Note: Descriptions are shown in the official language in which they were submitted.
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Title: Flax Seed Specific Promoters
FIELD OF THE INVENTION
The present invention relates to plant genetic engineering methods useful for
the
alteration of the constituents of plant seeds. More specifically, the
invention relates to
promoters that have been obtained from flax and are capable of directing
expression of
non-native genes in flax seeds as well as the seeds of other plants.
BACKGROUND OF THE INVENTION
Flax or linseed (Linum usitatissimum) is a commercially important oilseed
crop.
Flax oil and meal are valuable raw materials derived from flax seed. A further
economically significant raw material, flax fiber, is obtainable from the stem
of the plant.
The flax oil fraction is used for non-edible purposes, for example in the
manufacture of
varnish and paint, and has more recently become suited for use in the
manufacture of a range
of edible products, such as margarines and salad oils and dressings, by virtue
of newly bred
so called Linola cultivars (Green (1986) Can. J. Plant Sci, 66: 499-503). Flax
meal is used
primarily as a constituent of ruminant feeds while flax fibers are used in the
manufacture of
linen fabrics. Given its economic importance as a source for raw materials, it
is desirable to
further improve and diversify the available flax cultivar portfolio both with
respect to
agronomic performance, for example seed yield, resistance to pathogens and low
climatic
temperatures, and with respect to yield and quality of the raw materials to
suit
downstream applications. Although it is possible to obtain improved flax
cultivars
through conventional plant breeding, as evidenced by the development of the
Linola
cultivars, developing an elite agronomic plant line requires large investments
in plant
breeding due to the long timelines involved. Plant genetic engineering
technology allows
the isolation of genes directly from unrelated species and the transfer of
these genes into
elite agronomic backgrounds, thereby significantly reducing the time required
to develop
new cultivars. In addition plant genetic engineering permits the manufacture
of products not
naturally obtainable from flax, for example therapeutic agents.
In order to develop novel flax cultivars through plant genetic engineering,
control
over the expression of the introduced foreign or non-native gene is of
critical importance.
The desired expression characteristics for the non-native gene, such as the
level of
expression of the non-native gene, the particular plant tissue or organ in
which the
non-native gene is expressed, and the particular time in the growth cycle of
the plant at
which the non-native gene is expressed, will vary depending on the application
for which
the plant line is developed. For example, the modification of the seed oil
composition may
require low levels of seed-specific expression of an enzyme involved in fatty
acid
metabolism at an early stage in seed development (see for example US Patent
5,420,034).
On the other hand expression of a pharmaceutical protein could preferably
require high
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levels of leaf-specific expression upon harvesting of the plant leaves (see
for example, US
Patent 5,929,304).
In order to manipulate the expression characteristics of non-native genes
numerous
factors can be influenced. One factor is the choice of the transcriptional
promoter used. A
wide range of plant compatible promoters is currently available and some of
the better
documented promoters include constitutive promoters such as the 35-S CaMV
promoter
(Rothstein et al. (1987), Gene 53: 153-161) and the ubiquitin promoter (US
Patent 5,614,399),
tissue specific promoters such as seed-specific promoters, for example the
phaseolin
promoter (Sengupta-Gopalan et al., (1985), PNAS USA 82: 3320-3324) and
inducible
promoters, such as those inducible by heat (Czarnencka et al., (1989), Mol.
Cell. Biol. 9 (8):
3457-3464), UV light, elicitors and wounding (Lois et al., (1989) EMBO J. 8
(6): 1641-1648), or
chemicals such as endogenous hormones (Skriver et al. (1991), Proc: Natl.
Acad. Sci. USA
88(16): 7266-7270). Other factors that can be manipulated in order to control
the expression
characteristics of non-native gene in plants include transcriptional
modification factors
such as introns, polyadenylation sites and transcription termination sites.
The expression
characteristics of the non-native gene can further be manipulated by factors
that affect
translation, such as ribosomal binding sites and the codon bias that is
exhibited by the host.
Furthermore, the non-native gene itself may affect the viability of the
transgenic plant,
thus limiting particularly the levels of expression that can be attained. In
some cases it
may be possible to overcome this problem, by expressing the protein in a
tissue specific
manner, e.g. in the leaves or seed, or by restricting the accumulation of the
protein in
different subcellular compartments such as for example the cytoplasm, the
endoplasmic
reticulum or vacuoles, typically by the presence or the absence of specific
targeting
sequences capable of directing the protein to these compartments. Another
factor that will
affect the expression characteristics is the location in which the construct
inserts itself into
the host chromosome. This effect could provide an explanation as to why
different plants,
transformed with the same recombinant construct, can have fluctuating levels
of
recombinant protein expression.
To the best of the inventors' knowledge, expression of non-native genes in
flax
seeds is only documented in PCT Patent Application WO 98/18948. This
application
discloses two stearoyl-acyl carrier protein desaturase (SAD) genes derived
from flax. The
associated SAD promoter sequences are useful for the modification of flax and
other plants
for the expression of endogenous or foreign genes. However the methods taught
by WO
98/18948 are limited by the fact that the SAD promoters are not seed-specific
in flax and
confer expression to leaves, stems, flowers and seeds. Expression of non-
native genes thus
may result in undesirable side effects in non-seed tissues. In addition the
use of the SAD
promoters allows limited control over expression level and timing of
expression.
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There is a need in the art to further improve methods for the expression of
non-native genes in flax seeds and other plant seeds.
SUMMARY OF THE INVENTION
The present invention relates to improved methods for the seed-specific
expression of non-native genes in plants. In particular, the invention relates
to improved
methods for the seed-specific expression of non-native genes in flax.
Accordingly, in one aspect, the invention provides a method for the expression
of
a nucleic acid sequence of interest in flax seeds comprising:
(a) preparing a chimeric nucleic acid construct comprising in the 5' to 3'
direction of transcription as operably linked components
(1) a seed-specific promoter obtained from flax; and
(2) the nucleic acid sequence of interest wherein said nucleic acid of
interest is
non-native to said flax seed-specific promoter;
(b) introducing said chimeric nucleic acid construct into a flax plant cell;
and
(c) growing said flax plant cell into a mature flax plant capable of setting
seed,
wherein said nucleic acid sequence of interest is expressed in the seed under
the control of
said flax seed-specific promoter.
In a preferred embodiment of the invention, at least one expression
characteristic,
e.g. timing of expression in the plant's life cycle, conferred by the promoter
to the
non-native nucleic acid sequence is similar to that expression characteristic
when conferred
to a native nucleic acid sequence. In further preferred embodiments, the flax
seed-specific
promoter is an oleosin promoter, a 2S storage protein promoter or a legumin-
like seed storage
protein promoter.
In a further aspect, the present invention provides transgenic flax seeds
prepared
according to a method comprising:
(a) preparing a chimeric nucleic acid construct comprising in the 5' to 3'
direction of transcription as operably linked components:
(1) a seed-specific promoter obtained from flax; and
(2) a nucleic acid sequence of interest wherein said nucleic acid of interest
is
non-native to said seed-specific promoter;
(b) introducing said chimeric nucleic acid construct into a flax plant cell;
and
(c) growing said flax plant cell into a mature flax plant capable of setting
seed,
wherein said nucleic acid sequence of interest is expressed in the seed under
the control of
said seed-specific promoter.
In a further aspect the present invention provides flax plants capable of
setting
seed prepared by a method comprising:
(a) preparing a chimeric nucleic acid construct comprising in the 5' to 3'
direction of transcription as operably linked components:
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(1) a seed-specific promoter obtained from flax; and
(2) a nucleic acid sequence of interest wherein said nucleic acid of interest
is
non-native to said seed-specific promoter;
(b) introducing said chimeric nucleic acid construct into a flax plant cell;
and
(c) growing said flax plant cell into a mature flax plant capable of setting
seed,
wherein said nucleic acid sequence of interest is expressed in the seed under
the control of
said seed-specific promoter.
In yet a further aspect, the present invention provides novel flax seed
specific
promoters useful for the expression of non-native genes in flax seeds and the
seeds of other
plant species, useful for example for modification of the protein or oil
composition of the
seed.
In a preferred embodiment, the seed specific promoter comprises:
( a ) a nucleic acid sequence as shown in Figure 1 (SEQ.ID.NO.:1), Figure 2
(SEQ.ID.N0.:4), Figure 3 (SEQ.ID.N0.:6) or Figure 4 (SEQ.ID.N0.:8) wherein T
can also be
U;
(b) a nucleic acid sequence that is complimentary to a nucleic acid sequence
of
(a);
(c) a nucleic acid sequence that has substantial sequence homology to a
nucleic
acid sequence of (a) or (b);
(d) a nucleic acid sequence that is an analog of a nucleic acid sequence of
(a), (b)
or (c); or
(e) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a),
(b),
(c) or (d) under stringent hybridization conditions.
In another aspect, the invention provides chimeric nucleic acid sequences
comprising a first nucleic acid sequence obtained from flax operatively linked
to a second
nucleic acid sequence non-native to said first nucleic acid sequence wherein
said first nucleic
acid sequence comprises a novel flax seed-specific promoter.
Other features and advantages of the present invention will become readily
apparent from the following detailed description. It should be understood,
however, that
the detailed description and the specific examples while indicating preferred
embodiments
of the invention are given by way of illustration only, since various changes
and
modifications within the spirit and scope of the invention will become
apparent to those
skilled in the art of this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which:
Figure 1 shows the DNA sequence (SEQ.ID.NO.:l) of a flax genomic clone
encoding a 16.0 kDa oleosin protein (SEQ.ID.NOS.:2 and 3).
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Figure 2 shows the DNA sequence (SEQ.ID.N0.:4) of a flax genomic clone
encoding a 18.6 kDa oleosin protein (SEQ.ID.N0.:5).
Figure 3 shows the DNA sequence (SEQ.ID.N0.:6) of a flax genomic clone
encoding a 2S storage protein (SEQ.1D.N0.:7).
Figure 4 shows the DNA sequence (SEQ.ID.N0.:8) of a flax genomic clone
encoding a 54.5 kDa legumin-like storage protein (SEQ.ID.NOS.:9-12).
Figure 5 shows Southern blot analysis of flax genomic DNA probed with flax
oleosin DNA sequences.
Figure 6 shows a Northern blot analysis of the seed specific expression of
flax
oleosins.
Figure 7 shows a Northern blot analysis of the developmental expression of
flax
oleosins during seed development.
Figure 8 shows the GUS activity of flax embryos bombarded with flax oleosin
promoter-GUS-flax terminator gene constructs.
Figure 9 shows GUS expression in developing flax embryos and Arabidopsis seeds
of plants transformed with a 2S protein gene promoter GUS fusion.
Figure 10 shows the tissue-specific expression of GUS in transgenic flax
plants
transformed with a limn promoter-GUS-limn terminator gene construct.
Figure 11 shows the temporal expression of GUS in transgenic flax plants
transformed a limn promoter-GUS-limn terminator gene construct.
Figure 12 shows the expression of GUS in transgenic Brassica napus plants (L1
to
L9) transformed with a limn promoter-GUS-limn terminator gene construct.
Figure 13 shows the expression of GUS in transgenic Arabidopsis plants
transformed with a limn promoter-GUS-linin terminator gene construct at
different stages
of seed development.
DETAILED DESCRIPTION OF THE INVENTION
As hereinbefore mentioned, the present invention relates to improved methods
for
the expression of non-native genes in plants, in particular flax. The
invention provides
methods allowing the seed-specific expression of non-native genes in flax. The
methods of
the invention are advantageous in that improved control over the expression of
non-native
genes in flax seeds is obtained. Expression of the non-native gene is
restricted to the seeds,
thereby limiting potential undesirable effects resulting from the expression
in other plant
organs or tissues. In addition, the provided methodology allows improved
control over
expression characteristics, such as the expression level of the non-native
gene and timing of
expression of the non-native gene in the developmental cycle of the plant. The
methods of
the present invention are particularly useful in that in accordance with the
present
invention the seed composition with respect to valuable raw materials, such as
oil, protein
and polysaccharides, may be altered both qualitatively and quantitatively.
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Accordingly, in one aspect, the invention provides a method for the expression
of
a nucleic acid sequence of interest in flax seeds comprising:
(a) preparing a chimeric nucleic acid construct comprising in the 5' to 3'
direction of transcription as operably linked components;
(1) a seed-specific promoter obtained from flax; and
(2) the nucleic acid sequence of interest wherein said nucleic acid of
interest is
non-native to said flax seed-specific promoter;
(b) introducing said chimeric nucleic acid construct into a flax plant cell;
and
(c) growing said flax plant cell into a mature plant capable of setting seed,
wherein said nucleic acid sequence of interest is expressed in the seed under
the control of
said flax seed-specific promoter.
As used herein, the term "non-native" refers to any nucleic acid sequence,
including any RNA or DNA sequence, which is not normally associated with the
seed-specific promoter. This includes heterologous nucleic acid sequences
which are
obtained from a different plant species as the promoter as well as homologous
nucleic acid
sequences which are obtained from the same plant species as the promoter but
are not
associated with the promoter in the wild-type (non-transgenic) plant.
The non-native nucleic acid sequence when linked to a seed-specific promoter
obtained from flax results in a chimeric construct. The chimeric construct is
introduced into a
flax plant cell to create a transgenic flax plant cell which results in a
detectably different
phenotype of the flax plant cell or flax plant grown from it when compared
with a
non-transgenic flax plant cell or flax plant grown from it. A contiguous
nucleic acid sequence
identical to the nucleic acid sequence of the chimeric construct is not
present in the
non-transformed flax plant cell or flax plant grown from it. In this respect,
chimeric nucleic
acid sequences include those sequences which contain a flax promoter linked to
a nucleic acid
sequence obtained from another plant species or a nucleic acid sequence from
flax but
normally not associated with that promoter. Chimeric nucleic acid sequences as
used herein
further include sequences comprising a flax promoter and a nucleic acid
sequence that is
normally linked to the promoter but additionally containing a non-native
nucleic acid
sequence. For example, if the promoter is a flax seed-specific oleosin
promoter, sequences
"non-native" to the flax oleosin promoter also include a sequence comprising a
fusion
between the flax oleosin gene naturally associated with the oleosin promoter,
and a coding
sequence of interest that is not naturally associated with the promoter. The
term
non-native is also meant to include a fusion gene as hereinabove which
additionally
includes a cleavage sequence separating the nucleic acid sequence that is
normally linked to
the promoter sequence and the gene encoding the protein of interest.
The term "nucleic acid sequence" refers to a sequence of nucleotide or
nucleoside
monomers consisting of naturally occurring bases, sugars and intersugar
(backbone) linkages.
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The term also includes modified or substituted sequences comprising non-
naturally occurring
monomers or portions thereof, which function similarly. The nucleic acid
sequences of the
present invention may be ribonucleic (RNA) or deoxyribonucleic acids (DNA) and
may
contain naturally occurring bases including adenine, guanine, cytosine,
thymidine and
uracil. The sequences may also contain modified bases such as xanthine,
hypoxanthine,
2-aminoadenine, 6-methyl, 2-propyl, and other alkyl adenines, 5-halo uracil, 5-
halo
cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-
thiouracil,
8-halo adenine, 8-amino adenine, 8-thiol adenine, 8-thio-alkyl adenines, 8-
hydroxyl
adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-
thiol
guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other 8-substituted
guanines, other
aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-
trifluoromethyl
uracil and 5-trifluoro cytosine.
The term "seed-specific promoter", means that a gene expressed under the
control
of the promoter is predominantly expressed in plant seeds with no or no
substantial
expression, typically less than 5% of the overall expression level, in other
plant tissues.
In a further aspect, the present invention provides novel flax seed specific
promoters useful for the expression of non-native genes in flax seeds and the
seeds of other
plant species. The promoters may be used to modify for example the protein,
oil or
polysaccharide composition of the seeds. In a preferred embodiment, the seed
specific
promoter comprises:
( a ) a nucleic acid sequence as shown in Figure 1 (SEQ.ID.N0.:1), Figure 2
(SEQ.ID.N0.:4), Figure 3 (SEQ.ID.N0.:6) or Figure 4 (SEQ.ID.N0.:8) wherein T
can also be
U;
(b) a nucleic acid sequence that is complimentary to a nucleic acid sequence
of
(a);
(c) a nucleic acid sequence that has substantial sequence homology to a
nucleic
acid sequence of (a) or (b);
(d) a nucleic acid sequence that is an analog of a nucleic acid sequence of
(a), (b)
or (c); or
(e) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a),
(b),
(c) or (d) under stringent hybridization conditions.
The term "sequence that has substantial sequence homology" means those nucleic
acid sequences which have slight or inconsequential sequence variations from
the sequences
in (a) or (b), i.e., the sequences function in substantially the same manner
and are capable of
driving seed specific expression of non-native nucleic acid sequences. The
variations may be
attributable to local mutations or structural modifications. Nucleic acid
sequences having
substantial homology include nucleic acid sequences having at least 65%, more
preferably at
least 85%, and most preferably 90-95% identity with the nucleic acid sequences
as shown in
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Figure 1 (SEQ.ID.N0.:1), Figure 2 (SEQ.ID.N0.:4), Figure 3 (SEQ.ID.N0.:6) or
Figure 4
(SEQ.ID.N0.:8).
The term "sequence that hybridizes" means a nucleic acid sequence that can
hybridize to a sequence of (a), (b), (c) or (d) under stringent hybridization
conditions.
Appropriate "stringent hybridization conditions" which promote DNA
hybridization are
known to those skilled in the art, or may be found in Current Protocols in
Molecular Biology,
John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following may be
employed:
6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a
wash of 2.0 x SSC
at 50°C. The stringency may be selected based on the conditions used in
the wash step. For
example, the salt concentration in the wash step can be selected from a high
stringency of
about 0.2 x SSC at 50°C. In addition, the temperature in the wash step
can be at high
stringency conditions, at about 65°C.
The term "a nucleic acid sequence which is an analog" means a nucleic acid
sequence which has been modified as compared to the sequence of (a), (b) or
(c) wherein the
modification does not alter the utility of the sequence (i.e. as a seed
specific promoter) as
described herein. The modified sequence or analog may have improved properties
over the
sequence shown in (a), (b) or (c). One example of a modification to prepare an
analog is to
replace one of the naturally occurring bases (i.e. adenine, guanine, cytosine
or thymidine) of
the sequence shown in Figure 1, Figure 2, Figure 3 or Figure 4 with a modified
base such as
such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other
alkyl
adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-
aza thymine,
pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine,
8-thiolalkyl
adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo
guanines, 8 amino
guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other
8-substituted guanines, other aza and deaza uracils, thymidines, cytosines,
adenines, or
guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
Another example of a modification is to include modified phosphorous or oxygen
heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl
intersugar
linkages or short chain heteroatomic or heterocyclic intersugar linkages in
the nucleic acid
molecule shown in Figure 1, Figure 2, Figure 3 or Figure 4. For example, the
nucleic acid
sequences may contain phosphorothioates, phosphotriesters, methyl
phosphonates, and
phosphorodithioates.
A further example of an analog of a nucleic acid molecule of the invention is
a
peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate
backbone in the
DNA (or RNA), is replaced with a polyamide backbone which is similar to that
found in
peptides (P.E. Nielsen, et al Science 1991, 254, 1497). PNA analogs have been
shown to be
resistant to degradation by enzymes and to have extended lives in vivo and in
vitro. PNAs
also bind stronger to a complimentary DNA sequence due to the lack of charge
repulsion
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between the PNA strand and the DNA strand. Other nucleic acid analogs may
contain
nucleotides containing polymer backbones, cyclic backbones, or acyclic
backbones. For
example, the nucleotides may have morpholino backbone structures (U.S. Pat.
No.
5,034,506). The analogs may also contain groups such as reporter groups, a
group for
improving the pharmacokinetic or pharmacodynamic properties of nucleic acid
sequence.
In another aspect, the invention provides chimeric nucleic acid sequences
comprising a first nucleic acid sequence obtained from flax operatively linked
to a second
nucleic acid sequence non-native to said first nucleic acid sequence wherein
said first nucleic
acid sequence comprises a novel flax seed-specific promoter. Preferably, the
promoter is
selected from the group of promoters comprising Figure 1, Figure 2, Figure 3
and Figure 4 or a
nucleic acid sequence hybridizing thereto under stringent conditions.
In accordance with the present invention, the chimeric nucleic acid sequences
can
be incorporated in a known manner in a recombinant expression vector which
ensures good
expression in the seed cell. Accordingly, the present invention includes a
recombinant
expression vector comprising a chimeric nucleic acid sequence of the present
invention
suitable for expression in a seed cell.
The term "suitable for expression in a seed cell" means that the recombinant
expression vectors contain the chimeric nucleic acids sequence of the
invention, a regulatory
region and a termination region, selected on the basis of the seed cell to be
used for
expression, which is operatively linked to the nucleic acid sequence encoding
the
polypeptide of desirable amino acid composition. Operatively linked is
intended to mean
that the chimeric nucleic acid sequence encoding the polypeptide is linked to
a regulatory
sequence and termination region which allows expression in the seed cell. A
typical
construct consists, in the 5' to 3' direction of a regulatory region complete
with a promoter
capable of directing expression in a plant, a polypeptide coding region and a
transcription
termination region functional in plant cells. These constructs may be prepared
in accordance
with methodology well known to those of skill in the art of molecular biology
(see for
example: Sambrook et al. (1990), Molecular Cloning, 2T'd ed. Cold Spring
Harbor Press). The
preparation of constructs may involve techniques such as restriction
digestion, ligation, gel
electrophoresis, DNA sequencing and PCR. A wide variety of cloning vectors is
available to
perform the necessary cloning steps. Especially suitable for this purpose are
the cloning
vectors with a replication system that is functional in Escherichia coli such
as pBR322, the
pUC series Ml3mp series, pACYC184, pBluescript etc. Nucleic acid sequences may
be
introduced into these vectors and the vectors may be used to transform E. coli
which may be
grown in an appropriate medium. Plasmids may be recovered from the cells upon
harvesting
and lysing the cells. Final constructs may be introduced into plant vectors
compatible with
integration into the plant such as the Ti and Ri plasmids.
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The methods for the expression of non-native genes in flax seeds in accordance
with the present invention may be practiced using any flax seed-specific
promoter and are
not limited by the specific flax seed specific promoter that is selected. In
preferred
embodiments of the present invention, the flax seed-specific promoter confers
to the
non-native nucleic acid sequence at least one expression characteristic which
is similar or
identical to an expression characteristic conferred to the native nucleic acid
sequence by the
native promoter. The term "expression characteristic" as used herein refers to
any
measurable property or effect conferred by the flax seed-specific promoter to
the nucleic
acid sequence operably linked to the flax seed-specific promoter. Thus in
preferred
embodiments, timing of expression in the plant's life cycle, of the non-native
nucleic acid
sequence is similar or identical to timing of expression of the native nucleic
acid sequence. In
further preferred embodiments, the expression level of the heterologous
nucleic acid
sequence is similar or identical to the expression level of the native nucleic
acid sequence. In
yet further specific embodiments, the response of the non-native gene to
alterations in
lighting conditions, changes in wavelength or light intensity for example,
changes in
temperature, tissue wounding, changes in concentration of chemical agents,
such as for
example phytohormones and pesticides, is similar to the response of the native
nucleic acid
sequence to these stimuli. Other desired expression characteristics conferred
by a flax
seed-specific promoter may be recognized by those skilled in the art and a
flax seed-specific
promoter may be selected accordingly.
Flax-seed specific promoters that may be used in accordance with the present
invention include promoters associated with seed storage proteins, such as all
albumins and
globulins, including the vicilin and legumin-like proteins, as well as seed-
specific
promoters not associated with seed storage proteins, such as oleosins. Of
further particular
interest are promoters associated with fatty acid metabolism, such as acyl
carrier protein
(ACP), saturases, desaturases, elongases and the like.
In preferred embodiments of the present invention the seed specific promoter
used
is an oleosin promoter, a legumin-like seed storage protein promoter or a 2S
storage protein
promoter. In particularly preferred embodiments the seed specific promoter has
the
sequence shown in Figure 1, Figure 2, Figure 3 or Figure 4 or any nucleic acid
sequences
obtainable from flax and hybridizing to any one of these four nucleic acid
sequences under
stringent conditions.
Additional flax seed-specific promoters may be used in accordance with the
present invention. These promoters may be obtained in a number of ways. Where
a flax seed
protein has been isolated, it may be partially sequenced, so that a nucleic
acid probe may be
designed for identifying RNA specific to the seed. To further enhance the RNA
specifically associated with the seed, cDNA may be prepared from seed cells
and the
cDNA may be subtracted with mRNA or cDNA from non-seed cells. The remaining
seed
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cDNA may then be used to probe a genomic DNA library for complementary
sequences.
Sequences hybridizing to the cDNA may subsequently be obtained and the
associated
promoter region may be isolated. It is also possible to screen genomic DNA
libraries
prepared from flax seed tissues using known seed specific genes from other
plant species and
subsequently isolate their associated promoters. Due to the relative abundance
of
seed-storage proteins in seeds it is also be possible to obtain sequence
information through
random sequencing of flax seed cDNA libraries. Those cDNA sequences matching
sequence of
known seed-storage proteins could be used to identify the associated promoter.
Databases
containing sequence information from large scale sequencing from for example
Arabidopsis
and maize may be searched for known seed-specific proteins and/or promoters
and the
information may be used to identify promoter sequences in flax that share
sequence
similarity. Alternative methods to isolate additional flax seed specific
promoters may be
used and novel flax seed specific promoters may be discovered by those skilled
in the art
and used in accordance with the present invention.
The nucleic acid sequence of interest linked to the promoter may be any
nucleic
acid sequence of interest including any RNA or DNA sequence encoding a peptide
or protein
of interest, for example, an enzyme, or a sequence complementary to a genomic
sequence,
where the genomic sequence may be at least one of an open reading frame, an
intron, a
non-coding leader sequence, or any sequence where the complementary sequence
will inhibit
transcription, messenger RNA processing, for example splicing or translation.
The nucleic
acid sequence of interest may be synthetic, naturally derived or a combination
thereof. As
well, the nucleic acid sequence of interest could be a fragment of the natural
sequence, for
example just include the catalytic domain or a structure of particular
importance.
Depending upon the nature of the nucleic acid sequence of interest, it may be
desirable to
synthesize the sequence with plant preferred codons. The plant preferred
codons may be
determined from the codons of highest frequency in the proteins expressed in
the largest
amount in particular plant species of interest.
The nucleic acid sequence of interest may encode any of a variety of
recombinant
proteins. Examples of recombinant proteins which might be expressed by the
methods of
the present invention include proteins with a favorable catalytic function or
a valuable
protein that will accumulate to high levels and be extracted if desired.
Proteins with a
catalytic function, include, but are not limited to, proteins that confer a
new biochemical
phenotype on the developing seeds. New phenotypes could include such
modifications as
altered seed-protein or seed oil composition or seed polysaccharide
composition, enhanced
production of pre-existing desirable products or properties and the reduction
or even
suppression on an undesirable gene product using antisense, ribozyme or co-
supression
technologies (Izant and Weintraub (1984) Cell 26: 1007-1015, antisense;
Hazelhoff and
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Gerlach (1988) Nature 334: 585-591, ribozyme; Napoli et al. (1990) Plant Cell
2: 279-289,
co-suppression).
It is expected that the desired proteins would be expressed in all embryonic
tissues, although varying cellular expression may be detected in the different
embryonic
tissues such as the embryonic axis and cotyledons. The nucleic acid sequence
of interest may
be expressed at any stage in seed development. The timing of expression may
depend on the
particular use of the invention. Expression of enzymes involved in oil
modification may be
desirable early in seed development, for example before accumulation of seed
storage
protein.
Besides the promoter region and the nucleic acid sequence of interest, a
nucleic
acid sequence capable of terminating transcription is typically included in
expression
vectors. Transcriptional terminators are preferably about 200 to about 1,000
nucleotide base
pairs and may comprise any such sequences functional in plants, such as the
nopaline
synthase termination region (Bevan et al., (1983) Nucl. Acid. Res. 11: 369-
385), the
phaseolin terminator (van der Geest et al., (1994) Plant J. 6(3): 413-423),
the terminator for
the octopine synthase gene of Agrobacterium tumefaciens or other similarly
functioning
elements. These transcription terminator regions can be obtained as described
by An (1987),
Methods in Enzym. 153: 292 or are already present in plasmids available from
commercial
sources such as ClonTech, Palo Alto, California. The choice of the appropriate
terminator
may have an effect of the rate of transcription.
The chimeric construct may further comprise enhancers such as the AMV leader
(Jobling and Gehrke (1987), Nature 325: 622-625) or introns. It should be
understood that the
design of the expression vector may depend on such factors as the choice of
the plant species
and/or the type of polypeptide to be expressed.
The expression vectors will normally also contain a marker gene. Marker genes
comprise all genes that enable distinction of transformed plant cells from non-
transformed
cells, including selectable and screenable marker genes. Conveniently, a
marker may be a
resistance marker to a herbicide, for example, glyphosate or phosphinothricin,
or to an
antibiotic such as kanamycin, 6418, bleomycin, hygromycin, chloramphenicol and
the like,
which confer a trait that can be selected for by chemical means. Screenable
markers may be
employed to identify transformants through observation. They include but are
not limited
to the (3-glucuronidase or uidA gene, a (3-lactamase gene or a green
fluorescent protein (Niedz
et al. (1995) Plant Cell Rep. 14: 403).
In order to introduce nucleic acid sequences into plant cells in general a
variety of
techniques are available to the skilled artisan. Agrobacterium-mediated
transformation
for flax plant cells has been reported and flax transformants may be obtained
in accordance
with the methods taught by Dong and McHughen (1993) Plant Science 88: 61-77,
although a
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variety of other techniques (see below) may also be used to introduce the
chimeric DNA
constructs in flax cells if so desired.
Transformed flax plants grown in accordance with conventional agricultural
practices known to a person skilled in the art are allowed to set seed. Flax
seed may then be
obtained from mature flax plants and analyzed for desired altered properties
with respect
to the wild-type seed.
Two or more generations of plants may be grown and either crossed or selfed to
allow identification of plants and strains with desired phenotypic
characteristics
including production of the recombinant polypeptide. It may be desirable to
ensure
homozygosity in the plants to assure continued inheritance of the recombinant
trait.
Methods for selecting homozygous plants are well known to those skilled in the
art of plant
breeding and include recurrent selfing and selection and anther and microspore
culture.
Homozygous plants may also be obtained by transformation of haploid cells or
tissues
followed by regeneration of haploid plantlets subsequently converted to
diploid plants by
any number of known means (e.g. treatment with colchicine or other microtubule
disrupting
agents).
The present invention also includes transgenic flax seeds prepared according
to a
method comprising:
(a) preparing a chimeric nucleic acid construct comprising in the 5' to 3'
direction of transcription as operably linked components:
( 1 ) a seed-specific promoter obtained from flax; and
(2) a nucleic acid sequence of interest wherein said nucleic acid of interest
is
non-native to said seed-specific promoter;
(b) introducing said chimeric nucleic acid construct into a flax plant cell;
and
(c) growing said flax plant cell into a mature flax plant capable of setting
seed
wherein said nucleic acid sequence of interest is expressed in the seed under
the
control of said seed-specific promoter.
In preferred embodiments of the invention the seed-specific promoter is
selected
from the group of flax seed specific promoters consisting of, a 2S storage
protein promoter, a
globulin promoter, an oleosin promoter, and a legumin-like seed storage
protein promoter.
Specific promoter sequences are shown in Figure 1 (SEQ.ID.N0.:1), Figure 2
(SEQ.ID.N0.:4), Figure 3 (SEQ.ID.N0.:6) and Figure 4 (SEQ.ID.N0.:8).
The present invention further provides flax plants capable of setting seed
prepared by a method comprising:
(a) preparing a chimeric nucleic acid construct comprising in the 5' to 3'
direction of transcription as operably linked components:
(1) a seed-specific promoter obtained from flax; and
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(2) a nucleic acid sequence of interest wherein said nucleic acid of interest
is
non-native to said seed-specific promoter;
(b) introducing said chimeric nucleic acid construct into a flax plant cell;
and
(c) growing said flax plant cell into a mature flax plant capable of setting
seed
wherein said nucleic acid sequence of interest is expressed in the seed under
the
control of said seed-specific promoter.
The present invention further provides methods of use for the novel promoters
shown in Figure 1 (SEQ.ID.NO.:1), Figure 2 (SEQ.ID.N0.:4), Figure 3
(SEQ.1D.N0.:6) and
Figure 4 (SEQ.ID.N0.:8) in plant species other than flax. Accordingly, the
invention also
includes the preparation of chimeric nucleic acid constructs comprising a
promoter selected
from the group promoters shown in Figure 1, Figure 2, Figure 3 and Figure 4
and a nucleic acid
sequence of interest, and expression in a seed-specific manner of the nucleic
acid sequence of
interest in plant species other than flax and under the control of the flax
promoter.
In another aspect of the present invention there is provided a method for the
expression of a nucleic acid sequence of interest in plant seeds comprising:
(a) preparing a chimeric nucleic acid construct comprising in the 5' to 3'
direction of transcription as operably linked components;
(1) a seed-specific promoter selected from the group of seed-specific
promoters
consisting of
(i) a nucleic acid sequence as shown in Figure 1 (SEQ.ID.NO.:1), Figure 2
(SEQ.ID.N0.:4), Figure 3 (SEQ.ID.N0.:6) or Figure 4 (SEQ.ID.N0.:8)
wherein T can also be U;
(ii) a nucleic acid sequence that is complimentary to a nucleic acid
sequence of (i);
(iii) a nucleic acid sequence that has substantial sequence homology to a
nucleic acid sequence of (i) or (ii); and
(iv) a nucleic acid sequence that is an analog of a nucleic acid sequence of
(i), (ii) or (iii);
(v) a nucleic acid sequence that hybridizes to a nucleic acid sequence of
(i), (ii), (iii) or (iv) under stringent hybridization conditions; and
(2) said nucleic acid of interest;
(b) introducing the chimeric nucleic acid construct into a plant cell;
(c) growing said plant cell into a mature plant capable of setting seed,
wherein
said nucleic acid sequence of interest is expressed in the seed under the
control of said
seed-specific promoter.
A variety of techniques are available for the introduction of nucleic acid
sequences, in particular DNA, into plant host cells in general. For example,
the chimeric
DNA constructs may be introduced into host cells obtained from dicotelydenous
plants, such
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as tobacco, and oleoagenous species, such as Brassica napus using standard
Agrobacterium
vectors by a transformation protocol such as described by Moloney et al.
(1989), Plant Cell
Rep. 8: 238-242 or Hinchee et al. (1988) Bio/Technol. 6: 915-922; or other
techniques known
to those skilled in the art. For example, the use of T-DNA for transformation
of plant cells
has received extensive study and is amply described in EP 0 120 516, Hoekema
et al., (1985),
Chapter V In: The Binary Plant Vector System Offset-drukkerij Kanters BV,
Alblasserdam); Knauf et al. (1983), Genetic Analysis of Host Expression by
Agrobacterium,
p. 245, In: Molecular Genetics of Bacteria-Plant Interaction, Puhler, A. ed.
Springer-Verlag,
NY); and An et al., (1985), (EMBO J., 4: 277-284). Agrobacterium
transformation may also be
used to transform monocot plant species (US Patent 5,591,616).
Conveniently, explants may be cultivated with Agrobacterium tumefaciens or
Agrobacterium rhizogenes to allow for the transfer of the transcription
construct in the
plant host cell. Following transformation using Agrobacterium the plant cells
are dispersed
into an appropriate medium for selection, subsequently callus, shoots and
eventually plants
are recovered. The Agrobacterium host will harbour a plasmid comprising the
vir genes
necessary for transfer of the T-DNA to plant cells. For injection and
electroporation (see
below) disarmed Ti-plasmids (lacking the tumour genes, particularly the T-DNA
region)
may be introduced into the plant cell.
The use of non-Agrobacterium techniques permits the use of constructs
described
herein to obtain transformation and expression in a wide variety of
monocotyledonous and
dicotyledonous plant species. These techniques are especially useful for
transformation of
plant species that are intractable in an Agrobacterium transformation system.
Other
techniques for gene transfer include particle bombardment (Sanford, (1988),
Trends in
Biotechn. 6: 299-302), electroporation (Fromm et al., (1985), PNAS USA, 82:
5824-5828;
Riggs and Bates, (1986), PNAS USA 83: 5602-5606), PEG mediated DNA uptake
(Potrykus
et al., (1985), Mol. Gen. Genetics., 199: 169-177), microinjection (Reich et
al., Bio/Techn.
(1986) 4:1001-1004) and silicone carbide whiskers (Kaeppler et al. (1990)
Plant Cell Rep. 9:
415-418).
In a further specific applications such as to B. napus, the host cells
targeted to
receive recombinant DNA constructs typically will be derived from cotyledonary
petioles
as described by Moloney et al. (1989) Plant Cell Rep. 8: 238-242. Other
examples using
commercial oil seeds include cotyledon transformation in soybean explants
(Hinchee et al.,
(1988) Bio/Technol. 6: 915-922) and stem transformation of cotton (Umbeck et
al., (1987)
Bio/Technol. 5: 263-266).
Following transformation, the cells, for example as leaf discs, are grown in
selective medium. Once the shoots begin to emerge, they are excised and placed
onto rooting
medium. After sufficient roots have formed, the plants are transferred to
soil. Putative
transformed plants are then tested for presence of a marker. Southern blotting
is performed
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on genomic DNA using an appropriate probe, to show integration into the genome
of the host
cell.
The methods provided by the present invention can be used in conjunction a
broad
range of plant species. Particularly preferred plant cells employed in
accordance with the
present invention include cells from the following plants: soybean (Glycine
max), rapeseed
(Brassica napus, Brassica campestris), sunflower (Helianthus annuus), cotton
(Gossypium
hirsutum), corn (Zea mays), tobacco (Nicotiana tobacum), alfalafa (Medicago
sativa),
wheat (Triticum sp.), barley (Hordeum vulgare), oats (Avena sativa L.),
sorghum (Sorghum
bicolor), Arabidopsis thaliana, potato (Solanum sp.), flax/linseed (Linum
usitatissimum),
safflower (Carthamus tinctorius), oil palm (Eleais guineeis), groundnut
(Arachis
hypogaea), Brazil nut (Bertholletia excelsa) coconut (focus nucifera), castor
(Ricinus
communis), coriander (Coriandrum sativum), squash (Cucurbita maxima), jojoba
(Simmondsia chinensis) and rice (Oryza sativa).
The present invention has a variety of uses which include improving the
intrinsic
value of plant seeds by their accumulation of altered polypeptides or novel
recombinant
peptides or by the incorporation or elimination or a metabolic step. Use of
the invention
may result in improved protein quality (for example, increased concentrations
or essential or
rare amino acids), improved liquid quality by a modification of fatty acid
composition, or
improved or elevated carbohydrate composition. Examples include the expression
of
sulfur-rich proteins, such as those found in lupins or brazil nuts in a seed
deficient in
sulphurous amino acids. Improved protein quality could also be achieved by the
expression
of a protein or a fragment of a protein that is enriched in essential amino
acids including
lysine, cysteine, methionine and tryptophan. Alternatively, a fatty acyl
coenzyme A, a
transferase enzyme capable of modifying fatty acid ratios in triglycerides
(storage lipid)
could be expressed. In cases where a recombinant protein is allowed to
accumulate in the
seed, the protein could also be a peptide which has pharmaceutical or
industrial value. In
this case the peptide could be extracted from the seed and used in crude or
purified form as
appropriate for the intended use. As well, the polypeptides that are expressed
in the seeds
can be fragments or derivatives or the native protein. Pharmaceutically useful
proteins
may include, but are not limited to, anticoagulants, such as hirudin,
antibodies, including
monoclonal antibodies and antibody fragments, vaccines, cytokines or growth
factors such as
bovine growth factor, cholinergic differentiation factor (CDF), ciliary
neurotrophic factor
(CNTF), fibroblast growth factor (FGF), fish growth factor, gonadotropin,
granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage
colony-stimulating factor (GM-CSF), human growth hormone, interferon alpha
(IFN-a),
interferon beta (IFN-(3), interferon gamma (IFN-'y), interleukin 1-alpha (IL1-
a),
interleukin 1-beta (ILl-(3), interleukin-2 (IL-2), interleukin-3 (IL-3),
interleukin-4 (IL-4),
interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL-10), leukemia
inhibitory factor
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(LIF), thioredoxin, macrophage colony-stimulating factor (M-CSF),
myelomonocytic
growth factor, nerve growth factor (NGF), oncostatin M, platelet-derived
growth factor
(PDGF), prolactin, transforming growth factor alpha (TGF-a), transforming
growth factor
beta2 (TGF-(32), tumour necrosis factor alpha (TNF-a), and tumour necrosis
factor beta
(TNF-(3). Pharmaceutically useful proteins can also include mammalian
proteins, for
example, but not limited to a-1-antitrypsin, anti-obesity proteins, blood
proteins, collagen,
collagenase, elastin, elastase, enteropeptidase, fibrinogen, haemoglobin,
human serum
albumin, insulin, lactoferrin, myoglobin and pulmonary surfactant proteins.
Industrially useful peptides may include, but are not limited to a-amylase or
other amylases, amyloglucosidase, arabinase, catalase, cellobiohydrolase,
cellulases,
chitinases, chymotrypsin, dehydrogenases, endo-glucanase, chymosin, endo-
galactanase,
esterases, (3-galactosidase, a-galactosidase or other galactosidases, gastric
lipases,
glucanases, glucose isomerase, hemi-cellulases, hydrolases, isomerase,
ligninases, lipases,
lyases, lysozymes, oxidases, oxidoreductase, papain, pectinases, pectin lyase,
peroxidases,
phosphatases, phytase, proteases, pullulanases, reductases, serine proteases,
thioredoxin,
transferase, trypsin, and xylanase.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES
EXAMPLE 1
Isolation of Seed-Specific Flax Promoters
Seed specific cDNA clones were isolated form a flax seed specific cDNA-
library.
These cDNA clones were sequenced and the Basic Local Alignment Search Tool
(BLAST)
was used to compare these sequences against others in public databases such as
Genbank.
This comparison revealed that the deduced amino acid sequence of several of
the isolated
cDNAs had a high degree of similarity to both the low and high molecular
weight class of
oleosins, 2S-albumin and legumin-like storage proteins. Probes were prepared
individually
from (portions of) cDNAs encoding oleosins, 2S albumin and legumin-like
storage proteins
and these were used to screen a genomic library prepared from the flax line
Forge that is
homozygous for four rust resistance genes (Anderson et al. (1997), The Plant
Cell 9: 641-651).
Several positive lambda clones for each probe were identified after high-
stringency
screening. The inserts were subcloned into the plasmid vector pBluescript and
sequenced.
Sequence information revealed that we had isolated the genomic counterparts to
the
oleosins, 2S albumin and cDNAs legumin-like cDNAs. Sequence information of the
genomic
clones containing sequences encoding a high and low molecular weight oleosin
isoforms, 2S
albumin and a legumin-like gene are presented in Figures 1 to 4 respectively.
Figure 1 and SEQ.ID.N0.:1 shows the DNA sequence of a flax genomic clone
encoding a 16.0 kDa oleosin protein (low molecular weight or L-isoform).
Putative
regulatory elements are identified and indicated. These include inverted
repeats (base
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pairs 805 to 813 and 821 to 829; base pairs 1858 to 1866 and 1877 to 1885),
direct repeats (base
pairs 184 to 193 and 1102 and 1111; base pairs 393 to 402 and 1701 to 1710;
base pairs 683 to
692 and 1546 to 1555; base pairs 770 to 781 and 799 to 810; base pairs 955 to
964 and 1936 to
1945; base pairs 1483 to 1496 and 1513 to 1526), the abscisic acid responsive
element (ABRE)
(base pairs 1859 to 1866), CACA box (base pairs 1933 to 1936), TATA box (base
pairs 1925 to
1931) and CAT box (base pairs 1989 to 1993). As well, the poly adenylation
signal is
indicated (base pairs 3020 to 3025). The open reading frame is interrupted by
1 short intron
(which are marked) and the 2 exons are translated and indicated in IUPAC
single letter
amino-acid codes.
Figure 2 and SEQ.ID.N0.:4 shows the DNA sequence of a flax genomic clone
encoding a 18.6 kDa oleosin protein (high molecular weight or H-isoform).
Putative
regulatory elements are identified and indicated. These include direct repeats
(base pairs
14 to 25 and 1427 to 1438; base pairs 80 to 89 and 1242 to 1251; base pairs
177 to 186 and 837 to
846; base pairs 1281 to 1290 and 1242 to 1251; base pairs 1591 to 1600 and
1678 to 1287). The
open reading frame is not interrupted by introns and is translated and
indicated in IUPAC
single letter amino-acid codes.
Figure 3 and SEQ.ID.N0.:6 shows the DNA sequence of the flax genomic clone
encoding a 2S storage protein. Nucleotide sequencing of this clone revealed it
to have an
open reading frame of 174 amino acids that showed homology to the plant 2S
storage group
of proteins. The sequence encodes an open reading frame with 38% overall
similarity to a
Brassica oleracea 2S storage protein, including complete conservation of the
glutamine-rich
stretch QQQGQQQGQQQ (SEQ.ID.N0.:13). In addition, the 2S storage protein gene
promoter contained several putative promoter regulatory elements. These
include AT rich
repeats (base pairs 25-36, 97-108 and 167-190), RY-like repeat (base pairs 240-
247),
G-box-like element (base pairs 274-280), seed specific box-like motif (base
pairs 285-290)
and TATA box (base pairs 327-333).
Figure 4 and SEQ.ID.N0.:8 shows the DNA sequence of a flax genomic clone
encoding a 54.4 kDa flax legumin-like seed storage protein. This legumin-like
seed storage
protein gene will also be referred to as "liniri'. The deduced amino acid
sequence of the limn
gene was compared to the legumin-like protein from R. communis, the legumin
precursor
from M. salicifolia, Q.robur and G. hirsutum, the glutelin precursor from
O.sativa and a 12 S
seed storage protein from A. thaliana. The limn gene shows a sequence
identity/similarity
with the corresponding proteins from R. communis, M. salicifolia, Q.robur, G.
hirsutum,
O.sativa and A. thaliana of 59/15, 47/16, 50/17, 45/17, 43/18 and 43/18
percent
respectively. Putative regulatory elements in the promoter region are
identified and
indicated. These include inverted repeats (base pairs 265 to 276 and 281 to
292; base pairs
513 to 524 and 535 to 545), repeats (base pairs 1349 to 1360 and 1367 to 1378;
base pairs 1513 to
1529 and 1554 to 1572), the abscisic acid responsive element (ABRE) (base
pairs 1223 and
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1231), legumin box (RY repeats) (between base pairs 1223 and 1231), a possible
vicilin box
region (base pairs 1887 to 1894), CAAT box (base pairs 1782 to 1785) and TATA
box (base
pairs 1966 to 1970). As well, the signal peptide for ER membrane targeting is
indicated
(base pairs 2034-2080). The open reading frame is interrupted by 3 short
introns (which are
marked) and the 4 exons are translated and indicated in IUPAC single letter
amino-acid
codes.
Figure 5 shows Southern blot analysis of flax genomic DNA. 60 lZg of flax
genomic
DNA was isolated from leaves, digested with EcoRI (lane I), HindIII (lane 2)
and BamHI
(lane 3) and was loaded into the respective lanes. A) Hybridizations were
performed with
random primed 32P-labelled 3T cDNA (high molecular weight flax oleosin
isoform). B)
Hybridizations were performed with random primed 32P-labelled 7R cDNA (low
molecular
weight flax oleosin isoform). The results demonstrate that both 3T (high
molecular weight
oleosin isoform) and 7R (low molecular weight oleosin isoform) oleosin cDNAs
hybridize
with flax genomic DNA. More specifically the results indicate that 3T is
likely to
represent a 2-copy gene in flax, as seen by two bands in each lane of
digestion. Similarly, 7R
is likely to represent a multigene family in flax as multiple bands were
detected for each
digestion.
EXAMPLE 2
Seed specific expression of flax oleosin ~eenes
Figure 6 shows a Northern blot analysis of the seed specific expression of
flax
oleosins. Northern hybridization of the two oleosin mRNA in different tissues.
Ten ~g of
total RNA was extracted from different tissues, R, root; C, cotyledon; L,
leaf; S, seed
capsule; E, embryo. The membrane was probed with (A) cDNA encoding high
molecular
weight (H)-isoform (identical to coding sequence as presented in Figure 2) and
(B) cDNA
encoding low molecular weight (L) -isoform (identical to coding sequence as
presented in
Figure 1). Both the transcripts are expressed only in the embryo and seed
capsule, which
contains embryos.
EXAMPLE 3
Developmental expression of flax oleosin scenes during seed development
Figure 7 shows a Northern blot analysis of the developmental expression of
flax
oleosins during seed development. 15 ug per lane of total RNA was loaded in
each lane on
agarose/formaldehyde gel and blotted onto HybondN+ membrane. 10J: This
membrane was
probed using the 32P dCTP labeled flax oleosin cDNA clone (low molecular
weight isoform).
Stages indicated are the number of days past anthesis (DPA). 3T) 15 ug per
lane of total
RNA was loaded in each lane on agarose/formaldehyde gel and blotted onto
HybondN+
membrane. 3T: This membrane was probed using the 32P dCTP labeled flax oleosin
cDNA
clone (high molecular weight isoform). Both the transcripts were expressed
very early in
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development (6DPA, early cotyledonary stage). Expression is maximum at 16 to
20 DPA
(late cotyledonary stage) and declines at 22 DPA (mature embryos).
EXAMPLE 4
Transient Seed specific expression of (~~~lucuronidase (GUS) when under the
regulator3r
control of flax oleosin regulatory seduences
Two constructs were made using standard molecular biology techniques (eg see
Sambrook et al. (1990), Molecular Cloning, 2nd ed. Cold Spring Harbor Press,
including
restriction enzyme digestions, ligation and polymerase chain reaction (PCR).
Construct pSC54: The (3-glucuronidase reporter coding sequence from vector
GUSN358>S
(Clontech Laboratories) was placed between the promoter sequence from
nucleotide 21 to
1852 and terminator sequence from 2395 to 3501 (as described in Figure 1).
This insert was
cloned into pBluescript and the resulting vector is called pSC54
Construct pSC60: The (3-glucuronidase reporter coding sequence from vector
GUSN358>S
(Clontech Laboratories) was placed between the promoter sequence from
nucleotide 1 to 2023
and terminator sequence from 2867 to 3925 (as described in Figure 2). This
insert was cloned
into pBluescript and the resulting vector is called pSC60.
pSC54, pSC60 and a promoter-less GUS construct (Control) were introduced into
the flax embryos using particle bombardment using standard protocols (eg see
Abenes et al.
(1997) Plant Cell reports 17:1-7). Figure 8 shows the GUS activity of flax
embryos
bombarded with pSC54, pSC60 and a promoterless GUS construct measured 48 hours
after
particle bombardment. As can be seen the flax oleosin regulatory sequences are
sufficient to
drive the expression of GUS in flax embryos.
EXAMPLE 5
Stable seed specific expression of 3~-glucuronidase (GUS) in flax and
Arabidopsis when
under the regulatory control of flax 2S storag~protein $ene promoter
A GUS reporter gene construct was made by incorporating 5' and 3' regions from
the
DNA fragment described in Figure 3 into promoterless-GUS pBI101 vector as
follows.
A 400bp amplicon from the 5' end of the DNA fragment described in Figure 3 was
PCR amplified using the following primers (location shown in Fig 3):
5' primer(1): 5'-TCCACTATGTAGGTCATA-3' (SEQ.ID.N0.:14)
3' primer(1): 5'-CTTTAAGGTGTGAGAGTC-3' (SEQ.ID.N0.:15)
The PCR primers also contained restriction sites for HindIII and BamHI which
were used to clone the 400bp 5'UTR amplicon into the HindIII/BamHI sites of
the pBI101
vector in front of the GUS reporter gene. A 736bp amplicon from the 3'
untranslated region
(3'UTR) of the DNA fragment described in Figure 3 was PCR amplified using the
following
primers (location shown in Fig 3):
5' primer (2):5'-AGGGGTGATCGATTA-3' (SEQ.ID.N0.:16)
3' primer (2):5'-GATAGAACCCACACGAGC-3' (SEQ.ID.N0.:17)
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The PCR primers also contained restriction sites for SacI and EcoRI. The NOS
terminator region of the pBI101 vector was cut out with SacI/EcoRI digestion
and replaced
with the similarly digested 736bp 3'UTR amplicon of the DNA fragment described
in
Figure 3.
The GUS reporter construct was then electroporated into Agrobacterium
tumifaciens strain AGLI and transformation of flax (Finnegan et al. (1993)
Plant Mol Biol.
22(4): 625-633) and Arabidopsis (Valvekens et al. Proc. Natl. Acad. Sci. 85:
5536-5540)
carried out according to previously described protocols.
Various tissues from flax and Arabidopsis plants carrying the GUS reporter
construct were assayed histologically for evidence of GUS activity. In the
case of flax, leaf
tissue, root tissue and mid-maturity embryos dissected out of developing seeds
were stained
for GUS activity. For Arabidopsis, developing seeds were stained for GUS in
situ in their
siliques.
GUS staining was carried out by immersing the tissues in histochemical buffer
containing 0.5 mM X-glue, 0.5 M potassium phosphate buffer (pH 7.0), 1 mM
EDTA, 0.5 M
sorbital, 0.5 mM potassium ferricyanide and 0.5 mM potassium ferrocyanide. The
staining
reaction was carried out for 12-16 hrs at 37°C and the reaction was
stopped by adding 95%
ethanol. Tissues were subsequently cleared of chlorophyll by repeated washing
in 95%
ethanol prior to photography. Figure 9 shows clear evidence of strong GUS
activity in
developing flax embryos and Arabidopsis seeds, and no evidence of GUS reporter
gene
expression in flax roots or leaves, or in Arabidopsis silique walls.
EXAMPLE 6
Stable seed specific expression of 3~-;~lucuronidase (GUS) in flax Arabidopsis
and Brassica
nanus when under the regulator~r control of flax legumin-like storage protein
gene
resulatory seduences
A construct was made using standard molecular biology techniques, including
restriction enzyme digestions, ligation and polymerase chain reaction (PCR).
In order to
obtain a DNA fragment containing approximately 2 kilobases from the 5'
transcriptional
initiation region of the flax legumin-like seed storage protein in a
configuration suitable for
ligation to a GUS coding sequence, a PCR based approach was used. This
involved the use of
the polymerase chain reaction to amplify the precise sequence desired for the
expression
analysis. To perform the necessary PCR amplification, two oligonucleotide
primers were
synthesized (Beckman Oligo 1000M DNA synthesizer) have the following
sequences:
5' primer: 5'TATCTAGACTCAAGCATACGGACAAGGGT 3' (SJ-634) (SEQ.ID.N0.:18)
The italicized bases correspond to nucleotide positions 1 to 21 in the
sequence
reported in Figure 4. The additional nucleotides 5' of this sequence in the
primer are not
identical to the promoter sequence, but were included in order to place a XbaI
site at the 5'
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end of the amplification product. The XbaI (5'-TCTAGA-3') (SEQ.ID.N0.:19) site
is
underlined.
A second (3') primer was synthesized which had the following sequence:
3' primer 5'GGTTATCATTGTATGAACTGA3' (SJ-618) (SEQ.ID.N0.:20)
This primer contains the precise complement (shown in italics) to the sequence
reported in Figure 4 from bases 2343 to 2363. This primer was not designed
with an
additional restriction enzyme site due to the fact that a natural NcoI site
(5'-CCATGG-3')
(SEQ.ID.N0.:21) straddles the start codon between base pairs 2034 and 2039,
thereby
allowing for insertion of the storage protein promoter into the appropriate
cloning vector.
These two primers were used in a PCR amplification reaction to produce a DNA
fragment containing the sequence between nucleotides 1 and 2342 of the flax
seed storage
protein gene with a XbaI site at the 5' end and a NcoI site 302 base.pairs
from the 3' end.
PCR amplification was performed using the enzyme Pfu (Strategene) using
conditions
recommended by the enzyme manufacturer and a temperature program of
94°C
(denaturation) for 1 minute, 55°C (annealing) for 1 minute, and
72°C (elongation) for 3.5
minutes. The template was the legumin seed storage protein genomic clone shown
in Figure
4.
The resulting amplification product was subsequently digested with XbaI and
NcoI to remove the desired 2 kb promoter region. This promoter fragment was
cloned into
the XbaI and NcoI sites of a XbaI and NcoI digested plasmid designated
pGUS1318
(Plasmid pGUSN358S (Clontech Laboratories) was cut with NcoI and EcoRI and the
GUS
insert was cloned into pBluescriptKS+ (Stratagene) which was adapted to
contain an NcoI
site in the multiple cloning site.) The resulting plasmid containing the
promoter-GUS
fusion was called pPGUS1318. The terminator of the legumin seed storage
protein from flax
was also amplified from the above mentioned genomic clone. To perform the
necessary PCR
amplification, oligonucleotide primers were synthesized having the following
sequences:
5' primer: 5' GCAAGCTTAATGTGACGGTGAAATAATAACGG 3' (SJ620) (SEQ.ID.N0.:22)
The italicized bases correspond to nucleotide positions 3780 to 3803 in the
sequence
reported in Figure 4. The additional nucleotides 5' of this sequence in the
primer are not
identical to the promoter sequence, but were included in order to place a
HindIII site at the
5' end of the amplification product. The HindIII site (5'-AAGCTT-3')
(SEQ.ID.N0.:23) is
underlined.
A second (3') primer was synthesized which had the following sequence:
3' primer 5'TAGGTACCTGGCAGGTAAAGACTCTGCTC3' (SJ-618) (SEQ.1D.N0.:24)
This primer contains the precise complement (shown in italics) to the sequence
reported in Figure 4 from bases 4311 to 4290. The additional nucleotides 5' of
this sequence in
the primer are not identical to the promoter sequence, but were included in
order to place a
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KpnI site at the 5' end of the amplification product. The KpnI site (5'-GGTACC-
3')
(SEQ.ID.N0.:25) is underlined.
These two primers were used in a PCR amplification reaction to produce a DNA
fragment containing the sequence between nucleotides 3779 and 4311 of the flax
seed storage
protein gene terminator with a HindIII site at the 5' end and a KpnI site at
3' end.
Amplification using PCT was as described above. The above pPGUS1318 vector
that
contains the amplified promoter was digested with XhoI and treated with Klenow
to
create a blunt end. The vector was subsequently digested with KpnI and the
above
amplified terminator sequence was inserted so that it was located 3' of the
GUS coding
sequence. The resulting vector containing the flax seed storage protein
promoter, GUS and
the flax seed storage protein terminator is referred to as pPGUST.
The XbaI-KpnI insert of pPGUST which contains the limn .promoter-GUS coding
sequence-limn terminator sequence was ligated into the XbaI-KpnI sites of
pSBS3000 (This
vector is a derivative from the Agrobacterium binary plasmid pPZP221
(Hajdukiewicz et
al., 1994, Plant Molec. Biol. 25: 989-994). In pSBS3000 the plant gentamycin
resistance gene
of pPZP221 was replaced with parsley ubiquitin promoter-phosphinothricin
acetyl
transferase gene-parsley ubiquitin termination sequence to confer resistance
to the herbicide
glufosinate ammonium). The resulting vector is called pSBS2089. In addition
the
XbaI-KpnI insert of pPGUST which contains the limn promoter-GUS coding
sequence-linin
terminator sequence was ligated into the XbaI-KpnI sites of the Agrobacterium
binary
plasmid pCGN1559 (MacBride and Summerfield, 1990, Plant Molec. Biol. 14 269-
276,
confers resistance to the antibiotic kanamycin)). The resulting vector was
called pSBS2083.
Plasmids pSBS2089 and pSBS2083 were electroporated into Agrobacterium strain
EHA101.
Agrobacterium strain EHA101 (pSBS2089) was used to transform flax and
Arabidopsis,
Agrobacterium strain EHA101 (pSBS2083) was used to transform Brassica napus.
Flax
transformation was performed essentially as described in Jordan and McHughen
(1988)
Plant cell reports 7: 281-284, except transgenic shoots were selected on 10 uM
L-phosphinothricine instead of kanamycin. Arabidopsis transformation was done
essentially as described in "Arabidopsis Protocols; Methods in Molecular
Biology" Vol 82.
Edited by Martinez-Zapater JM and Salinas J. ISBN 0-89603-391-0 pg 259-266
(1998) except
the putative transgenic plants were selected on agarose plates containing 80uM
L-phosphinothricine. Brassica napus transformation was done essentially as
described in
Moloney et al. (1989). Plant Cell Reports. 8: 238-242.
Figure 10 shows the tissue-specific expression of GUS in transgenic flax
plants
transformed with a limn-GUS gene construct (pSBS2089). GUS expression was
measured in
roots (R), stems (S), leaves (L), Buds (B) and embryo (E). Some expression was
seen in buds,
and maximal expression was achieved in embryo tissues. No detectable
expression was seen
in any of the untransformed (WT) tissues.
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Figure 11 shows the temporal expression of GUS in transgenic flax plants
transformed with a limn-GUS gene construct (pSBS2089). As can be seen, maximum
expression is achieved in mature (pre-dessicated) flax embryos.
Figure 12 shows the absolute expression of GUS in transgenic Brassica napus
plants (L1 to L9) transformed with a limn-GUS gene construct (pSBS2083). As
can be seen
high level expression can be achieved in Brassica napus plants. When comparing
individual transgenic plants, a typical variation in expression due to
position effect can
also be seen.
Figure 13 shows expression of GUS in transgenic Arabidopsis siliques
(transformed
with a limn-GUS gene construct (pSBS2089)) during seed development. As can be
seen high
level expression can also be achieved in Arabidopsis seed tissues. Maximum
expression is
achieved at stage 4 (mature but not fully dessicated) of seed development. No
detectable
expression is observed in non-seed tissues such as leaves, stems, roots and
silique walls
(results not shown).
While the present invention has been described with reference to what are
presently considered to be the preferred examples, it is to be understood that
the invention
is not limited to the disclosed examples. To the contrary, the invention is
intended to cover
various modifications and equivalent arrangements included within the spirit
and scope of
the appended claims.
All publications, patents and patent applications are herein incorporated by
reference in their entirety to the same extent as if each individual
publication, patent or
patent application was specifically and individually indicated to be
incorporated by
reference in its entirety.
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SEQUENCE LISTING
<110> Chaudhary, Sarita
van Rooijen, Gijs
Moloney, Maurice
Singh, Surinder
SemBioSys Genetics Inc.
Commonwealth Scientific and Industrial Research Organisation
<120> Flax Seed Specific Promoters
<130> 9369-147
<140>
<141>
<150> 60/151044
<151> 1999-08-27
<15G> 60/161,722
<151> 1999-10-27
<160> 25
<170> PatentIn Ver. 2.0
<210> 1
<211> 4305
<212> DNA
<213> Linum usitatissimum
<400> 1
ttcaaaaccc gattcccgag gcggccctat tgaagatatg ggggaagttc gacgagatcg 60
atgtcgggtc gagtgctatg gtgatggtgc cgtttggggg gaggatgagc gagatagcca 120
agactagcat tccgttccca cacagagttg ggaatttgta ccaaatccaa cacttgtcgt 180
attggagcga cgatagggac gcggaaaaac acatccgttg gatcagggag ttgtacgatg 240
atctcgagcc ttatgtgtcg aagaatccga ggtatgctta cgtgaactac agggatctcg 300
acatcgggat gaatggagga ggtgaagggg atgagaaggg tacttatggt gaggctaagg 360
tgtgggggga gaagtacttt ggggtcaact ttgatcggtt ggttcgggtg aagacgattg 420
ttgatcccaa taatgtgttt cgaaacgagc agagcattcc ctcaattcca actcggttat 480
aaggatcaat gatcaatgag aattttcctt tccaatgtga ttacaagttc tattgggtca 540
gctttctcaa ctgctcctat tcatttagat taattcataa caactattaa tttaccagcc 600
ttttatccgg cccgttggcc gatttatttt cttaagtttt agatgaaatg aaaccgattt 660
agtttttatt gagatgagat taatcttaat ttgcttgaaa tttactcacg gttgatgtga 720
tatttggaat taactaaaat gataaatatc ggataaaaat aaaaatattt aaaataaata 780
acataaacat aagaacaata aaataaataa atttaatttt aatttatttc cttgttttct 840
ttctgtatca tacatctctt ctcttacttc ttaaaggctt ttcaattatc acttaattaa 900
atacaataga taaatcgtta attctataac attaacctat acacttgcac ggtgaacaat 960
caatatgata atataataat aatataataa ttcaattatt aatctacaat tttttaatta 1020
taaagtttat gcggtcagtt tctgcaagct ccgagctcct tgtcatcgtt agtttctgcg 1080
gtctcaaggt ataacgactc ggagcgacga gccctttgct tccaatggac gggttgcatt 1140
tctgccgtcg ttgagctcga ttggcgtgtc atgctggagt cagagttcct acaaaaaaac 1200
cctaaactag agggtgatta gggtgaaatt agggtgttgg cctgggttcc attgtccaaa 1260
gttttagtca acttaaaaac agacttaaat tttatgcttc aaaatagttt atctgttatt 1320
atattagcgt gtaattagtc ttgacaatgg ggccggacgg gtacggattc gggaccccga 1380
tccccgccca tagtgtaatg gctcaactgc caagtcagca ttggaccgaa attattggac 1440
acgaagtact aatgtgaaaa actttacatt tgttattttc tactttaata ctatgctatt 1500
ttcaaaattt gaactttaat actatgtttt tatatagttt agtatatctt aatttttatg 1560
caaattcatc taattgtatt aaactatttt cgatccgtag ctaattattt cgaaggcaag 1620
tcaaagtgtt attgtggact atgtgagcta atattgaacc tttatctctc ccaaccactc 1680
aagttaattg aaccaaactc gatcggttgg gtttcgagct atttcgagcc attgttgtta 1740
tatgcacgtg agatatcaag attgacccga acactttatt atgataatgt agaaaaagaa 1800
aacatattct aagactacat gcatgcaaag tgcaacccct gcatggaaag ctgctcaaca 1860
cgtggcatag actcccgcca cgtgtccatt ccacctcatc acctcacccc caccgttcac 1920
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ctcttattat atcacaacaa tcaatcaatc ctactcctcc atactcgaac aaatccgacc 1980
aacttatacc aatattccca aacttgatta atttctcagc aatatggatc agacgcacca 2040
gacatacgcc ggaaccacgc agaacccgag ctatggcggc gggggcacaa tgtaccagca 2100
gcagcagccg aggtcttacc aggcggtgaa ggcggccact gcagccaccg cgggtggatc 2160
cctcatcgtt ctgtccggtc tcatccttac ggccaccgtc atttcactca tcatagccac 2220
ccctctcctt gtcatcttca gccctgttct tgtcccggct ctcatcaccg tcgggctctt 2280
gatcaccggg tttcttgctt ccggtgggtt cggagtcgcc gccgtcaccg tcttgtcctg 2340
gatctatagg tatgtataag ctttggactt tagtattgtt ataaaataca taagctgatt 2400
tatgaacatg gatctcccaa caagagttat ttaaatgcat tctcggtctg actcgatcgg 2460
ttgggttttg agctactcgg tcacaatggt cgggtcggct ctggatctgt tatactaata 2520
tttggaagcc tgaagtttca ttgttctgcc ccaacttccc actacctttt gagggtgtta 2580
agaagccata caaactaatt atgaatccct cccaacaact cagaactcga gtcagtgggt 2640
tgtgacggtt ctctataaac atttcgaaaa tctttgttca atgaacgtag aaatgaccat 2700
gcttgatgat tgtgggtctt ataaggtacg tgaccggcgg gcacccggcg ggaggggatt 2760
cgctggacca ggctaggtcg aagctggccg gaaaggccag ggaggtgaag gacagggcgt 2820
cggagttcgc acagcagcat gtcacaggtg gtcaacagac ctcttaaaga gagtcctcta 2880
gttaaattgg tcttcgtttc tgtttcgtgg cggcttgtaa actctctttt aagtgtgctg 2940
ttttcctttt gtctcgtgtg ttgtaagtga aagtgtaatc gaagttccaa gttggagatg 3000
tttgtaacga tgatgttttc taataatcag agatattaaa agggttgcta atttagtatt 3060
gcgtctgatc tcggaccaaa ctcgcaagta aaattgcaga ggatgagttg tacagaacaa 3120
gcgtgcattg ttctggaagt tcatctcctt ggagccgacc ttgttgcttg cagtttcgcc 3180
aagtccacta gacaatgtta cgagttaagc ctctgtcaaa cagatcgctc tagcgtccca 3240
gaaaacacca gatttttcga aaaccatcgg ggatcaattt tcgattcaat tccgatcttg 3300
gaagtacttg aacagaagca tgatgctaaa agataataga aaatcgaagc ctagaaaagt 3360
tgtacagaaa gcaacaagtc aaaaatatag atcaacttca aaggttcaaa ttacatctta 3420
cagaccccaa aaaatgacag ttaacagaag tcgactaaac agaaaccagc cagcttcacc 3480
tggaatgaag gagctttgat caatccatcc tagcttcatt cccctttgaa attgcagaca 3540
gagctctcat cctgctaaag ctggtggctt attcttaacc ctgcaatcaa taagcatgaa 3600
ctaacattgg acaccttcat cggcggattg ctcgaaaatc agtgagcgag ggatttacct 3660
gtgtgtgtag taacctctct ccttgtacat aaaatctgga aattccggca tcaactactg 3720
ccacctttct gcttaaggtg attttatcac caaggctgag cgtgattcct tgcgtcttgc 3780
tccgaatcct gatgtatcca ctgagctttc catctccttc cttctccagg cttatgttca 3840
ccaatgcgtc ctcgccgaac acactcttgg cgtacaagtt cgcagccagg aatccacact 3900
ctccatcaag tgcagacctg caaaccccaa ataagaacac aaactccaaa gtcaacgatc 3960
aattctccgc cttttatgaa gaaaaggaaa cttctgggta cttacggtgc cgtcagacac 4020
ttcatatttg tagacttgat gatatggtcc aggaattcct tctcgttctg aattgttgtg 4080
ttaacagcaa cctgacagac agaaagatat cgcaaattta agatactggg atgactaggc 4140
acagagaaat gaaatctaat tctagaagta aaaccttatt ttcccattca aattctgccc 4200
acatagtccg gaacgcagca tccgagcaag aagcaggaga gatgtaatcc atgatatcga 4260
tgtggatatc gttgaggacg acaactgaac gttccatcac attgg 4305
<210> 2
<211> 109
<212> PRT
<213> Linum usitatissimum
<400> 2
Met Asp Gln Thr His Gln Thr Tyr Ala Gly Thr Thr Gln Asn Pro Ser
1 5 10 15
Tyr Gly Gly Gly Gly Thr Met Tyr Gln Gln Gln Gln Pro Arg Ser Tyr
20 25 30
Gln Ala Val Lys Ala Ala Thr Ala Ala Thr Ala Gly Gly Ser Leu Ile
35 40 45
Val Leu Ser Gly Leu Ile Leu Thr Ala Thr Val Ile Ser Leu Ile Ile
50 55 60
Ala Thr Pro Leu Leu Val Ile Phe Ser Pro Val Leu Val Pro Ala Leu
65 70 75 80
Ile Thr Val Gly Leu Leu Ile Thr Gly Phe Leu Ala Ser Gly Gly Phe
85 90 95
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Gly Val Ala Ala Val Thr Val Leu Ser Trp Ile Tyr Arg
100 105
<210> 3
<211> 46
<212> PRT
<213> Linum usitatissimum
<400> 3
Tyr Val Thr Gly Gly His Pro Ala Gly Gly Asp Ser Leu Asp Gln Ala
1 5 10 15
Arg Ser Lys Leu Ala Gly Lys Ala Arg Glu Val Lys Asp Arg Ala Ser
20 25 30
Glu Phe Ala Gln Gln His Val Thr Gly Gly Gln Gln Thr Ser
35 40 45
<210> 4
<211> 3501
<212> DNA
<213> Linum usitatissimum
<400> 4
tctagacatt tgacataaac cgaattcaaa gaacacaaca ttgactaaca ccaaaaagaa 60
atagagtagt gaaatttgga agattaaaaa atagaaacaa actgattctt agaaagaaga 120
gatgattagg tgctttcagt tcggtctgtc aggaaatcga gatgttcact tatttacatt 180
gtcgattcat ctcccaattg tcctggttcc tttactgtcc gacgcttttt tgaatcccag 240
ttaattccca tcaagtcttc cttcagctgc gtagcactgc tagctccaac atggagcgtg 300
gagtctactc gttcatgggg catcgcaaag gtttgccttc atgttctgct accagccagc 360
gcccaccgcc tcttggttgt gtggacaatt gcggtgaagc gcgcaagttg acatcccata 420
gtctcgacac ttcaccatat ggatgtttaa aacgtatatc acgagtgcga tctacatgtc 480
ccatcacacc acatataaag caatagtttg ggagcttttc atatttgaaa cgggcattga 540
cgacttgccc tctcgataat ttaatctttt tttctcttca gctgattgtg tgcatccatt 600
cgggctcaga agcacatcaa agggatctct ccatcgtagt attgggtcgt gtcgtatgat 660
acgaagcagt cgatgaagtt tcctaatgtg cgagctacag gctccgcaaa gaacccgcga 720
ggtagatcgt atgctagtac ccaaaaatca gtttgtcgta gcggaatcaa cactagagac 780
tcaccctaat gcatctcatg tgtgatgaac agtttatcat ttgtgagtct aggggtcatt 840
gtcgatgacc caatgcacat tgagcttatg atagaatttg aataggaagc gttttccacc 900
cagatcacga atagctaccc ctttttcggg cgccaaattt ccggcatcct atcttccacc 960
acaacttaaa gatgcgatcg gtaaggaact caccgaccac acacatcgaa taatcttcgg 1020
tgaccggttc ctgttgatca agtccctcaa tttcctcaac ctagtcttca atcgccgcta 1080
gcgttatccc ccgcatatgg actttcatag cgcggagcgt agccggagac gacgagcaag 1140
aaggatgagc ggcggcagat tgcggctaaa gaaacgagct tcctgccttg ctctatggag 1200
gcagatttct gagttgatgg tgatggattt gtgatgtgga cacttttaat ttaagttgat 1260
tttttagcac ttcattcacg taattaaata aataatttcc agtattttat atttatttcc 1320
ttacgttatc taattttttg aaagattaaa actttgatat aggcaagatc atgacacgtc 1380
gaagttaagt gaatgagact cctaacaagg taataacaaa gcagttcata aaccgaatga 1440
ccttgatctt tactaagctt gagatcattg aacatataat taaatacgtt aatgaaagat 1500
aagaacttta atataaaaat cattcaaaac gagaaactga taacaaaaac aaagcaaacg 1560
gccaacaaaa taatagacgg tggaaggatg atgcagagcc atccaccctt ttttcccagt 1620
ttccttactg cttacttctc tatgcatatc acaagacgcc cttgaaactt gttagtcatg 1680
cagagccctt actcgccagg tcaccgcacc acgtgttact ctatcacttc tcctcccttt 1740
cctttaaaga accaccacgc cacctccctc tcacaaacac tcataaaaaa accacctctt 1800
gcatttctcc caagttcaaa ttagttcaca gctaagcaag aactcaacaa caatggcgga 1860
tcgtacaaca cagccacacc aagtccaggt ccacacccag caccactatc ccaccggcgg 1920
ggctttcggc cgttatgaag gtggactcaa aggcggtcca catcaccagc aaggatcagg 1980
cagcggccca tcagcttcca aggtgttagc agtcatgacc gcgctcccca tcggcgggac 2040
cctccttgcc ttggccggga taaccttggc tgggacgatg atcgggctgg cgatcaccac 2100
cccgattttt gtcatctgca gccctgttct agtcccggcc gctctgctca tcgggtttgc 2160
cgtgagcgcg tttctggcct cggggatggc cgggctgaca gggctgacct cgctgtcgtg 2220
gtttgcgagg tatctgcagc aggctgggca gggagttgga gtgggggtgc cggatagttt 2280
cgagcaggcg aagaggcgca tgcaggatgc tgctgggtat atggggcaga agaccaagga 2340
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agttgggcag gagatccaga ggaagtctca ggatgtgaaa gcatcagaca aataaggtga 2400
taataagggg ttttgggttc gtgtgtaaac tggtaaaatg gaaattctgg gttttactgt 2460
acttttgcat gtagtggaat gaatgagttc ttgttctctt ttgtctttta atcataaagt 2520
aagaagcagc atttcatgtt ctggttgaat attgtcaaga attcgcaaca aatttagcta 2580
aaccagttca atcttaccgg ttagacgact tcccagtaag aaacattcca ggtccatccc 2640
ggtataagag tctggacttc tgaaaccttt agaccttgga tttggaaaaa agatgaaacc 2700
tttagaataa attacaacga tggcagattg tacaaaactg gagtcgagat catgtaaatt 2760
agcccataac taagaaccgg cgatgacaac aattactagg aatatggttg ttgggctggt 2820
cggcggctag cggtgatgat ttggaagaat cggggatcca gaatgtgaga accgaatcat 2880
cgacgaacat tacccggcga ggagcccatt tcaagcaact ttggaactcc tatatggctg 2940
ttccagcagg ccacctgctc aagaaagaaa gaagccatgt cagaaatcct tacgaaatct 3000
aactggatgc tgatatgaat ccgccaggtg tgcggagttc tttacaggca ggatctataa 3060
agaagaaaca tgttttgtat tggcattgtt gatgttccaa gcacgcagcg atctatctcc 3120
ggatcctaac aacaaaaata cggattctgt aagaaacaag cgcagaaaac ttctgcaacg 3180
aaaccactcg tatatttggt tctgagttgg agaaagatga ccatactact gtatttggtt 3240
gaacttggat tggaaccgaa attttgagtt gaaaagcgag tgatcgtata taaatttcag 3300
attcagatta ggatatccta tgagagaagg tagagttacc tgatactaca tactgcccat 3360
caggggtaaa agttgcctcg atggttgtgt ttggagatgg ttccaggcta aatccacaac 3420
gctgaacaaa ttaaaagatg aatggatcaa tcttcaaccc ttacttctgc atttatgagg 3480
attggctcaa ggctctctag a 3501
<210> 5
<211> 180
<212> PRT
<213> Linum usitatissimum
<400> 5
Met Ala Asp Arg Thr Thr Gln Pro His Gln Val Gln Val His Thr Gln
1 5 10 15
His His Tyr Pro Thr Gly Gly Ala Phe Gly Arg Tyr Glu Gly Gly Leu
20 25 30
Lys Gly Gly Pro His His Gln Gln Gly Ser Gly Ser Gly Pro Ser Ala
35 40 45
Ser Lys Val Leu Ala Val Met Thr Ala Leu Pro Ile Gly Gly Thr Leu
50 55 60
Leu Ala Leu Ala Gly Ile Thr Leu Ala Gly Thr Met Ile Gly Leu Ala
65 70 75 80
Ile Thr Thr Pro Ile Phe Val Ile Cys Ser Pro Val Leu Val Pro Ala
85 90 95
Ala Leu Leu Ile Gly Phe Ala Val Ser Ala Phe Leu Ala Ser Gly Met
100 105 110
Ala Gly Leu Thr Gly Leu Thr Ser Leu Ser Trp Phe Ala Arg Tyr Leu
115 120 125
Gln Gln Ala Gly Gln Gly Val Gly Val Gly Val Pro Asp Ser Phe Glu
130 135 140
Gln Ala Lys Arg Arg Met Gln Asp Ala Ala Gly Tyr Met Gly Gln Lys
145 150 155 160
Thr Lys Glu Val Gly Gln Glu Ile Gln Arg Lys Ser Gln Asp Val Lys
165 170 175
Ala Ser Asp Lys
180
<210> 6
CA 02383376 2002-02-27
WO 01/16340 PCT/CA00/00988
5/11
<211> 1676
<212> DNA
<213> Linum usitatissimum
<400> 6
tccactatgt aggtcatatc catcatttta atttttgggc accattcaat tccatcttgc 60
ctttagggat gtgaatatga acggccaagg taagagaata aaaataatcc aaattaaagc 120
aagagaggcc aagtaagata atccaaatgt acacttgtca tcgccgaaat tagtaaaata 180
cgcggcatat tgtattccca cacattatta aaataccgta tatgtattgg ctgcatttgc 240
atgaataata ctacgtgtaa gcccaaaaga acccacgtgt agcccatgca aagttaacac 300
tcacgacccc attcctcagt ctccactata taaacccacc atccccaatc ttaccaaacc 360
caccacacga ctcacaactc gactctcaca ccttaaagaa ccaatcacca ccaaaaaatg 420
gcaaagctga tgagcctagc agccgtagca acgcagttcc tcttcctgat cgtggtggac 480
gcatccgtcc gaaccacagt gattatcgac gaggagacca accaaggccg cggtggaggc 540
aaggtggcag ggacagcagc agtctgcgag cagcagatcc agcagcgaga cttcctgagg 600
agctgccagc agttcatgtg ggagaaagtc cagaggggcg gccacagcca ctattacaac 660
cagggccgtg gaggaggcga acagagccag tacttcgaac agctgtttgt gacgacctta 720
agcaattgcg caccgcggtg caccatgcca ggggacttga agcgtgccat cggccaaatg 780
aggcaggaaa tccagcagca gggacagcag cagggacagc agcaggaagt tcagaggtgg 840
atccagcaag ctaaacaaat cgctaaggac ctccccggac agtgccgcac ccagcctagc 900
caatgccagt tccagggcca gcagcaatct gcatggtttt gaaggggtga tcgattatga 960
gatcgtacaa agacactgct aggtgttaag gatggataat aataataata atgagatgaa 1020
tgtgttttaa gttagtgtaa cagctgtaat aaagagagag agagagagag agagagagag 1080
agagagagag agagagagag agaggctgat gaaatgttat gtatgtttct tggtttttaa 1140
aataaatgaa agcacatgct cgtgtggttc tatcgaatta ttcggcggtt cctgtgggaa 1200
aaagtccaga agggcggccg cagctactac tacaaccaag gccgtggagg agggcaacag 1260
agccagcact tcgatagctg ctgcgatgat cttaagcaat tgaggagcga gtgcacatgc 1320
aggggactgg agcgtgcaat cggccagatg aggcaggaca tccagcagca gggacagcag 1380
caggaagttg agaggtggtc ccatcaatct aaacaagtcg ctagggacct tccgggacag 1440
tgcggcaccc agcctagccg atgccagctc caggggcagc agcagtctgc atggttttga 1500
agtggtgatc gatgagatcg tataaagaca ctgctaggtg ttaaggatgg gataataaga 1560
tgtgttttaa gtcattaacc gtaataaaaa gagagagagg ctgatggaat gttatgtatg 1620
tatgtttctt ggtttttaaa attaaatgga aagcacatgc tcgtgtgggt tctatc 1676
<210> 7
<211> 174
<212> PRT
<213> Linum usitatissimum
<400> 7
Met Ala Lys Leu Met Ser Leu Ala Ala Val Ala Thr Gln Phe Leu Phe
1 5 10 15
Leu Ile Val Val Asp Ala Ser Val Arg Thr Thr Val Ile Ile Asp Glu
20 25 30
Glu Thr Asn Gln Gly Arg Gly Gly Gly Lys Val Ala Gly Thr Ala Ala
35 40 45
Val Cys Glu Gln Gln Ile Gln Gln Arg Asp Phe Leu Arg Ser Cys Gln
50 55 60
Gln Phe Met Trp Glu Lys Val Gln Arg Gly Gly His Ser His Tyr Tyr
65 70 75 80
Asn Gln Gly Arg Gly Gly Gly Glu Gln Ser Gln Tyr Phe Glu Gln Leu
85 90 95
Phe Val Thr Thr Leu Ser Asn Cys Ala Pro Arg Cys Thr Met Pro Gly
100 105 110
Asp Leu Lys Arg Ala Ile Gly Gln Met Arg Gln Glu Ile Gln Gln Gln
115 120 125
Gly Gln Gln Gln Gly Gln Gln Gln Glu Val Gln Arg Trp Ile Gln Gln
CA 02383376 2002-02-27
WO 01/16340 PCT/CA00/00988
6/11
130 135 140
Ala Lys Gln Ile Ala Lys Asp Leu Pro Gly Gln Cys Arg Thr Gln Pro
145 150 155 160
Ser Gln Cys Gln Phe Gln Gly Gln Gln Gln Ser Ala Trp Phe
165 170
<210> 8
<211> 4999
<212> DNA
<213> Linum usitatissimum
<400> 8
ctcaagcata cggacaaggg taaataacat agtcaccaga acataataaa caaaaagtgc 60
agaagcaaga taaaaaaatt agctatggac attcaggttc atattggaaa catcattatc 120
ctagtcttgt gaccatcctt cctcctgctc tagttgagag gccttgggac taacgagagg 180
tcagttggga tagcagatcc ttatcctgga ctagcctttc tggtgtttca gagtcttcgt 240
gccgccgtct acatctatct ccattaggtc tgaagatgac tcttcacacc aacgacgttt 300
aaggtctcta tcctactcct agcttgcaat acctggcttg caatacctgg agcatcgtgc 360
acgatgattg gatactgtgg aggaggagtg tttgctgatt tagagctccc ggttgggtga 420
tttgacttcg atttcagttt aggcttgttg aaatttttca ggttccattg tgaagccttt 480
agagcttgag cttccttcca tgttaatgcc ttgatcgaat tctcctagag aaaagggaag 540
tcgatctctg agtattgaaa tcgaagtgca catttttttt caacgtgtcc aatcaatcca 600
caaacaaagc agaagacagg taatctttca tacttatact gacaagtaat agtcttaccg 660
tcatgcataa taacgtctcg ttccttcaag aggggttttc cgacatccat aacgacccga 720
agcctcatga aagcattagg gaagaacttt tggttcttct tgtcatggcc tttataggtg 780
tcagccgagc tcgccaattc ccgtccgact ggctccgcaa aatattcgaa cggcaagtta 840
tggacttgca accataactc cacggtattg agcaggacct attgtgaaga ctcatctcat 900
ggagcttcag aatgtggttg tcagcaaacc aatgaccgaa atccatcaca tgacggacgt 960
ccagtgggtg agcgaaacga aacaggaagc gcctatcttt cagagtcgtg agctccacac 1020
cggattccgg caactacgtg ttgggcaggc ttcgccgtat tagagatatg ttgaggcaag 1080
acccatctgt gccactcgta caattacgag agttgttttt tttgtgattt tcctaagttt 1140
ctcgttgatg gtgagctcat attctacatc gtatggtctc tcaacgtcgt ttcctgtcat 1200
ctgatatccc gtcatttgca tccacgtgcg ccgcctcccg tgccaagtcc ctaggtgtca 1260
tgcacgccaa attggtggtg gtgcgggctg ccctgtgctt cttaccgatg ggtggaggtt 1320
gagtttgggg gtctccgcgg cgatggtagt gggttgacgg tttggtgtgg gttgacggca 1380
ttgatcaatt tacttcttgc ttcaaattct ttggcagaaa acaattcatt agattagaac 1440
tggaaaccag agtgatgaga cggattaagt cagattccaa cagagttaca tctcttaaga 1500
aataatgtaa cccctttaga ctttatatat ttgcaattaa aaaaataatt taacttttag 1560
actttatata tagttttaat aactaagttt aaccactcta ttatttatat cgaaactatt 1620
tgtatgtctc ccctctaaat aaacttggta ttgtgtttac agaacctata atcaaataat 1680
caatactcaa ctgaagtttg tgcagttaat tgaagggatt aacggccaaa atgcactagt 1740
attatcaacc gaatagattc acactagatg gccatttcca tcaatatcat cgccgttctt 1800
cttctgtcca catatcccct ctgaaacttg agagacacct gcacttcatt gtccttatta 1860
cgtgttacaa aatgaaaccc atgcatccat gcaaactgaa gaatggcgca agaacccttc 1920
ccctccattt cttatgtggc gaccatccat ttcaccatct cccgctataa aacaccccca 1980
tcacttcacc tagaacatca tcactacttg cttatccatc caaaagatac ccaccatggc 2040
tagatcatca agccctttgc ttctctcact ctgcattttc gccattctct tccactcttc 2100
tctgggtagg cagcaattcc agcaggggaa cgagtgccag atcgacagga tcgacgcatc 2160
cgagccggac aaaaccatcc aggcagaagc tggcaccatc gaggtatggg accagaaccg 2220
ccagcaattc cagtgcgctg gtgttgccgt tgtaaggcgc accattgagc ccaaaggtct 2280
tctcttgcct ttctacagca acacccctca gctcatctac atcgttcaag gtataaatta 2340
aatcagttca tacaatgata accaccactt cgaatgtatt tatcaaatat caatgatcga 2400
tgcacctgta tgtgttgtgt atattcaggt aggggagtta caggaatcat gttcccakga 2460
tgtccagaga cattcgagga atcccagcag caaggacaac agggccaaca gggtagttcc 2520
caagaccagc accagaagat ccgccgcttc cgtgaaggtg acgtcattgc cgtccctgcc 2580
ggtgtagccc actggtccta caacgatggc aacgaaccag tcatggccat tgttgtccat 2640
gacacttcca gccacctcaa ccaactggac aacaacccca gggtatataa gcattgccgt 2700
agttgctaat aaattgcaca caattggaac tctattttca gtatctaata actttttcct 2760
tttttggcag aacttctact tggcaggaaa cccgagagac gagttcgaac aatcgcagca 2820
aggaggcagg ctgagccgtg gggagagtga aggtggacga ggacgcaggg aacctcttca 2880
acctgcaaca acctcttctt gcggaatcga ctccaagctc atcgcggagg cgttcaatgt 2940
cgacgagaac gtggcaagga ggctacagag cgagaacgac aacagaggcc agatcgtccg 3000
CA 02383376 2002-02-27
WO 01/16340 PCT/CA00/00988
7/11
agtcgaaggc gagctcgaca tcgtcagacc tccgaccagt atccaggagg agtcacagga 3060
gcagggaggt cgtggtggtg gccgctacta ctccaatgga gtggaggaga ccttctgctc 3120
catgagacta attgagaaca tcggcgatcc ttctcgggca gacattttca ctccagaagc 3180
cggccgcgtt agatccctca acagccacaa cctccccgtc ctgcaatgga tccagcttag 3240
cgccgagaga ggcgttctct acaatgtata gatctcactc acgcaccaac tctaaattga 3300
atccctaatt atttaattca ccgatatctg accgaccggt ttgaattttg taggaagcga 3360
tcaggctgcc gcactggaac atcaacgcac acagcatagt gtacgcgatc agaggacaag 3420
ccagagtcca gatcgtgaac gaggaaggga attcggtgtt cgatggagtg ctgcaggaag 3480
gacaggtggt gacggtgccg cagaacttcg cggtggtaaa gagatcccag agcgagaggt 3540
ttgagtgggt ggcgttcaag accaacgaca acgcgatggt gaactcgcta gccgggagga 3600
catcggcagt aagggcgatc cccgcggatg tactggctaa cgcctggagg gtgtcgccgg 3660
aggaggcgag gagggtgaag ttcaacaggc aggagactca cttggctagc accaggggcc 3720
agtccaggtc gcccgggagg ttgaatgtcg tcaaggaggt gatcaacttg cttatgtaaa 3780
atgtgacggt gaaataataa cggtaaaata tatgtaataa taataataat aaagccacaa 3840
agtgagaatg aggggaaggg gaaatgtgta atgagccagt agccggtggt gctaattttg 3900
tatcgtattg tcaataaatc atgaattttg tggtttttat gtgttttttt aaatcatgaa 3960
ttttaaattt tataaaataa tctccaatcg gaagaacaac attccatatc catggatgtt 4020
tctttaccca aatctagttc ttgagaggat gaagcatcac cgaacagttc tgcaactatc 4080
cctcaaaagc tttaaaatga acaacaagga acagagcaac gttccaaaga tcccaaacga 4140
aacatattat ctatactaat actatattat taattactac tgcccggaat cacaatccct 4200
gaatgattcc tattaactac aagccttgtt ggcggcggag aagtgatcgg cgcggcgaga 4260
agcagcggac tcggagacga ggccttggat gagcagagtc tttacctgcc agggcgtgaa 4320
ggggaagagc ggccttctgg agtaggagtt cagcaagcgg cggttccttg gcggagtaag 4380
cggacgtaag ggtggntgtc gacgtcntcg tttcnggagg cgnattcatg aagggttaaa 4440
gtcanatctg tagctctcga gtgctcaggg agccnaaaga cgttgggaaa ccgtcgncgt 4500
ttggggcatc agtcngcggg gcacgcttcc ctcctgctgc tccanaancn angtanattt 4560
aaaaganatg ggaaattaan taatggnaat nannaggagg attgnaacgg tcnganccgn 4620
angaanagtt tttannggtt taaatactgg gggagtngna gccngccnct ggttccngtg 4680
tagangaaac caagnnccgg gaggnttnca nnngnnaggg agaaaaagga nncatttnan 4740
nangcngagg gacatgaanc ggtacngagc tgnggttcan nnancggcgn nnggnagtcc 4800
cnngggaccn ggntggggtn anaagggaan ggaacattng gtngnangga naanaccntt 4860
ttacnattgc ctttgcaggn nngtntnggc ncntncgggt nacatnccgc tgcatgggct 4920
ttggggngcc nanaggnagc cncangggna nncngccncc ttgtncangn cgctnaagtt 4980
cnattgtana tggncgttg 4999
<210> 9
<211> 96
<212> PRT
<213> Linum usitatissimum
<400> 9
Met Ala Arg Ser Ser Ser Pro Leu Leu Leu Ser Leu Cys Ile Phe Ala
1 5 10 15
Ile Leu Phe His Ser Ser Leu Gly Arg Gln Gln Phe Gln Gln Gly Asn
20 25 30
Glu Cys Gln Ile Asp Arg Ile Asp Ala Ser Glu Pro Asp Lys Thr Ile
35 40 45
Gln Ala Glu Ala Gly Glu Val Trp Asp Gln Asn Arg Gln Gln Phe Gln
50 55 60
Cys Ala Gly Val Ala Val Val Arg Arg Thr Ile Glu Pro Lys Gly Leu
65 70 75 80
Leu Leu Pro Phe Tyr Ser Asn Thr Pro Gln Leu Ile Tyr Ile Val Gln
85 90 95
<210> 10
<211> 85
<212> PRT
<213> Linum usitatissimum
CA 02383376 2002-02-27
WO 01/16340 PCT/CA00/00988
8/11
<400> to
Gly Arg Gly Val Thr Gly Ile Met Phe Pro Xaa Cys Pro Glu Thr Phe
1 5 10 15
Glu Glu Ser Gln Gln Gln Gly Gln Gln Gly Gln Gln Gly Ser Ser Gln
20 25 30
Asp Gln His Gln Lys Ile Arg Arg Phe Arg Glu Gly Asp Val Ile Ala
35 40 45
Val Pro Ala Gly Val Ala His Trp Ser Tyr Asn Asp Gly Asn Glu Pro
50 55 60
Val Met Ala Ile Val Val His Asp Thr Ser Ser His Leu Asn Gln Leu
65 70 75 80
Asp Asn Asn Pro Arg
<210> 11
<211> 165
<212> PRT
<213> Linum usitatissimum
<400> 11
Asn Phe Tyr Leu Ala Gly Asn Pro Arg Asp Glu Phe Glu Gln Ser Gln
1 5 10 15
Gln Gly Gly Arg Leu Ser Arg Gly Glu Ser Glu Gly Gly Arg Gly Arg
20 25 30
Arg Glu Pro Leu Gln Pro Ala Thr Thr Ser Ser Cys Gly Ile Asp Ser
35 40 45
Lys Leu Ile Ala Glu Ala Phe Asn Val Asp Glu Asn Val Ala Arg Arg
50 55 60
Leu Gln Ser Glu Asn Asp Asn Arg Gly Gln Ile Val Arg Val Glu Gly
65 70 75 80
Glu Leu Asp Ile Val Arg Pro Pro Thr Ser Ile Gln Glu Glu Ser Gln
85 90 95
Glu Gln Gly Gly Arg Gly Gly Gly Arg Tyr Tyr Ser Asn Gly Val Glu
100 105 110
Glu Thr Phe Cys Ser Met Arg Leu Ile Glu Asn Ile Gly Asp Pro Ser
115 120 125
Arg Ala Asp Ile Phe Thr Pro Glu Ala Gly Arg Val Arg Ser Leu Asn
130 135 140
Ser His Asn Leu Pro Val Leu Gln Trp Ile Gln Leu Ser Ala Glu Arg
145 150 155 160
Gly Val Leu Tyr Asn
165
<210> 12
<211> 141
<212> PRT
<213> Linum usitatissimum
CA 02383376 2002-02-27
WO 01/16340 PCT/CA00/00988
9/11
<400> 12
Glu Ala Ile Arg Leu Pro His Trp Asn Ile Asn Ala His Ser Ile Val
1 5 10 15
Tyr Ala Ile Arg Gly Gln Ala Arg Val Gln Ile Val Asn Glu Glu Gly
20 25 30
Asn Ser Val Phe Asp Gly Val Leu Gln Glu Gly Gln Val Val Thr Val
35 40 45
Pro Gln Asn Phe Ala Val Val Lys Arg Ser Gln Ser Glu Arg Phe Glu
50 55 60
Trp Val Ala Phe Lys Thr Asn Asp Asn Ala Met Val Asn Ser Leu Ala
65 70 75 80
Gly Arg Thr Ser Ala Val Arg Ala Ile Pro Ala Asp Val Leu Ala Asn
85 90 95
Ala Trp Arg Val Ser Pro Glu Glu Ala Arg Arg Val Lys Phe Asn Arg
100 105 110
Gln Glu Thr His Leu Ala Ser Thr Arg Gly Gln Ser Arg Ser Pro Gly
115 120 125
Arg Leu Asn Val Val Lys Glu Val Ile Asn Leu Leu Met
130 135 140
<210> 13
<211> 11
<212> PRT
<213> Linum usitatissimum
<400> 13
Gln Gln Gln Gly Gln Gln Gln Gly Gln Gln Gln
1 5 10
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 14
tccactatgt aggtcata 18
<210> 15
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 15
ctttaaggtg tgagagtc 18
<210> 16
<211> 15
<212> DNA
<213> Artificial Sequence
CA 02383376 2002-02-27
WO 01/16340 PCT/CA00/00988
10/11
<220>
<223> Description of ArtificialSequence: Primer
<400> 16
aggggtgatc gatta 15
<210> 17
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: Primer
<400> 17
gatagaaccc acacgagc 18
<210> 18
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: Primer
<400> 18
tatctagact caagcatacg gacaagggt 29
<210> 19
<211> 6
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: Xbal site
<400> 19
tctaga 6
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: Primer
<400> 20
ggttatcatt gtatgaactg a 21
<210> 21
<211> 6
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: NcoI site
<400> 21
ccatgg
6
<210> 22
<211> 32
<212> DNA
<213> Artificial Sequence
CA 02383376 2002-02-27
WO 01/16340 PCT/CA00/00988
11/11
<220>
<223> Description of ArtificialSequence:Primer
<400> 22
gcaagcttaa tgtgacggtg aaataataacgg 32
<210> 23
<211> 6
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence:HindIII Site
<400> 23
aagctt 6
<210> 24
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence:Primer
<400> 24
taggtacctg gcaggtaaag actctgctc 29
<210> 25
<211> 6
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence:KpnI Site
<400> 25
ggtacc
6
