anti-sense stearoyl-acyl ACP desaturase sequence are desired.
Such a first DNA construct will contain a DNA sequence
encoding the plant acyl-ACP thioesterase of interest under the
control of regulatory elements capable of preferentially
directing the expression of the plant acyl-ACP thioesterase in
seed tissue as compared with other plant tissues when such a
construct is expressed in a transgenic plant. The second DNA
construct will contain a DNA sequence encoding a plant
stearoyl-acyl ACP desaturase element positioned in an anti-
sense orientation under the control of regulatory elementscapable of directing the transcription of the plant stearoyl-
acyl ACP desaturase in the plant host cell.
In a different aspect, this inve~ntion relates to methods
of using a DNA sequence encoding a plant acyl-ACP thioesterase
for modifying the composition of triglycerides, i.e., plant
oils, produced by a plant seed. Such modified oil
compositions may be obtained either by expression of the acyl-
ACP thioesterase alone, or by expression of the acyl-ACP
thioesterase in combination with a second construct which
provides for reduction of the level of the native stearoyl-ACP
desaturase of the target plant species. Plants and plant
parts, particularly seeds and oils extracted from such seeds,
having such a modified fatty acid composition are contemplated
herein.
Also provided in the present invention is a novel
antisense desaturase plant expression construct which provides
regions from two different classes of desaturase genes from a
multigene family. Expression of this construct in transgenic
plant seeds provides an improved method for increasing
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stearate levels in plant seeds by antisense methods. Higher
levels of stearate are obtained as a result of this improved
method, as well as higher percentages of first generation
transformants which display the desired increased stearate
phenotype.
DE~TT-Fn DESCRl~llu~. OF THE lNVI ~lU ~
By this invention, a mech~n;~m for the increased
accumulation o~ stearate (C18:0) in plants is provided. As
described herein, plant acyl-ACP thioesterases having
substantial activity toward 18:0-ACP substrates are involved
in the accumulation o~ stearate in at least some plant species
and may be expressed in transgenic plant seeds to provide an
increase in the levels of stearate fatty acids. Furth~rmore,
it is demonstrated that such 18:0-ACP thioesterases are
members of the Class I group of plant acyl-ACP thioesterases.
Class I type thioesterases have been found in essentially
all plant sources examined to date, and are suggested to be
essential "housekeeping" enzymes (Jones et al. The Plant Cell
(1995) 7:359-371) required for membrane biosynthesis. Class I
type thioesterases have previously been shown to have activity
primarily on 18:1 acyl-ACP substrates, with some lesser amount
of activity on 16:0 substrates, and only little or no activity
on 18:0 su~strates. Examples of Class I thioesterases from
safflower, Cuphea hoo~eriana and Brassica rapa (campestrls),
which have activity primarily on 18:1-ACP substrate, have been
described (WO 92/20236 and WO 94/10288). Other 18:1
thioesterases have been reported in Arabidopsis thaliana
(Dormann et al. (1995) Arch. Biochem. Biophys. 316:612-618),
Brasslca napus (Loader et al. (1993) Plant Mol. Biol. 23:769-
778) and cor; ~n~ (Dormann et al. (1994) Biochem. Biophys.
Acta 1212:134-136). A Class I thioesterase from soybean (WO
92/11373) was reported to provide 10- and 96-fold increases in
16:0-ACP and 18:1-ACP activity upon expression in E. coli, and
a smaller (3-4 fold) increase in 18:0-ACP activity.
A second class of plant thioesterases, Class II tor FatB)
thioesterases, include enzymes that primarily utilize fatty
acids with shorter chain-lengths, from C8:0 to C14:0 (medium
chain fatty acids) as well as C16:0. Class II thioesterases
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preferably catalyze the hydrolysis of substrates cont~;n;ng
saturated fatty acids. Class II (or FatB) thioesterase genes
have been isolated from California Bay, elm, Cuphea
hookeriana, Cuphea palustris, Cuphea lanceolata, nutmeg,
Arabidopsis thaliana, mango, leek and c~mrh~r. A FatB
thioesterase gene was also identified in mangosteen in the
gene isolation experiments described herein. Expression of
the FatB gene in E. coli ~emo~ctrated hydrolysis activity
primarily on 16:0 substrates.
In the following examples, isolation of genes encoding
Class I acyl-ACP thioesterases from mangosteen is described.
Two different types of Class I thioesterase genes were
discovered. One mangosteen thioesterase gene, (GarmFatA2), is
shown herein to be an 18:1-ACP specific Class I thioesterase
similar to those discovered previously in other plant tissues.
However, a second type of mangosteen Class I thioesterase
gene, represented by clone GarmFatAl, was discovered which
~monctrates 18:1-ACP thioesterase activity (100-fold increase
upon expression in E. coli), but also ~mon~trates substantial
activity on 18:0-ACP substrates. The 18:0 activity of
GarmFatAl is a~L~imately 25% of the 18:1 activity, whereas
in most Class I thioesterases analyzed to date, the 18:1
activity is highly pre~m;nAnt, with activity on 16:0 and 18:0
substrates detectable at less than 5% of the 18:1 activity
levels. Additionally, most plant Class I thioesterases
~m~ctrate approximately equal activity on 16:0 and 18:0
substrates, whereas the GarmFatAl mangosteen thioesterase of
the present invention ~m~.ctrates a clear preference for
hydrolysis of 18:0 substrates over 16:0 substrates.
The novel mangosteen GarmFatAl thioesterase clone will
thus be useful for production of the 18:1/18:0 thioesterase in
host cells, and particularly for expression in plant seed
cells for modification of TAG fatty acid composition to
provide increased levels of C18:0 fatty acyl groups.
Furth~rmore, the mangosteen GarmFatAl clone will be useful in
plant genetic engineering application in conjunction with
plants cont~;n;ng elevated levels of C18:0 (stearate) fatty
acids. Such plants may be obtained by antisense gene
regulation of stearoyl-ACP desaturase in Brassica seeds as
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WO97112047 PCT~S96/16078
described by Knutzon et al. ~Proc. Nat. Acad. Sci. (1992)
89:2624-2628), and may also be obtained by co-suppression
using sense expression constructs of the stearoyl-ACP
desaturase gene, or by conventional mutation and plant
breeding programs.
The det~rmin~tion that Class I type plant thioesterases
are active in the in vivo accumulation of 18:0 fatty acids
suggests several possibilities for additional plant sources of
genes which encode thioesterase proteins having substantial
activity on 18:0 substrates. Stearate is found in some
natural plant species, particularly tropical plant species, in
abundance. For example, other species in the genus Garcinia
~c~llmlll~te triglycerides cont~inin~ stearate in their seeds,
e.g., kokum. Other natural plant source of Cl8:0 fatty acids
include plants of the Mangifera family: e.g., mango,
Butyrospermum (shea), Pentadesma (tallow tree), Illipe (illipe
butter), Theobroma (cocoa), Simarou~a (tree of paradise) and
Shorea ( sal).
A plant acyl-ACP thioesterase DNA sequence useful for
alteration of stearate levels as described herein encodes ~or
amino acids, in the ~orm o~ a protein, polypeptide or peptide
fragment, which amino acids ~mnnctrate substantial activity
on 18:0 acyl-ACP substrates to form 18:0 free fatty acid
(i.e., stearate) under plant enzyme reactive conditions. By
"enzyme reactive conditions" is meant that any necessary
conditions are available in an environment (i.e., such factors
as temperature, pH, lack of inhibiting substances) which will
permit the enzyme to function.
One skilled in the art will readily recognize that
antibody preparations, nucleic acid probes (DNA and RNA) and
the like may be prepared and used to screen and recover
"homologous" or "related" Class I acyl-ACP thioesterases from
a variety of plant sources. Typically, nucleic acid probes
are labeled to allow detection, preferably with radioactivity
although enzymes or other methods may also be used. As plant
thioesterase genes are known to contain extensive sequence
homology, various DNA screening methods such as PCR or DNA
hybridization methods may be used to identify related Class I
thioesterases. Plant thioesterase genes show at least
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WO97/12047 PCT~S96/16078
approximately 50% sequence identity at the nucleic acid level.
Between members of Class I thioesterases the percentage of
sequence identity is as high as 70-80%, and Class II
thioesterases typically demonstrate sequence identity of at
least 60%.
Thus, in order to obtain additional stearoyl-ACP
thioesterases, a genomic or other appropriate library prepared
from the candidate plant source of interest is probed with
conserved sequences from one or more Class I plant
thioesterases to identify homologously related sequences.
Positive clones are then analyzed by restriction enzyme
digestion and/or sequencing. When a genomic library is used,
one or more sequences may be identified providing both the
coding region, as well as the transcriptional regulatory
elements of the thioesterase gene from such plant source.
Probes can also be considerably shorter than the entire
se~uence. Oligonucletides may be used, for example, but
should be at least about 10, preferably at least about 15,
more preferably at least 20 nucleotides in length. When
shorter length regions are used for comparison, a higher
degree of sequence identity is required than for longer
sequences. Shorter probes are often particularly useful for
polymerase chain reactions (PCR), especially when highly
conserved sequences can be identified. (See, Gould, et al.,
PNAS USA (1989) 86:1934-1938.)
When longer nucleic acid fragments are employed (>100 bp)
as probes, especially when using complete or large cDNA
sequences, one can still screen with moderately high
stringencies (for example using 50% formamide at 37C with
m;n;mAl w~h;ng) in order to obtain signal from the target
sample with 20-50% deviation, i.e., homologous sequences.
(For additional information regarding screening techniques see
Beltz, et al. Methods in Enzymology (1983) 100:266-285.).
Once the related acyl-ACP thioesterase sequence is
obtained, expression of the plant acyl-ACP thioesterase in a
host cell may be obtained to further characterize the activity
of the thioesterase. In this manner, additional plant acyl-
ACP thioesterases having substantial activity on stearoyl-ACP
may be identified for use in the methods of the present
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WO97/12047 PCT~S96/16078
invention. As demonstrated herein by increasing the amount of
stearoyl-ACP preferring thioesterase available to the plant
FAS complex, an increased percentage o~ stearate may be
, - provided. Additionally, by decreasing the amount o~ stearoyl-
ACP desaturase available to the plant FAS complex in
conjunction with an increase o~ the amount o~ stearoyl-ACP
thioesterase available, a more marked increased percentage of
stearate may be obtA; ne~ .
The nucleic acid se~uences which encode plant stearoyl-
ACP thioesterases may be used in various constructs, ~or
example, as probes to obtain further sequences.
Alternatively, these sequences may be used in conjunction with
appropriate regulatory sequences to increase levels of the
respective thioesterase of interest in a host cell for
recovery or study o~ the enzyme in vi tro or in vivo or to
decrease levels o~ the respective thioesterase o~ interest ~or
some applications when the host cell is a plant entity,
including plant cells, plant parts (including but not limited
to seeds, cuttings or tissues) and plants.
A nucleic acid sequence encoding a plant acyl-ACP
thioesterase of this invention which has substantial activity
on 18:0 acyl groups may include genomic, cDNA or mRNA
sequence. A cDNA sequence may or may not contain pre-
processing sequences, such as transit peptide sequences.
Transit peptide sequences ~acilitate the delivery o~ the
protein to a given organelle and are cleaved from the amino
acid moiety upon entry into the organelle, releasing the
~mature~ sequence. The use o~ the precursor plant acyl-ACP
thioesterase DNA sequence cont~;ning the transit peptide and
mature protein encoding sequences is preferred in plant cell
expression cassettes. Other plastid transit peptide
sequences, such as a transit peptide o~ seed ACP, may be
employed to translocate the plant stearoyl-ACP thioesterase of
this invention to various organelles o~ interest.
Furth~rmnre, as discussed above the complete genomic
sequence of the plant stearoyl-ACP thioesterase may be
obtained by the screening o~ a genomic library with a probe,
such as a cDNA probe, and isolating those sequences which
regulate expression in seed tissue. In this m~nn~r, the
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WO97/12047 PCT~S96/16078
transcription and translation initiation regions, introns,
and/or transcript t~rm-n~tion regions of the plant stearoyl-
ACP thioesterase may be obt~' ne~ for use in a variety of DNA
constructs, with or without the thioesterase structural gene.
Once the desired plant stearoyl-ACP thioesterase nucleic
acid sequence is obtained, it may be manipulated in a variety
of ways. Where the sequence involves non-coding flanking
regions, the flanking regions may be subjected to resection,
mutagenesis, etc. Thus, transitions, transversions,
deletions, and insertions may be performed on the naturally
occurring sequence. In addition, all or part of the sequence
may be synthesized. In the structural gene, one or more
codons may be modified to provide for a modified amino acid
sequence, or one or more codon mutations may be introduced to
provide for a convenient restriction site or other purpose
involved with construction or expression. The structural gene
may be further modified by employing synthetic adapters,
linkers to introduce one or more convenient restriction sites,
or the like.
The nucleic acid or amino acid sequences encoding a plant
stearoyl-ACP thioesterase of this invention may be combined
with other non-native, or "heterologous~, sequences in a
variety of ways. By "heterologous" sequences is meant any
sequence which is not naturally found joined to the plant
stearoyl-ACP thioesterase, including, for example,
combinations of nucleic acid sequences from the same plant
which are not naturally found ~oined together.
The DNA sequence encoding a plant stearoyl-ACP
thioesterase of this invention may be employed in conjunction
with all or part of the gene sequences normally associated
with the thioesterase. In its component parts, a DNA
thioesterase encoding sequence is combined in a DNA construct
having, in the 5I to 3' direction of transcription, a
transcription initiation control region capable o~ promoting
transcription and translation in a host cell, the DNA sequence
encoding plant stearoyl-ACP thioesterase and a transcription
and translation t~rm;n~tion region.
Potential host cells include both prokaryotic and
eukaryotic cells. A host cell may be unicellular or found in
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WO97/12047 PCT~S96/16078
a multicellular differentiated or undifferentiated organism
dep~n~; ng upon the intended use. Cells of this invention may
be distin~l;che~ by having a plant stearoyl-ACP thioesterase
foreign to the wild-type cell present therein, for example, by
having a recombinant nucleic acid construct encoding a plant
stearoyl-ACP thioesterase therein.
Depen~; n~ upon the host, the regulatory regions will
vary, including regions from viral, plasmid or chromosomal
genes, or the like. For expression in prokaryotic or
eukaryotic microorg~n; Sm~, particularly unicellular hosts, a
wide variety of constitutive or regulatable promoters may be
employed. Expression in a microorganism can provide a ready
source of the plant enzyme.
For the most part, the constructs will involve regulatory
regions functional in plants which provide ~or modi~ied
production of plant stearoyl-ACP thioesterase, and
modification of the fatty acid composition. The open re~;ng
frame, coding ~or the plant stearoyl-ACP thioesterase or
functional ~ragment thereof will be joined at its 5~ end to a
transcription initiation regulatory region such as the wild-
type sequence naturally found 5' upstream to the thioesterase
structural gene, or to a heterologous regulatory region from a
gene naturally expressed in plant tissues. Examples of useful
plant regulatory gene regions include those from T-DNA genes,
such as nopaline or octopine synthase, plant virus genes, such
as CaMV 35S, or from native plant genes.
For such applications when 5' upstream non-coding regions
are obtained from other genes regulated during seed
maturation, those preferentially expressed in plant embryo
tissue, such as ACP and napin-derived transcription initiation
control regions, are desired. Such "seed-specific promoters~
may be obt~;ne~ and used in accordance with the te~hlngs of
USPN 5,4~0,034 having a title "Seed-Specific Transcriptional
Regulation". Transcription initiation regions which are
preferentially expressed in seed tissue, i.e., which are
undetectable in other plant parts, are considered desirable
for fatty acid modifications in order to min;m;ze any
disruptive or adverse effects of the gene product.
CA 0220~6~7 1997-0~-20
WO97112047 PCT~S96/16078
Regulatory transcript termination regions may be provided
in DNA constructs of this invention as well. Transcript
t~rm-n~tion regions may be provided by the DNA sequence
encoding the plant stearoyl-ACP thioesterase or a convenient
transcription t~rm;n~tion region derived from a different gene
source, for example, the transcript tPrm-n~tion region which
is naturally associated with the transcript initiation region.
Where the transcript t~rm;n~tion region is from a different
gene source, it will contain at least about 0.5 kb, preferably
about 1-3 kb of sequence 3I to the structural gene ~rom which
the t~rm;n~tion region is derived.
Plant expression or transcription constructs having a
plant stearoyl-ACP thioesterase as the DNA sequence of
interest for increased or decreased expression thereof may be
employed with a wide variety of plant life, particularly,
plant life involved in the production of vegetable oils for
edible and industrial uses. Most especially preferred are
temperate oilseed crops. Plants of interest include, but are
not limited to, rapeseed (Canola and High Erucic Acid
varieties), sunflower, safflower, cotton, Cuphea, soybean,
peanut, coconut and oil palms, and corn. Depen~;n~ on the
method for introducing the recombinant constructs into the
host cell, other DNA se~uences may be required. Importantly,
this invention is applicable to dicotyledons and
monocotyledons species alike and will be readily applicable to
new and/or improved transformation and regulation techniques.
The method of transformation is not critical to the
instant invention; various methods of plant transformation are
currently available. As newer methods are available to
transform crops, they may be directly applied her~lm~er. For
example, many plant species naturally susceptible to
Agrobacterium infection may be successfully transformed via
tripartite or binary vector methods of Agrobacterium mediated
transformation. In addition, techniques of microinjection,
DNA particle bombardment, electroporation have been developed
which allow for the transformation of various monocot and
dicot plant species.
In de~eloping the DNA construct, the various components
of the construct or fragments thereof will normally be
14
CA 0220~6~7 1997-0~-20
WO97/12047 PCT~S96/16078
inserted into a convenient cloning vector which is capable of
replication in a bacterial host, e.g., E. coli. Numerous
vectors exist that have been described in the literature.
After each cloning, the plasmid may be isolated and subjected
to further manipulation, such as restriction, insertion of new
fragments, ligation, deletion, insertion, resection, etc., so
as to tailor the components of the desired sequence. Once the
construct has been completed, it may then be transferred to an
appropriate vector for further manipulation in accordance with
the manner of transformation of the host cell.
Normally, included with the DNA construct will be a
structural gene having the necessary regulatory regions for
expression in a host and providing for selection of
transformant cells. The gene may provide for resistance to a
cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc.,
complementation providing prototrophy to an auxotrophic host,
viral ;mmlm;ty or the like. Dep~n~;ng upon the number of
different host species the expression construct or components
thereof are introduced, one or more markers may be employed,
where different conditions for selection are used for the
different hosts. A number of markers have been developed for
use for selection of transformed plant cells, such as those
which provide resistance to various antibiotics, herbicides,
or the like. The particular marker employed is not essential
to this invention, one or another marker being preferred
dep~n~;ng on the particular host and the manner of
construction.
As mentioned above, the m~nne~ in which the DNA construct
is introduced into the plant host is not critical to this
invention. Any method which provides for efficient
transformation may be employed. Various methods for plant
cell transformation include the use of Ti- or Ri-plasmids,
microinjection, electroporation, DNA particle bombardment,
liposome fusion, DNA bombardment or the like. In many
instances, it will be desirable to have the construct bordered
on one or both sides by T-DNA, particularly having the left
and right borders, more particularly the right border. This
is particularly useful when the construct uses A. tumefaciens
CA 0220~6~7 1997-0~-20
W097/l2047 PCT~S96/16078
or A. rhizogenes as a mode for transformation, although the T-
DNA borders may find use with other modes of transformation.
Once a transgenic plant is obtained which is capable of
producing seed having a modified fatty acid composition,
traditional plant breeding techniques, including methods of
mutagensis, may be employed to further manipulate the fatty
acid composition. Alternatively, additional foreign fatty
acid modifying DNA sequence may be introduced via genetic
engineering to further manipulate the fatty acid composition.
One may choose to provide for the transcription or
transcription and translation of one or more other sequences
of interest in concert with the expression of a plant
stearoyl-ACP thioesterase in a plant host cell. In
particular, the reduced expression of stearoyl-ACP desaturase
in combination with expression of a plant stearoyl-ACP
thioesterase may be preferred in some applications.
Stearoyl-ACP thioesterases may also be used in
combination with other thioesterase genes with differing
specificities. For example, a transgenic oilseed crop
expressing both a lauroyl-ACP thioesterase (WO 92/20236) and a
stearoyl-ACP thioesterase during seed development will produce
an oil enriched in both lauric and stearic acids. Similarly,
a transgenic oilseed crop expressing both a palmitoyl-ACP
thioesterase (WO 95/13390) and a stearoyl-ACP thioesterase
during seed development will produce an oil enriched in both
palmitic and stearic acids. These thioesterase gene
constructs may be linked to each other in the genome of the
transgenic plant or may be unlinked. Conversely the
thioesterase genes may be combined in the sa-m-e transgenic
plant by generating plants with one or the other thioesterase
gene and subsequently crossing two plants, one of each type.
By selecting parents for such crosses, it is possible to
further manipulate the relative ratios of desired fatty acids
in a seed oil.
When one wishes to provide a plant transformed for the
c~mh;nP~ effect of more than one nucleic acid sequence of
interest, typically a separate nucleic acid construct will be
provided for each. The constructs, as described above contain
transcriptional or transcriptional and translational
16
CA 0220~6~7 1997-0~-20
WO97/12047 PCT~S96/16078
regulatory control regions. The constructs may be introduced
into the host cells by the same or different methods,
including the introduction of such a trait by crossing
transgenic plants via traditional plant breeding methods, so
long as the resulting product is a plant having both
characteristics integrated into its genome.
By decreasing the amount of stearoyl-ACP desaturase, an
increased percentage of saturated fatty acids is provided.
Using anti-sense, transwitch, ribozyme or some other stearoyl-
ACP desaturase reducing technology, a decrease in the amountof stearoyl-ACP desaturase available to the plant cell is
produced, resulting in a higher percentage of saturates such
as one or more of laurate (Cl2:0), myristate (C14:0),
palmitate (Cl6:0), stearate (Cl8:0), arachidate (C20:0),
behenate (C22:0) and lignocerate (C24:0). In rapeseed r~
stearoyl-ACP desaturase results in increased stearate levels
and total saturates (Knutzon et al . ~1992) Proc. Nat. Acad.
Sci 89:2264-2628). A novel construct is also provided herein
which may be used in antisense reduction of stearoyl-ACP
desaturase to increase both the percentage of stearate which
may be obt~;ne~ and the percentage of primary transformants
expressing the increased stearate trait.
Of special interest is the produ~tion of triglycerides
having increased levels of stearate or palmitate and stearate.
In addition, the production of a variety of ranges of such
saturates is desired. Thus, plant cells having lower and
higher levels of stearate fatty acids are contemplated. For
example, fatty acid compositions, including oils, having a 10%
level of stearate as well as compositions designed to have up
to an appropriate 60~ level of stearate or other such modified
fatty acid(s) composition are contemplated.
Oils with increased percentages of stearate, are desired.
Increased stearate percentages (by weight) ranging from native
levels to increases of up to 25 fold are described. By
manipulation of various aspects of the DNA constructs (e.g.,
choice of promoters, number of copies, etc.) and traditional
breeding methods, one skilled in the art may achieve even
greater levels of stearate. By combination of the plant
stearoyl-ACP desaturase sequence in combination with the
CA 0220~6~7 1997-0~-20
WO97~12047 PCT~S96/16078
expression of a plant stearoyl-ACP thioesterase in seed
tissue, an increased percentage o~ stearate can be achieved in
rapeseed and other plant species. DNA sequence of C.
tinctorius stearoyl-ACP desaturase gene, as well as DNA
sequences of stearoyl-ACP desaturase genes from a Ricinus, a
Brassica and a Simmnn~.sia plant are found in WO 91/13972.
The invention now being generally described, it will be
more readily understood by reference to the following examples
which are included for purposes of illustration only and are
not intended to limit the present invention.
EXAMPLES
ExamPle 1 Mangosteen Thioesterase Gene Sequences
A cDNA bank is prepared from seeds extracted from mature
mangosteen fruit using the methods as described in Stratagene
Zap cDNA synthesis kit (Stratagene; La Jolla, CA). Oil
analysis of the mangosteen tissues used for RNA isolation
reveals 18:0 levels o~ approximately 50%. Oil analysis of
seeds from less mature mangosteen fruit reveals 18:0 levels of
20-40%. Total RNA is isolated from the mangosteen seeds by
modifying the CTAB DNA isolation method of Webb and Knapp
(Plant Mol. Biol. Reporter (1990) 8:180-195). Buffers include:
REC: 50 mM TrisCl pH 9, 0.7 M NaCl, 10 mM EDTA pH8,
0.5% CTAB.
REC+: Add B-mercaptoethanol to 1% i mme~i ately prior
to use.
RECP: 50 mM TrisCl pH9, 10 mM EDTA pH8, and 0.5%
CTAB.
RECP+: Add B-mercaptoethanol to 1% i mmP~i ately prior
to use.
For extraction of 1 g of tissue, 10ml of REC+ and 0.5 g
of PVPP is added to tissue that has been ground in liquid
nitrogen and homogenized. The homogenized material is
centrifuged for 10 min at 12000 rpm. The supernatant is poured
through miracloth onto 3ml cold chloroform and homogenized
CA 02205657 1997-05-20
W O 97/12047 PCT~US96/16078
again. After centrifugation, 12,000 RPM for 10 min, the upper
phase is taken and its volume determ; n~ . An equal volume of
RECP+ is added and the mixture is allowed to stand ~or 20 min.
at room temperature. The material is centrifuged ~or 20 min.
at 10,000 rpm twice and the supernatant is discarded after
c each spin. The pellet is dissolved in 0.4 ml of 1 M NaCl
(DEPC) and extracted with an equal volume of
phenol/chloroform. Following ethanol precipitation, the
pellet is dissolved in 1 ml o~ DEPC water.
Brie~ly, the cloning method for cDNA synthesis is as
follows. First strand cDNA synthesis is according to
Stratagene Instruction M~n~ with some modi~ications
according to Robinson, et al. (Methods in Molecular and
Cellular Blology (1992) 3:118-127). In particular,
approximately 57~g of LiCl precipitated total RNA was used
instead of 5~g o~ poly(A)+ RNA and the reaction was incubated
at 45C rather than 37C for 1 hour.
Probes for library screening are prepared by PCR from
mangosteen cDNA using oligonucleotides to conserved plant
acyl-ACP thioesterase regions. Probe Garm 2 and Garm 106 are
prepared using the following oligonucleotides. The nucleotide
base codes ~or the below oligonucleotides are as ~ollows:
A = ~ni n~ C = cytosine
25 T = thymine U = uracil
G = gll~n;ne S = guanine or cytosine
K = guanine or thymine W = ~n;ne or thymine
M = ~n;ne or cytosine R = adenine or guanine
Y = cytosine or thymine
30 B = gl~n;n~, cytosine or thymine
H = ~n;ne, cytosine or thymine
N = ~nine, cytosine, gli~n;n~ or thymine
Garm 2
4874: 5' CUACUACUACUASYNTVNGYNATGATGAA 3' (SEQ ID NO:9)
4875: 5' CAUCAUCAUCAURCAYTCNCKNCKRTANTC 3' (SEQ ID NO:10)
Primer 4874 is a sense primer designed to correspond to
possible encoding sequences ~or conserved peptide
CA 0220~6~7 l997-0~-20
WO97/12047 PCT~S96/16078
V/L/A W/S/Y V/A M M N, where the one letter amino acid code is
used and a slash between amino acids indicates more than one
amino acid is possible for that position. Primer 4875 is an
antisense primer designed to correspond to possible encoding
sequences for peptide D/E Y R R E C.
Garm lO6
5424: 5' AUGGAGAUCUCUGAWCRBTAYCCTAMHTGGGGWGA 3' (SEQ ID NO:ll)
5577: 5l ACGCGUACUAGUTTNKKNCKCCAYTCNGT 3' (SEQ ID NO:12)
Primer 5424 is a sense primer designed to correspond to
possible encoding sequences for peptide E/D H/R Y P K/T W G D.
Primer 5577 is an antisense primer designed to correspond to
possible encoding sequences for peptide T E W R K/P K.
The DNA fragments resulting from the above reactions are
amplified for use as probes by cloning or by further PCR and
radiolabeled by random or specific priming.
Approximately 800,000 plaques are plated according to
manufacturer's directions. For screening, plaque filters are
prehybridized at room temperature in 50% formamide, 5X SSC,
lOX Denhardt's, O.1% (w/v) SDS, 5mM Na2EDTA, O.lmg/ml
denatured salmon sperm DNA. Hybridization with a mixture of
the Garm 2 and Garm 106 probes is conducted at room
temperature in the same buffer as above with added 10%(w/v)
dextran sulfate and probe. Plaque purification and phagemid
excision were conducted as described in Stratagene Zap cDNA
Synthesis Kit instructions.
A~oximately 90 acyl-ACP thioesterase clones were
identified and sorted as to thioesterase type by DNA
sequencing and/or PCR analysis. Of the analyzed clones, at
least 28 were Class I (FatA) types, and 59 were Class II
(FatB) types. Two subclasses o~ FatA type clones were
observed, the most prominent type is termed GarmFatAl and the
single clone of the second subclass is termed GarmFatA2. DNA
and translated amino acid sequence o~ ~armFatAl clone C14-4
(pCGN5252) (SEQ ID NO:7) is presented in Figure 2. DNA
sequence and translated amino acid sequence of the FatA2 clone
Cl4-3 (SEQ ID NO:8) is presented in Figure 3.
Constructs for expression of the Figure 2 Garm FatAl
clone in E. coli are prepared as follows. Restriction sites
CA 02205657 l997-05-20
W097/~2047 PCT~S96/16078
are inserted by PCR mutagenesis at amino acid 49 (SacI), which
is near the presumed mature protein amino t~rm; n~.s, and
following the stop codon for the protein encoding region
(BamHI) . The mature protein encoding region is inserted as a
SacI/BamHI ~ragment into pBC SK (Stratagene; La Jolla, CA)
resulting in pCGN5247, which may be used to provide for
expression of the mangosteen thioesterase as a lacZ fusion
protein.
Results o~ thioesterase activity assays on mangosteen
Class I thioesterase clone GarmFatA1 using 16:0, 18:0 and 18:1
acyl-ACP substrates are shown below.
Acyl-ACP Thioesterase activity (cpm/min)
16:0 18:0 18:1
Control 1400 3100 1733
GarmFatA1 4366 23916 87366
The GarmFatA1 cone ~m~strates preferential activity on C18:1
acyl-ACP substrate, and also ~m~nstrates substantial activity
(approximately Z5% of the 18:1 activity) on C18:0 acyl-ACP
substrates. Only a small increase in Cl6:0 activity over
activity in control cells is observed, and the 16:0 activity
represents only approximately 3~ of the 18:1 activity.
Expression of GarmFatA2 thioesterase in E. coli and assay
of the resultant thioesterase activity ~m~nstrates that C18:1
is highly preferred as the acyl-ACP substrate. The
thioesterase activity on 16:0 and 18:0 acyl-ACP substrates are
approximately equal and represent less than 5% of the observed
18:1 activity.
Expression of Class II type mangosteen thioesterase
clones in E. coli ~monstrates that 16:0 is highly preferred
over other acyl-ACP substrates.
Exam~le 2 Plant Transformation methods
A. Agrobacterium-mediated Transformation
Methods which may be used for Agrobacterium-mediated
transformation of Brassica are described by Radke et al.
21
CA 0220~6~7 l997-0~-20
W097/12047 PCT~S96/16078
(Theor. Appl. Genet. (1988) 75:685-694; Plant Cell Reports
(1992) 11: 499-505).
Transgenic Arabidopsis thaliana plants may be obt~i n~ by
Agrobacterium-mediated transformation as described by
Valverkens et al., (Proc. Nat. Acad. Sci. (1988) 85:5536-
5540).
B. Particle Bombardment
DNA sequences of interest may be introduced as expression
cassettes, comprising at least a promoter region, a gene of
interest, and a t~rm;n~tion region, into a plant genome via
particle bombardment as described for example in European
Patent Application 332 855 and in co-p~n~; ng application USSN
07/225,332, filed July 27, 1988.
Briefly, tungsten or gold particles of a size ranging
from O.5mM-3mM are coated with DNA of an expression cassette.
This DNA may be in the form of an aqueous mixture or a dry
DNA/particle precipitate. Tissue used as the target for
hnmhArdment may be from cotyle~on~ry explants, shoot
meristems, immature leaflets, or anthers.
The bombardment of the tissue with the DNA-coated
particles is carried out using a Biolistics~O particle gun
(Dupont; Wilmington, DE). The particles are placed in the
barrel at variable distances ranging from lcm-14cm from the
barrel mouth. The tissue to be bombarded is placed beneath
the stopping plate; testing is performed on the tissue at
distances up to 2Ocm. At the moment of discharge, the tissue
is protected by a nylon net or a combination of nylon nets
with mesh ranging from lOmM to 300mM.
Following bombardment, plants may be regenerated
following the method of Atreya, et al., (Plant Science Letters
(1984) 34:379-383). Briefly, embryo axis tissue or cotyledon
segments are placed on MS medium (Murashige and Skoog, Physio.
Plant. (1962) 15:473) (MS plus 2.0 mg/l 6-benzyl~n~ne (BA)
for the cotyledon segments) and incubated in the dark for 1
week at 25 + 2C and are subsequently transferred to
continuous cool white fluorescent light (6.8 W/m2). On the
10th day of culture, the plantlets are transferred to pots
cont~;n;ng sterile soil, are kept in the shade for 3-5 days
are and finally moved to greenhouse.
CA 02205657 l997-05-20
WO97/12047 PCT~S96/16078
The putative transgenic shoots are rooted. Integration
of exogenous DNA into the plant genome may be confirmed by
various methods know to those skilled in the art.
Exam~le 3 Construct ~or Expression of Garm FatA1
Thioesterase in Plant Seeds
- The napin expression cassette, pCGN1808, is described in
USPN 5,420,034 which is incorporated herein by reference.
pCGN1808 is modi~ied to contain flanking restriction sites to
allow movement of only the expression se~uences and not the
antibiotic resistance marker to binary vectors such as
pCGN1557 (McBride and Summerfelt (1990) Plant Mol. Biol.
14:269-276). Synthetic oligonucleotides cont~inin~ KpnI, NotI
and HindIII restriction sites are ~nn~l ed and ligated at the
unique HindIII site of pCGN1808, such that only one HindIII
site is recovered. The resulting plasmid, pCGN3200 contains
unique HindIII, NotI and KpnI restriction sites at the 3'-end
of the napin 3'-regulatory sequences as confirmed by se~uence
analysis.
The majority of the napin expression cassette is
subcloned from pCGN3200 by digestion with ~in~TTI and SacI and
ligation to ~in~TTI and SacI digested pIC19R (Marsh, et al.
(1984) Gene 32:481-485) to make pCGN3212. The extreme 5'-
sequences of the napin promoter region are reconstructed by
PCR using pCGN3200 as a template and two primers flanking the
SacI site and the junction of the napin 5'-promoter and the
pUC backbone of pCGN3200 from the pCGN1808 construct. The
forward primer contains ClaI, HindIII, NotI, and KpnI
restriction sites as well as nucleotides 408-423 of the napin
5'-sequence (from the EcoRV site) and the reverse primer
contains the complement to napin sequences 718-739 which
include the unique SacI site in the 5'-promoter. The PCR was
performed using in a Perkin Elmer/Cetus t~rm~cycler according
to manufacturer's specifications. The PCR fragment is
subcloned as a blunt-ended fragment into pUC8 (Vieira and
Messing (1982) Gene 19:259-268) digested with HincII to give
pCGN3217. Sequence of pCGN3217 across the napin insert
verifies that no improper nucleotides were introduced by PCR.
The napin 5-sequences in pCGN3217 are ligated to the r~m~;n~e~
-
CA 0220~6~7 1997-0~-20
WO97/12047 PCT~S96/16078
of the napin expression cassette by digestion with ~laI and
SacI and ligation to pCGN3212 digested with ClaI and SacI.
The resulting expression cassette pCGN3221, is digested with
HindIII and the napin expression sequences are gel purified
away and ligated to pIC20H (Marsh, supra) digested with
~in~TTT. The final expression cassette is pCGN3223, which
contains in an ampicillin resistant background, essentially
identical 1.725 napin 5' and 1.265 3' regulatory sequences as
found in pCGN1808. The regulatory regions are flanked with
HindIII, NotI and KpnI restriction sites and unique SalI,
BglII, PstI, and XhoI cloning sites are located between the 5'
and 3' noncoding regions. pCGN3223 is also descri~ed in WO
92/20236 which is incorporated herein by reference.
Mangosteen acyl-ACP thioesterase clone pCGN5252 (Garm
FatAl) is digested with PstI and X~oI and cloned into
PstI/X~oI digested pCGN3223 resulting in pCGN5253 cont~;n;n~
the mangosteen thioesterase encoding region positioned for
transcriptional control from the napin promoter. The PstI
site in pCGN5252 is located in the cloning vector at the 5'
end of the cDNA clone, and the XhoI site is located in the 3'
untranslated region at nucleotides 1233-1238.
For Agrobacterium-mediated plant transformation, pCGN5253
is digested with Asp718 and cloned into Asp718 digested
pCGN1578, a binary vector for plant transformation, resulting
in clone pCGN5255. To insert a repeat of the napin 5~/garm
FatAl/napin 3' into the binary vector, pCGN5253 is digested
with Asp718 and the napin 5'/Garm FatAl/napin 3' region gel
purified. The gel purified fragment is then ligated into
Asp718 digested pCGN5255, resulting in clone pCGN5266
pCGN5255 is transformed into Agrobacterium tumefaciens
strain EHA101 and used to transform a high oleic acid line of
Brassica napus. pCGN5266 is transformed into Agrobacterium
tumefaciens strain EHA101 and used to transform B. napus
variety Quantum (SP30021) and a low linolenic B. napus line,
Q04 (LP004).
24
CA 0220~6~7 1997-0~-20
WO97/12047 PCT~S96/16078
Exam~le 4 Analysis of Transgenic Plants Expressing
Mangosteen Thioesterase
A. Analysis of 5255-Transformants
Pooled segregating mature seeds ~rom pCGN5255
transformants are analyzed to determine fatty acid composition
by GC as described in WO 92/20236. In the 22 plants analyzed,
18:0 (stearate) levels ranged ~rom 2.8 to 14.2 weight percent,
compared to background levels in a nontransformed plant of 2.2
percent. Analyses of pooled seed samples from additional
transformants were conducted, and 18:0 levels of 16.6 weight
percent were detected in transformant 5255-29. Levels of 16:0
fatty acids are not affected by expression of the Garm FatAl
thioesterase.
Half seeds from transgenic plants 5255-20 and 5255-3 were
similarly analyzed to determine the levels o~ 18:0 fatty acids
obtained in individual seeds. For event 5255-20, half seeds
with 18:0 ~atty acid contents of up to 22.6 weight percent
were obtained, and from event 5255-3, half seeds with 18:0
fatty acid contents of up to 10.0 weight percent were
obt~lne~. S;m;l~r data were obtained with other 5255
transgenic events and revealed 18:0 fatty acid levels of up to
26.6% in half seeds of 5255-29 and up to 15~ in half seeds of
5255-19.
The highest half-seed plants were grown to maturity and
T3 pooled seed was analyzed for fatty acid composition.
Stearate levels up to 26~ in pooled seed were observed in
selections from transformant 5255-29. Half-seeds from the T3
pools of event 5255-20 showed individuals with stearate
contents up to 39%. Selections from event 5255-3, which
contains the transgene in only one locus, had T3 pooled seed
with the same stearate content (11%) as the half-seeds from
which they were selected, as would be expected if the
insertion were homozygous. The trait is being inherited in a
M~n~l; an fashion and the stearate levels are being
maintained.
B. Analysis of 5266-Transformants
Pooled segregating mature seeds from pCGN5266
transformants are analyzed to determine fatty acid composition
by GC as described in WO 92/20236. Results of these assays
CA 0220~6~7 1997-0~-20
WO97/12047 PCT~S96/16078
for the 10 transformants in each variety with the highest
stearate levels are presented in Figure 4 as weight percent
fatty acid composition. In plants analyzed to date, 18:0
levels range from approximately 4 to 22 weight percent,
compared to background levels in nontransformed plants of 1.4
(QO4) and 1.8 (Quantum) percent.
Half seeds from transgenic plants 5266-LP004-2 and 5266-
SP30021-29 were similarly analyzed to det~rm;ne the levels of
18:0 fatty acids obtained in individual seeds. Fatty acid
composition data from the ten seeds of those analyzed which
contained the highest levels of stearate are presented in
Figure 5. For event 5266-LP004-2, half seeds with 18:0 fatty
acid contents of up to 33.13 weight percent were obtained, and
from event 5266-SP30021-29, half seeds with 18:0 fatty acid
contents of up to 38.86 weight percent were obtained. Similar
data were obtained with other 5266 transgenic events.
Exam~le 5 Construct for Antisense Regulation of Stearoyl-ACP
Desaturase in Brassica
Brassica napus stearoyl-ACP desaturase clones were
isolated as follows. A cDNA library was constructed from RNA
isolated from mid maturation (30 days after pollination) seeds
of Brassica napus cultivar 212/86. The library was
constructed using the lambda Uni-ZAP (Stratagene) vector kit
according to the manufacturers directions with the following
modification: lOO~g of total RNA was used for cDNA synthesis,
and first strand cDNA synthesis was carried out at 42 C. The
cDNA library was screened with the coding region of a delta-9
desaturase gene isolated from Brassica campestris (Knutzon et
al. (1992) ~roc. Natl Acad. Sci. 89:2624-2628). Partial DNA
sequence was obtained from 42 clones that hybridized with the
probe. The clones fell into 5 classes. DNA sequence was
obtained from the largest clone from each class. BND9 and
BND16 were most closely related and 78.6% of the cDNA clones
were of this class. BND11 and BND53 were related, and 19% of
the cDNA clones were of this class.
An antisense gene was constructed to generate antisense
RNA homologous to both of the major classes of desaturase
genes, BND9 and BND11. A fragment cont~;n;ng the ma~ority of
26
CA 0220~6~7 1997-0~-20
WO97/12047 PCT~S96/16078
the coding region of BND9 was excised using the enzymes
XindIII and PvuII. The ends were filled in with the klenow
fragment of DNA Polymerase I and deoxynucleotides. Plasmid
pCGN3223, cont~;n;n~ a napin expression cassette, was
linearized with BglII and the ends were filled in with the
klenow ~ragment of DNA Polymerase I and deoxynucleotides.
Ligation of the pCGN3223 vector and the BND9 fragment resulted
in pCGN7826. pCGN7826 was digested with XhoI and the ends
were filled in with the klenow fragment of DNA Polymerase I
and deoxynucleotides. The filled in fragment isolated from
BNDll after digestion with BglII and PvuII was ligated to
pCGN7826 to yield pCGN7690. pCGN7690 contains a napin
promoter positioned for expression of an antisense transcript
homologous to both the BND9 and BNDll mRNAs. The antisense
gene was excised from pCGN7690 using Asp718 and cloned into
Asp718 digested binary plant transformation vector
pCGN1559PASS to yield pCGN7696. pCGN1559PASS is a binary
vector such as those described by McBride et al. (supra) and
is prepared from pCGN1559 by substitution of the pCGN1559
linker region with a linker region contA; n; ng the following
restriction digestion sites: Asp718/AscI/PacI/XbaI/
BamHI/SwaI/Sse8387(PstI)/~in~TTI. AscI, PacI, SwaI and
Sse8387 have 8-base restriction recognition sites and are
available from New England BioLabs: AscI, PacI; Boehringer
25 Manheim: SwaI and Takara (Japan): Sse8387.
Exam~le 6 Analysis of Transgenic Plants Expressing
an Antisense Desaturase Construct
Approximately 70 transgenic plants cont~;n;ng pCGN7696
were generated. Pools of 50 seeds were analyzed using gas
chromatography to determine the fatty acid composition of the
seed oils. The average level of stearate in pooled seeds from
these transgenic plants was 7.7%. Pooled seeds from plant
7696-31 contained 14.7% (by weight) stearate, pooled seeds
from plant 7696-54 contained 14.1% stearate, pooled seeds from
plant 7696-69 contained 11.4% stearate, pooled seeds from
plant 7696-36 cont~;ne~ 11.3% stearate, and pooled seeds from
plant 7696-45 contained 9.5% stearate. By comparison, pooled
seeds from untransformed control plants contain less than 3.0%
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stearate. Also, in pooled seeds from 20 transgenic plants
cont~;n;ng an antisense construct cont~;n;ng only the BND9
desaturase sequence under control of the napin promoter, an
average of 3.6% stearate in the pooled seeds was observed.
This data demonstrates the dual antisense desaturase gene
construct, pCGN7696, provides for improved stearate production
as compared to a single gene construct Additional data
confirm these results as the highest stearate level observed
in pooled seed from a BND9 transformant was 7.6 % as compared
to 14.7% with 7696 transformants. Similarly, the highest
single seed level of stearate observed with BND9 transformants
was 10.1% in comparison to levels of up to 29% obt~;ne~ in
7696 transformants.
Segregation analysis of the oil composition of single
seeds suggested that 7696-54 and 7696-31 contained two T-DNA
loci, while 7696-36, 7696-59 and 7696-45 contained single T-
DNA loci. Individual seeds were analyzed to det~rm;ne
stearate ranges for individual transformants. Stearate levels
in single seeds from 7696-54 and 7696-31 ranged from 2 to 29
weight percent while single seeds from the single locus plants
contained stearate contents ranging between 2 and 20 percent
by weight.
Exam~le 7 Crosses and Analysis of Plants Cont~;n;ng Both
Antisense Desaturase and Mangosteen Thioesterase
Constructs
Half-seed analysis of T2 seeds from 5255 and 7696
transformants were used to select individual transformants to
be crossed for combination of the stearoyl-ACP thioesterase
and antisense desaturase traits. Transgenic plants were grown
from the r~m~;n;ng half-seed where desirable levels of 18:0
and/or numbers of GarmFatA1 gene inserts were observed. 18:0
contents of representative half seeds for 5255-3, 5255-19,
5255-29 and 5255-20 events and 7696-31, 7696-36, 7696-45 and
7696-54 events selected for crossing experiments are provided
in Table 1 below.
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TA~3LE 1
1/2 Seed 1/2 Seed
5255 Event 18:0 % 7696 Event 18:0 %
5255-29-7 21.31 7696-54-11 29.30
5255-20-1120.33 *7696-45-1 17.65
*5255-3-29 8.11 *7696-36-1 29.62
*5255-19-1 14.95 7696-31-1 28.12
* Single insertions by segregation analysis
Data from various Fl crosses of the transgenic events
indicated above are provided in Table 2. The highest stearate
levels observed by hal~-seed analysis o~ seeds ~rom selected
Fl crosses are shown as weight percent of total fatty acids.
TABLE 2
Parent 5255-29-7 5255-20-11 5255-3-29 5255-19-1
7696-54-11 47.14 42.40 35.46 40.28
7696-45-1 18.77 - 7.61 13.39
7696-36-1 34.03 - 21.33
7696-31-1 31.84
The above results ~mnn~trate the existence of numerous
half seeds with stearate contents above 30% (as high as 47%)
from crosses of multiple loci plants from each transgene as
well as ~rom single locus events. The combination of
antisense desaturase and mangosteen TE gives higher stearate
than either hemizygous parent. In some cases the Fl's have
higher stearate than seen so ~ar in either homozygous parent
suggesting the genes are acting synergistically.
29
=
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WO97/12047 PCT~S96/16078
Exam~le 8 Transformation and Transaenic Plant Analvsis with
a Construct Cont~i n; nq Antisense Desaturase and
Manaosteen Thioesterase Constructs
As an alternative to separate transformations and plant
breeding as described above to obtain transgenic plants
cont~;ning both antisense stearoyl-ACP desaturase and stearoyl
thioesterase genes, such transgenic plants may be obtained by
transformation with a single construct cont~;n;n~ both
antisense stearoyl-ACP desaturase and stearoyl thioesterase
genes. One such construct is the binary transformation vector
pCGN7748. pCGN7748 contains 2 copies of napin expression
cassettes with antisense desaturase BNDg and BNDll genes. It
also contains one copy of an expression cassette cont~; n; ng
antisense desaturase BND9 and BNDll genes under the regulatory
control of a stearoyl-ACP desaturase gene promoter, and one
copy of a napin expression cassette for expression of the
mangosteen thioesterase gene.
pCGN7748 was constructed by cloning two copies of the
napin/antisense BND9/ antisense BNDll gene from pCGN7690 (see
Example 5) as Asp718 fragments into Asp718 digested binary
vector pCGN1559PASS to yield pCGN7859. The napin/mangosteen
thioesterase gene was excised from pCGN5253 (see Example 3)
using Asp718 and the ends filled in with the klenow fragment
of DNA polymerase one and all 4 dNTPs. The resulting blunt
ended DNA fragment was cloned into SwaI digested pCGN7859 to
yield plasmid pCGN7743. The DNA fragment cont~- n; ng
BND9/BNDll from pCGN7690 was excised with SalI and XhoI, and
treated with the klenow fragment of DNA polymerase one and the
nucleotides dCTP and TTP and ligated to the desaturase
expression cassette of pCGN5207, which had been digested with
BamHI and treated with klenow fragment of DNA polymerase one
and the nucleotides dGTP and d~TP. [pCGN5207 contains
approximately 1.5Kb of the 5' regulatory region and 1.3 kB of
the 3' regulatory region of the Brassica rapa stearoyl-ACP
desaturase gene (Knutzon et al. (1992) Proc. Na~. Acad. Sci.
89:2624-2628). A polylinker cont~;n;ng BamHI-Ps~I-NotI-XbaI-
NaeI-EcoRI-ClaI restriction sites separates the 5' and 3'
regulatory regions in pCGN5207.] The clone resulting from
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WO97/12047 PCT~S96/16078
insertion of the BND9/BND11 fragment in the desaturase
expression cassette was pCGN7745. The SmaI ~ragment from
pCGN7745 was cloned into pCGN7743 digested with Sse8387 (after
treating with the klenow fragment of DNA polymerase one and
all 4 dNTPs) to yield pCGN7748.
pCGN7748 is transformed into Agrobacterium tumefaciens
strain EHA101 and used to transform B. napus variety Quantum
(SP30021).
T2 pooled seed from a plant transformed with pCGN7748 was
analyzed to determine fatty acid composition and A~m~n~trated
the ~ollowing fatty acid composition profile (weight percent
fatty acids):
7748-SP30021-1
%16:0 = 4.8 %18:0 - 43.8 %20:0 = 6.4
%16:1 = 0.1 %18:1 = 16.6 %20:1 = 0.2
%18:2 = 19.5 ~2Z:0 = 1.4
%18:3 = 7.2
These results demonstrate that mangosteen thioesterase
clone GarmFatA1 may be used to increase the 18:0 content of
seed oils from transgenic plants, and that improved vegetable
oils having a stearic acid content of greater than 30 weight
percent may be obt~;n~ from such seeds following cr~ ;ng and
fatty acid extraction procedures. Furth~rmore, in plants
expressing the mangosteen thioesterase in combination with an
antisense desaturase construct, levels of 18:0 can be further
increased and may exceed 50% of the total percentage of fatty
acids in the plant seed oil.
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WO97/12047 PCT~S96/16078
All publications and patent applications mentioned in
this specification are indicative of the level of skill of
those skilled in the art to which this invention pertains.
All publications and patent applications are herein
incorporated by reference to the same extent as if each
individual publication or patent application was specifically
and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in
some detail by way of illustration and example for purposes of
clarity of underst~n~;ng, it will be obvious that certain
changes and modifications may be practiced within the scope of
the appended claims.
