CA2310304C - Flax seed specific promoters - Google Patents

Abstract

Novel methods for the expression of non-native genes in flax seeds and the seeds of other plant species are provided. The methods involve the use of seed-specific promoters obtained from flax. Additionally provided are novel flax seed-specific promoters, chimeric nucleic acid constructs comprising novel flax seed-specific promoters, transgenic plant cells, transgenic plants and transgenic plant seeds containing novel flax seed-specific promoters. The promoters and methods are useful, for example, for altering the seed oil and protein composition in flax seed or other plant seeds.

Description

B&P File No. 9369-144/MG
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-

-2-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 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

-3-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.
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 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 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

-4-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:
(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) from nucleotides 1 to 2023, Figure 2 (SEQ ID NO:4) from nucleotides 1 to 1852,

-5-Figure 8 (SEQ ID NO:6) from nucleotides 1 to 417, or Figure 6 (SEQ ID
NO:8) from nucleotides 1 to 2035 wherein T can also be U;
(b) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a) under stringent hybridization conditions;
(c) a nucleic acid sequence that is complementary to a nucleic acid sequence of (a); or (d) a nucleic acid sequence that has substantial sequence homology to a nucleic acid sequence of (a).

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: 1) of a flax genomic clone encoding a 16.0 kDa oleosin protein (SEQ ID NOS:2 and 3).
Figure 2 shows the DNA sequence (SEQ ID NO:4) of a flax genomic clone encoding a 16.2 kDa oleosin protein (SEQ ID NO:5).
Figure 3 is a bar graph showing the transient expression of GUS in flax embryos when a flax oleosin promoter was used to drive the expression.
Figure 4 shows Southern blot analysis of flax genomic DNA.
Figure 5 shows Northern blot analysis of the developmental expression of flax oleosins during seed development.

Figure 6 shows the DNA sequence (SEQ ID NO:8) of a flax genomic clone encoding a 54.4 kDa legumin-like seed storage protein (SEQ ID NOS:9 to 12), this sequence is referred to as limn.

-6-Figure 7 is a graph showing the transient expression of GUS in flax embyos when the legumin-like seed-storage protein promoter and phaseolin seed-specific promoters were used to drive GUS expression.
Figure 8 shows the DNA sequence (SEQ ID NO:6) of a flax genomic clone encoding an 2S storage protein promoter (SEQ ID NO:7).
Figure 9 shows histological assays of GUS activity in various tissues from flax and Arabidopsis plants carrying a 2S storage protein promoter GUS reporter construct. (a) flax seed with 2S-GUS construct (b) control (untransformed) flax seeds (c) flax stems and leaves with 2S-GUS construct (d) Arabidopsis siliques and seeds with 2S-GUS
construct.
1 o 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.
Accordingly, in one aspect, the invention provides 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 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 plant cell; and

-7-(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 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 plant cell to create a transgenic plant cell which results in a detectably different phenotype of the plant cell or plant grown from it when compared with a non-transgenic plant cell or 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 plant cell or 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 the same species 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 naturally associated with the oleosin promoter, and a coding sequence of interest that is not naturally associated with the promoter. Non-native can also include a fusion as described above which also includes a cleavage sequence separating the promoter sequence and the protein sequence 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. 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

-8-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 NO:1) from nucleotides 1 to 2023, Figure 2 (SEQ ID NO:4) from nucleotides 1 to 1852, Figure 8 (SEQ ID NO:6) from nucleotides I to 417, or Figure 6 (SEQ ID
NO:8) from nucleotides 1 to 2035 wherein T can also be U;
(b) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a) under stringent hybridization conditions;
(c) a nucleic acid sequence that is complementary to a nucleic acid sequence of (a); or (d) a nucleic acid sequence that has substantial sequence homology to a nucleic acid sequence of (a).
The term "sequence that has substantial sequence homology" means those nucleic acid sequences which have slight or inconsequential sequence variations from these sequences, i.e., the sequences function in substantially the same manner to drive seed specific expression of a non-native nucleic acid sequence. The variations may be attributable to local mutations or structural modifications. Nucleic acid sequences having

-9-substantial homology include nucleic acid sequences having at least 85%, preferably 90-95% identity with the nucleic acid sequence as shown in Figure 1 (SEQ ID
NO:1), Figure 2 (SEQ ID NO:4), Figure 6 (SEQ ID NO:8) or Figure 8 (SEQ ID NO:6).
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.

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 (SEQ ID
NO:1) from nucleotides 1 to 2023, Figure 2 (SEQ ID NO:4) from nucleotides i to 1852, Figure 8 (SEQ
ID NO:6) from nucleotides 1 to 417, or Figure 6 (SEQ ID NO:8) from nucleotides 1 to 2035 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

- 10-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, 2"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 are 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. Sequence 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.
The methods 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 to an expression characteristic conferred to the native nucleic acid sequence by the native promoter. Thus in preferred embodiments, timing of expression in the plant's life cycle, of the non-native nucleic acid sequence is similar 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 to the expression level of the native nucleic acid sequence. 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 seed storage proteins, such as all albumins and globulins, including the vicilin and legumin-like proteins, as well as non-seed storage protein promoters 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

-11-embodiments the seed specific promoter is Figure 1 (SEQ ID NO:1) from nucleotides 1 to 2023, Figure 2 (SEQ ID NO:4) from nucleotides 1 to 1852, Figure 8 (SEQ ID
NO:6) from nucleotides 1 to 417, or Figure 6 (SEQ ID NO:8) from nucleotides 1 to 2035 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 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 promoters from other plant species. 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

- 12-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 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 different cellular expression may be detected in different seed 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.
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

- 13-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-b), interferon gamma (IFN-g), interleukin 1-alpha (IL 1-a), interleukin 1-beta (IL
1-b), 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 (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-b2), tumour necrosis factor alpha (TNF-a), and tumour necrosis factor beta (TNF-b).
Pharmaceutically useful proteins can also include mammalian proteins, for example, but not limited to a-l-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, b-galactosidase, -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.

-14-A nucleic acid sequence capable of terminating transcription is typically included in expression vectors. Transcriptional terminators are preferably includes from 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 transciption 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.
In addition to the promoter region and nucleic acid sequence of interest, 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, G418, 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 0-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 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 and are allowed to set seed known to a person of skill in the art.
Flax seed may

-15-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 promoters are shown in Figure 1 (SEQ ID NO:1) from nucleotides 1 to 2023, Figure 2 (SEQ ID NO:4) from nucleotides I to 1852, Figure 8 (SEQ ID NO:6) from nucleotides 1 to 417 and Figure 6 (SEQ ID NO:8) from nucleotides 1 to 2035.
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:

- 16-(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.
The present invention further provides methods of use for the novel promoters of Figure 1 (SEQ ID NO:1) from nucleotides 1 to 2023, Figure 2 (SEQ ID NO:4) from 1o nucleotides 1 to 1852, Figure 8 (SEQ ID NO:6) from nucleotides 1 to 417 and Figure 6 (SEQ ID NO:8) from nucleotides 1 to 2035 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 of promoters shown in Figure 1 (SEQ ID NO:1) from nucleotides I to 2023, Figure 2 (SEQ ID NO:4) from nucleotides 1 to 1852, Figure 8 (SEQ
ID NO:6) from nucleotides 1 to 417 and Figure 6 (SEQ ID NO:8) from nucleotides 1 to 2035 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.
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 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 By, 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).

- 17-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 calus, 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 monocotyledenous and dicotelydenous 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 biolistics (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 application such as to B. napus, the host cells targeted to receive recombinant DNA constructs typically will be derived from cotelydenary petioles as described by Moloney et al. (1989) Plant Cell Rep. 8: 238-242. Other examples using commercial oil seeds include cotelydon 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 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 over 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

-18-(Brassica napus, Brassica campestris), sunflower (Helianthus annuus), cotton (Gossypium hirsulum), 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 (Cocus nucifera), castor (Ricinus communis), coriander (Coriandrum sativum), squash (Cucurbita maxima), jojoba (Simmondsia chinensis) and rice (Oryza sativa).
The following non-limiting examples are illustrative of the present invention:
1 o EXAMPLES

Isolation of Seed-Specific Flax Promoters To isolate flax oleosin genomic clones, a mixture of Arabidopsis, maize and carrot eDNA was used to probe a genomic library derived from the flax line Forge that is homozygous for four rust resistance genes (Anderson et al. (1997), The Plant Cell 9: 641-651). Three positive clones, two oleosin genomic clones, encoding low molecular weight isoforms and 1 genomic clone, encoding high molecular weight isoforms were analyzed.
The sequence of one low molecular weight isoform and one high molecular weight isoform oleosin genomic clones are shown in Figures 1 and 2, respectively.
Figure 1 shows the DNA sequence (SEQ ID NO:1) of a flax genomic clone encoding a 16.0 kDa oleosin protein (SEQ ID NOS:2 and 3). Putative regulatory elements are identified and indicated. These include inverted repeats (base 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.

- 19-Figure 2 shows the DNA sequence (SEQ ID NO:4) of a flax genomic clone encoding a 18.6 kDa oleosin protein (SEQ ID NO:5). 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 shows the transient expression of GUS, using biolostics, in flax embyos 1 o wherein the GUS expression is directed by the flax oleosin promoter, as shown in Figure 1 (pSC54) (SEQ ID NO:1) from nucleotides 1 to 2023, with a promoter-less-GUS
construct (p1318) and unshot flax embryos as controls. The constructs were introduced into the flax embryos using biolistics. Embryos shot with pSC54 were also incubated in the absence (pSC54-U) or presence (pSC54-A) of 10 m ABA. GUS activity is expressed in pmol product/min/mg protein. Experiments are repeated in triplicate. These results indicate that the flax oleosin promoter, as shown in Figure 1 (SEQ ID NO:1) from nucleotides 1 to 2023, is a strong promoter that contains sufficient information to direct expression of a reporter gene in a transient manner in flax embryos. As well, treatment with ABA
increases the activity of the promoter 68% as compared to the lack of ABA treatment.

Figure 4 shows Southern blot analysis of flax genomic DNA. 60 g of flax genomic DNA was isolated from leaves, digested with EcoRl (lane I), Hindlll (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.

-20-Figure 5 shows Northern blot analysis of the developmental expression of flax oleosins during seed development. 1OJ) 15 gg per lane of total RNA was loaded in each lane on agarose/formaldehyde gel and blotted onto HybondTMN+ membrane. The membrane was probed using the 32P dCTP labeled flax oleosin cDNA clone (low molecular weight isoform), IOJ. Stages indicated are the number of days past anthesis (DPA). 3T) 15 g per lane of total RNA was loaded in each lane on agarose/formaldehyde gel and blotted onto HybondTMN+
membrane. The membrane was probed using the 32P dCTP labeled flax oleosin cDNA
clone (high molecular weight isoform), 3T. Both the transcripts were expressed very early in development (6DPA, early cotyledonary stage). Expression is maximum at 16 to 20 DPA (late cotyledonary stage) and declines at 22 DPA (mature embryos).

Figure 6 shows the DNA sequence (SEQ ID NO:8) of a flax genomic clone encoding a 54.4 kDa flax legumin-like seed storage protein (SEQ ID NOS:9 to 12).
Putative regulatory elements 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 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.
The genomic clone was isolated as follows. The above mentioned cDNA library was probed with oleosins from Arabidopsis, maize and carrot. Some clones were detected that did not contain oleosin sequences but a legumin-like seed storage protein. The cDNA
clone containing the sequence, cDNA 2/P was used to probe the above mentioned flax genomic library. The probe was synthesized using random hexamers and the Klenow fragment from E. coli and purified using the BioGe1TM P-60 system (BioRad).
Membranes were allowed to hybridize overnight. Following exposure to X-ray film (20 minutes at -80C) positive plaques were isolated.
The DNA was amplified using a liquid lysis culture followed by isolation of the phage DNA. (Sambrook et al. (1990), Molecular Cloning, 2nd ed. Cold Spring Harbor

-21-Press). The DNA was digested with Xbal and sub-cloned into the vector pBS
(Bluescript II
KS+).

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' TATCTAGACTCAA GCA TA CGGA CAA GGGT 3' (SJ-634) (SEQ ID NO:18) The italicized bases correspond to nucleotide positions 1 to 21 in the sequence reported in Figure 6. 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' end of the amplification product. The XbaI (5'-TCTAGA-3') (SEQ ID NO:19) site is underlined.
A second (3') primer was synthesized which had the following sequence:
3' primer: 5' GGTTATCATTGTATGAACTGA 3' (SJ-618) (SEQ ID NO:20) This primer contains the precise complement (shown in italics) to the sequence reported in Figure 6 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 NO: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

-22-minutes. The template was the legumin seed storage protein genomic clone shown in Figure 6.
The resulting amplification product was subsequently digested with Xbal and Ncol to remove the desired 2 kb promoter region. This promoter fragment was cloned into the Xbal and NcoI sites of a XbaI and Ncol digested plasmid designated pGUS 1318 (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 NO:22).
The italicized bases correspond to nucleotide positions 3780 to 3803 in the sequence reported in Figure 6. 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 HindlIl site at the 5' end of the amplification product. The HindIIl site (5'-AAGCTT-3') (SEQ ID
NO:23) is underlined.
A second (3') primer was synthesized which had the following sequence:
3' primer: 5' TAGGTACCTGGCAGGTAAAGACTCTGCTC 3' (SJ-618) (SEQ ID
NO:24) This primer contains the precise complement (shown in italics) to the sequence reported in Figure 6 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 KpnI site at the 5' end of the amplification product. The KpnI site (5'-GGTACC-3') (SEQ ID NO: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 HindlIl 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

-23-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 plasmid pPGUST was transferred into flax embryos by the method of biolistics (Sanford (1988), Trends in Biotechn. 6: 299-302). The resultant flax embryos left for 3 days and subsequently GUS expression assays (Jefferson (1987) Plant Mol. Biol.
Rep. 5:
387-405) were performed on the embryos. As a control, GUS assays were performed on unshot 3 day old flax embryos. The GUS expression data is found in Figure 7.
The flax legumin-like seed storage protein promoter directed expression of GUS was compared to the seed-specific phaseolin promoter (Sengupta-Gopalan et al., (1985), PNAS
USA 82:
3320-3324) directed expression of GUS. Within standard error, expression of the legumin-like seed storage protein promoter is equal to the phaseolin promoter.
These results indicate that the flax legumin seed storage protein gene promoter is a strong promoter that contains sufficient information to direct expression of a reporter gene in a transient manner in flax embryos, 3 days after integration.

A highly abundant seed-specific cDNA clone was isolated from a flax seed-specific cDNA library. 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.
More specifically, the isolated flax cDNA sequence encoded 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 NO:13).
The first 200 bp of the 5' end of the eDNA was used as a probe to screen the flax genomic library described in Example 1. Several positive lambda clones were identified after high stringency screening, one of which was shown by probing to have both the 5' and 3' ends of the cDNA, and therefore harboured the genomic counterpart of the full length cDNA. The insert from this lambda clone was subcloned into plasmid vector Bluescript as EcoRl fragments. Nucleotide sequencing of one of the EcoRl fragments showed it to have a perfect match for the full-length cDNA sequence. In addition, this EcoRl fragment contained several putative promoter regulatory elements as shown in Figure 8.
These include AT rich repeats (base pairs 25-36, 97-108 and 167-190), RY-like repeat (base pairs

-24-240-247), G-box-like element (base pairs 274-280), seed specific box-like motif (base pairs 285-290) and TATA box (base pairs 327-333).

A GUS reporter gene construct was made by incorporating 5' and 3' regions from the EcoRl fragment described in Example 7 into promoterless-GUS pBI101 vector as follows.
A 400bp amplicon from the 5' end of the EcoRl fragment was PCR amplified using the following primers (location shown in Fig 8):
5' primer(l): 5'-TCCACTATGTAGGTCATA-3' (SEQ ID NO:14) 3' primer(l): 5'-CTTTAAGGTGTGAGAGTC-3' (SEQ ID N 0:15) The PCR primers also contained restriction sites for HindIIl and BamHI which were used to clone the 400bp 5'UTR amplicon into the HindIII/BamHI sites of the pBI
101 vector in front of the GUS reporter gene. A 736bp amplicon from the 3' untranslated region (3'UTR) of the EcoRl fragment was PCR amplified using the following primers (location shown in Fig 8):
5' primer (2): 5'-AGGGGTGATCGATTA-3' (SEQ ID NO:16) 3' primer (2): 5'-GATAGAACCCACACGAGC-3' (SEQ ID NO:17) The PCR primers also contained restriction sites for Sacl and EcoRI. The NOS
terminator region of the pBI 101 vector was cut out with Sacl/EcoRl digestion and replaced with the similarly digested 736bp 3'UTR amplicon of the EcoRl fragment.
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-gluc, 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

-25-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.
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.

-26-SEQUENCE LISTING
GENERAL INFORMATION

APPLICANTS: SemBioSys Genetics Inc.
Commonwealth Scientific and Industrial Research Organisation TITLE OF INVENTION: Flax Seed Specific Promoters NUMBER OF SEQUENCES: 25 CORRESPONDENCE ADDRESS: Bereskin & Parr Scotia Plaza 40 King St W, 40th Floor Toronto, Ontario Canada M5H 3Y2 COMPUTER READABLE FORM:
MEDIUM TYPE: Floppy disk COMPUTER: iMac - Using Virtual PC
OPERATING SYSTEM: Windows '98 SOFTWARE: Patentln Ver. 2.0 CURRENT APPLICATION DATA:
APPLICATION NUMBER: CA 2,310,304 FILING DATE: 30-May-2000 PRIOR APPLICATION DATA:
APPLICATION NUMBER: US 60/161,722 FILING DATE: 27-Oct-1999 PRIOR APPLICATION DATA:
APPLICATION NUMBER: US 60/151,044 FILING DATE: 27-Aug-1999 ATTORNEY/AGENT INFORMATION:
(A) NAME: Bereskin & Parr (B) REGISTRATION NUMBER: 2800 (C) REFERENCE/DOCKET NUMBER: 9369-144 INFORMATION FOR SEQ ID NO:1:
SEQUENCE CHARACTERISTICS
LENGTH: 4305 TYPE: nucleic acid MOLECULE TYPE: DNA
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO: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

-27-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 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

-28-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 INFORMATION FOR SEQ ID NO:2:
SEQUENCE CHARACTERISTICS
LENGTH: 109 TYPE: amino acid MOLECULE TYPE: PRT
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Asp Gln Thr His Gln Thr Tyr Ala Gly Thr Thr Gln Asn Pro Ser Tyr Gly Gly Gly Gly Thr Met Tyr Gln Gln Gln Gln Pro Arg Ser Tyr Gln Ala Val Lys Ala Ala Thr Ala Ala Thr Ala Gly Gly Ser Leu Ile Val Leu Ser Gly Leu Ile Leu Thr Ala Thr Val Ile Ser Leu Ile Ile Ala Thr Pro Leu Leu Val Ile Phe Ser Pro Val Leu Val Pro Ala Leu Ile Thr Val Gly Leu Leu Ile Thr Gly Phe Leu Ala Ser Gly Gly Phe Gly Val Ala Ala Val Thr Val Leu Ser Trp Ile Tyr Arg INFORMATION FOR SEQ ID NO:3:
SEQUENCE CHARACTERISTICS
LENGTH: 46 TYPE: amino acid MOLECULE TYPE: PRT
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO:3:

Tyr Val Thr Gly Gly His Pro Ala Gly Gly Asp Ser Leu Asp Gln Ala Arg Ser Lys Leu Ala Gly Lys Ala Arg Glu Val Lys Asp Arg Ala Ser Glu Phe Ala Gln Gln His Val Thr Gly Gly Gln Gln Thr Ser

-29-INFORMATION FOR SEQ ID NO:4:
SEQUENCE CHARACTERISTICS
LENGTH: 3501 TYPE: nucleic acid MOLECULE TYPE: DNA
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO: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 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

-30-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 attggatcaa tcttcaaccc ttacttctgc atttatgagg 3480 attggctcaa ggctctctag a 3501 INFORMATION FOR SEQ ID NO:5:
SEQUENCE CHARACTERISTICS
LENGTH: 180 TYPE: amino acid MOLECULE TYPE: PRT
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Ala Asp Arg Thr Thr Gln Pro His Gln Val Gln Val His Thr Gln His His Tyr Pro Thr Gly Gly Ala Phe Gly Arg Tyr Glu Gly Gly Leu Lys Gly Gly Pro His His Gln Gln Gly Ser Gly Ser Gly Pro Ser Ala Ser Lys Val Leu Ala Val Met Thr Ala Leu Pro Ile Gly Gly Thr Leu Leu Ala Leu Ala Gly Ile Thr Leu Ala Gly Thr Met Ile Gly Leu Ala Ile Thr Thr Pro Ile Phe Val Ile Cys Ser Pro Val Leu Val Pro Ala Ala Leu Leu Ile Gly Phe Ala Val Ser Ala Phe Leu Ala Ser Gly Met Ala Gly Leu Thr Gly Leu Thr Ser Leu Ser Trp Phe Ala Arg Tyr Leu Gln Gln Ala Gly Gln Gly Val Gly Val Gly Val Pro Asp Ser Phe Glu Gln Ala Lys Arg Arg Met Gln Asp Ala Ala Gly Tyr Met Gly Gln Lys Thr Lys Glu Val Gly Gln Glu Ile Gln Arg Lys Ser Gln Asp Val Lys Ala Ser Asp Lys

-31-INFORMATION FOR SEQ ID NO:6:
SEQUENCE CHARACTERISTICS
LENGTH: 1676 TYPE: nucleic acid MOLECULE TYPE: DNA
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO: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 INFORMATION FOR SEQ ID NO:7:
SEQUENCE CHARACTERISTICS
LENGTH: 174 TYPE: amino acid MOLECULE TYPE: PRT
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met Ala Lys Leu Met Ser Leu Ala Ala Val Ala Thr Gln Phe Leu Phe Leu Ile Val Val Asp Ala Ser Val Arg Thr Thr Val Ile Ile Asp Glu

-32-Glu Thr Asn Gln Gly Arg Gly Gly Gly Lys Val Ala Gly Thr Ala Ala Val Cys Glu Gln Gln Ile Gln Gln Arg Asp Phe Leu Arg Ser Cys Gln Gln Phe Met Trp Glu Lys Val Gln Arg Gly Gly His Ser His Tyr Tyr Asn Gln Gly Arg Gly Gly Gly Glu Gln Ser Gln Tyr Phe Glu Gin Leu Phe Val Thr Thr Leu Ser Asn Cys Ala Pro Arg Cys Thr Met Pro Gly Asp Leu Lys Arg Ala Ile Gly Gln Met Arg Gln Glu Ile Gln Gln Gln Gly Gln Gln Gln Gly Gln Gln Gln Glu Val Gln Arg Trp Ile Gln Gln Ala Lys Gln Ile Ala Lys Asp Leu Pro Gly Gin Cys Arg Thr Gln Pro Ser Gln Cys Gln Phe Gln Gly Gin Gln Gln Ser Ala Trp Phe INFORMATION FOR SEQ ID NO:8:
SEQUENCE CHARACTERISTICS
LENGTH: 4999 TYPE: nucleic acid MOLECULE TYPE: DNA
ORGANISM: Linum usitatissimum FEATURE
NAME/KEY: misc feature LOCATION: (4396)..(4396) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4407)..(4407) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4415)..(4415) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc_feature LOCATION: (4423)..(4423) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4445)..(4445) OTHER INFORMATION: n is a, C. g, or t

- 33 -NAME/KEY: misc feature LOCATION: (4475)..(4475) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4497)..(4497) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4515)..(4515) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4545)..(4545) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4548)..(4548) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4550)..(4550) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4552)..(4552) OTHER INFORMATION: n is a. C, g, or t NAME/KEY: misc feature LOCATION: (4556)..(4556) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4567)..(4567) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4580)..(4580) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4587)..(4587) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4591)..(4591) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc_feature LOCATION: (4593)..(4594) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4605)..(4605) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc_feature LOCATION: (4613)..(4613) OTHER INFORMATION: n is a, c, g, or t

-34-NAME/KEY: misc_feature LOCATION: (4616)..(4616) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4620)..(4620) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4622)..(4622) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc_feature LOCATION: (4626)..(4626) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4635)..(4636) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4657)..(4657) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4659)..(4659) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4664)..(4664) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4668)..(4668) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4677)..(4677) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4685)..(4685) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4695)..(4696) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc_feature LOCATION: (4705)..(4705) OTHER INFORMATION: n is a, Cl g, or t NAME/KEY: misc feature LOCATION: (4708)..(4708) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4711)..(4713) OTHER INFORMATION: n is a, C. g, or t

- 35 -NAME/KEY: misc feature LOCATION: (4715)..(4716) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc_feature LOCATION: (4731)..(4732) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4738)..(4738) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4740)..(4741) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4743)..(4743) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4746)..(4746) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc_feature LOCATION: (4759)..(4759) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4766)..(4766) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4773)..(4773) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4780)..(4782) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4784)..(4784) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4790)..(4792) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4795)..(4795) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4802)..(4803) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4810)..(4810)

-36-OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4813)..(4813) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4820)..(4820) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4822)..(4822) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc_feature LOCATION: (4830)..(4830) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4839)..(4839) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc_feature LOCATION: (4843)..(4843) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4845)..(4845) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4847)..(4847) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc_feature LOCATION: (4851)..(4851) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4854)..(4854) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4858)..(4858) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc_feature LOCATION: (4865)..(4865) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4880)..(4882) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4885)..(4885) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature

-37-LOCATION: (4887)..(4887) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4891)..(4891) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4893)..(4893) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4895)..(4895) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4901)..(4901) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4906)..(4906) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4927)..(4927) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc_feature LOCATION: (4931)..(4931) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4933)..(4933) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4937)..(4937) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc_feature LOCATION: (4942)..(4942) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4945)..(4945) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4949)..(4949) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4951)..(4952) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4954)..(4954) OTHER INFORMATION: n is a, c, g, or t

-38-NAME/KEY: misc_feature LOCATION: (4958)..(4958) OTHER INFORMATION: n is a, C, g, or t NAME/KEY: misc feature LOCATION: (4965)..(4965) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4968)..(4968) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4970)..(4970) OTHER INFORMATION: n is a, c, g, or t NAME/KEY: misc feature LOCATION: (4975)..(4975) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4982)..(4982) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4989)..(4989) OTHER INFORMATION: n is a, C. g, or t NAME/KEY: misc feature LOCATION: (4994)..(4994) OTHER INFORMATION: n is a, c, g, or t SEQUENCE DESCRIPTION: SEQ ID NO: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

-39-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 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

-40-INFORMATION FOR SEQ ID NO:9:
SEQUENCE CHARACTERISTICS
LENGTH: 96 TYPE: amino acid MOLECULE TYPE: PRT
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO:9:

Met Ala Arg Ser Ser Ser Pro Leu Leu Leu Ser Leu Cys Ile Phe Ala Ile Leu Phe His Ser Ser Leu Gly Arg Gln Gln Phe Gln Gln Gly Asn Glu Cys Gln Ile Asp Arg Ile Asp Ala Ser Glu Pro Asp Lys Thr Ile Gln Ala Glu Ala Gly Glu Val Trp Asp Gln Asn Arg Gln Gln Phe Gln Cys Ala Gly Val Ala Val Val Arg Arg Thr Ile Glu Pro Lys Gly Leu Leu Leu Pro Phe Tyr Ser Asn Thr Pro Gln Leu Ile Tyr Ile Val Gln INFORMATION FOR SEQ ID NO:10:
SEQUENCE CHARACTERISTICS
LENGTH: 85 TYPE: amino acid MOLECULE TYPE: PRT
ORGANISM: Linum usitatissimum FEATURE
NAME/KEY: misc_feature LOCATION: (11)..(11) OTHER INFORMATION: Xaa is any amino acid SEQUENCE DESCRIPTION: SEQ ID NO:10:

Gly Arg Gly Val Thr Gly Ile Met Phe Pro Xaa Cys Pro Glu Thr Phe Glu Glu Ser Gln Gln Gln Gly Gln Gln Gly Gln Gln Gly Ser Ser Gln Asp Gln His Gln Lys Ile Arg Arg Phe Arg Glu Gly Asp Val Ile Ala Val Pro Ala Gly Val Ala His Trp Ser Tyr Asn Asp Gly Asn Glu Pro

-41-Val Met Ala Ile Val Val His Asp Thr Ser Ser His Leu Asn Gln Leu Asp Asn Asn Pro Arg INFORMATION FOR SEQ ID NO:11:
SEQUENCE CHARACTERISTICS
LENGTH: 165 TYPE: amino acid MOLECULE TYPE: PRT
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO:11:

Asn Phe Tyr Leu Ala Gly Asn Pro Arg Asp Glu Phe Glu Gln Ser Gln Gln Gly Gly Arg Leu Ser Arg Gly Glu Ser Glu Gly Gly Arg Gly Arg Arg Glu Pro Leu Gln Pro Ala Thr Thr Ser Ser Cys Gly Ile Asp Ser Lys Leu Ile Ala Glu Ala Phe Asn Val Asp Glu Asn Val Ala Arg Arg Leu Gln Ser Glu Asn Asp Asn Arg Gly Gln Ile Val Arg Val Glu Gly Glu Leu Asp Ile Val Arg Pro Pro Thr Ser Ile Gln Glu Glu Ser Gln Glu Gln Gly Gly Arg Gly Gly Gly Arg Tyr Tyr Ser Asn Gly Val Glu Glu Thr Phe Cys Ser Met Arg Leu Ile Glu Asn Ile Gly Asp Pro Ser Arg Ala Asp Ile Phe Thr Pro Glu Ala Gly Arg Val Arg Ser Leu Asn Ser His Asn Leu Pro Val Leu Gln Trp Ile Gln Leu Ser Ala Glu Arg Gly Val Leu Tyr Asn INFORMATION FOR SEQ ID NO:12:
SEQUENCE CHARACTERISTICS
LENGTH: 141 TYPE: amino acid

-42-MOLECULE TYPE: PRT
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO:12:

Glu Ala Ile Arg Leu Pro His Trp Asn Ile Asn Ala His Ser Ile Val Tyr Ala Ile Arg Gly Gln Ala Arg Val Gln Ile Val Asn Glu Glu Gly Asn Ser Val Phe Asp Gly Val Leu Gln Glu Gly Gln Val Val Thr Val Pro Gln Asn Phe Ala Val Val Lys Arg Ser Gln Ser Glu Arg Phe Glu Trp Val Ala Phe Lys Thr Asn Asp Asn Ala Met Val Asn Ser Leu Ala Gly Arg Thr Ser Ala Val Arg Ala Ile Pro Ala Asp Val Leu Ala Asn Ala Trp Arg Val Ser Pro Glu Glu Ala Arg Arg Val Lys Phe Asn Arg Gln Glu Thr His Leu Ala Ser Thr Arg Gly Gln Ser Arg Ser Pro Gly Arg Leu Asn Val Val Lys Glu Val Ile Asn Leu Leu Met INFORMATION FOR SEQ ID NO:13:
SEQUENCE CHARACTERISTICS
LENGTH: 11 TYPE: amino acid MOLECULE TYPE: PRT
ORGANISM: Linum usitatissimum SEQUENCE DESCRIPTION: SEQ ID NO:13:

Gln Gln Gln Gly Gln Gln Gln Gly Gln Gln Gln INFORMATION FOR SEQ ID NO:14:
SEQUENCE CHARACTERISTICS
LENGTH: 18 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (synthetic DNA primer) SEQUENCE DESCRIPTION: SEQ ID NO:14:

tccactatgt aggtcata 18

-43-INFORMATION FOR SEQ ID NO:15:
SEQUENCE CHARACTERISTICS
LENGTH: 18 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (synthetic DNA primer) SEQUENCE DESCRIPTION: SEQ ID NO:15:

ctttaaggtg tgagagtc 18 INFORMATION FOR SEQ ID NO:16:
SEQUENCE CHARACTERISTICS
LENGTH: 15 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (synthetic DNA primer) SEQUENCE DESCRIPTION: SEQ ID NO:16:

aggggtgatc gatta 15 INFORMATION FOR SEQ ID NO:17:
SEQUENCE CHARACTERISTICS
LENGTH: 18 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (synthetic DNA primer) SEQUENCE DESCRIPTION: SEQ ID NO:17:

gatagaaccc acacgagc 18 INFORMATION FOR SEQ ID NO:18:
SEQUENCE CHARACTERISTICS
LENGTH: 29 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (synthetic DNA primer) SEQUENCE DESCRIPTION: SEQ ID NO:18:

tatctagact caagcatacg gacaagggt 29 INFORMATION FOR SEQ ID NO:19:
SEQUENCE CHARACTERISTICS
LENGTH: 6 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (XbaI site) SEQUENCE DESCRIPTION: SEQ ID NO:19:

tctaga 6

-44-INFORMATION FOR SEQ ID NO:20:
SEQUENCE CHARACTERISTICS
LENGTH: 21 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (synthetic DNA primer) SEQUENCE DESCRIPTION: SEQ ID NO:20:

ggttatcatt gtatgaactg a 21 INFORMATION FOR SEQ ID NO:21:
SEQUENCE CHARACTERISTICS
LENGTH: 6 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (NcoI site) SEQUENCE DESCRIPTION: SEQ ID NO:21:

ccatgg 6 INFORMATION FOR SEQ ID NO:22:
SEQUENCE CHARACTERISTICS
LENGTH: 32 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (synthetic DNA primer) SEQUENCE DESCRIPTION: SEQ ID NO:22:

gcaagcttaa tgtgacggtg aaataataac gg 32 INFORMATION FOR SEQ ID NO:23:
SEQUENCE CHARACTERISTICS
LENGTH: 6 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (HindIII site) SEQUENCE DESCRIPTION: SEQ ID NO:23:

aagctt 6 INFORMATION FOR SEQ ID NO:24:
SEQUENCE CHARACTERISTICS
LENGTH: 29 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (synthetic DNA primer) SEQUENCE DESCRIPTION: SEQ ID NO:24:

-45-taggtacctg gcaggtaaag actctgctc 29 INFORMATION FOR SEQ ID NO:25:
SEQUENCE CHARACTERISTICS
LENGTH: 6 TYPE: nucleic acid MOLECULE TYPE: artificial sequence (KpnI site) SEQUENCE DESCRIPTION: SEQ ID NO:25:

ggtacc 6

Claims (15)

WE CLAIM:

1. 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) said 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, wherein said flax seed-specific promoter is selected from the group of promoters consisting of oleosin promoters, 2S storage protein promoters and legumin-like seed-storage protein promoters.

2. The method according to claim 1 wherein at least one expression characteristic conferred by said seed-specific promoter to its native nucleic acid sequence is conferred to said non-native nucleic acid sequence.

3. The method according to claim 2 wherein said expression characteristic is timing of expression or level of expression.

4. The method according to any one of claims 1 to 3 wherein said flax seed-specific promoter comprises:
(a) a nucleic acid sequence comprising:
i) nucleotides 1 to 2023 as shown in SEQ ID NO: 1;
ii) nucleotides 1 to 1852 as shown in SEQ ID NO:4;
iii) nucleotides 1 to 417 as shown in SEQ ID NO:6; or iv) nucleotides 1 to 2035 as shown in SEQ ID NO:8, wherein T
can also be U;
(b) a nucleic acid sequence that hybridizes to the nucleic acid sequence of (a) under stringent hybridization conditions wherein said stringent hybridization conditions are 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 X SSX at 50°C;
(c) a nucleic acid sequence that is complementary to the nucleic acid sequence of (a); or (d) a nucleic acid sequence that has at least 65% sequence identity to the nucleic acid sequence of (a).

5. The method according to any one of claims 1 to 4 wherein expression of said nucleic acid sequence of interest results in an alteration in protein or fatty acid composition in said seed.

6. A transgenic flax cell 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, wherein said seed-specific promoter is a seed storage protein promoter, an oleosin promoter, a 2S storage protein promoter or a legumin-like seed-storage protein promoter.

7. The transgenic flax cell according to claim 6 wherein at least one expression characteristic conferred by said seed-specific promoter to its native nucleic acid sequence is conferred to said non-native nucleic acid sequence.

8. The transgenic flax cell according to claim 7 wherein said expression characteristic is timing of expression or level of expression.

9. Transgenic flax cell according to any one of claims 6 to 8 wherein said seed specific promoter comprises:
(a) a nucleic acid sequence comprising:

i) nucleotides 1 to 2023 as shown in SEQ ID NO: 1;
ii) nucleotides 1 to 1852 as shown in SEQ ID NO:4;
iii) nucleotides 1 to 417 as shown in SEQ ID NO:6; or iv) nucleotides 1 to 2035 as shown in SEQ ID NO:8, wherein T
can also be U;
(b) a nucleic acid sequence that hybridizes to the nucleic acid sequence of (a) under stringent hybridization conditions wherein said stringent hybridization conditions are 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 X SSX at 50°C;
(c) a nucleic acid sequence that is complementary to the nucleic acid sequence of (a); or (d) a nucleic acid sequence that has at least 65% sequence identity to the nucleic acid sequence of (a).

10. The transgenic flax cell according to any one of claims 6 to 9 wherein expression of said non-native gene of interest results in an alteration in the seed protein or fatty acid composition.

11. An isolated nucleic acid molecule capable of directing seed-specific expression in a plant comprising:

(a) a nucleic acid sequence comprising:
i) nucleotides 1 to 2023 as shown in SEQ ID NO: 1;
ii) nucleotides 1 to 1852 as shown in SEQ ID NO:4;
ii) nucleotides 1 to 417 as shown in SEQ ID NO:6; or iv) nucleotides I to 2035 as shown in SEQ ID NO:8, wherein T
can also be U;
(b) a nucleic acid sequence that hybridizes to the nucleic acid sequence of (a) under stringent hybridization conditions wherein said stringent hybridization conditions are 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 X SSX at 50°C;

(c) a nucleic acid sequence that is complementary to the nucleic acid sequence of (a); or (d) a nucleic acid sequence that has at least 65% sequence identity to the nucleic acid sequence of (a).

12. An isolated chimeric nucleic acid molecule comprising:
(a) a first nucleic acid sequence comprising a seed-specific promoter obtained from flax comprising:
(1) a nucleic acid sequence comprising:
i) nucleotides 1 to 2023 as shown in SEQ ID NO:1;
ii) nucleotides 1 to 1852 as shown in SEQ ID NO:4;
iii) nucleotides 1 to 417 as shown in SEQ ID NO:6; or iv) nucleotides 1 to 2035 as shown in SEQ ID NO:8, wherein T can also be U;
(2) a nucleic acid sequence that hybridizes to the nucleic acid sequence of (1) under stringent hybridization conditions wherein said stringent hybridization conditions are 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 X SSX at 50°C;
(3) a nucleic acid sequence that is complementary to the nucleic acid sequence of (1); or (4) a nucleic acid sequence that has at least 65% sequence identity to the nucleic acid sequence of (1); and (b) a second nucleic acid sequence non-native to said flax seed-specific promoter.

13. A method for the expression of a nucleic acid sequence of interest in a plant seed comprising:
(a) introducing the chimeric nucleic acid sequence according to claim 12 into a plant cell; and (b) growing said plant cell into a mature plant capable of setting seed wherein the second nucleic acid sequence is expressed in the seed under the control of the seed specific promoter.

14. A method according to claim 13 wherein said plant cell is selected from the group of plants consisting of 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 (Cocus nucifera), castor (Ricinus communis), coriander (Coriandrum sativum), squash (Cucurbita maxima), jojoba (Simmondsia chinensis) and rice (Oryza sativa).

15. A plant cell prepared according to the method of claim 13 or 14.