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CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
TRANS-2-ENOYL-COA REDUCTASE GENE OF EUGLENA GRACILIS
Description
The invention relates to the use of a nucleic acid sequence SEQ ID NO : 1, SEQ
ID NO
4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10 that when expressed will
increase the total amount of lipids (i.e. triacylglycerols, diacylglycerols,
monoacylglyc-
erols, phospholipids, waXesters and/or fatty acids) that is produced in
transgenic
organisms.
More specifically this invention describes the identification of a nucleic
acid sequence
SEQ ID NO : 1 from Euglena gracilis encoding a trans-2-enoyl-CoA reductase
(TER -
E.C.1.3.1.44).
In another embodiment, this invention is directed to a protein comprising an
amino acid
sequence as set forth in SEQ ID NO : 2, SEQ ID NO : 5, SEQ ID NO : 7, SEQ ID
NO
9 or SEQ ID NO : 11 or a functional fragment, derivative, variant, or
ortologue thereof.
The present invention further includes the nucleotide sequence as set forth in
SEQ ID NO: 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10 as
well as portions of the genomic sequence, the cDNA sequence, allelic variants,
synthetic variants and mutants thereof. This includes sequences that are to be
used as
probes, vectors for transformation or cloning intermediates.
SEQ ID NO : 2, SEQ ID NO : 5, SEQ ID NO : 7, SEQ ID NO : 9 or SEQ ID NO : 11
are
the deduced amino acid sequences of the open reading frames SEQ ID NO : 1, SEQ
ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10.
Another aspect of the present invention relates to those polypeptides, which
have at
least 60% identity to SEQ ID NO : 2, SEQ ID NO : 5, SEQ ID NO : 7, SEQ ID NO :
9 or
SEQ ID NO : 11.
The invention furthermore relates to expression constructs for expressing a
nucleic
acid sequence SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or
SEQ
ID NO : 10 encoding a TER gene in plants, preferably in plant seeds,
transgenic plants
expressing a TER gene and to the use of said transgenic plants for the
production of
food, feed, seed, pharmaceuticals or fine chemicals, in particular for the
production of
oils.
In oil crops like e.g. rape, sunflower, oil palms etc., the oil (i.e.
triacylglycerols) is the
most valuable product of the seeds or fruits and other compounds such as
starch,
protein and fiber is regarded as by-products with less value. Enhancing the
quantity of
CONFIRMATION COPY
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WO 2005/040366 PCT/EP2004/011294
2
lipids per weight basis at the expense of other compounds in oil crops would
therefore
increase the value of the crop. If proteins that promote the allocation of
reduced carbon
into the production of lipids can be up regulated by overexpression, the cells
will accu-
mulate more lipids at the expense of other products. This approach could not
only be
used to increase the lipids content in already high oil producing organisms
such as oil
crops, they could also lead to significant lipid production in moderate or low
oil contain-
ing crops such as soy, oat, maize, potato, sugar beats, and turnips as well as
in
microorganisms.
Increasing the lipid content in plants and, in particular, in plant seeds is
of great interest
for traditional and modern plant breeding and in particular for plant
biotechnology.
Owing to the increasing consumption of vegetable oils for nutrition or
industrial
applications, possibilities of increasing or modifying vegetable oils are
increasingly the
subject of current research (for example Topfer et al. (1995) Science 268:681-
686). Its
aim is in particular increasing the fatty acid content in seed oils.
The fatty acids which can be obtained from the vegetable oils are also of
particular
interest. They are employed, for example, as bases for plasticizers,
lubricants,
surfactants, cosmetics and the like and are employed as valuable bases in the
food
and feed industries. Thus, for example, it is of particular interest to
provide rapeseed
oils with fatty acids with medium chain length since these are in demand in
particular in
the production of surfactants. With regard to medical ramifications, the long
chain fatty
acids (C18 and longer) found in many seed oils have been linked to reductions
in
hypercholesterolemia and other clinical disorders related to coronary heart
disease
(Brenner 1976, Adv. Exp. Med. Biol. 83:85-101 ). Therefore, consumption of a
plant
having increased levels of these types of fatty acids may reduce the risk of
heart
disease. Enhanced levels of seed oil content also increase large-scale
production of
seed oils and thereby reduce the cost of these oils.
The targeted modulation of plant metabolic pathways by recombinant methods
allows
the modification of the plant metabolism in an advantageous manner which, when
using traditional breeding methods, could only be achieved after a complicated
procedure or not at all. Thus, unusual fatty acids, for example specific
polyunsaturated
fatty acids, are only synthesized in certain plants or not at all in plants
and can
therefore only be produced by expressing the relevant gene in transgenic
plants (for
example Millar et al. (2000) Trends Plant Sci 5:95-101 ).
Triacylgylcerols, diacylglycerols, monoacylglycerols, phospholipids, waxesters
and
other lipids are synthesized from fatty acids. Fatty acid biosynthesis and
triacylglycerol
biosynthesis can be considered as separate biosynthetic pathways owing to the
compartmentalization, but as a single biosynthetic pathway in view of the end
product.
Lipid synthesis can be divided into two part-mechanisms, one which might be
termed
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3
"prokaryotic" and another which may be termed "eukaryotic" (Browse et al.
(1986)
Biochemical J 235:25-31; Ohlrogge & Browse (1995) Plant Cell 7:957-970). The
prokaryotic mechanism is localized in the plastids and encompasses the
biosynthesis
of the free fatty acids which are exported into the cytosol, where they enter
the
eukaryotic mechanism in the form of fatty acid acyl-CoA esters. In this
pathway the
fatty acids are esterified by glycerol-3-phosphate acyltransferase and
lysophosphatidic
acid acyltransferase to the sn-1 and sn-2 positions of glycerol-3-phosphate,
respec-
tively, to yield phosphatidic acid (PA). The PA is the precursor for other
polar and
neutral lipids, the latter being formed in the Kennedy pathway (Voelker 1996,
Genetic
Engineering ed.:Setlow 18:111-113; Shanklin & Cahoon 1998, Annu. Rev. Plant
Physiol. Plant Mol. Biol. 49:611-641; Frentzen 1998, Lipids 100:161-166;
Millar et al.
2000, Trends Plant Sci. 5:95-101 ).
The last step in the synthesis of triacylglycerols has been shown to occur by
two
different enzymatic reactions, an acyl-CoA dependent reaction catalyzed by an
acyl-
CoA : diacylglycerol acyltransferase (Cases, S. et al., (1998) Proc. Natl.
Acad. Sci.,
USA 95, 13018-13023.; Lardizabal, et al., 2001 ) and the acyl-CoA independent
reaction catalyzed by an phospholipid : diacylglyerol acyltransferase
(Dahlqvist, et al.,
2000).
In higher plants waxesters are synthesized from the fatty acids exported from
the
plastid and their derivatives, particular very long chain fatty acids and
their derivatives
(Dusty Post-Beittenmiller:Biochemistry and molecular biology of wax production
in
plants. In Annu. Rev. Plant PhysioLPlant Mol. Biol.(1996); Editor Jones, R L.,
Vol 47,
pp405-430 ).
In Euglena gracilis waxesters are formed when grown under anaerobic
conditions.
Transfer of Euglena from aerobic to anaerobic conditions causes rapid
formation of
waxesters at the expense of the reserve polysaccharide paramylon. This
anaerobic
formation of waxesters is coupled by a net synthesis of ATP (Inui, H. et al.
(1982)
FEBS Lett. 150: 89-93) and, as such, the phenomenon is called waxester
fermentation.
In Euglena, four systems of fatty acid synthesis have been described: 1 )
involving
multifunctional fatty acid synthetase in the cytosol, 2) acyl-carrier-protein-
dependent
systems in the chloroplasts and 3) involving a fatty acid synthetase in
microsomes
(Kitaoka, S.(1989) Enzymes and their functional location. pp. 2-135 in D.E.
Buetow,
ed. The biology of Euglena, Vol. 4. Subcellular biochemistry and molecular
biology.
Academic Press, San Diego).
In addition a 4t" novel system see figure 1, was discovered: In mitochondria
of Euglena
gracilis, a de novo fatty acid synthesis system was found that is different
from the
systems in the cytosol (FAS I) and in the chloroplasts (FAS II) (Inui et al.
1982, 1984).
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4
The reaction is the reverse of the (3-oxidation mechanism of fatty acids with
Trans-2-
enoyl-CoA reductase (TER) (E.C. 1.3.1.44) replacing acyl-CoA dehydrogenase of
the
degradative system (Inui, H. et al., Eur. J. Biochem. 142(1984):121-126).
Trans-2-
enoyl-CoA reductase (TER) is the key enzyme of this fatty acid biosynthetic
pathway
since it catalyses the last step of the cycle and creates the end product,
fatty acyl CoA.
Although the enzyme's activity was measured and a partial purification of the
protein
achieved, protein sequence information for TER was not gained by Inui and co-
workers. Without information on the peptide or nucleotide sequence the
polypeptide
chain, gene and mRNA for this enzyme remained unknown. An enzyme of such
function was not described for any other organisms so far.
It is an object of the present invention to provide additional methods for
increasing
the lipid content in plants.
1'5 We have found that this object is achieved by the present invention.
In the present invention we show, that a polynucleotide SEQ ID NO : 1, SEQ ID
NO : 4,
SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10 present in Euglena gracilis and
when over-expressed in plants enhances the amount of triacylglycerols,
diacylglyc-
erols, monoacylglycerols, phospholipids, waxesters andlor fatty acids that
accumulates
in plants.
The biochemical purification of traps-2-enoyl-CoA reductase (TER) from Euglena
gracilis strain Z was carried out as described in example 1. A series of
chromato-
graphic purifications steps was undertaken, see table 4. These included ion
exchange
chromatography (DEAE-Fraktogel), hydrophobic interactions (phenylsapharose),
affinity chromatography (Reaktive Red 120) and hydroxyapatite chromatography.
The
corresponding purification levels can be seen in table 4. Furthermore, an
additional
ion exchange chromatography (Mono Q), purification over a preparative gel and
a final
gel filtration through Superdex 200 completed the purification scheme. This
scheme
achieved more than 1600 fold purification. When submitted to a SDS-PAGE using
standard protocols, the final enzyme preparation showed a major and a thin
minor
band very closely together at about 44 kDa, see figure 2. Enzyme activity was
mea-
sured as described by Inui and co-workers (Inui et al., 1984) and was
associated with
the major, upper band.
The major and the minor band were cut from the gel separately and digested
with
trypsin using standard protocols. The resulting peptides were extracted from
the gel
and analysed using ESI-Q-TOF MS/MS using standard protocols. Both bands were
shown to yield solely identical peptides, confirming the complete purification
of the TER
as a single subunit enzyme in contrast to the description of Inui and co-
workers (Inui,
H. et al., (1986) J. Biochem. 100:995-1000).
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WO 2005/040366 PCT/EP2004/011294
RNA was isolated from Euglena gracilis cells according to the procedure as
described
in example 2.
5 The cDNA library of Euglena gracilis was constructed as described in example
3.
From the cDNA Bank pBluescript phagemides were generated by in-vivo-excision
using the ExAssist helper phage for further analysis.
Degenerate primers were designed according to these peptides (see example 4)
and used for PCR with cDNA as template. A 837 by fragment was amplified with
the
following primers: 5'-GGITGGTAYAAYACIGTIGC-3' (referring to peptide 7) and
5'-GTYTCRTAICCIGCRAARTC-3' (referring to peptide 9). This fragment was cloned
into pBluescript SK+lHincll and sequenced (SEQ ID NO : 3). The translated
sequence
contained several peptides of the purified protein and therefore the 837 by
fragment
was used as hybridisation probe to screen a cDNA library constructed with mRNA
from
Euglena cells. Screening of 250.000 recombinant phages resulted in six
independent
clones. cDNA inserts varied between 1600 by and 1900 bp. Sequencing of all six
clones from both ends revealed that all clones represented the same transcript
and
varied only in length. The longest clone was sequenced completely double-
stranded
via deletion by exonuclease III. The clone had a length of 1912 by and encodes
an
open reading frame of 1620 by coding for 539 as (SEQ ID NO : 1 and SEQ ID NO :
2).
At both ends it had adaptors consisting of a Notl and EcoRl restriction site
and was
inserted into the EcoRl site of the vector pBluescript SK+, see figure 3.
The clone was liberated from pBluscript SK+ using EcoRl. 5' overhangs were
removed
with mung bean nuclease (Roche) using the manufacturer's protocol) and a blunt
end
ligation using standard protocols performed with the binary vector pSUN 300 or
ST593.
The results of the expression studies of Euglena gracilis TER in E, coli - see
example 6 - can be taken as consideration that the N-terminal part of the cDNA
clone
may constitute a mitochondria) targeting signal, which has to be cleaved to
yield the
mature and active TER protein. Nevertheless the possibility that the N-
terminal part
of the cDNA clone constitutes a transmembran domain can not be excluded. This
possible transmembran domain could be lost during biochemical purification of
TER
from Euglena gracilis (see example 1 ). If expressed in E. coli this domain
may possibly
disrubt acticity measurement with the C4-substrat (see example 1 ) due to
incorrect
convolution or missing membran-linkage.
A first subject matter of the invention comprises a method of increasing the
total lipid
content in a plant organism or a tissue, organ, part, cell or propagation
material thereof,
comprising
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a) the transgenic expression of SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6,
SEQ ID NO : 8 or SEQ ID NO : 10 in said plant organism or in a tissue, organ,
part, cell or propagation material thereof, and
b) the selection of plant organisms in which - in contrast to or comparison
with
the starting organism - the total lipid content in said plant organism or in a
tissue,
organ, part, cell or propagation material thereof is increased.
Other proteins resulting in the same effect as the protein set forth in SEQ ID
NO : 2,
SEQ ID NO : 5, SEQ ID NO : 7, SEQ ID NO : 9 or SEQ ID NO : 11 are obtainable
from
the specific sequences provided herein. Furthermore, it will be apparent that
one
can obtain natural and synthetic TERs, including those with modified amino
acid
sequences and starting materials for synthetic-protein modeling from the
exempli-
Pied TERs and from TERs which are obtained through the use of such exemplified
sequences. Modified amino acid sequences include sequences that have been
mutated, truncated, increased and the like, whether such sequences were
partially
or wholly synthesized.
Further, the nucleic acid probes (DNA or RNA) derived from the SEQ ID NO : 1,
SEQ
ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10 of the present
invention
can be used to screen and recover homologous or related sequences from a
variety of
plant and microbial sources.
The over-expression of SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO
: 8
or SEQ ID NO : 10 in yeast or plants will increase the content in total lipids
and
enhances the amount of triacylglycerols, diacylglycerols, monoacylglycerols,
phospholipids, waxesters and/or fatty acids that accumulate compared to wild
type
yeasts or plants.
The present invention can be characterized by the following aspects:
Example 1 describes the biochemical purification of trans-2-enoyl-CoA
reductase
(TER) from Euglena gracilis.
Example 2 describes the isolation of total RNA and poly-(A)+ RNA from Euglena
gracilis cells.
Example 3 describes the Euglena gracilis cDNA library construction.
Example 4 describes the identification of the TER protein sequence by peptide
fingerprinting and subsequent cloning of the corresponding cDNA.
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Example 5 describes the triacylglycerol accumulation in yeast cells expressing
the
TER gene.
Example 6 describes the functional expression of traps-2-enoyl-CoA reductase
(TER)
in E. coli
Example 7 describes TER constructs for overexpression in Arabidopsis.
Example 8 describes the plasmids for plant transformation.
Example 9 describes the transformation of Arabidopsis.
Example 10 describes the In vitro analysis of the function of the TER gene in
trans-
genic plants.
Example 11 describes the lipid content in transgenic Arabidopsis plants over-
expressing the TER gene.
Example 12 describes the amino acid characteristic for traps-enoyl activity
based on
sequence comparison.
The invention can furthermore be characterized by:
use of the nucleic acid sequence SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6,
SEQ
ID NO : 8 or SEQ ID NO : 10 encoding a protein SEQ ID NO : 2, SEQ ID NO : 5,
SEQ
ID NO : 7, SEQ ID NO : 9 or SEQ ID NO : 11 that enhances the production of
triacyl-
glycerols, diacylglycerols, monoacylglycerols, phospholipids, waxesters and/or
fatty
acids;
genetic transformation of an oil-producing organism with said sequence in
order
to be expressed in this organism, resulting in an active protein that
increases the
lipid content of the organism.
The nucleic acid sequence is derived from the sequence shown in SEQ ID NO : 1
from the Euglena gracilis TER gene (genomic clone or cDNA) or from a nucleic
acid
sequence or cDNA that contains a nucleotide sequence coding for a protein with
an
amino acid sequence that is 60% or more identical to the amino acid sequence
as
presented in SEQ ID NO: 2.
The gene product, which we refer to as traps-2-enoyl-CoA reductase (TER) is
not
itself catalyzing the synthesis of triacylglycerols, diacylglycerols,
monoacylglycerols,
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8
phospholipids and/or waxesters but its presence elevates the amount of fatty
acids
synthesized.
The instant invention pertains to a gene construct comprising a said
nucleotide
sequence SEQ ID NO: 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID
NO : 10 of the instant invention, which is operably linked to a
heterologous nucleic acid.
The term "operably linked" means a serial organization e.g. of a promoter,
coding
sequence, terminator and/or further regulatory elements whereby each element
can
fulfill its original function during expression of the nucleotide sequence.
Further, a vector comprising the said nucleotide sequence SEQ ID NO: 1, SEQ ID
NO
4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10 of the instant invention is
contemplated in the instant invention. This includes also an expression vector
which
can harbor a selectable marker gene and/or nucleotide sequences for the
replication
in a host cell and/or the integration into the genome of the host cell.
Furthermore, this invention relates to a method for producing a TER encoded by
the nucleic acid sequence SEQ ID NO: 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID
NO
8 or SEQ ID NO : 10 in a host cell or progeny thereof including genetically
engineered
oil seeds, yeast and moulds or any other oil-accumulating organism, via the
expression
of a construct in the cell. Of particular interest is the expression of the
nucleotide
sequence of the present invention from transcription initiation regions that
are preferen-
tially expressed in plant seed tissues. It is further contemplated that an
artificial gene
sequence encoding a TER may be synthesized, especially to provide plant-
preferred
codons. Transgenic cells containing a TER as a result of the expression of a
TER
encoding sequence are also contemplated within the scope of the invention.
Further, the invention pertains a transgenic cell or organism containing a
said nucleo-
tide sequence and/or a said gene construct and/or a said vector. The object of
the
instant invention is further a transgenic cell or organism which is an
eucaryotic cell or
organism. Preferably, the transgenic cell or organism is a yeast cell or a
plant cell or
a plant. The instant invention further pertains said transgenic cell or
organism having
an increased biosynthetic pathway for the production of substrates for the
synthesis of
triacylglycerol. A transgenic cell or organism having increased lipid content
is also
contemplated within the scope of this invention.
Further, the invention pertains a transgenic cell or organism wherein the
activity of a
TER encoded by the nucleic acid sequence SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID
NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10 is increased in said cell or organism.
The
increased activity of TER is characterized by an alteration in gene
expression, catalytic
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9
activity andlor regulation of activity of the enzyme. Moreover, a transgenic
cell or
organism is included in the instant invention, wherein the increased
biosynthetic
pathway for the production of substrates for the production of triacylglycerol
is charac-
terized e.g. by the increased supply of metabolic precursors of the fatty acid
biosynthe-
sis.
In a different embodiment, this invention also relates to methods of using a
nucleic acid
sequence SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID
NO : 10 for increasing the lipid content within the cells of different
organisms.
Further, the invention makes possible a process for elevating the production
of triacyl-
glycerol, which comprises growing transgenic cells or organisms under
conditions
whereby the nucleic acid sequence SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6,
SEQ ID NO : 8 or SEQ ID NO : 10 is expressed in order to produce a TER protein
in these cells which results in the enhanced production of triacylglycerols,
diacylglyc-
erols, monoacylglycerols, phospholipids, waxesters and/or fatty acids.
Corresponding genes coding for TER can be isolated from other organisms,
especially
of the phylum Euglenida, the order Euglenales, the genus Euglena and/or the
subgen-
era Euglena, Calliglena or Discoglena. Species belonging to the subgenus
Calliglena
e.g. Euglena sanguinea, Euglena mutabilis, Euglena clara, Euglena velata,
Euglena
agilis, Euglena caudate, Euglena polymorpha, Euglena granulata, Euglena
rostrifera,
Euglena repulsans, Euglena anabaena and Euglena satelles. Species belonging to
the
subgenus Euglena : e.g. Euglena viridis, Euglena stellata, Euglena geniculata,
Euglena
tristella and Euglena chadefaudii. Species belonging to the subgenus
Discoglena : e.g.
Euglena acus, Euglena texts, Euglena tripteris, Euglena desces, Euglena
oxyuris,
Euglena spirogyra, Euglena helicoidea, Euglena proxima and Euglena
ehrenbergii.
Corresponding genes coding for TER can be isolated from closely related genera
like
Colacium, Eutreptia, Eutreptiella, Phacus, Lepocinclis, Cryptoglena,
Trachelomonas,
Strombomonas, Ascoglena, Klebsiella, Astasia, Rhabdomonas, Menoidium,
Peranema,
Anisonema, Tetreutreptia, Hyalophacus, Khawkinea, Distigma, Cyclidiopsis, Ento-
siphon, Ploeotia, Gyropaigne, Notoselenus, Petalomonas, Parmidium.
Furthermore the invention pertains transgenic organisms comprising, in their
genome
or on a plasmid, a nucleic acid sequence SEQ ID NO :1, SEQ ID NO : 4, SEQ ID
NO
6, SEQ ID NO : 8 or SEQ ID NO : 10 according to the above, transferred by
recombi-
nant DNA technology. One important type of transgenic organism covered by this
invention are commercially relevant plants in which said nucleic acid sequence
preferably would be expressed under the control of a storage organ specific
promoter.
Alternatively, the nucleic acid sequence could also be expressed under the
control of a
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WO 2005/040366 PCT/EP2004/011294
seed-specific promoter or any other promoter suitable for tissue-specific high-
level
expression in plants.
The invention also pertains a protein encoded by a DNA molecule according to
5 SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10
or a
functional biologically active fragment thereof having TER activity in
transgenic
organisms. Alternatively, the invention pertains a protein produced in an
organism,
which has the amino acid sequence set forth in SEQ ID NO : 2, SEQ ID NO : 5,
SEQ
ID NO : 7, SEQ ID NO : 9 or SEQ ID NO : 11 or an amino acid sequence with at
least
10 60% homology to said amino acid sequence having TER activity.
The protein can be is isolated from Euglena gracilis- as described in example
1 - but
also from other organisms.
An TER can be recovered from plant material by various methods well known in
the art.
Organs of plants can be separated mechanically from other tissue or organs
prior to
isolation of the seed storage compound from the plant organ. Following
homogeni-
zation of the tissue, cellular debris is removed by centrifugation and the
supernatant
fraction containing the soluble proteins is retained for further purification
of the desired
compound. If the product is secreted from cells grown in culture, then the
cells are
removed from the culture by low-speed centrifugation and the supernatant
fraction is
retained for further purification.
The supernatant fraction from either purification method is subjected to
chromato-
graphy with a suitable resin, in which the desired molecule is either retained
on a
chromatography resin while many of the impurities in the sample are not, or
where
the impurities are retained by the resin while the sample is not. Such
chromatography
steps may be repeated as necessary, using the same or different chromatography
resins. One skilled in the art would be well-versed in the selection of
appropriate
chromatography resins and in their most efficacious application for a
particular
molecule to be purified. The purified product may be concentrated by
filtration or
ultrafiltration, and stored at a temperature at which the stability of the
product is
maximized.
There are a wide array of purification methods known to the art and the
preceding
method of purification is not meant to be limiting. Such purification
techniques are
described, for example, in Bailey J.E. & Ollis D.F. 1986, Biochemical
Engineering
Fundamentals, McGraw-HiII:New York). The purification protocol described
herein
for purification from Euglena gracilis can be taken as an example and can
easily be
adapted to other organisms by those skilled in the art.
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11
The identity and purity of the isolated compounds may be assessed by
techniques
standard in the art. These include high-performance liquid chromatography
(HPLC),
spectroscopic methods, staining methods, thin layer chromatography, analytical
chromatography such as high performance liquid chromatography, NIRS, enzymatic
assay, or microbiologically. Such analysis methods are reviewed in: Patek et
al.
(1994, Appl. Environ. Microbiol. 60:133-140), Malakhova et al. (1996,
Biotekhnologiya
11:27-32) and Schmidt et al. (1998, Bioprocess Engineer 19:67-70), Ulmann's
Ency-
clopedia of Industrial Chemistry (1996, Vol. A27, VCH: Weinheim, p. 89-90, p.
521-540,
p. 540-547, p. 559-566, 575-581 and p. 581-587) and Michal G. (1999,
Biochemical
Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons;
Fallon, A. et al. 1987, Applications of HPLC in Biochemistry in: Laboratory
Techniques
in Biochemistry and Molecular Biology, vol. 17).
The invention additionally pertains the use of a protein according to SEQ ID
NO : 2,
SEQ ID NO : 5, SEQ ID NO : 7, SEQ ID NO : 9 or SEQ ID NO : 11 or derivatives
of that
protein having TER activity and resulting in increased production of
triacylglycerols,
diacylglycerols, monoacylglycerols, phospholipids, waxesters and/or fatty
acids.
Surprisingly, it has been found that the heterologous expression of the TER
gene
of Euglena gracilis leads to a significantly increased triacylglycerol,
diacylglycerol,
monoacylglycerol, phospholipid, waxester and/or fatty acid content ( storage
oil) in
the seeds of Arabidopsis thaliana as described in example 11. The lipid
content was
increased by approximately 5%, in one transgenic line even by 10%, compared
with
wild-type control plants. The over-expression of the nucleic acid sequence
encoding
a TER protein has no adverse effects on the growth or other properties of the
trans-
formed plants.
The method according to the invention can be applied in principle to all plant
species,
in addition to the species Arabidopsis thaliana, which is employed as model
plant. The
method according to the invention is preferably applied to lipid crops whose
oil content
is already naturally high and/or for the industrial production of oils.
Plant organism or tissue, organ, part, cell or propagation material thereof is
generally
understood as meaning any single- or multi-celled organism or a cell, tissue,
part or
propagation material (such as seeds or fruit) of same which is capable of
photo-
synthesis. Included for the purpose of the invention are all genera and
species of
higher and lower plants of the Plant Kingdom. Annual, perennial,
monocotyledonous
and dicotyledonous plants are preferred. Also included are mature plants,
seeds,
shoots and seedlings, and parts, propagation material (for example tubers,
seeds
or fruits) and cultures derived from them, for example cell cultures or callus
cultures.
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Plant encompasses all annual and perennial monocotyledonous or dicotyledonous
plants and includes by way of example, but not by limitation, those of the
genera
Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis,
Trifolium,
Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis,
Brassica,
Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,
Nicotiana,
Solarium, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca,
Bromus,
Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum,
Pennisetum,
Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,
Phaseolus,
Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea and
Populus.
Preferred plants are those from the following plant families: Amaranthaceae,
Aster-
aceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae,
Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae,
Malv-
aceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanaceae,
Sterculi-
aceae, Tetragoniaceae, Theaceae, Umbelliferae.
Preferred monocotyledonous plants are selected in particular from the
monocotyle-
donous crop plants such as, for example, the Gramineae family, such as rice,
maize,
wheat or other cereal species such as barley, millet and sorghum, rye,
triticale or oats,
and sugar cane, and all grass species.
The invention is applied very particularly preferably to dicotyledonous plant
organisms.
Preferred dicotyledonous plants are selected in particular from the
dicotyledonous
crop plants such as, for example,
Asteraceae such as Heliantus annuus (sunflower), tagetes or calendula and
others,
Compositae, especially the genus Lactuca, very particularly the species sativa
(lettuce) and others,
Cruciferae, particularly the genus Brassica, very particularly the species
napus
(oilseed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv
Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other
cabbages;
and the genus Arabidopsis, very particularly the species thaliana, and cress
or canola and others,
Cucurbitaceae such as melon, pumpkin/squash or zucchini and others,
- Leguminosae, particularly the genus Glycine, very particularly the species
max
(soybean), soya, and alfalfa, pea, beans or peanut and others,
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13
Rubiaceae, preferably the subclass Lamiidae such as, for example Coffea
arabica or Coffea liberica (coffee bush) and others,
Solanaceae, particularly the genus Lycopersicon, very particularly the species
esculentum (tomato), the genus Solanum, very particularly the species tubero-
sum (potato) and melongena (aubergine) and the genus Capsicum, very particu-
larly the genus annuum (pepper) and tobacco or paprika and others,
Sterculiaceae, preferably the subclass Dilleniidae such as, for example, Theo-
broma cacao (cacao bush) and others,
Theaceae, preferably the subclass Dilleniidae such as, for example, Camellia
sinensis or Thea sinensis (tea shrub) and others,
- Umbelliferae, particularly the genus Daucus (very particularly the species
carota
(carrot)) and Apium (very particularly the species graveolens dulce (celeary))
and
others;
and linseed, cotton, hemp, flax, cucumber, spinach, carrot, sugar beet and the
various
tree, nut and grapevine species, in particular banana and kiwi fruit.
Also encompassed are ornamental plants, useful or ornamental trees, flowers,
cut
flowers, shrubs or turf plants which may be mentioned by way of example but
not by
limitation are angiosperms, bryophytes such as, for example, Hepaticae
(liverworts)
and Musci (mosses); pteridophytes such as ferns, horsetail and clubmosses;
gymno-
sperms such as conifers, cycades, ginkgo and Gnetatae; algae such as Chlorophy-
ceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophy-
ceae (diatoms) and Euglenophyceae. Plants within the scope of the invention
comprise
by way of example and not by way of limitation, the families of the Rosaceae
such as
rose, Ericaceae such as rhododendron and azalea, Euphorbiaceae such as
poinsettias
and croton, Caryophyllaceae such as pinks, Solanaceae such as petunias,
Gesneria-
ceae such as African violet, Balsaminaceae such as touch-me-not, Orchidaceae
such
as orchids, Iridaceae such as gladioli, iris, freesia and crocus, Compositae
such as
marigold, Geraniaceae such as geranium, Liliaceae such as dracena, Moraceae
such
as ficus, Araceae such as cheeseplant and many others.
Furthermore, plant organisms for the purposes of the invention are further
organisms
capable of being photosynthetically active such as, for example, algae,
cyanobacteria
and mosses. Preferred algae are green algae such as, for example, algae from
the
genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Syne-
chocystis is particularly preferred.
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14
Most preferred are oil crops. Oil crops are understood as being plants whose
lipid
content is already naturally high and/or which can be used for the industrial
production
of oils. These plants can have a high lipid content and/or else a particular
fatty acid
composition which is of interest industrially. Preferred plants are those with
a lipid
content of at least 1 % by weight. Oil crops encompassed by way of example:
Borvago
officinalis (borage); Brassica species such as B. campestris, B. napus, B.
raps
(mustard, oilseed rape or turnip rape); Cannabis sativa (hemp); Carthamus
tinctorius
(safflower); Cocos nucifera (coconut); Crambe abyssinica (crambe); Cuphea
species
(Cuphea species yield fatty acids of medium chain length, in particular for
industrial
applications); Elaeis guinensis (African oil palm); Elaeis oleifera (American
oil palm);
Glycine max (soybean); Gossypium hirisutfum (American cotton); Gossypium bar-
badense (Egyptian cotton); Gossypium herbaceum (Asian cotton); Helianthus
annuus
(sunflower); Linum usitatissimum (linseed or flax); Oenothera biennis (evening
primrose); Olea europaea (olive); Oryza sativa (rice); Ricinus communis
(castor);
Sesamum indicum (sesame); Triticum species (wheat); tea mays (maize), and
various nut species such as, for example, walnut or almond.
"Total lipid content" refers to the sum of all oils, preferably to the sum of
the triacyl-
glycerols, diacylglycerols, monoacylglycerols, phospholipids, waxesters andlor
fatty
acids.
"Lipids" encompasses neutral and/or polar lipids and mixtures of these. Those
mentioned in Table 1 may be mentioned by way of example, but not by
limitation.
Neutrale lipidsTriacylglycerol (TAG)
Diacylglycerol (DAG)
Monoacylglycerol (MAG)
Polar lipids Monogalactosyldiacylglycerol (MGDG)
Digalactosyldiacylglycerol (DGDG)
Phosphatidylglycerol (PG)
Phosphatidylcholine (PC)
Phosphatidylethanolamine (PE)
Phosphatidylinositol (PI)
Phosphatidylserine (PS)
Sulfoquinovosyldiacylglycerol
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Table 1: Classes of plant lipids
Neutral lipids preferably refers to triacylglycerols. Both neutral and polar
lipids may
comprise a wide range of various fatty acids. The fatty acids mentioned in
Table 2
5 may be mentioned by way of example, but not by limitation.
Nomenclature' Name
16:0 Palmitic acid
16:1 Palmitoleic acid
16:3 Roughanic acid
18:0 Stearic acid
18:1 Oleic acid
18:2 Linoleic acid
18:3 Linolenic acid
y-18:3-18:3 Gamma-linolenic acid
20:0 Arachidic acid
22:6 Docosahexaenoic acid (DHA)
20:2 Eicosadienoic acid
20:4 Arachidonic acid (AA)
20:5 Eicosapentaenoic acid (EPA)
22:1 Erucic acid
Table 2: Overview over various fatty acids (selection)
' Chain length: number of double bonds
10 * not naturally occurring in plants
Oils preferably relates to seed oils.
"Increase in" the total lipid content refers to the increased lipid content in
a plant or a
15 part, tissue or organ thereof, preferably in the seed organs of the plants.
In this context,
the lipid content is at least 5%, preferably at least 10%, particularly
preferably at least
15%, very particularly preferably at least 20%, most preferably at least 25%
increased
under otherwise identical conditions in comparison with a starting plant which
has not
been subjected to the method according to the invention, but is otherwise
unmodified.
Conditions in this context means all of the conditions which are relevant for
germina-
tion, culture or growth of the plant, such as soil conditions, climatic
conditions, light
conditions, fertilization, irrigation, plant protection treatment and the
like.
"TER" generally refers to all those proteins which are capable of having the
enzymatic
activity of a trans-2-enoyl-CoA reductase and resulting if a corresponding
nucleic acid
SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10
is
expressed in an increased lipid content in oil producing organisms, especially
microor-
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16
ganisms, yeast, fungi and plants and said proteins are identical to SEQ ID NO
: 2, SEQ
ID NO : 5, SEQ ID NO : 7, SEQ ID NO : 9 or SEQ ID NO : 11 or have homology to
SEQ ID NO : 2, SEQ ID NO : 5, SEQ ID NO : 7, SEQ ID NO : 9 or SEQ ID NO : 11.
TER refers in particular to the polypeptide sequence SEQ ID NO : 2, SEQ ID NO
: 5,
SEQ ID NO : 7, SEQ ID NO : 9 or SEQ ID NO : 11.
Most preferably, TER refers to the Euglena gracilis protein TER as shown in
SEQ ID
NO : 2 and functional equivalents or else functionally equivalent portions of
the above.
Trans-2-enoly-CoA-reductase can be recovered from different organisms or plant
material by various methods well known in the art. Organs of plants can be
separated
mechanically from other tissue or organs prior to isolation of the seed
storage com-
pound from the plant organ. Following homogenization of the tissue, cellular
debris is
removed by centrifugation and the supernatant fraction containing the soluble
proteins
is retained for further purification of the desired compound. If the product
is secreted
from cells grown in culture, then the cells are removed from the culture by
low-speed
centrifugation and the supernatant fraction is retained for further
purification.
The supernatant fraction from either purification method is subjected to
chromato-
graphy with a suitable resin, in which the desired molecule is either retained
on a
chromatography resin while many of the impurities in the sample are not, or
where
the impurities are retained by the resin while the sample is not. Such
chromatography
steps may be repeated as necessary, using the same or different chromatography
resins. One skilled in the art would be well-versed in the selection of
appropriate
chromatography resins and in their most efficacious application for a
particular
molecule to be purified. The purified product may be concentrated by
filtration or
ultrafiltration, and stored at a temperature at which the stability of the
product is
maximized.
There is a wide array of purification methods known in the art and the
preceding
method of purification is not meant to be limiting. Such purification
techniques are
described, for example, in Bailey J.E. & Ollis D.F. 1986, Biochemical
Engineering
Fundamentals, McGraw-HiII:New York). The purification protocol described
herein
for purification from Euglena gracilis can be taken as an example and can
easily be
adapted to other organisms by those skilled in the art.
"Functional equivalents" refers in particular to natural or artificial
mutations of the
Euglena gracilis protein TER as shown in SEQ ID NO : 2, SEQ ID NO : 5, SEQ ID
NO
7, SEQ ID NO : 9 or SEQ ID NO : 11 and homologous polypeptides from other
organisms belong e.g. to the phylum Euglenida which have the same essential
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17
characteristics of TER as defined above. Mutations encompass substitutions,
additions,
deletions, inversions or insertions of one or more amino acid residues.
The TER to be employed advantageously within the scope of the present
invention can
be found readily by database searches or by screening gene or cDNA libraries
using
the polypeptide sequence shown in SEQ ID NO : 2, which is given by way of
example,
or the nucleic acid sequence as shown in SEQ ID NO : 1, which encodes the
latter, as
search sequence or probe.
Said functional equivalents preferably have at least 60%, particularly
preferably at least
70%, particularly preferably at least 80%, most preferably at least 90%
homology with
the protein of SEQ ID NO : 2.
Furthermore the nucleic acid sequence SEQ ID NO : 3 can be used in order to
identify
and clone genes encoding a TER from organisms having at least 60 % homology to
SEQ ID NO : 3.
The functional equivalent of SEQ ID NO : 2 has an identity of at least 50%, 51
%, 52%,
53%, 54%, 55%, 56%, 57% preferably at least 58%, 59%, 60%, 61 %, 62%, 63%,
64%,
65%, 66%, 67%, 68%, 69%, and 70% more preferably 71 %, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85% most preferably at least
86%,
87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with
the SEQ ID NO : 2.
Furthermore the following nucleic acid sequences can be used in order to
identify and
clone genes encoding a TER from difFerent organisms and having at least 40%
identity
to SEQ ID NO : 2:
Sequences from SwissProt/ TREMBL (Code & species):
Q87QB9 Vibrio parahaemolyticus
Q8D8Y6 Vibrio vulnificus
Q8PE66 Xanthomonas campestris (pv.
campestris)
Q83EP5 Coxiella burnetii.
Q8PR25 Xanthomonas axonopodis (pv.
citri)
Q8EG14 Shewanella oneidensis
Q87CN3 Xylella fastidiosa
Q88E33 Pseudomonas putida
Q8XIP1 Clostridium perfringens
Q8D795 Vibrio vulnificus.
Q93HE4 Streptomyces avermitilis
Q87HT6 Vibrio parahaemolyticus
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18
Sequences from PIR (Code & species):
A83277 Pseudomonas aeruginosa
H82630 Xylella fastidiosa
AD0498 Yersinia pestis
B82164 Vibrio cholerae
B82418 Vibrio cholerae
696956 Clostridium acetobutylicum
Sequences from Genbank REFSEQ (Code & species):
ZP00116993 Cytophaga hutchinsonii
ZP00064975 Microbulbifer degradans
ZP00033810 Burkholderia fungorum
Table 3 shows the amino acid sequence of TER compared to the amino acid
sequence
of these homologous nucleic acids and the corresponding conserved amino acid
residues. Conserved residues are shaded in grey. The consensus for these
residues is
given below the sequences: capital letters denote mandatory residues; regular
letters
give the most prominent amino acid of a mandatory similarity group at this
location.
By comparision with functionally characterised sequences putative functional
sites of
SEQ ID NO: 2 may be assigned to the following stretches of amino acids:
30
~ NADH binding site
amino acids 190 to196 of SEQ ID NO: 2
(Jornvall et al. (1995) "Short-chain dehydrogenases/reductases. (SDR)".
Biochem.
34: 6003-6013.)
~ Short chain dehydrogenase/reductase catalytic sites
amino acids 231 to 238 and 279 to 285 of SEQ ID NO: 2
(Des et al.( 2000) "Molecular cloning and expression of mammalian peroxisomal
trans-2-enoyl-coenzyme A reductase cDNAs." J. Biol. Chem. 275: 24333-24340).
~ FAD binding site
amino acids 515 to 520 of SEQ ID NO: 2
(Chang & Hammes (1989) "Homology analysis of the protein sequences of fatty
acid syntheses from chicken liver, rat mammary gland, and yeast." Proc. Natl.
Acad. Sci. USA 86: 8373-8376.)
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WO 2005/040366 PCT/EP2004/011294
19
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CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
24
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CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
Homology between two polypeptides is understood as meaning the identity of the
amino acid sequence over the entire sequence length which is calculated by
compari-
son with the aid of the program algorithm GAP (Wisconsin Package Version 10.0,
5 University of Wisconsin, Genetics Computer Group (GCG), Madison, USA),
setting
the following parameters:
Gap Weight: 8 Length Weight: 2
10 Average Match: 2,912 Average Mismatch: -2,003
For example, a sequence with at least 80% homology with the sequence SEQ ID
NO: 2
at the protein level is understood as meaning a sequence which, upon
comparison with
the sequence SEQ ID NO: 2 with the above program algorithm and the above para-
15 meter set has at least 80% homology.
Functional equivalents - for example - also encompass those proteins which are
encoded by nucleic acid sequences which have at least 60%, particularly
preferably at
least 70%, particularly preferably at least 80%, most preferably at least 90%
homology
20 with the nucleic acid sequence SEQ ID NO : 1.
Homology between two nucleic acid sequences is understood as meaning the
identity
of the two nucleic acid sequences over the entire sequence length which is
calculated
by comparison with the aid of the program algorithm GAP (Wisconsin Package
Version
25 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison,
USA),
setting the following parameters:
Gap Weight: 50 Length Weight: 3
Average Match: 10 Average Mismatch: 0
For example, a sequence which has at least 80% homology with the sequence SEQ
ID
NO : 1 at the nucleic acid level is understood as meaning a sequence which,
upon
comparison with the sequence SEQ ID NO: 1 with+n the above program algorithm
with
the above parameter set has a homology of at least 80%.
Functional equivalents also encompass those proteins which are encoded by
nucleic
acid sequences which hybridize under standard conditions with a nucleic acid
se-
quence described by SEQ ID NO : 1 or a nucleic acid sequence which is
complemen-
tary thereto or parts of the above and which have the essential
characteristics of an
TER as characterised by SEQ ID NO : 2.
CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
26
Natural examples of TERs and the corresponding genes can furthermore readily
be
found in various organisms whose genomic sequence is unknown, by hybridization
techniques in a manner known per se, for example starting from the nucleic
acid
sequences SEQ ID NO : 1 or SEQ ID NO : 3.
The hybridization may be carried out under moderate (low stringency) or,
preferably,
under stringent (high stringency) conditions.
Such hybridization conditions are described, inter alia, in Sambrook, J.,
Fritsch, E.F.,
Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd edition, Cold
Spring
Harbor Laboratory Press, 1989, pages 9.31-9.57 or in Current Protocols in
Molecular
Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
By way of example, the conditions during the washing step may be selected from
the range of conditions which is limited by those with low stringency (with 2X
SSG at
50°C) and those with high stringency (with 0.2X SSC at 50°C,
preferably at 65°C)
(20X SSC: 0.3 M sodium citrate, 3 M sodium chloride, pH 7.0).
In addition, the temperature may be raised during the washing step from
moderate
conditions at room temperature, 22°C, to stringent conditions at
65°C.
Both parameters, salt concentration and temperature, may be varied
simultaneously
and it is also possible to keep one of the two parameters constant and to vary
only the
other one. It is also possible to use denaturing agents such as, for example,
formamide
or SDS during hybridization. In the presence of 50% formamide, the
hybridization is
preferably carried out at 42°C.
Some exemplary conditions for hybridization and washing step are listed below:
(1.) hybridization conditions with, for example
(i) 4X SSC at 65°C, or
(ii) 6X SSC at 45°C, or
(iii) 6X SSC at 68°C, 100 mg/ml denatured fish sperm DNA, or
(iv) 6X SSC, 0.5% SDS, 100 mg/ml denatured fragmented salmon sperm
DNA at 68°C, or
(v) 6X SSC, 0.5% SDS, 100 mg/ml denatured fragmented salmon sperm
DNA, 50% formamide at 42°C, or
CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
27
(vi) 50% formamide, 4X SSC at 42°C, or
(vii) 50% (vol/vol) formamide, 0.1 % bovine serum albumin; 0.1 % Ficoll,
0.1 % polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5,
750 mM NaCI, 75 mM sodium citrate at 42°C, or
(viii) 2X or 4X SSC at 50°C (moderate conditions), or
(ix) 30 to 40% formamide, 2X or 4X SSC at 42.°C (moderate conditions).
(2.) Washing steps of 10 minutes each with, for example
(i) 0.015 M NaCI/0.0015 M sodium citrate/0.1 % SDS at 50°C, or
(ii) 0.1X SSC at 65°C, or
(iii) 0.1X SSC, 0.5% SDS at 68°C, or
(iv) 0.1X SSC, 0.5% SDS, 50% formamide at 42°C, or
(v) 0.2X SSC, 0.1 % SDS at 42°C, or
(vi) 2X SSC at 65°C (moderate conditions).
30
The invention furthermore relates to transgenic expression constructs which
can
ensure a transgenic expression of a TER as characterised by SEQ ID NO : 2, SEQ
ID
NO : 5, SEQ ID NO : 7, SEQ ID NO : 9 or SEQ ID NO : 11 or in a plant organism
or a
tissue, organ, part, cells or propagation material of said plant organism.
The definition given above applies to a TER, with the transgenic expression of
a
nucleic acid encoding TER and described by the sequence with the SEQ ID NO : 1
being particularly preferred.
In said transgenic expression constructs, a nucleic acid molecule encoding a
TER is
preferably in operable linkage with at least one genetic control element (for
example a
promoter) which ensures expression in a plant organism or a tissue, organ,
part, cell or
propagation material of same.
Especially preferred are transgenic expression cassettes wherein the nucleic
acid
sequence encoding a TER is described by
CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
28
a) a sequence SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or
SEQ ID NO : 10 or
b) a sequence derived from a sequence SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID
NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10 or in accordance with the degeneracy
of the genetic code
c) a sequence which has at least 60% identity with a sequence SEQ ID NO : 1,
SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO~ : 10.
Operable linkage is understood as meaning, for example, the sequential
arrangement
of a promoter with the nucleic acid sequence encoding a TER which is to be
expressed
(for example the sequence as shown in SEQ ID NO : 1 ) and, if appropriate,
further
regulatory elements such as, for example, a terminator in such a way that each
of the
regulatory elements can fulfil its function when the nucleic acid sequence is
expressed
recombinantly. Direct linkage in the chemical sense is not necessarily
required for
this purpose. Genetic control sequences such as, for example, enhancer
sequences
can also exert their function on the target sequence from positions which are
further
removed or indeed from other DNA molecules. Preferred arrangements are those
in
which the nucleic acid sequence to be expressed recombinantly is positioned
behind
the sequence acting as promoter so that the two sequences are linked
covalently
to each other. The distance between the promoter sequence and the nucleic acid
sequence to be expressed recombinantly is preferably less than 200 base pairs,
particularly preferably less than 100 base pairs, very particularly preferably
less than
50 base pairs.
Operable linkage and a transgenic expression cassette can both be effected by
means
of conventional recombination and cloning techniques as they are described,
for
example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in
Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions,
Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Ausubel FM et al.
(1987)
Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley
Inter-
science and in Gelvin et al. (1990) In: Plant Molecular Biology Manual.
However,
further sequences which, for example, act as a linker with specific cleavage
sites
for restriction enzymes, or of a signal peptide, may also be positioned
between the
two sequences. Also, the insertion of sequences may lead to the expression of
fusion proteins. Preferably, the expression cassette composed of a promoter
linked
to a nucleic acid sequence to be expressed can be in a vector-integrated form
and
can be inserted into a plant genome, for example by transformation.
CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
29
However, a transgenic expression cassette is also understood as meaning those
constructs where the nucleic acid sequence encoding an TER is placed behind an
endogenous plant promoter in such a way that the latter brings about the
expression
of the TER.
Promoters which are preferably introduced into the transgenic expression
cassettes
are those which are operable in a plant organism or a tissue, organ, part,
cell or
propagation material of same. Promoters which are operable in plant organisms
is
understood as meaning any promoter which is capable of governing the
expression of
genes, in particular foreign genes, in plants or plant parts, plant cells,
plant tissues or
plant cultures. In this context, expression may be, for example, constitutive,
inducible or
development-dependent.
The following are preferred:
a) Constitutive promoters
"Constitutive" promoters refers to those promoters which ensure expression in
a
large number of, preferably all, tissues over a substantial period of plant
develop-
ment, preferably at all times during plant development (Benfey et al.(1989)
EMBO J 8:2195-2202). A plant promoter or promoter originating from a plant
virus is especially preferably used. The promoter of the CaMV (cauliflower
mosaic virus) 35S transcript (Franck et al. (1980) Cell 21:285-294; Odell et
al.
(1985) Nature 313:810-812; Shewmaker et al. (1985) Virology 140:281-288;
Gardner et al. (1986) Plant Mol Biol 6:221- 228) or the 19S CaMV promoter
(US 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J 8:2195-2202) are
especially preferred. Another suitable constitutive promoter is the Rubisco
small
subunit (SSU) promoter (US 4,962,028), the IeguminB promoter (GenBank Acc.
No. X03677), the promoter of the nopalin synthase from Agrobacterium, the TR
dual promoter, the OCS (octopine synthase) promoter from Agrobacterium, the
ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29:637-649), the
ubi-
quitin 1 promoter (Christensen et al. (1992) Plant Mol Biol 18:675-689; Bruce
et al. (1989) Proc Natl Acad Sci USA 86:9692-9696), the Smas promoter, the
cinnamyl alcohol dehydrogenase promoter (US 5,683,439), the promoters of the
vacuolar ATPase subunits, the promoter of the Arabidopsis thaliana nitrilase-1
gene (GenBank Acc. No.: U38846, nucleotides 3862 to 5325 or else 5342) or
the promoter of a proline-rich protein from wheat (WO 91/13991 ), and further
promoters of genes whose constitutive expression in plants is known to the
skilled worker. The CaMV 35S promoter and the Arabidopsis thaliana nitrilase-1
promoter are particularly preferred.
b) Tissue-specific promoters
CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
Furthermore preferred are promoters with specificities for seeds, such as, for
example, the phaseolin promoter (US 5,504,200; Bustos MM et al. (1989) Plant
Cell 1 (9):839-53), the promoter of the 2S albumin gene (Joseffson LG et al.
5 (1987) J Biol Chem 262:12196- 12201 ), the legumine promoter (Shirsat A et
al.
(1989) Mol Gen Genet 215(2):326-331 ), the USP (unknown seed protein) pro-
moter (Baumlein H et al. (1991 ) Mol Gen Genet 225(3):459-67), the napin gene
promoter (US 5,608,152; Stalberg K et al. (1996) L Planta 199:515-519), the
promoter of the sucrose binding proteins (WO 00/26388) or the legumin B4 pro-
10 moter (LeB4; Baumlein H et al. (1991) Mol Gen Genet 225: 121-128; Baumlein
et al. (1992) Plant Journal 2(2):233-9; Fiedler U et al. (1995) Biotechnology
(NY) 13(10):1090f), the Arabidopsis oleosin promoter (WO 98/45461 ), and the
Brassica Bce4 promoter (V1IO 91/13980).
15 Further suitable seed-specific promoters are those of the gene encoding
high-
molecular weight glutenin (HMWG), gliadin, branching enyzme, ADP glucose
pyrophosphorylase or starch synthase. Promoters which are furthermore
preferred are those which permit a seed-specific expression in monocots such
as
maize, barley, wheat, rye, rice and the like. The promoter of the Ipt2 or Ipt1
gene
20 (V1/O 95/15389, WO 95/23230) or the promoters described in WO 99/16890
(promoters of the hordein gene, the glutelin gene, the oryzin gene, the
prolamin
gene, the gliadin gene, the glutelin gene, the zein gene, the casirin gene or
the
secalin gene) can advantageously be employed.
25 c) Chemically inducible promoters
The expression cassettes may also contain a chemically inducible promoter
(review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol
48:89-108), by means of which the expression of the exogenous gene in the
30 plant can be controlled at a particular point in time. Such promoters such
as, for
example, the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22:361-366),
a salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-
inducible promoter (EP 0 388 186), a tetracyclin-inducible promoter (Gatz et
al.
(1992) Plant J 2:397-404), an abscisic acid-inducible promoter EP 0 335 528)
or
an ethanol-cyclohexanone-inducible promoter (VllO 93/21334) can likewise be
used. Also suitable is the promoter of the glutathione-S transferase isoform
II
gene (GST-II-27), which can be activated by exogenously applied safeners such
as, for example, N,N-diallyl-2,2-dichloroacetamide (WO 93/01294) and which is
operable ~in a large number of tissues of both monocots and dicots.
Particularly preferred are constitutive promoters, very particularly preferred
seed-
specific promoters, in particular the napin promoter and the USP promoter.
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31
In addition, further promoters which make possible expression in further plant
tissues
or in other organisms such as, for example, E.coli bacteria, may be linked
operably with
the nucleic acid sequence to be expressed. Suitable plant promoters are, in
principle,
all of the above-described promoters.
The nucleic acid sequences present in the transgenic expression cassettes
according
to the invention or transgenic vectors can be linked operably with further
genetic control
sequences besides a promoter. The term genetic control sequences is to be
under-
stood in the broad sense and refers to all those sequences which have an
effect on
the establishment or the function of the expression cassette according to the
invention.
Genetic control sequences modify, for example, transcription and translation
in
prokaryotic or eukaryotic organisms. The transgenic expression cassettes
according
to the invention preferably encompass a plant-specific promoter 5'-upstream of
the
nucleic acid sequence to be expressed recombinantly in each case and, as
additional
genetic control sequence, a terminator sequence 3'-downstream, and, if
appropriate,
further customary regulatory elements, in each case linked operably with the
nucleic
acid sequence to be expressed recombinantly.
Genetic control sequences also encompass further promoters, promoter elements
or
minimal promoters capable of modifying the expression-controlling properties.
Thus,
genetic control sequences can, for example, bring about tissue-specific
expression
which is additionally dependent on certain stress factors. Such elements are,
for
example, described for water stress, abscisic acid (Lam E and Chua NH, J Biol
Chem
1991; 266(26): 17131-17135) and thermal stress (Schoffl F et al. (1989) Mol
Gen
Genetics 217(2-3):246-53).
Further advantageous control sequences are, for example, in the Gram-positive
promoters amy and SP02, and in the yeast or fungal promoters ADC1, MFa, AC, P-
60,
CYC1, GAPDH, TEF, rp28, ADH.
In principle all natural promoters with their regulatory sequences like those
mentioned
above may be used for the method according to the invention. In addition,
synthetic
promoters may also be used advantageously.
Genetic control sequences further also encompass the 5'-untranslated regions,
introns
or nonencoding 3'-region of genes, such as, for example, the actin-1 intron,
or the
Adh1-S intron 1, 2 and 6 (for general reference, see: The Maize Handbook,
Chapter
116, Freeling and Walbot, Eds., Springer, New York (1994)). It has been
demonstrated
that these may play a significant role in regulating gene expression. Thus, it
has been
demonstrated that 5'-untranslated sequences can enhance the transient
expression
of heterologous genes. Translation enhancers which may be mentioned by way of
CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
32
example are the tobacco mosaic virus 5' leader sequence (Gallie et al. (1987)
Nucl
Acids Res 15:8693-8711 ) and the like. They may furthermore promote tissue
specificity
(Rouster J et al. (1998) Plant J 15:435-440).
The transient expression cassette can advantageously contain one or more of
what are
known as enhancer sequences in operable linkage with the promoter, and these
make
possible an increased recombinant expression of the nucleic acid sequence.
Additional
advantageous sequences such as further regulatory elements or terminators may
also
be inserted at the 3' end of the nucleic acid sequences to be expressed
recombinantly.
One or more copies of the nucleic acid sequences to be expressed recombinanly
may
be present in the gene construct.
Polyadenylation signals which are suitable as control sequences are plant poly-
adenylation signals, preferably those which correspond essentially to
Agrobacterium
tumefaciens T-DNA polyadenylation signals, in particular those of gene 3 of
the T-DNA
(octopine synthase) of the Ti plasmid pTiACHS (Gielen et al. (1984) EMBO J
3:835
et seq.) or functional equivalents thereof. Examples of particularly suitable
terminator
sequences are the OCS (octopin synthase) terminator and the NOS (nopaline syn-
thase) terminator.
Control sequences are furthermore understood as those which make possible homo-
logous recombination or insertion into the genome of a host organism, or
removal
from the genome. In the case of homologous recombination, for example, the
coding
sequence of the specific endogenous gene can be exchanged in a directed
fashion for
a sequence encoding a dsRNA. Methods such as the cre/lox technology permit the
tissue-specific, possibly inducible, removal of the expression cassette from
the genome
of the host organism (Saner B (1998) Methods. 14(4):381-92). Here, certain
flanking
sequences are added to the target gene.(lox sequences), and these make
possible
removal by means of cre recombinase at a later point in time.
A recombinant expression cassette and the recombinant vectors derived from it
may
comprise further functional elements. The term functional element is to be
understood
in the broad sense and refers to all those elements which have an effect on
generation,
replication or function of the expression cassettes, vectors or transgenic
organisms
according to the invention. Examples which may be mentioned, but not by way of
limitation, are:
a) Selection markers which confer resistance to a metabolism inhibitor such as
2-deoxyglucose-6-phosphate (V110 98/45456), antibiotics or biocides,
preferably
herbicides, such as, for example, kanamycin, G 418, bleomycin, hygromycin, or
phosphinothricin and the like. Particularly preferred selection markers are
those
which confer resistance to herbicides. The following may be mentioned by way
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33
of example: DNA sequences which encode phosphinothricin acetyltransferases
(PAT) and which inactivate glutamine synthase inhibitors (bar and pat gene),
5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes),
which confer resistance to Glyphosate (N-(phosphonomethyl)glycine), the gox
gene, which encodes Glyphosate-degrading enzyme (Glyphosate oxidoreduc-
tase), the deh gene (encoding a dehalogenase which inactivates dalapon),
sulfonylurea- and imidazolinone-inactivating acetolactate synthases, and bxn
genes which encode nitrilase enzymes which degrade bromoxynil, the aria
gene, which confers resistance to the antibiotic apectinomycin, the
streptomycin
phosphotransferase (SPT) gene, which permits resistance to streptomycin, the
neomycin phosphotransferase (NPTII) gene, which confers resistance to kana-
mycin or geneticidin, the hygromycin phosphotransferase (HPT) gene, which
confers resistance to hygromycin, the acetolactate synthase gene (ALS), which
confers resistance to sulfonylurea herbicides (for example mutated ALS
variants
with, for example, the S4 and/or Hra mutation).
b) Reporter genes which encode readily quantifiable proteins and which allow
the
transformation efficacy or the expression site or time to be assessed via
their
color or enzyme activity. Very particularly preferred in this context are
reporter
proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(1 ):29-44) such
as the "green fluorescent protein" (GFP) (Sheen et al. (1995) Plant Journal
8(5):777-784), chloramphenicol transferase, a luciferase (Ow et al. (1986)
Science 234:856-859), the aequorin gene (Prasher et al. (1985) Biochem Bio-
phys Res Commun 126(3):1259-1268), f3-galactosidase, with (3-glucuronidase
being very particularly preferred (Jefferson et al. (1987) EMBO J 6:3901-
3907).
c) Replication origins which allow replication of the expression cassettes or
vectors
according to the invention in, for example, E. coli. Examples which may be
mentioned are ORI (origin of DNA replication), the pBR322 on or the P15A
on (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2"d ed. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
d) Elements which are required for agrobacterium-mediated plant transformation
such as, for example, the right or left border of the T-DNA, or the vir
region.
To select cells which have successfully undergone homologous recombination or
else
cells which have succesfully been transformed, it is generally required
additionally
to introduce a selectable marker which confers resistance to a biocide (for
example
a herbicide), a metabolism inhibitor such as 2-deoxyglucose-6-phosphate
(UVO 98/45456) or an antibiotic to the cells which have successfully undergone
recombination. The selection marker permits the selection of the transformed
cells
from untransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81-84).
CA 02541141 2006-03-31
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34
In addition, said recombinant expression cassette or vectors may comprise
further
nucleic acid sequences which do not encode a nucleic acid sequence SEQ ID NO:
1,
SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10 and whose recom-
binant expression leads to a further increase in fatty acid biosynthesis. By
way of
example, but not by limitation, such a proOIL nucleic acid sequence which
is additionally expressed recombinantly can be selected from among nucleic
acids
encoding acetyl-CoA carboxylase (ACCase), glycerol-3-phosphate acyltransferase
(GPAT), lysophosphatidate acyltransferase (LPAT), diacylglycerol
acyltransferase
(DAGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). Such sequences
are
known to the skilled worker and are readily accessible from databases or
suitable
cDNA libraries of the respective plants.
An expression cassette according to the invention can advantageously be
introduced
into an organism or cells, tissues, organs, parts or seeds thereof (preferably
into plants
or plant cells, tissues, organs, parts or seeds) by using vectors in which the
recombi-
nant expression cassettes are present. The invention therefore furthermore
relates to
said recombinant vectors which encompass a recombinant expression cassette for
a
nucleic acid sequence SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO :
8
or SEQ ID NO : 10.
For example, vectors may be plasmids, cosmids, phages, viruses or else
agrobacteria.
The expression cassette can be introduced into the vector (preferably a
plasmid vector)
via a suitable restriction cleavage site. The resulting vector is first
introduced into E.coli.
Correctly transformed E.coli are selected, grown, and the recombinant vector
is
obtained with methods known to the skilled worker. Restriction analysis and
sequenc-
ing may be used for verifying the cloning step. Preferred vectors are those
which make
possible stable integration of the expression cassette into the host genome.
The invention furthermore relates to transgenic plant organisms or tissues,
organs,
parts, cells or propagation material thereof which comprise a SEQ ID NO : 1,
SEQ ID
NO : 4, SEQ ID NO : 6, SEQ ID N.O : 8 or SEQ ID NO : 10 as defined above, a
transgenic expression cassette for a SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO :
6,
SEQ ID NO : 8 or SEQ ID NO : 10 or a transgenic vector encompassing such an
expression cassette.
Such a transgenic plant organism is generated, for example, by means of trans-
formation or transfection of the corresponding proteins or nucleic acids. The
generation
of a transformed organism (or a transformed cell or tissue) requires
introducing the
DNA in question (for example the expression vector), RNA or protein into the
host cell
in question. A multiplicity of methods is available for this procedure, which
is termed
transformation (or transduction or transfection) (Keown et al. (1990) Methods
in
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Enzymology 185:527-537). Thus, the DNA or RNA can be introduced for example
directly by microinjection or by bombardment with DNA-coated microparticles.
The cell
may also be permeabilized chemically, for example with polyethylene glycol, so
that the
DNA may reach the cell by diffusion. The DNA can also be introduced by
protoplast
5 fusion with other DNA-comprising units such as minicells, cells, lysosomes
or lipo-
somes. Electroporation is a further suitable method for introducing DNA; here,
the
cells are permeabilized reversibly by an electrical pulse. Soaking plant parts
in DNA
solutions, and pollen or pollen tube transformation, are also possible. Such
methods
have been described (for example in Bilang et al. (1991 ) Gene 100:247-250;
Scheid
10 et al. (1991 ) -Mol Gen Genet 228:104-112; Guerche et al. (1987) Plant
Science 52:111-
116; Neuhaus et al. (1987) Theor Appl Genet 75:30-36; Klein et al. (1987)
Nature
327:70-73; Howell et al. (1980) Science 208:1265; Horsch et al.(1985) Science
227:1229-1231; DeBlock et al. (1989) Plant Physiology 91:694-701; Methods for
Plant
Molecular Biology (Weissbach and Weissbach, eds.) Academic Press Inc. (1988);
and
15 Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic
Press Inc.
(1989)).
For plant transformation binary vectors such as pBinAR can be used (Hofgen &
Willmitzer 1990, Plant Sci. 66:221-230). Construction of the binary vectors
can be
20 performed by ligation of the cDNA in sense or antisense orientation into
the T-DNA.
5-prime to the cDNA a plant promoter activates transcription of the cDNA. A
polyadenylation sequence is located 3 prime to the cDNA. Tissue-specific
expression
can be achieved by using a tissue specific promoter. For example, seed-
specific
expression can be achieved by cloning the napin or LeB4 or USP promoter 5-
prime to
25 the cDNA. Also any other seed specific promoter element can be used. For
constitutive expression within the whole plant the CaMV 35S promoter can be
used.
The expressed protein can be targeted to a cellular compartment using a signal
peptide, for example for plastids, mitochondria or endoplasmic reticulum
(Kermode
1996, Crit. Rev. Plant Sci. 15:285-423). The signal peptide is cloned 5-prime
in frame
30 to the cDNA to achieve subcellular localization of the fusion protein.
Further examples for plant binarywectors are the pSUN300 vectors into which
the TER
gene candidates is cloned. These binary vectors contain an antibiotics
resistance gene
driven under the control of the Nos-promotor and a USP seed-specific promoter
in front
35 of the candidate gene with the OCS terminator, see figure 4. TER cDNA is
cloned into
the multiple cloning site of the plant binary vector in sense orientation
behind the USP
seed-specific promoter. The recombinant vector containing the gene of interest
is
transformed into DhSa cells (Invitrogen) using standard conditions.
Transformed cells
are selected for on LB agar containing 50,ug/ml kanamycin grown overnight at
37°C.
Plasmid DNA is extracted using the QIAprep Spin Miniprep Kit (Qiagen)
following
manufacturer's instructions. Analysis of subsequent clones and restriction
mapping is
performed according to standard molecular biology techniques (Sambrook et al.
1989,
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36
Molecular Cloning, A Laboratory Manual. 2"d Edition. Cold Spring Harbor
Laboratory
Press. Cold Spring Harbor, NY).
In plants, the methods which have been described for transforming and
regenerating
plants from plant tissues or plant cells are exploited for transient or stable
transforma-
tion. Suitable methods are, in particular, protoplast transformation by
polyethylene
glycol-induced DNA uptake, the biolistic method with the gene gun, what is
known as
the particle bombardment method, electroporation, the incubation of dry
embryos in
DNA-containing solution, and microinjection.
In addition to these "direct" transformation techniques, transformation may
also be
effected by bacterial infection by means of Agrobacterium tumefaciens or Agro-
bacterium rhizogenes and the transfer of corresponding recombinant Ti plasmids
or Ri plasmids by infection with transgenic plant viruses. Agrobacterium-
mediated
transformation is best suited to cells of dicotyledonous plants. The methods
are
described, for example, in Horsch RB et al. (1985) Science 225: 1229f.
When agrobacteria are used, the expression cassette is to be integrated into
specific
plasmids, either into a shuttle vector or into a binary vector. If a Ti or Ri
plasmid is to be
used for the transformation, at least the right border, but in most cases the
right and
left border, of the Ti or Ri plasmid T-DNA is linked to the expression
cassette to be
introduced as flanking region.
Binary vectors are preferably used. Binary vectors are capable of replication
both
in E.coli and in Agrobacterium. As a rule, they contain a selection marker
gene and
a linker or polylinker flanked by the right and left T-DNA border sequence.
They
can be transformed directly into Agrobacterium (Holsters et al. (1978) Mol Gen
Genet
163:181-187). The selection marker gene, which is, for example, the nptll
gene, which
confers resistance to kanamycin, permits a selection of transformed
agrobacteria.
The Agrobacterium which acts as host organism in this case should already
contain
a plasmid with the vir region. The latter is required for transferring the T-
DNA to the
plant cells. An Agrobacterium transformed in this way can be used for
transforming
plant cells. The use of T-DNA for the transformation of plant cells has been
studied
intensively and described (EP 120 516; Hoekema, In: The Binary Plant Vector
System,
Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J
4:277-
287). Various binary vectors, some of which are commercially available, such
as, for
example, pB1101.2 or pBIN19 (Clontech Laboratories, Inc. USA), are known.
Further promoters which are suitable for expression in plants have been
described
(Rogers et al. (1987) Meth in Enzymol 153:253-277; Schardl et al. (1987) Gene
61:1-
11; Berger et al. (1989) Proc Natl Acad Sci USA 86:8402-8406).
CA 02541141 2006-03-31
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37
Direct transformation techniques are suitable for any organism and cell type.
In cases
where DNA or RNA are injected or electroporated into plant cells, the plasmid
used
need not meet any particular requirements. Simple plasmids such as those from
the
pUC series may be used. If intact plants are to be regenerated from the
transformed
cells, it is necessary for an additional selectable marker gene to be present
on the
plasmid.
Stably transformed cells, i.e. those which contain the inserted DNA integrated
into
the DNA of the host cell, can be selected from untransformed cells when a
selectable
marker is part of the inserted DNA. By way of example, any gene which is
capable of
conferring resistance to antibiotics or herbicides (such as kanamycin, G 418,
bleo-
mycin, hygromycin or phosphinothricin and the like) is capable of acting as
marker (see
above). Transformed cells which express such a marker gene are capable of
surviving
in the presence of concentrations of such an antibiotic or herbicide which
kill an un-
transformed wild type..Examples are mentioned above and preferably comprise
the
bar gene, which confers resistance to the herbicide phosphinothricin (Rathore
KS et al.
(1993) Plant Mol Biol 21 (5):871-884), the nptll gene, which confers
resistance to
kanamycin, the hpt gene, which confers resistance to hygromycin, or the EPSP
gene,
which confers resistance to the herbicide Glyphosate. The selection marker
permits
selection of transformed cells from untransformed cells (McCormick et al.
(1986)
Plant Cell Reports 5:81-84). The plants obtained can be bred and hybridized in
the
customary manner. Two or more generations should be grown in order to ensure
that the genomic integration is stable and hereditary.
The above-described methods are described, for example, in Jenes B et
al.(1993)
Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and
Utili-
zation, edited by SD Kung and R Wu, Academic Press, pp.128-143, and in
Potrykus
(1991 ) Annu Rev Plant Physiol Plant Molec Biol 42:205-225). The construct to
be
expressed is preferably cloned into a vector which is suitable for
transforming Agro-
bacterium tumefaciens, for example pBin19 (Bevan et al. (1984) Nucl Acids Res
12:8711 f).
Agrobacterium mediated plant transformation with the TER nucleic acid
described
herein can be performed using standard transformation and regeneration
techniques
(Gelvin, Stanton B. & Schilperoort R.A, Plant Molecular Biology Manual, 2nd
ed.
Kluwer Academic Publ., Dordrecht 1995 in Sect., Ringbuc Zentrale Signatur:BT11-
P;
Glick, Bernard R. and Thompson, John E. Methods in Plant Molecular Biology and
Bio-
technology, S. 360, CRC Press, Boca Raton 1993). For example, Agrobacterium
mediated transformation can be performed using the GV3 (pMP90) (Koncz &
Schell,
1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tume-
faciens strain.
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38
Arabidopsis thaliana can be grown and transformed according to standard
conditions
(Bechtold 1993, Acad. Sci. Paris. 316:1194-1199; Bent et al. 1994, Science
265:1856-
1860). Additionally, rapeseed can be transformed with the TER nucleic acid of
the
present invention via cotyledon or hypocotyl transformation (Moloney et al.
1989,
Plant Cell Report 8:238-242; De Block et al. 1989, Plant Physiol. 91:694-701
). Use of
antibiotica for Agrobacterium and plant selection depends on the binary vector
and
the Agrobacterium strain used for transformation. Rapeseed selection is
normally
performed using kanamycin as selectable plant marker. Additionally,
Agrobacterium
mediated gene transfer to flax can be performed using, for example, a
technique
described by Mlynarova et al. (1994, Plant Cell Report 13:282-285).
Transformation of soybean can be performed using for example a technique
described
in EP 0 424 047, U.S. 5,322,783 (Pioneer Hi-Bred International) or in EP 0 397
687,
U.S. 5,376,543 or U.S. 5,169,770 (University Toledo). Soybean seeds are
surface
sterilized with 70% ethanol for 4 minutes at room temperature with continuous
shaking,
followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20
minutes with
continuous shaking. Then the seeds are rinsed 4 times with distilled water and
placed
on moistened sterile filter paper in a Petri dish at room temperature for 6 to
39 hours.
The seed coats are peeled off, and cotyledons are detached from the embryo
axis. The
embryo axis is examined to make sure that the meristematic region is not
damaged.
The excised embryo axes are collected in a half-open sterile Petri dish and
air-dried to
a moisture content less than 20% (fresh weight) in a sealed Petri dish until
further use.
Agrobacterium tumefaciens culture is prepared from a single colony in LB solid
medium
plus appropriate antibiotics (e.g. 100 mg/I streptomycin, 50 mg/I kanamycin)
followed
by growth of the single colony in liquid LB medium to an optical density at
600 nm of
0.8. Then, the bacteria culture is pelleted at 7000 rpm for 7 minutes at room
tempera-
ture, and re-suspended in MS (Murashige & Skoog 1962, Physiol. Plant. 15:473-
497)
medium supplemented with 100 mM acetosyringone. Bacteria cultures are
incubated
in this pre-induction medium for 2 hours at room temperature before use. The
axis of
soybean zygotic seed embryos at approximately 44% moisture content are imbibed
for
2 h at room temperature with the pre-induced Agrobacterium suspension culture.
(The
imbibition of dry embryos with a culture of Agrobacterium is also applicable
to maize
embryo axes).
The embryos are removed from the imbibition culture and are transferred to
Petri
dishes containing solid MS medium supplemented with 2% sucrose and incubated
for
2 days, in the dark at room temperature. Alternatively, the embryos are placed
on top
of moistened (liquid MS medium) sterile filter paper in a Petri dish and
incubated under
the same conditions described above. After this period, the embryos are
transferred to
either solid or liquid MS medium supplemented with 500 mg/I carbenicillin or
300 mg/I
cefotaxime to kill the agrobacteria.
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39
The method of plant transformation is also applicable to Brassica and other
crops. In
particular, seeds of canola are surface sterilized with 70% ethanol for 4
minutes at
room temperature with continuous shaking, followed by 20% (v/v) Clorox
supplemented
with 0.05 % (v/v) Tween for 20 minutes, at room temperature with continuous
shaking.
Then, the seeds are rinsed 4 times with distilled water and placed on
moistened sterile
filter paper in a Petri dish at room temperature for 18 hours. The seed coats
are
removed and the seeds are air dried overnight in a half-open sterile Petri
dish. During
this period, the seeds lose approximately 85% of their water content. The
seeds are
then stored at room temperature in a sealed Petri dish until further use
similarly as
described in the procedure for soybean embryos.
Once a transformed plant cell has been generated, an intact plant can be
obtained
using methods known to the skilled worker. For example, callus cultures are
used as
starting material. The development of shoot and root can be induced in this as
yet
undifferentiated cell biomass in the known fashion. The plantlets obtained can
be
planted out and used for breeding.
The skilled worker is familiar with such methods for regenerating plant parts
and intact
plants from plant cells. Methods which can be used for this purpose are, for
example,
those described by Fennell et al. (1992) Plant Cell Rep. 11: 567-570; Stoeger
et al
(1995) Plant Cell Rep. 14:273-278; Jahne et al. (1994) Theor Appl Genet 89:525-
533.
"Transgenic", for example in the case of a TER, refers to a nucleic acid
sequence, an
expression cassette or a vector comprising said TER nucleic acid sequence or
to an
organism transformed with said nucleic acid sequence, expression cassette or
vector
or all those constructs established by recombinant methods in which either
a) the nucleic acid sequence encoding a TER or
b) a genetic control sequence, for example a promoter which is functional in
plant
organisms, which is linked operably with said nucleic acid sequence under a)
are not in their natural genetic environment or have been modified by
recombinant
methods, it being possible for the modification to be, for example, a
substitution,
addition, deletion, inversion or insertion of one or more nucleotide residues.
Natural
genetic environment refers to the natural chromosomal locus in the source
organism or
the presence in a genomic library. In the case of a genomic library, the
natural genetic
environment of the nucleic acid sequence is preferably retained, at least to
some
extent. The environment flanks the nucleic acid sequence at least on one side
and has
a sequence length of at least 50 bp, preferably at least 500 bp, particularly
preferably
at least 1000 bp, very particularly preferably at least 5000 bp. A naturally
occurring
CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
expression cassette, for example the naturally occurring combination of the
promoter
of a gene coding for an TER with the corresponding TER gene, becomes a
transgenic
expression cassette when the latter is modified by non-natural, synthetic
("artificial")
methods such as, for example, a mutagenization. Such methods are described in
5 US 5,565,350; WO 00/15815; see also above.
Host or starting organisms which are preferred as transgenic organisms are,
above
all, plants in accordance with the above definition. Included for the purposes
of the
invention are all genera and species of higher and lower plants of the Plant
Kingdom,
10 in particular plants which are used for obtaining oils, such as, for
example, oilseed
rape, sunflower, sesame, safflower, olive tree, soya, maize, wheat and nut
species.
Furthermore included are the mature plants, seed, shoots and seedlings, and
parts,
propagation material and cultures, for example cell cultures, derived
therefrom. Mature
plants refers to plants at any desired developmental stage beyond the seedling
stage.
15 Seedling refers to a young, immature plant at an early developmental stage.
The transgenic organisms can be generated with the above-described methods for
the
transformation or transfection of organisms.
20 The invention furthermore relates to the use of the transgenic organisms
according to
the invention and to the cells, cell cultures, parts - such as, for example,
in the case
of transgenic plant organisms roots, leaves and the like - and transgenic
propagation
material such as seeds or fruits which are derived therefrom for the
production of food-
stuffs or feedstuffs, pharmaceuticals or fine chemicals, in particular oils,
fats, fatty acids
25 or derivatives of these.
Besides influencing the lipid content, the transgenic expression of SEQ ID NO
: 1, SEQ
ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10 or derivatives
thereof in
plants may mediate yet further advantageous effects such as, for example,
30 an increased stress resistance. Such osmotic stress occurs for example in
saline soils
and water and is an increasing problem in agriculture. Increased stress
tolerance
makes it possible, for example, to. use areas in which conventional arable
plants
are not capable of thriving for agricultural usage.
35 The determination of activities and kinetic parameters of enzymes is well
established
in the art. TER activity can be measured according to Inui et al., (1984),
European
J. Biochem. 142:121-126.
The activity of a recombinant gene product in the transformed host organism
can be
40 measured on the transcriptional or/and on the translational level. A useful
method
to ascertain the level of transcription of the gene (an indicator of the
amount of mRNA
available for translation to the gene product) is to perform a Northern blot
(for reference
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41
see, for example, Ausubel et al. 1988, Current Protocols in Molecular Biology,
Wiley:
New York), in which a primer designed to bind to the gene of interest is
labeled with a
detectable tag (usually radioactive or chemiluminescent), such that when the
total RNA
of a culture of the organism is extracted, run on gel, transferred to a stable
matrix and
incubated with this probe, the binding and quantity of binding of the probe
indicates the
presence and also the quantity of mRNA for this gene. This information at
least partially
demonstrates the degree of transcription of the transformed gene. Total
cellular RNA
can be prepared from plant cells, tissues or organs by several methods, all
well-known
in the art, such as that described in Bormann et al. (1992, Mol. Microbiol.
6:317-326).
To assess the presence or relative quantity of protein translated from this
mRNA,
standard techniques, such as a Western blot, may be employed (see, for
example,
Ausubel et al. 1988, Current Protocols in Molecular Biology, Wiley: New York).
In
this process, total cellular proteins are extracted, separated by gel
electrophoresis,
transferred to a matrix such as nitrocellulose, and incubated with a probe,
such as an
antibody, which specifically binds to the desired protein. This probe is
generally tagged
with a chemiluminescent or colorimetric label which may be readily detected.
The
presence and quantity of label observed indicates the presence and quantity of
the
desired mutant protein present in the cell.
The effect of the genetic modification in plants on a desired seed storage
compound
e.g. triacylglycerols, diacylglycerols, monoacylglycerols, phospholipids,
waxesters
and/or fatty acids can be assessed by growing the modified plant under
suitable
conditions and analyzing the seeds or any other plant organ for increased
production
of the desired product. Such analysis techniques are well known to one skilled
in the
art, and include spectroscopy, thin layer chromatography, staining methods of
various
kinds, enzymatic and microbiological methods, and analytical chromatography
such as
high perFormance liquid chromatography (see, for example, Ullman 1985,
Encyclopedia
of Industrial Chemistry, vol. A2, pp. 89-90 and 443-613, VCH: Weinheim;
Fallon, A.
et al. 1987, Applications of HPLC in Biochemistry in: Laboratory Techniques in
Biochemistry and Molecular Biology, vol. 17; Rehm et al., 1993 Product
recovery and
purification, Biotechnology, vol. 3i Chapter III, pp. 469-714, VCH: Weinheim;
Better,
P.A. et al., 1988 Bioseparations: downstream processing for biotechnology,
John Wiley
& Sons; Kennedy J.F. & Cabral J.M.S. 1992, Recovery processes for biological
materials, John Wiley and Sons; Shaeiwitz J.A. & Henry J.D. 1988, Biochemical
separations in: Ulmann's Encyclopedia of Industrial Chemistry, Separation and
purification techniques in biotechnology, vol. B3, Chapter 11, pp. 1-27, VCH:
Wein-
heim; and Dechow F.J. 1989).
Besides the above-mentioned methods, plant lipids are extracted from plant
material as
described by Cahoon et al. (1999, Proc. Natl. Acad. Sci. USA 96, 22:12935-
12940) and
Browse et al. (1986, Anal. Biochemistry 442:141-145). Qualitative and
quantitative lipid
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4a
or fatty acid analysis is described in Christie, William W., Advances in Lipid
Metho-
dology. Ayr/Scotland :Oily Press. - (Oily Press Lipid Library; Christie,
William W., Gas
Chromatography and Lipids. A Practical Guide - Ayr, Scotland :Oily Press, 1989
Repr.
1992. - IX,307 S. - (Oily Press Lipid Library; and "Progress in Lipid
Research, Oxford
:Pergamon Press, 1 (1952) -16 (1977) Progress in the Chemistry of Fats and
Other
Lipids CODEN.
Unequivocal proof of the presence of fatty acid products can be obtained by
the
analysis of transgenic plants following standard analytical procedures: GC, GC-
MS or
TLC as variously described by Christie and references therein (1997 in:
Advances on
Lipid Methodology 4t" ed.: Christie, Oily Press, Dundee, pp. 119-169; 1998).
Detailed
methods are described for leaves by Lemieux et al. (1990, Theor. Appl. Genet.
80:234-
240) and for seeds by Focks & Benning (1998, Plant Physiol. 118:91-101 ).
Positional analysis of the fatty acid composition at the C-1, C-2 or C-3
positions of the
glycerol backbone is determined by lipase digestion (see, e.g., Siebertz &
Heinz 1977,
Z. Naturforsch. 32c:193-205, and Christie 1987, Lipid Analysis 2"d Edition,
Pergamon
Press, Exeter, ISBN 0-08-023791-6).
A typical way to gather information regarding the influence of increased or
decreased
protein activities on lipid and sugar biosynthetic pathways is for example via
analyzing
the carbon fluxes by labeling studies with leaves or seeds using '4C-acetate
or'4C-
pyruvate (see, e.g. Focks & Benning 1998, Plant Physiol. 118:91-101; Eccleston
&
Ohlrogge 1998, Plant Cell 10:613-621 ). The distribution of carbon-14 into
lipids and
aqueous soluble components can be determined by liquid scintillation counting
after
the respective separation (for example on TLC plates) including standards
like'4C-
sucrose and '4C-malate (Eccleston & Ohlrogge 1998, Plant Cell 10:613-621).
Material to be analyzed can be disintegrated via sonification, glass milling,
liquid
nitrogen and grinding or via other applicable methods. The material has to be
centri-
fuged after disintegration. The sediment is re-suspended in distilled water,
heated for
10 minutes at 100°C, cooled on ice and centrifuged again followed by
extraction in
0.5 M sulfuric acid in methanol containing 2% dimethoxypropane for 1 hour at
90°C
leading to hydrolyzed oil and lipid compounds resulting in transmethylated
lipids. These
fatty acid methyl esters are extracted in petrolether and finally subjected to
GC analysis
using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m,
0.32 mm) at a temperature gradient between 170°C and 240°C for
20 minutes and
5 min. at 240°C. The identity of resulting fatty acid methylesters is
defined by the use
of standards available form commercial sources (i.e., Sigma).
In case of fatty acids where standards are not available, molecule identity is
shown via
derivatization and subsequent GC-MS analysis. For example, the localization of
triple
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43
bond fatty acids is shown via GC-MS after derivatization via 4,4-Dimethoxy-
oxazolin-
Derivaten (Christie, Oily Press, Dundee, 1998).
For example, yeast expression vectors comprising the nucleic acids disclosed
herein,
or fragments thereof, can be constructed and transformed into Saccharomyces
cerevisiae using standard protocols. The resulting transgenic cells can then
be
assayed for alterations in sugar, oil, lipid or fatty acid contents.
Similarly, plant expression vectors comprising the nucleic acid SEQ ID NO : 1,
SEQ ID
NO : 4, SEQ ID NO : 6, SEQ ID NO : 8 or SEQ ID NO : 10 disclosed herein, or
fragments thereof, can be constructed and transformed into an appropriate
plant cell
such as Arabidopsis, soybean, rape, maize, wheat, Medicago truncatula, etc.,
using
standard protocols. The resulting transgenic cells and/or plants derived
therefrom can
then be assayed for alterations in oil, lipid or fatty acid contents.
Additionally, the sequence SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID
NO
8 or SEQ ID NO : 10 disclosed herein, or fragments thereof, can be used to
generate
knockout mutations in the genomes of various organisms, such as bacteria,
mammal-
ian cells, yeast cells, and plant cells (Girke at al. 1998, Plant J. 15:39-
48). The resultant
knockout cells can then be evaluated for their composition and content in seed
storage
compounds, and the effect on the phenotype and/or genotype of the mutation.
For
other methods of gene inactivation include US 6,004,804 "Non-Chimeric
Mutational
Vectors" and Puttaraju et al. (1999, "Spliceosome-mediated RNA trans-splicing
as a
tool for gene therapy" Nature Biotech. 17:246-252).
The invention now having been generally described will be more readily
understood by
reference to the following examples, which are included for the purpose of
illustration
only, and are not intended to limit scope of the present invention.
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44
Examples
General methods
General Cloning Processes:
Cloning processes such as, for example, restriction cleavages, agarose gel
electro-
phoresis, purification of DNA fragments, transfer of nucleic acids to
nitrocellulose and
nylon membranes, linkage of DNA fragments, transformation of Escherichia coli
and
yeast cells, growth of bacteria and sequence analysis of recombinant DNA were
carried out as described in Sambrook et al. (1989, Cold Spring Harbor
Laboratory
Press: ISBN 0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994, "Methods
in
Yeast Genetics", Cold Spring Harbor Laboratory Press: ISBN 0-87969-451-3).
Chemicals:
The chemicals used were obtained, if not mentioned otherwise in the text, in
p.a.
quality from the companies Fluka (Neu-Ulm), Merck (Darmstadt), Roth
(Karlsruhe),
Serva (Heidelberg) and Sigma (Deisenhofen). Solutions were prepared using
purified,
pyrogen-free water, designated as H20 in the following text, from a Milli-Q
water
system water purification plant (Millipore, Eschborn). Restriction
endonucleases, DNA-
modifying enzymes and molecular biology kits were obtained from the companies
AGS
(Heidelberg),Amersham (Braunschweig), Biometra (Gottingen), Boehringer (Mann-
heim), Genomed (Bad Oeynnhausen), New England Biolabs (Schwalbach/ Taunus),
Novagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Pharmacia
(Freiburg), Qiagen (Hilden) and Stratagene (Amsterdam, Netherlands). They were
used, if not mentioned otherwise, according to the manufacturer's
instructions.
Plant Growth:
Arabidopsis thaliana
Plants were either grown on Murashige-Skoog medium as described in Ogas et al.
(1997, Science 277:91-94; 1999, Proc. Natl. Acad. Sci. USA 96:13839-13844) or
on
soil under standard conditions as described in Focks & Benning (1998, Plant
Physiol.
118:91-101).
Example 1
Biochemical purification of trans-2-enoyl-CoA reductase (TER) from Euglena
gracilis
One kilogram of Euglena gracilis strain Z cells grown under aerobic conditions
for one
week was used as starting material.
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Euglena cultures were performed in a BIOSTAT B 10L fermenter (Braun Biotech).
The culturing conditions were as follows: culturing volume of 7 liters, light
intensity of
5000 Ix continuously, temperature of 28°C, stirring at 200 rpm. A
defined medium as
5 described by Ogbonna, J.C. et al. (1981) J. Appl. Phycol. 10:67-74 and
Yamane, Y
et al. (2001) Biotech. Lett. 23: 1223-1228 was modified and used. One liter
medium
was composed of 12 g glucose; 0,8 g KH~PO~.; 1,5 g (NH4)aS04; 0,5 g
MgS04x7H20;
0,2 g CaC03; 0,0144 g H3B03; 2,5 mg vitamin B~; 20 ~ug vitamin B12; 1 ml trace
element solution; 1 ml Fe-solution. The trace element solution was composed of
4,4 g
10 ZnS04x7H20; 1,16 g MnSO~xH~O; 0,3 g Na2MoO4x2H~0; 0,32 g CuS04x5H20; 0,38 g
CoS04x5H20 per 100 ml of destilled water and the Fe-solution consisted of 1,14
g
(NH4)2S04Fe(S04)ax6H20 and 1 g EDTA per 100 ml of destilled water. The pH of
the
medium was kept at 2,8 during the cultivation and the cultures were fumigated
with
2 liters/min air.
After the culture harvest using standard techniques the cells were disrupted
using a
French Press. A 30% ammonium sulphate cut was used to remove cell debris.
After
dialysis, a series of chromatographic purifications steps was undertaken, see
table 4.
These included ion exchange chromatography (DEAE-Fraktogel), hydrophobic
interactions (phenylsapharose), affinity chromatography (Reaktive Red 120) and
hydroxyapatite chromatography. The c-orresponding purification levels can be
seen in
table 4. Furthermore, an additional ion exchange chromatography (Mono Q),
purifica-
tion over a preparative gel and a final gel filtration through Superdex 200
completed the
purification scheme. This scheme achieved more than 1600 fold purification.
The details of the purification procedure were as follows: all chromatographic
steps
were carried out using a FPLC system (Amersham Biosciences). All columns were
packed according to the manufactors instructions or prepacked columns were
used. All
buffers were filtrated through 0,45,um nitrocellulosefilters (Sartorius,
Gottingen) and
degassed. After French Press disruption and 30 % ammonium sulphate cut the
first
chromatographic step was carried out with DEAE-Fraktogel EMD 650 S (Merck).
10 runs on a XK 26 column (Amersham Biosciences) with the dimensions of 2,6 x
12 cm were performed and elution of proteins was achieved with a linear
gradient from
0 to 1 M KCI in 25 mM potassiumphosphate buffer pH 6,8 with 1 mM EDTA, 1 mM
DTT
and 1 ,uM FAD. Active fractions were pooled and supplied in 4 runs to Phenyl
Sepha
rose 6 Fast Flow low sub (Amersham Biosciences) in a XK 26 column (2,6 x 14
cm).
Elution was performed with a linear gradient from 1 M NH4(S04)2 descending to
0 M
NH4(S04)~ in 10mM Tris-HCI pH 8,0 with 1 mM EDTA, 1 mM DTT and 1,uM FAD.
Active fractions were pooled and supplied in 5 runs to Reaktive Red 120
(Sigma) in a
XK 16 column (1,6 x 9 cm). Elution was performed with a linear gradient from 0
to 1 M
KCI in 25 mM potassiumphosphate buffer pH 6,8 with 1 mM EDTA, 1 mM DTT and
1 ,uM FAD. Active fractions were pooled again and supplied in 8 runs to
Hydroxyapatit
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46
matrix using Eco-Pac CHT II Cartridge (Bio-Rad, Miinchen). Elution was
performed
with a linear gradient from 10 mM potassiumphosphate buffer pH 6,8 with 1 mM
DTT
and 1,uM FAD to 500 mM potassiumphosphate buffer pH 6,8 with 1 mM DTT and 1 NM
FAD. Active fractions were pooled and supplied in 3 runs to Mono Q HR 5/5
column
(Amersham Biosciences). Elution was performed with a linear gradient from 0 to
1 M
KCI in 10 mM Tris-HCI pH 8,0 with 1 mM EDTA, 1 mM DTT and 1,uM FAD.
Active fractions were pooled and supplied in 6 runs to a 6 % continous native
poly-
acrylamid gel in a Mini Prep Cell (Bio-Rad) according to the manufactors
instructions.
Active fractions were pooled and supplied in 3 runs to a Superdex 200 HR 10/30
column (Amersham Biosciences). Elution was performed with 10 mM Tris-HCI pH
8,0
containing 150 mM NaCI, 1 mM DTT, 1 mM EDTA and 1,uM FAD. Active fractions
were
submitted to a SDS-PAGE using standard protocols. The final enzyme preparation
showed a major and a thin minor band very closely together at about 44kDa, see
figure 2.
When submitted to a SDS-PAGE gel (12%) using standard protocols, the final
enzyme
preparation showed a major and a thin minor band very closely together at
about
44kDa.
Enzyme activity was measured as described by Inui, H. et al. (1984) Eur. J.
Biochem.
142: 121-126 with the following modifications. The assay mixture contained 100
mM
potassiumphosphate buffer pH 6,2; 0,75 mM Crotonyl-CoA (Sigma); 0,4 mM NADH
and 2,uM FAD and enzyme. The assay mixture without substrate was preincubated
for
10 min at 30°C. The reaction was also performed at 30°C and
started with the addition
of substrate. The activity was determined by the decrease of absorbance at 340
nm.
The final assay volume was 1 ml (Ultrospec 2000 Spectrophotometer, Amersham
Biosciences) or 200,u1 (GENios microplate reader, Tecan Instruments,
Maennedorf,
Switzerland).
Figure 2 shows the protein pattern at different purification steps of TER in a
SDS-
PAGE gel (12%). Enzyme activity. was associated with the major, upper band.
CA 02541141 2006-03-31
WO 2005/040366 PCT/EP2004/011294
47
0o d-
M M ~
d O O T r O O O
N O O O O
(fl ~- O O O
L
O
V
O
M CO M r M
N
v
w=
.i
a
as
N
t4
v .~
G
N
O v ~ . I~ N
~ ~
N I' M CD O ~ ~,rj00
~ O ~
C V i.n N t~ ~ O M M ~ O
N D O
V G~ ~ N M ~- M
O'
O
C
O
O
N
C
c0 t
L .N
~3
o
O . ~ M Cfl d' M
O
~' O ~ '~' ~ "' ~
E
~ O
~ ~ " .~t'N d' '- O
tC.
,
.
N
w:. O
'L 1-
3
a
:~ ~ f' M (O 00
N
7 O ~ t' t' O ~ I' O N O
t~
N M N ~ l
i~
t M ~ C~ r r .-.
OC N ~.
O
H
N
N
U ~ O O ~ '
~ r
Q
C, -
.
L >
N X tB -
N N Q > ~ ~ a
(a
N ~ L
~
C N cSf~_ i' _t'n~ O O ~ Q.
d r
~! ~ -
~' tn
V W ~ W ~ ~ = a
v
_d
a
a
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4t3
Example 2
Isolation of Total RNA and poly-(A)+ RNA from Euglena gracilis cells
For the investigation of transcripts, both total RNA and poly-(A)+ RNA were
isolated.
RNA is isolated from Euglena gracilis cells according to the following
procedure:
The details for the isolation of total DNA relate to the working up of five
gram fresh
weight of Euglena. The material was triturated under liquid nitrogen in a
mortar to give
a fine powder and covered with 20 ml Resuspension buffer (50 mM Tris-HCI pH
8,0;
100 mM NaCI; 10 mM EDTA; 30 mM 2-mercaptoethanol; 2 % (w/v) SDS; 4 M Guani-
dinumthiocyanate; 5 % (w/v) Polyclar). The homogenate was transferred to a
conical
tube and extracted by shaking with the same volume of
phenol/chloroformlisoamyl
alcohol. For phase separation, centrifugation was carried out at 50008 and RT
for .
5 min. Nucleic acids were precipitated at -20°C for 60 min using ice-
cold isopropanol
and then sedimented at 4°C and 6000 g for 20 min and resuspended in 5
ml of
TE buffer which contained 10 pg/ml Proteinase K. Precipitation of RNA was
carried
out with 1,25 ml 10 M LiCI at -20°C overnight, followed by
sedimentation at 4°C and
10000 g for 30 min. The RNA pellet was resuspended in 5 ml DEPC-treated water
and
for further purification precipitated again with ethanol for 2 h at -
20°C. Final sedimen-
tation of RNA was achieved by centrifugation at 4°C and 10000 g for 20
min. The total
RNA was diluted in 2 ml TE and the concentration was determined.
The mRNA is prepared from total RNA, using the Amersham Biosciences mRNA puri-
fication kit, which utilizes oligo(dT)-cellulose columns.
Example 3
cDNA Library Construction
For cDNA library construction, first strand synthesis was achieved using
Moloney
Murine Leukemia Virus reverse transcriptase (Amersham Biosciences, Freiburg,
Ger-
many) and oligo-d(T)-primers, second strand synthesis by incubation with DNA
poly-
merase I, Klenow enzyme and RNAseH digestion at 12°C (30 min) and
22°C (1 h). The
reaction was stopped by incubation at 65°C (10 min) and subsequently
transferred to
ice. Nucleotides were removed by phenol/chloroform extraction and Sepharose CL-
4B.
EcoRl/ Notl adapters (Amersham Biosciences, Freiburg) were ligated to the cDNA
ends by T4-DNA-ligase (Amersham Biocsciences, 12°C, overnight) and
phosphory-
lated by incubation with polynucleotide kinase (Amersham Biosciences,
37°C, 30 min).
Excessive adaptors were removed by Sepharose CL-4Bc.DNA molecules were ligated
to vector arms and packed into lambda ZAPII phages using the Gigapacklll Gold
Kit
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49
(Stratagene, Amsterdam, Netherlands) using material and following the
instructions
of the manufacturer.
From the cDNA Bank pBluescript phagemides can be generated by in-vivo-excision
using the ExAssist helper phage ( Stratagene, Netherlands) for further
analysis.
Example 4
Identification of TER protein sequence by peptide fingerprinting and
subsequent clo-
ning of the corresponding cDNA
SDS-PAGE of the biochemically purified TER resulted in a double band of 44
kDa. The
major and the minor band were cut from the gel separately and digested with
trypsin
using standard protocols. The resulting peptides were extracted from the gel
and ana-
lysed using ESI-Q-TOF MS/MS using standard protocols. Both bands were shown to
yield solely identical peptides, confirming the complete purification of the
TER as a
single subunit enzyme in contrast to the description of Inui and co-workers
(Inui et al.
(1986) J. Biochem. 100: 995-1000). These were the peptides identified with ESI-
Q-
TOF MS/MS:
peptide 1 ACLKPLGATYTNR . '
peptide 2 AALEAGLYAR
peptide 3 VLVLGCSTGYGLSTR
peptide 4 TDPAT
peptide 5 SLDGDAFDSTTK
peptide 6 DLWSQVNTANLK
peptide 7 AGWYNTVAFEK
peptide 8 RVQEELAYAR
peptide 9 DLSDFAGYQTEFLR
peptide 10 LYPGDGSPLVDEAGR
peptide 11 LTQQYGCPAYPWAK
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peptide 12 VDDWEMAEDVQQAVK
peptide 13 STGYG(AMVR/LSEK)
5 peptide 14 AHPPTSPGPK
peptide 15 ALSEAGVLAEQK
peptide 16 ((GT)/(AS))HEGCLEQMVR
peptide 17 LYPENGAPLVDEQR
Degenerate primers were designed according to these peptides and used for PCR
with cDNA as template. Due to the high GC-content of E. gracilis an initial
denaturation
of 98°C for 10 min was accomplished prior to PCR. PCR conditions were
as
follows: 30 cycles with 94°C for 30 sec; 50°C for 30 sec and
72°C for 90 sec; ,
final extension at 72°C for 5 min. A 837 by fragment was amplified with
the
following primers: 5'-GGITGGTAYAAYACIGTIGC-3' (referring to peptide 7) and
5'-GTYTCRTAICCIGCRAARTC-3' (referring to peptide 9). This fragment was cloned
into pBluescript SIC+/Hincll and sequenced (SEQ ID NO : 3). The translated
sequence
contained several 'peptides of the purified protein and therefore the 837 by
fragment .
was used as hybridisation probe to screen a cDNA library constructed with mRNA
from aerobically grown Euglena cells as described in example 2 and 3.
Screening of
250.000 recombinant phages resulted in six independent clones. cDNA inserts
varied
between 1600 by and 1900 bp. Sequencing of all six clones from both ends
revealed
that all clones represented the same transcript and varied only in length. The
longest
clone was sequenced completely double-stranded via deletion by exonuclease
III. The
clone had a length of 1912 by and encodes an open reading frame of 1620 by
coding
for 539 as (SEQ ID NO : 1 and SEQ ID NO : 2). At both ends it had adaptors
consisting
of a Notl and EcoRl restriction site and was inserted into the EcoRl site of
the vector
pBluescript SKP. Figure 3 shows the map of the TER clone in the vector
pBluescript
SKP.
Example 5
Triacylglycerol accumulation in yeast cells expressing the TER gene
The TER gene can be excised from the cloning vector pBluescript SK+ by EcoRl
diges-
tion and cloned into the EcoRl behind the strong inducible GAL1 promotor in
the multi-
copy plasmid pYES2 (Invitrogen), thus generating the plasmid pYTER. The wild
type
yeast strain By4742 (MATa his301, leu2 O0, lys2 O0, ura3 DO), transformed with
the
pYTER is cultivated at 30°C on a rotary shaker in synthetic medium
(Sherman, F. et al.,
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51
(1986) Laboratory Course Manual for Methods in Yeast Genetics, Cold Spring
Harbor
Lab. Press, Plainview, NY. ) lacking uracil and supplemented with 2 %
(vol/vol) glycerol
and 2 % (vol/vol) ethanol. The GAL1 promoter is induced after 24 hours of
growth by
the addition of 2 % (wt/vol) final concentration of galactose. Cells are
harvested after
an additional 24 or 28 hours of growth. Wild type cells By4742, transformed
with the
empty vector (pYES2) and cultivated under identical conditions, are used as a
control.
In order to quantify the total lipid content, 3 x 5 ml aliquots from yeast
cultures are
harvested by centrifugation, and the resulting pellets are washed with
distilled water
and lyophilised. The weight of the dried cells is determined, and the fatty
acid content is
quantified by conventional gas-liquid chromatography (GLC) analyses after
conversion
to methyl esters (Dahlqvist et al. (2000) Proc. Natl. Acad. Sci. USA 97, 6487-
6492).
The lipid content is calculated as nmol fatty acids per mg dry weight. The
lipid compo-
sition of the yeast is determined in cells harvested from 35-ml liquid
cultures. The
harvested yeast cells are re-suspended in 15 ml glass tubes in water to~a
final volume
of 0.6 ml, to which 3.75 ml chloroform: methanol (1:2), 50 pl acetic acid, and
2 ml of
glass beads (0.45 to 0.50 mm). are added. The ,yeast cells are disrupted by
vigorous
agitation (5 x 1 min) and the lipids are extracted into chloroform according
to standard
method (Bligh, E.G. and Dyer, W.J., Can. J. Biochem. Physiol. 37(1959), 911-
917).
The collected lipid fraction is divided in two parts and separated'by TLC on
Silica Gel
60 plates (Merck) in hexarie / diethyl ether / acetic acid (70:30:1 ) for the
quantification
of neutral lipids, i.e. unesterified fatty acids (FA)., diacylglycerols (DAG),
triacylglycerols
(TAG), and steryl esters (SE), and in chloroform / methanol / acetic acid:
water
(85: 15: 10: 3.5) for the quantification of the major polar lipids, i.e.
phosphatidylinositol
(PI), phosphatidylserine (PS), phosphatidylcholine (PC), and
phosphatidylethanolamine
(PE). The lipid areas are located by brief exposure to 12 vapors and
identified by means
of appropriate standards. The different lipid classes are excised from the
plates and
fatty acid methyl. esters are prepared by heating the excised material at
85°C for 60 min
in 2% (vol/vol) sulfuric acid in dry methanol. The methyl esters are extracted
and quan
tified by conventional gas-liquid chromatography (GLC) analyses as described
in
Dahlqvist et al., 2000.
Example 6
Functional expression of trans-2-enoyl-CoA reductase (TER) in E.coli
Two different parts of the TER cDNA clone were choosen for heterologous
expression
in E. coli. The first construct (ter1) encompassed the complete open reading
frame (see
SEQ ID NO : 1 ).~ The second construct (ter2) comprised part of the open
reading frame
beginning with as 136. ter2 has a length of 1215 by and a calculated molecular
mass
of 45 kDa that is equivalent to the molecular mass of purified TER protein
from Euglena
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52
gracilis (see figure 2). The constructs ter1 and ter2 were amplified from
Euglena
gracilis cDNA using the following primers
TER1 Ndefor 5'-TAT ACA TAT GTC GTG CCC CGC CTC GCC GTC. TG-3'
Nde I
TER1 Bglfor 5'-TAT AGA TCT TAT GTC GTG CCC CGC CTC GCC GTC TG-3'
Bgl I I
TER2Ndefor 5'-TAT ACA TAT GTT CAC CAC CAC AGC GAA GGT CAT CC-3'
Nde I
TERXhorev 5'-TAT CTC GAG CTA CTG CTG GGC AGC ACT GG-3'
Xho I
ter2 was inserted via the restriction sites Nde I and Xho I into the vector
pET28a
(Novagen, Darmstadt, Germany) and expressed in the E. coli expression strain
BL21 (DE3) (Novagen).
50 to 100 ml of LB-medium with antibiotics were inoculated with a single E,
coli colony.
,. The cultures were grown under shaking at 37°C until OD600 0,6-1. The
induction was
'carried out with a finaI~IPTG concentration of 0,4 mM overnight at
16°C. Subsequently
the expression cultures were harvested via centrifugation at 4000 g for 10
min. The
pellet was resuspended in lysis buffer (50 mM NaHaPO4 pH 8,0; 300 mM NaCI; 10
mM
imidazol) and incubated at 4°C for 30 min after the addition of 1 mg/ml
lysozyme. Cell
disruption was carried with sonication in 6 cycles at each time for 10 sec
with 80 W.
RNase A (10 pg/ml) and DNase I (5 pg/ml) were added and the probes were
incubated
for another 10 min at 4°C. The supernatant of centrifugation at 10000 g
for 30 min
comprised the soluble protein fraction.
4 ml of soluble protein fraction was supplied with 1 ml of 50 % Ni-NTA agarose
(Qiagen, Hilden, Germany) and incubated under shaking for 1 h at 4°C.
Subsequently
the whole sample was added to a Polypropylene column (Qiagen) and the flow-.
through was saved. Up to three washing steps with every 4 ml washing buffer
(50 mM
NaH2P04 pH 8,0; 300 mM NaCI; 20 mM imidazol) were carried,out. The elution of
pro-
teins was carried out in four steps with every 0,5 ml of elution buffer (50 mM
NaH2P04
pH 8,0; 300 mM NaCI; 250 mM ~imidazol).
SDS-PAGE of soluble fraction of E.coli cells transformed with ter2-pET28 shows
a
major band at the expected size of 45 kDA in contrast to control cells (see
figure 5).
The ter2-protein was purified with the help of the added His-Tag via Ni-NTA
agarose
and 12% SDS-PAGE showed strong enrichment of ter2-protein (see figure 5).
Western
blot analysis and immunodetection were carried out using standard protocols
(Sam-
brook et al., 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
As first
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53
antibody a monoclonal mouse IgG His-Tag antibody (Novagen, Darmstadt, Germany)
was used and the second antibody was a HRP-conjugated anti-mouse antibody from
goat (Amersham Biosciences, Freiburg, Germany). Detection of secondary
antibody
was carried out with ECL Western Blotting Analysis Kit (Amersham Biosciences).
The
Western blot analysis of ter2-pET28 construct in BL21 (DE3) with subsequent
immu-
nodetection of the His-Tag revealed specific signals of the overexpressed ter2-
protein
(see figure 6 and 7). Specific activity of purified ter2-protein was 1510
nmol~mg-1
min-1.
ter1 was inserted in the vector pET28a (Novagen) and pET32a (Novagen) via the
resriction sites Nde I and Xho I and respectively Bgl II and Xho I (see
figures 7 and 8).
No expression could be shown for ter1 in E. coli BL21 (DE3) see figure 8
western blot
of ter1-pET32 construct in Rosetta(DE3). Therefore the E. coli expression host
strains
Origami(DE3) and Rosetta(DE3) (both Novageri) were tested for expression of
ter1. In
the SDS-PAGE for the ter1-constructs in pET28a no expression could be shown.
How-
ever, Western blotting with subsequent immunodectection of the His-Tag showed
a
specific signal for the construct ter1-pET32 in E. coli Rosetta(DE3) (see
figure 8). The
signal showed the expected size of 78 kDa of the ter1-pET32 construct
including the
thioredoxin-Tag of 19 kDa. A TER-specific activity for the expressed ter1-
protein could
not be measured. ,
The results of the expression studies of Euglena gracilis TER in E. coli can
be taken as
consideration that the N-terminal part of the cDNA clone may constitute a
mitochondrial
targeting signal, which has to be cleaved to yield the mature and active TER
protein.
Nevertheless the possibility that the N-terminal part of the cDNA clone
constitutes a
transmembrane domain can not be excluded. This possible transmembrane domain
could be lost during biochemical purification of TER from Euglena gracilis
(see example
1)..If expressed in E. coli this domain may possibly disrupt activity
measurement with
the C4-substrat (see example 1 ) due to incorrect convolution or missing
membrane-
linkage.
Example 7
Construction of a set of TER constructs for overexpression in Arabidopsis
Since the shortened version ter2 of the Ter gene was active in E.coli,
shortened and
modified, but functional versions of the TER may be produced. As an example
two
shortened versions of the Ter gene could be generated by PCR, sequenced and
also
transformed into plants: One with an ORF given by SEQ ID NO : 4 resulting in
the pro-
tein sequence given by SEQ ID NO : 5. This protein sequence is shortened
compared
to SEQ ID NO: 2 but identical to amino acid residues 126 and the following of
SEQ ID
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54
NO : 2. However before this identical region it has a 28 amino acid N-terminal
stretch
differing to SEQ ID NO : 2. This 28 amino acids is predicted to result in
mitochondrial
targeting without the need for the 125 amino acid long putative mitochondria)
targeting
sequence of SEQ ID NO : 2.
The second sequence (SEQ ID NO : 6) results in the amino acid sequence SEQ ID
NO
7 and corresponds to the shortened version described above as ter2 and shown
to be
active in E.coli. The protein resulting from this sequence is not predicted to
be targeted
to the mitochondrium.
As another example of variations ter 1 (SEQ ID NO : 1 ) and ter2 (SEQ ID NO :
6) may
be cloned into the binary vector ST593 (see figure 10) so that a new N-
terminal se-
quence is added to the ORF, resulting in SEQ ID NO : 8 and 10 respectively.
The cor-
responding protein sequences (SEQ ID NO : 9 and 11 ) will be the plastid
targeting
peptide of the small subunit of Rubisco fused to ter1 and ter 2 respectively.
These DNA sequences could be cloned into the binary vectors as exemplified
below in
example 8.
Example 8
Plasmids for Plant Transformation
For plant transformation binary vectors such as pBinAR can be used (Hofgen &
Willmitzer 1990, Plant Sci. 66:221-230). Construction of the binary vectors
can be
performed by ligation of the cDNA in sense or antisense orientation into the T-
DNA.
5-prime to the cDNA a plant promoter activates transcription of the cDNA. A
poly-
adenylation sequence is located 3'-prime to the cDNA. Tissue-specific
expression can
be achieved by using a tissue specific promoter. For example, seed-specific
expression
can be achieved by cloning the napin or LeB4 or USP promoter 5-prime to the
cDNA.
Also any other seed specific promoter element can be used. For constitutive
expres-
sion within the whole plant the CaMV 35S promoter can be used. The expressed
pro-
tein can be targeted to a cellular compartment using a signal peptide, for
example for
plastids, mitochondria or endoplasmic reticulum (Kermode 1996, Crit. Rev.
Plant Sci.
15:285-423). The signal peptide is cloned 5-prime in frame to the cDNA to
achieve
subcellular localization of the fusion protein.
Further examples for plant binary vectors are the pSUN300 vectors into which
the TER
gene candidates is cloned, see figure 4. These binary vectors contain an
antibiotic re-
sistance gene driven under the control of the Nos-promotor, and a USP seed-
specific
promoter in front of the candidate gene with the OCS terminator. TER cDNA is
cloned
into the multiple cloning site of the plant binary vector in sense or
antisense orientation
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behind the USP seed-specific promoter. The recombinant vector containing the
gene of
interest is transformed into DhSa (Invitrogen) using standard conditions.
Transformed
cells are selected for on LB agar containing 100 Ng/ml streptomycin grown
overnight at
37°C. Plasmid DNA is extracted using the QIAprep Spin Miniprep Kit
(Qiagen) following
5 manufacturer's instructions. Analysis of subsequent clones and restriction
mapping is
performed according to standard molecular biology techniques (Sambrook et al.
1989,
Molecular Cloning, A Laboratory Manual. 2nd Edition. Cold Spring Harbor
Laboratory
Press. Cold Spring Harbor, NY).
10 Example 9
Transformation of Arabidopsis
One skilled in the art may choose from various methods, the following one is
given as
15 an example:
Using floral dip essentially as described by Clough and Bent, 1998, plants are
trans-
formed with Agrobacterium tumefaciens C58C1 harboring the plasmid pSUN300-USP-
TER-OCS respectively. Entire plants (inflorescence and rosette) are submerged
for
20 20 - 30 sec in the infiltration media consisting of 5% sucrose, 0.02%
Silwet L-77~ (Osi
Specialties, Danbury, CT) and re-suspended transformed A. tumefasciens cells.
Plants
are then transferred to a growth chamber with a photoperiod of 16 h of light
at 21°C
and 8 h of dark at 18°C (70% humidity) or a similarly air-conditioned
greenhouse. The
T1 seeds are collected from mature plants. Subsequently transformed plants
~ivere
25 identified on selection media by growing T1 seeds on MS-agar plates
supplemented
with kanamycin (50 pgiml).
Example 10
In vitro Analysis of the Function of the TER gene in Transgenic Plants
The determination of activities and kinetic parameters of enzymes is well
established
in the art. TER activity can be measured according to Inui and co-workers
(Inui et al.,
1984).
Example 11
Lipid content in transgenic Arabidopsis plants over-expressing the TER gene.
Plant lipids were extracted from plant material as described by Cahoon et al.
(1999,
Proc. Natl. Acad. Sci. USA 96, 22:12935-12940) and Browse et al. (1986, Anal..
Bio-
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56
chemistry 442:141-145). Qualitative and quantitative lipid or fatty acid
analysis is de-
scribed in Christie, William W., Advances in Lipid Methodology. AyriScotland
:Oily
Press. - (Oily Press Lipid Library; Christie, William W., Gas Chromatography
and Lip-
ids. A Practical Guide - Ayr, Scotland :Oily Press, 1989 Repr. 1992. -
IX,307).
The total lipid content of dry T2 generation seeds from Arabidopsis thaliana
plants
over-expressing SEQ ID NO : 1, SEQ ID NO : 4 or SEQ ID NO : 6 will be
increased
compared to the empty vector Arabidopsis thaliana control plants not
expressing SEQ
ID NO : 1, SEQ ID NO : 4 or SEQ ID NO : 6. As an example the T2 seed lipid
content
determined by conventional gas chromatography as mg fatty acids per mg dry
seeds of
the plants overexpressing SEQ ID NO: 4 is given in table 4. Each value is the
mean of
two separate extractions of the seed of the given line. The control value is
the mean of
the seeds of 4 empty vector control plants grown simultaneously, each
extracted and
measured also in duplicate.
Table 4: Total seed lipids after overexpression of SEQ ID NO: 4
Line % fatty acids
22 31,4 0,9
10 32,4 1,1
7 35,5 3,2
controls 28,3 0,4
When expressed as a relative seed lipid content compared to the control plants
carry-
ing the empty pSun300-USP vector, a significant increase was seen as shown in
figure
9. Figure 9 describes seed oil content of Arabidopsis thaiiana T2 (grey bars)
of plants
expressing SEQ ID NO : 4 under the control of a seed specific promoter. The
white bar
represents the T2 controls. Error bars represent the standard deviation of 2
independ-
ent extractions for T2 seeds. All values are shown as percentage of the
average of the
corresponding control plants. The independent lines showed an increase in seed
stor-
age lipids of at least 10 % compared to the controls.
Example 12
Amino acid residues characteristic for traps-enoyl-activity based on sequence
comparison
Table 3 shows an alignment of the TER protein sequence with the closest
sequences
found. On the other hand sequence comparison with plastid enoyl-ACP-reductase
se-
quences from public databases shows little overall homology, but hints to some
amino
acid residues that may be conserved. Table 4 below lists the amino. acid
residues of
the TER (SEQ ID NO : 2) that were found to be conserved in plant plastidial
enoyl-
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57
ACP-reductases as well as in Euglena traps-enoyl-CoA-reductase and the
sequence
homologues shown in table 3. The amino acid residues shown in table 5 appear
con-
nected to the enoyl reductase function independent of ACP or CoA dependency.
The
relative positions are shown, but one skilled in the art has to expect that
variations of 5
to 10 amino acids, in some cases 20 to 30 amino acids in the relative
positions of these
key residues will .occur in some members of the enzyme family.
Table 5: Amino acid residues connected to enoyl reductase function
Amino acid residuePosition relative positions
in TER
SEQ ID NO
: 1
G 194 0
(R/K) 248 54
(UI) 250 56
K/R 260 66
V/I 275 81
(T/S) 291 97
(V/I/L) 311 117
Y 367 173
Y 377 183
(G) 380 ~ 186
V 408 214
(S) 417 223
Amino acids separated by a slash are interchangeably found at this position.
Amino
acids listed in brackets are found in the large majority of sequences.
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