CA2268745A1 - Flax promoters for manipulating gene expression - Google Patents

Abstract

The invention relates to promoter sequences obtained from flax and useful for modification of flax and other plants for expression of endogenous or foreign genes. The promoters are the SEQ ID NO. 3 and SEQ ID NO. 4 and are obtained from newly illucidated structures of two SAD genes in flax, namely SEQ ID NO. 1 and SEQ ID NO. 2. The promoters have been inserted into cloning plasmids and deposited at the American Type Culture Collection as plasmids pCDC220 and pCDC214 under deposit numbers ATCC 98192 and ATCC 98193, respectively. The promoters may be used in conjunction with genes to modify characteristics of flax and other plants. The invention includes the SAD genes themselves and DNA sequences substantially homologous to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4, as well as significant parts thereof.

Description

FLAX PROMOTERS FOR MANIPULATING GENE EXPRESSION
TECHNICAL FIELD
This invention relates to gene promoters useful for the genetic manipulation of plants. More particularly, the invention relates to gene promoters isolated from flax useful, for example, for manipulating the expression of indigenous genes or transgenes in flax and other plants to modify l0 endogenous characteristics or to introduce new ones.
BACKGROUND ART
Flax (hinum usitatissimum) is the second most important oilseed crop in Canada and an important crop worldwide. Unfortunately, the use of flax seed oil is limited by the narrow range of natural fatty acids present in it. Therefore, there is a need to create new cultivars with a wider range of fatty acid composition to supplement the existing food and confections markets (Rowland et al., 1995 - please refer to the "References" section below for full reference identification details). Also, there is a commercial interest in using flax as a vehicle for biofarming of pharmaceutical-related products by molecular genetic manipulation of appropriate transgenes (Moloney and van Rooijen, 1996). A need for flax varieties tolerant to various abiotic and biotic stresses has also been recognized (Rowland et al., 1995). For example, herbicide-tolerant flax varieties would be very useful in crop rotation programs. There is always, of course, a need for promoters useful for expressing foreign genes in various other plants.
Molecular genetic manipulation of flax seed composition or other characteristics, such as stress tolerance, can be achieved by expressing appropriate transgenes using seed-specific or constitutive gene promoters. While a cDNA sequence corresponding to a flax gene has been reported (Singh et al., 1994), no promoter has yet been characterized from flax. There is, therefore, a need to identify and isolate one or more genes and promoters from flax to facilitate genetic manipulation of the flax plant and other plants.
DISCLOSURE OF INVENTION
An object of the invention is to identify and isolate one or more genes and promoter sequences from flax and to utilize such sequences in the genetic manipulation of plants.
Another object of the invention is to provide a vector containing a promoter sequence from flax for introducing an indigenous gene or a transgene into flax or other plants.
Another object of the invention is to provide a method of modifiying flax and other plants to change characteristics thereof.
Stated in general terms, the present invention is based on the isolation, purification and characterization by the inventors of the present invention of two genes from flax and two promoters from those genes. The sequences obtained are used for regulating the expression of a heterologous gene (foreign, reporter or transgene) in flax and other plant species. This can result in flax plants having different range of fatty acids than natural flax and can result in the development of transgenic plants suitable for the production of specific products or having new and useful characteristics. Such plants and products are of commercial and industrial interest.

According to one aspect of the present invention, there is provided isolated and purified deoxyribonucleic acid of SEQ ID N0:1 or SEQ ID N0:2.
These sequences relate to the novel flax genes isolated and characterized by the inventors of the present invention.
These identified and isolated genes are useful in themselves for making antisense or sense constructs based on the derived sequences. Both types of contruct l0 can be used to reduce the levels of similar mRNA during expression of the natural genes. This would result in an increase in 18:0 fatty acid in membrane or storage lipids in flax and other plant species. Sense constructs may also be used in enhancing the levels of mRNA. Such enhancement will result in the increase of 16:1 or 18:1 fatty acids in membranes or storage lipids in.flax and other plant species. Such plants will be of increased commercial interest and value.
Thus, according to another aspect of the invention, there is provided a method of changing fatty acids of membrane and storage lipids of plants, characterized by making an antisense or sense construct based on SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3 or SEQ
ID N0:4, ligating the constuct into a plant transformation vector, using the vector to transform the genome of a plant or plant seed, and then growing the plant or plant seed and extracting membrane or storage lipids from the plants.
. According to another aspect of the invention, there is provided isolated and purified deoxyribonucleic acid of SEQ ID N0:3 or SEQ ID N0:4 (deposited as plasmids ATCC 98193 and 98192, respectively, see details below). These are the promoters that are useful for enhancing or enabling the expression of genes introduced into flax or other plants.
According to another aspect of the invention, there is provided a gene expression cassette comprising a sequence according to SEQ ID N0:1, SEQ ID N0:2, SEQ
ID N0:3 or SEQ ID N0:4. The gene expression cassette is useful in itself as this part of the plasmids mentioned above can be used to construct other plasmid suitable to transform other plant species.
l0 According to yet another aspect of the invention, there is provided a vector for introduction of a gene into a plant cell, the vector comprising a promotor of SEQ ID N0:3 or SEQ ID N0:4.
The invention also relates to transgenic plants and plant seeds having a genome containing an introduced promoter sequence of SEQ ID N0:3 or SEQ ID
N0:4 regulating the expression of an introduced gene, and a method of producing such plants and plant seeds.
The invention also relates to substantially homologous DNA sequences (e.g. greater than or equal to 40o homology, more preferably greater than or equal to 70o homology) isolated and/or characterized by known methods using the sequence information of SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3 or SEQ ID N0:4, and to parts of reduced length of promoter sequences SEQ ID N0:3 or SEQ ID N0:4 that are still able to function as promoters of gene expression. It will be appreciated by persons skilled in the art that small changes in the identities of nucleotides in a specific promoter sequence may result in reduced or enhanced effectiveness of the promoters and that partial promoter sequences often work as effectively as the full length versions. The ways in which promoter sequences can be varied or shortened are well known to persons skilled in the art, as are ways of testing the effectiveness of promoters. All such variations of the promoters are therefore claimed as part of the present invention.

5 It should be noted that.the term "promoter" in this disclosure includes the core promoter elements (TATA box and initiator) and upstream regulatory elements (enhancers)(Datla et al., 1997).
As will be appreciated from the description above, the promoters of the invention are beneficial in manipulating the expression of genes in flax and other crops.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows genomic DNA sequence of the SAD1 [SEQ
ID N0:1; identified in Fig. 1 as LUSAD1.SEQ] and SAD2 [SEQ ID N0:2; identified in Fig. 1 as LUSAD2.SEQ] genes and the corresponding SAD cDNA sequence [SEQ ID N0:5;
identified in Fig. 1 as LUCDNA]. Nucleotides (nt) are represented by capital letters. Nucleotides different from the cDNA sequence are shaded, including those of introns. Differences between SAD1 and SAD2 are shown in shaded lower case letters. Gaps in the sequences are presented by dashes. The start and stop codons on the cDNA sequence are boxed.
Fig. 2A is a partial restriction map of the SADl gene, and Fig. 2B shows the result of a DNA blot analysis identifying the regulatory sequences of SAD1 and SAD2.
Fig. 3 shows an outline of the scheme employed to isolate the promoter regions of the two SAD genes.
Position and direction of the primers used in IPCR are indicated by arrowheads. Various abbreviations are as follows: E, exon; I, Intron; RE, 5'- regulatory elements (promoters); and UT, untranslated regions.

Fig. 4 discloses nucleotide sequences [SEQ ID N0:3 (SAD1) and SEQ ID N0:4 (SAD2)] of the 5'- regulatory regions of the two SAD genes. Homologous nt are represented by a dash (-), gaps by a dot (.), and additions by lower case letters. A putative transcriptional site is indicated by +l, and a TATA box is overlined. Key restriction sites are also shown.
Fig. 5 shows salient features of the plasmids CDC214 and pCDC220. Various abbreviations are as follows: flax promoter I, SAD1 gene promoter; flax promoter II, SAD2 gene promoter; GUS (uidA), gene for (3-glucuronidase enzyme; nos-T, transcriptional terminator of the nopaline synthase gene; nptll, neomycin phospho-transferase expression cassette. The arrowheads indicate the direction of transcription.
Key restriction sites are shown. Regions outside the left and right border (LB and RB) are that of a previously described binary plant transformation vector, pRD410 (Datla et al., 1992).
Fig. 6 shows the expression of a heterologous gene (uidA) by the two SAD gene promoters in various tissues of flax. Different tissues are abbreviated as YL+A, young leaves and apices; ML, mature leaves: S, stems;
R, roots; B, buds; 1/2 OF, half open flower; F1, Flower; and MS, seeds at about mid-development. Data presented are from one generation of two plants transformed with a tandem 35s promoter (2x35s), two generations of two plants transformed with pCDC214 (SAD1), and one generation of two plants transformed with pCDC220(SAD2).
Fig. 7 shows the expression of a heterologous gene(uidA) by the two SAD gene promoters during flax seed development and in relation to,fatty acid and protein biosyntheses. For GUS assays, data represent one generation of two plants transformed with a tandem 35s promoter (2x35s), two generations of two plants transformed with pCDC214(SAD1), and two generations of a plant transformed with pCDC220 (SAD2). For fatty acids, three individual embryos of var. McGregor were analyzed. For protein content, data are from two transgenic plants transformed with pCDC214 and 220.
Fig. 8 shows the expression of a heterologous gene(uidA) by the two SAD gene promoters in tobacco leaves and mid-developmental seeds. Data represent 5 to 8 transgenic plants transformed with pCDC214 (SADl), pCDC220 (SAD2), pRD410 (35s), and pRD420 (uidA alone).
Fig. 9 shows the expression of a heterologous gene(uidA) by the two SAD gene promoters during tobacco seed development. Various developmental stages of tobacco seeds were identified according to de Silva et al. (1992) and are abbreviated as W, white; LB, light brown; B, brown; DB, dark brown; and M, mature. Data represent 5 to 8 transgenic plants transformed with pCDC214 (SADl), pCDC220 (SAD2), pRD410 (35s), and pRD420 (uidA alone).
Fig. 10 shows the expression of a heterologous gene(uidA) by the two SAD gene promoters in canola leaves and mature seeds. Data represent 2 to 5 plants transformed with pCDC214 (SADl), pCDC220 (SAD2), pRD410 (35s), and untransformed plants (UT).
BEST MODES FOR CARRYING OUT THE INVENTION
In flax, endogenous SAD activity can be detected from about 10 days after pollination (dap) to seed maturity, suggesting a promoter of this gene would be useful in manipulating gene expression during seed development. Moreover, SAD has been found to be the key enzyme in manipulating the levels of saturated fatty acids in rapeseed and soybean triacylglycerols (Knutzon et al., 1992; see Topfer et al., 1995).
During studies carried out by the inventors aimed at diversifying flax as a crop, it was discovered that there are two SAD genes in flax. The isolation, purification and characterization of these genes and their promoters is disclosed below, as well as the expression capabilities of the promoters in flax and other plant species.
The promoters developed according to the present invention can be used to modify an endogenous characteristic of flax or another plant species, or to to add a new characteristic. An example of a modification of an endogenous characteristic of flax is, for example, the alteration of levels of different types of fatty acids in the seed oils. The introduction of a new characteristic is, for example, the production of a thermoplastic polymer in plants that normally do not produce thermoplastics. While it is normally easy to detect added characteristics, it is sometimes difficult to detect altered characteristics because of natural variation of characteristics in plants. The alterations can, however, be detected by comparing the average characteristics of a statistically significant number of the plants under examination with a statistically significant number of genomically-unmodified plants of the same genotype, grown under identical environmental conditions at the same time. If there is an appreciable difference in the measured characteristic, then it can be said that there has been an alteration of that characteristic and that the alteration is a result of the genomic-modification.
In the case of an added characteristic, again the comparison can be made with genomically-unmodified plants of the same genotype, again grown under identical environmental conditions at the same time.
The promoters of the present invention belong to a two-member gene family encoding the enzyme D9 desaturase (Stearoyl-acyl carrier protein desaturase;
SAD; EC 1.14.99.6). Stearoyl-acyl carrier protein desaturase is the first enzyme in the fatty acid desaturation pathway, and it catalyzes the conversion of stearoyl-ACP(18:0-ACP) to oleoyl-ACP(18:1~9-ACP).
The promoters were isolated using the inverse polymerase chain reaction (IPCR) technique. They are capable of expressing a foreign gene, e.g. uidA (which encodes(3-glucuronidase: GUS), in various tissues with high level of expression in seeds.
In developing seeds, both promoters showed a similar temporal expression pattern for uidA (measured as GUS activity). The GUS activity could be detected as early as 4 dap in developing seeds and in desiccated seeds (~50 dap) of transgenic flax. In developing seeds, the ability of the promoters to effect uidA gene expression correlated well with both fatty acid and protein biosyntheses and the maximum activity of GUS
preceded the maximal accumulation of fatty acids and proteins.
The promoters of the invention are useful in manipulating transgene expression in a variety of tissues including seeds. Some of the products which are possible using these promoters include, but are not limited to, the following: plants with enhanced herbicide, pest, pathogen, and stress resistance;
plants containing oil, protein, and carbohydrate of altered composition and content; plants with reduced anti-nutritional substances: plants producing pharmaceutical compounds such as antibodies, neuropeptides, recombinant proteins, and biodegradable thermoplastics (Bennett, 1993; Moloney and van Rooijen, 1996; Datla et al., 1997).
5 The effectiveness of the promoters of the present invention is predictable from the effectiveness of known promoters. For example, it is well established that promoters such as cauliflower mosaic virus (CaMV) are capable of expressing a wide variety of genes in a 10 wide varity of plant species. Napin promoter (from rapeseed) has been used to express a variety of genes in canola/rapeseed (Knutzon et al., 1992; Jones et al., 1995; Dahesh et al., 1996). Phaseolin gene promoter (from bean) has also been used to express several genes in rapeseed (Ritz et al., 1995). The (3-conglycinin promoter (from soyabean) has been used to express genes not only in soyabean but also in Petunia (Kinney, 1997;
Chen et al., 1986).
Moreover, by testing the promoters in two very diverse plant species, as will become apparent from the experimental detail below, the inventors have demonstrated that the promoters would function in other diverse plant species as well.
Further demonstration of this principle can be obtained from Chen ZL, Schuler MA, Beachy RN. 1986;
Dehesh K, Jones A, Knutzon DS, Voelker TA. 1996; Hitz WD, Mauvis CJ, Ripp KG, Reiter RJ, DeBonte L, Chen, Z.
1995; Jones A, Davies HM, Voelker TA. 1995; Kinney, AJ.
1997; and Knutzon et al., 1992.
It is believed that the present invention can now best be described by presenting experimental details forming a specific illustration. It should be kept in mind, however, that the present invention is not limited to these details.

EXPERIMENTAL DETAILS
Molecular Biological Techniques Isolation of plasmid DNA, restriction digestion, modification and ligation of DNA, PCR, gel electrophoresis, and transformation and culture of E.
coli strains were carried out according to standard procedures (Sambrook et al., 1989). Nucleotide sequencing was performed using double stranded plasmid DNA by the dideoxy chain termination method (Sanger et al . , 1977 ) using a Taq DYEDEOXYTM terminator cycle sequencing kit (available from Applied Biosystems) and an Applied Biosystems Model 370A Sequencer (available from Applied Biosystems). The oligodeoxy-ribonucleotides used in nucleotide sequencing, and PCR
techniques were synthesised using a phosphoramidate synthesis procedure in a Biosearch 8750 DNA synthesizer (New Brunswick Scientific Co.), and purified by HPLC-based protocols (Gait, 1984). IPCR was done according to Ochman et al. (1993) and Warner et al. (1993).
Plant DNA was extracted using the protocol of Dellaporta et al. (Dellaporta et al., 1983) except that RNA was removed by adding 100~,g of RNAase B (Sigma) followed by incubation at 65°C for 20 min. The DNA was extracted once with an equal volume of phenol: chloroform (1:1, v/v) and once with an equal volume of chloroform:isoamyl alcohol (24:1, v/v). Five ~g of DNA was digested with the appropriate restriction enzyme, fractionated on a 0.8% agarose gel, and pressure-blotted onto Hybond-NTM nylon membranes (Amersham) using the PosiBlotTM apparatus (Stratagene) after depurination, denaturation and neutralization of the DNA (Sambrook et al., 1989). The blotting solution contained 0.02 M NaOH and 1 M NH9-acetate. The DNA was immobilized on the membrane by baking the membranes at 80°C for 1 h.
A radioactive probe for identifying promoters was prepared by annealing 10 ng of oligo-29A and 30A (Table 1 below) and then filling in the ends using the Klenow fragment of DNA polymerase and random primer kit solutions (GIBCO BRL).
Table 1 l0 Nucleotide sequence of various oligonucleotides (OL) used OL-24 (-) 5'-GAAI3mATGCCATCAT-ACTCCAATCAT-3' [SEQ ID N0:6]
OL-25 (+) 5'-GAAIZOCCTTCAACAAC-AATGGCTCTC-3' [SEQ ID N0:7]
OL-29A (+) 5'-izoCCTTCAACAACAATGGCTCTCAAGC-TCAACCCAGTCACCACCTT-3' [SEQ ID N0:8]
OL-30A (-) 5'-is9GGAGAAGTTGTTGAGGGAGCGTGTT-GAAGGGAAGGTGGTGACTGGGTTGA-3' [SEQ ID N0:9]
OL-39 (-) 5'-zs3TTGGTGGAGGTGGAACTGAA-3' [SEQ ID N0:10]
OL-110 (+) 5'-zs3AGCTAAAGAAGTCACATGGAC-3' [SEQ ID NO:11]
NOTE: The number in subscript corresponds to the nucleotide residue in the SAD cDNA sequence (Singh et al., 1994). + and -indicate coding and non-coding strand.
The sequence of oligo-29A corresponded to nt 120-163 of SAD cDNA (reported by Singh et al., 1994). The sequence of oligo-30A corresponded to nt 145-194. In this way, radioactive probe fragments spanning 75 bps in the 5' end region of SAD cDNA were obtained.
Prehybridization was done at 65°C for 3 h in 5x SSPE, 5x Denhardts solution, 0.5o SDS, and 500~g of l3 Salmon sperm DNA (amersham) . Hybridizat,i on s~ras done at 55C for h. The membrane was washed at room temperaturein 2x SSPE and 0.1~ SDS for 15 a~d 5 min and then 50 C in lx SSPE and 0.1% for 10 min.
at SDS

At this point the membrane was free of backgrou~:d signal. Autoradiograms were obtained by expcsing t:e membranes for variable lengths of tire to Kodak X-OMATT'"' AR films wi th intensifying screens at -70°C.
Reporter Gene Constructs l0 A 1.747 kb DNA fragment containing only t::e regulatory region and a part of the untranslated region of the SAD1 gene ~,~as amplified by PCR and cloned into the pCRII vector (Invitrogen Corp). The same f=agr.:en~
was retrieved as an EccRI fragment from the pCR=I
i5 vector and subseauently cloned into pBluescrioW"' =_ (Stratagene) to gain some cloning sites. The __'_a-%a:' 5' - regulatory regi on, approximately 1 . 257 '.{a, ..~ _:~.e SAD2 gene was PCR-amplified but using the piv ~'~a polymerase (Stratagene) , and cloned into an cc?.'~ ==to 20 of the pBluescrip~. II SK vector.
The SAD1 and SAD2 gene 5' regulatory eleme.~.ts r~==a cloned into pRD420 as a SalI-Sc~aI fragment i:: -= J.. _ the uidA. The plasmid pRD420 was obtained f=o~
R.S.S. Datla, NRC Plant Biotechnology Instit~.:te, _=
25 Gymnasium Place, Saskatoon, Saskatchewan, Carat , ~,'i OW9 (Dada et al., 1992). The resulting const=v~~~
were labeled as pCDC214 and pCDC220. These con~_=v=~s were deposited on October 3, 1996 (tested for -%'_~t___ty on October 9, 1996, deposit receipt dated October -30 1996) under the terms of the Budapest Treaty at _.._ American Type Culture Collection, 10801 Univers_=~:
Blvd., Manassas VA 20110-2209, USA, under depos:= ~-:s.
ATCC 98193 and 98192, respectively. The plasmi~s ~a CDC214 and 220 were transferred directly to Agrobacterium strain GV3101 containing helper plasmid pMP90 (Koncz and Sc'nel 1 , 196) using a freeze-taw method of transformation (An et al., 1983).
Plant Transformation Flax seeds were surface sterilized by stirring in 70 o ethanol for ? minutes, followed by three i0 :,li.~.uta washes in 0.5=~ sodium hypochlorite (freshly uilute~
from the commercial product), and 5 rinses i:~ steri'_e l0 distilled wa~ar. Seeds were germinated on basal medium consisting of Murashige and Skoog (MS) major and minor salts and Gamborg vitamins (Sigma 0404), 3° sucrose and 0.8~ agar. '_'he per: of the medium was adjusted to 5.8 before autoclaving. About 10 surface-sterilizes seeds l5 were placed in each 100x15 mm plate. The p1_tes were sealed with paraf_lm and placed in the dark at 22°C =or 5 to 7 days.
Derivatives of Agrobacteria tumefaciens stra'_~.
GV3101/pMP90 carrning pCDC214 and pCDC220 were :rown c::
20 solidified 2x YT :~.edi um (Sambrook et a1. 1 989) supplemented vita. 50 ug/m1 kanamycin and 50 ug/~!' gentamycin sulfate. Single colonies trom ~ to .. aay-old culture plates were used to inoculate 10 m_ '__~wid 2x YT medium containing antibiotics as above an2~~ uM
?5 acetosyringone. Cul Lures were grown at 28°C wi=_~
rotary agitation nor about 24 hours. Prior to inoculation of flax tissues, the cell concentra=_o:, of the suspension was adjusted to 1x109 cells/ml.
The following methods for obtaining transfor:~ed .lax 30 callus were modified from Mlynarova et al. (19:s?.
Hypocotyls of 5-7 day aseptic flax seedlings were cut into segments 3-4 mm long. To avoid dehydratic::, _'.'.~.e segments were maintained in a small volume of l:~u_d basal medium until all the hypocotyls were cut. The hypocotyl segments were immersed in bacterial suspension (1x109 cells/ml) for 30 minutes with occasional swirling. The suspension was removed by 5 aspiration and the hypocotyl segments were transferred to sterile filter paper to remove excess liquid. The segments were placed on agar-solidified (0.80) basal medium supplemented with 4.44~,M 6-benzylaminopurine and 0.54~.M naphthaleneacetic acid (MSD4x2 medium;
l0 Basiran et al., 1987). Maltose (30) replaced sucrose as the carbohydrate source. About 25 explants were placed in each 100x15 mm Petri dish and maintained at 22-24°C, with a 16 h photoperiod and photon density of approximately 50 ~,mol/m2/s. After 2 days the segments 15 were transferred to the same medium supplemented with 100~g/ml kanamycin for selection of transformed cells and 200~,g/ml cefotaxime to eliminate Agrobacteria.
The explants were maintained under the same growth conditions for 3 weeks. As a control, non-inoculated segments were treated in the same way.
Green callus formed at the cut ends of most of the inoculated hypocotyl segments, whereas little or no callus appeared on non-inoculated segments and they were completely bleached after 3 weeks on the selection medium. Callus was excised and transferred to basal medium (3o maltose) supplemented with S~,M zeatin and antibiotics as above. Shoots regenerated from some of the calli within 3-4 weeks.
When the shoots had elongated to 0.5 to 1.0 cm, they were removed from the callus and placed in capped glass tubes (100x25 mm) containing 8 ml rooting medium: 1/2 strength MS salts, 3o sucrose, 0.1 ~,M IAA, 0.8o agar, pH 5.8, and 30~,g/ml kanamycin for selection of transformed shoots. The shoots were maintained under low light (<25~.mo1/m2/s) for 5-8 days by which time some of the shoots had roots about 2-3 mm long. The plantlets were transferred to pots in the growth chamber within 10-14 days, when roots had elongated to about 2 cm and the shoots were 3-5 cm tall. Transgenic plants were grown under 18 h of light (300-500 ~.mol/m2/s) and day/night temperature of 20/17°C. The plants were fertilized just before flowering with a solution containing 27 g of 15N:30P:15K supplemented with 0.9 g CuS04 in 9 liters of water.
Transformation of canola and tobacco were performed according to Moloney et al. (1989) and Horsch et al.
(1985), respectively.
Tissue Samplin Various tissues and developing seeds at different stage of development were harvested and immediately frozen in liquid N2 and stored at -80°C until analyzed.
In progeny generations, these tissues were combined from a total of 8 plants .
Fluorimetric GUS Enzyme Assay Fluorimetric GUS assay was done essentially according to Jefferson (1987). The assays were done in a micro well titer plate and fluorescence of the reactions was measured by CytoFluorTM II multi-well fluorescence plate reader (PerSeptive Biosystems}.
Determination of Fatty Acid and Protein Content in Seeds The fatty acid content of seeds of different ages was determined by fatty acid methyl ester analysis of seed homogenates as described previously (Taylor et al., 1992).
The same protein extracts which were used for GUS

assays were used far protein estimation. Protein concentration was determined using a modified Bradford assay method (Bio-Rad protein assay) and BSA as the standard.
RESULTS
Isolation and characterization of the two SAD genes The inventors of the present invention have found that three lines of evidence prove there are two SAD
genes in flax, namely: the amplification of two different sized DNA fragments by PCR, the results of restriction analysis of cloned PCR products, and the results of DNA blot analysis of flax genomic DNA.
The genomic sequences of the two SAD genes were amplified by PCR. Several oligonucleotide primers were synthesized based on the nucleotide sequence of the published SAD cDNA sequence (Singh et al., 1994).
These primers were used in all possible combinations with flax genomic DNA as the template to amplify different segments of SAD genes. The molecular size of the PCR products was determined by agarose gel electrophoresis: in most reactions two products of very similar molecular size were detected, suggesting the possibility of two SAD genes in flax. Amplification with oligo-25 and 24 (Table 1) yielded a fragment of about 2.6 kb. This fragment contained the whole SAD
gene as determined by sequence data.
The amplified SAD gene fragments were cloned into pCRII vector (Invitrogen Corp.). The identity of the amplified gene products was confirmed by comparison of their nucleotide sequences with the SAD cDNA sequence (Singh et al., 1994). Sequence analyses indicated that the SAD1 and SAD2 genes have 97.2% similarity with each other in the coding region and 96.2% and 93.7% with the published flax cDNA sequence, respectively (Fig. 1).

It is clear that the mRNA for SAD cDNA, reported by Singh et al. (1994), was transcribed from the SAD1 gene. Some general features of the flax SAD genes have been deduced from sequence analysis. As expected on the basis of the cDNA sequence, the coding region of the gene is 1192 bps. This consists of three exons interrupted by two introns of approximately 0.6 to 0.7 kb. Exon 1 consists of 123 bp, whereas exons 2 and 3 are 507 by and 561 by long, respectively.
Verification for the presence of two SAD genes in flax comes from the analyses of two independent clones, each containing the full length gene. Although the nucleotide sequences of the coding regions are almost identical, there are several base changes. One of these has altered a restriction enzyme site, NcoI, resulting in the observation that the two clones have different restriction digestion patterns. The two clones also differ significantly in their intron sequences (Fig. 1). The different intron sequences are presumably responsible for the slight difference in the molecular size of the two PCR products generated by the same primer combination.
Identification of SAD Gene Promoter Sequences in Flax Genome Genomic DNA was extracted from 7-10 days old seedlings of flax var. McGregor (obtained from Dr. G.
Rowland, Crop Development Centre, 51 Campus Dr., Saskatoon, Saskatchewan S7N 5A8), digested with restriction enzyme, BamHI, BclI, BglII, NdeI or SstI, gel-fractionated and blotted onto nylon membrane for probing. These restriction enzymes would cut within the flax SAD genomic sequence as indicated in Figure 2A
and elsewhere in the flax genome. When the DNA blot was hybridized with the probe, DNA fragments containing the 5'- upstream region and a part of the 5'-untranslated and coding region of the SAD gene were expected to hybridize (Figure 2A).
The result of one such experiment is shown in Figure 2B. In each lane, two different size fragments hybridized with the probe indicating the existence of two SAD genes in flax. Singh et al. (1994) have shown only one SAD gene in flax. Since both the genes might be active, the inventors decided to isolate the 5' regulatory DNA sequences of both SAD genes.
Isolation and Characterization of Promoter Elements 5'- regulatory DNA sequences of the two SAD genes were amplified using the IPCR technique.
DNA blot analysis of the flax genome indicated that the two fragments obtained from the digestion of flax DNA with the restriction enzyme SstI would contain about 1.7 and 1.2 kb of 5' flanking regions of the SADl and SAD2 gene, respectively (Figs. 2B, 3 and 4). These fragments are expected to contain sufficient 5'-regulatory elements required for gene expression. SstI
was used to cut the flax genomic DNA, and the circularized DNA template required for IPCR was prepared. An outline of the promoter isolation scheme is shown in Fig. 3, and is believed to be self-explanatory.
Flax genomic DNA was digested with the restriction enzyme SstI and gel fractionated. DNA fragments were isolated from a region of the agarose gel where the two promoter fragments that hybridized with the SAD probe were expected (Fig. 2B and 3). These DNA fragments were ligated at a concentration favoring the circularization of single DNA molecules (Ochman et al., 1993; Warner et al., 1993). The circularized DNA was then used as a template in the IPCR with two primers (oligo-39 and oligo 110; Table 1). The orientation of the each member of the primer set used in the IPCR is opposite to that normally used in a regular PCR (Fig.
3). Two distinct fragments of the expected sizes, 2.2 5 kb and 1.7 kb, were amplified using IPCR. The untranslated region and parts of the exon 1 and exon 2 constituted the additional approximately 0.5 kb (Fig.3). The two fragments could also be digested with SstI indicating the authenticity of the PCR product.
10 The two DNA fragments were cloned in the pCRII
vector (Invitrogen Corp.) and sequenced. The DNA
sequence of the 5'- regulatory regions of the two SAD
genes was compiled and compared (Fig. 4). The two SAD
promoters are quite homologous. A large deletion of 15 368 by in the SAD2 gene promoter (corresponding to nt 759 to 391 in the SAD1 promoter) is very conspicuous.
There are a few short deletions, some substitutions and minor gaps in both the promoters. Based on the sequence data, 3'- regions of these DNA fragments were 20 matched with the 5'- coding regions of the two SAD
genes, and thereby assigned the promoters to their respective SAD genes.
Expression of the~3-qlucuronidase Gene by Flax Promoters in Transgenic Plants The ability of a promoter to regulate expression of a gene spatially and temporally can be demonstrated by using it to express a heterologus gene. To achieve this here, first, reporter gene constructs were made by fusing the promoter of the SAD1 or SAD2 gene with the uidA gene (Fig. 5). These expression constructs were then used to transform flax, canola and tobacco, and independent transgenic plants of these species were obtained.

Different tissues were sampled and assayed for GUS
activity to determine spatial or tissue-specific expression. Developing seeds were also collected at various stages of development to analyse the temporal expression pattern of the two promoters during seed development.
These promoters were capable of expressing the uidA
gene in various tissues, with high level of expression in seeds (Fig. 6). In developing seeds, both the l0 promoters showed similar temporal expression patterns for GUS (Fig. 7). The GUS activity could be detected as early as 4 dap in developing seeds and in desiccated seeds (approximately 50 dap) of transgenic flax with higher activities around mid-development (14 to 28 dap ) .
In tobacco, GUS activity in leaf was insignificant with both the promoters whereas in seeds GUS activity could be detected easily (Fig. 8). In developing tobacco seeds, GUS activity was highest at about mid-development (Fig. 9). In canola, GUS activity could be detected easily in both leaves and seeds (Fig. 10).
Utility of the Flax Promoters in Regulating Gene Expression The utility of the flax promoters disclosed here is demonstrated by comparing their effect on uidA gene expression with both lipid and protein biosynthesis in developing flax seeds. In developing seeds, uidA
expression correlated well with both fatty acid and protein biosynthesis (Fig. 7). Tn seeds, maximum expression of the uidA gene controlled by the SAD gene promoters preceded the maximum accumulation of fatty acids and proteins. Also, in tobacco the temporal pattern of uidA gene expression correlated well with the lipid biosynthesis (de Silva et al., 1992).

Therefore, these promoters are useful in manipulating gene expression in seeds. Since these promoters are also active in other tissues they are useful in manipulating gene expression in a variety of tissues.
Utility of SAD Genes The utility of the genes can be demonstrated by carrying out the following predictive experiments (similar experiments have been reported in Knutzen et al., 1992; Topfer et al., 1995). Firstly, antisense or sense constructs are made using the disclosed or other promoters. For example, these genes or their parts can be ligated into a SmaI restriction site of pCDC 214 or 220 (Fig. 5) or any other convenient cloning site of another plant transformation vector. These recombinant plasmids can then be mobilized, for example, into an Agrobacterium strain which can then be used to transform a variety of plant species. Any changes in fatty acids of membrane and storage lipids can be evaluated by routine methods described in this application.
Both type of constructs are expected to reduce the levels of similar mRNA during expression of the natural genes resulting in an increase of 18:0 fatty acid in membrane or storage lipids. Sense constructs can also be used in enhancing the levels of mRNA. Such enhancement will likely result in the increase of 16:1 or 18:1 fatty acids in membranes or storage lipids of plants. Such plants will be of increased commercial interest and value.
It will be appreciated by persons skilled in the art that various modifications and alterations may be made to the present invention without departing from the general scope of the invention as defined by the following claims. All such variations and modifications should be considered part of this invention.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Ravinder Kumar Jain (B) STREET: 2913 Irvine Avenue (C) CITY: Saskatoon (D) STATE: Saskatchewan (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): S7J 2A9 (A) NAME: Roberta Gail Thompson (B) STREET: 117 Capilano Court (C) CITY: Saskatoon (D) STATE: Saskatchewan (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): S7K 4B9 (A) NAME: David Charles Taylor (B) STREET: 622 Wollaston Bay (C) CITY: Saskatoon (D) STATE: Saskatchewan (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): S7J 9C3 (A) NAME: Gordon Grant Rowland (B) STREET: 213 Lake Crescent (C) CITY: Saskatoon (D) STATE: Saskatchewan (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): S7H 3A1 (A) NAME: Alan Gordon McHUghen (B) STREET: 35 Cathedral Bluffs Road (C) CITY: Saskatoon (D) STATE: Saskatchewan (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): S7P lAl (A) NAME: Samuel Leonard MacKenzie (B) STREET: 17 Cambridge Crescent (C) CITY: Saskatoon (D) STATE: Saskatchewan (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): S7H 3P9 (ii) TITLE OF INVENTION: Flax Promoters For Manipulating Gene Expression (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/029,916 (B) FILING DATE: 30-OCT-1996 (2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2701 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum (B) STRAIN: McGregor (xi) SEQUENCE DESCRIPTION:
SEQ ID N0: 1:

TTTTTCCAAT

CTCAGCCAGC

GCTCAACCCA

TCCTCGCACC

TCCTCCTCCT

GGTTGCTTAC

TTATGTCATT

AATCAATTCC TTTGTGTTTCGTTTTGCGCT GTAATTGATT~GTCAGTGTTT980 ACGATTCTGG

TTTCCTGTAG

CCTCATTCCA

ACCATGGGGA

ACGAAACCGC

TGCTGGCACT

ATGGAGAAGA

ATACTGCCAA

CTTTTACGGG

CGAGGATTCG

TCCCGTTCAG

AGAGTGTGGA

T

(2) INFORMATION FOR SEQ ID
NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2705 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: l.lnear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Linum usitatissimum (B) STRAIN: Mc6regor (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:

CACCTTCCCT TCGACCCGCT

CATGGCTGCT TCCACTTTCA

TTCCTCGGAA TCTCCGCCGT

TAGCGTTCAT TCAAATCTGC

CATTATGTTA TTGTTCGTTT

ACGAGTCTGG ATTTTGCGCT

ACCTCCGTCC ATCAAATGCA

TCTGTCTCTG TATCTACTAT

TGAACCAAGT GGCTTACAAT

CTCAAAAGAG GTTTTTCTAT

TCCTGGGAAG AATGATTAAT

TGATAATTCA ATATAGGGGT

AAAGAAGTCA CATGGACCAC

ACAGAAGCTG GAGATCTTTA

CCTGAAGCCG GTTGAGAAAT

TGGGTTCGAG GAGCAAGTGA

TTTTGTTGTG CTGGTTGGGG

GCTCAACACC CTTGACGGGG

CTGGACAAGG GCGTGGACCG

TTACCTCTCT GGAAGGGTGG

CTCTGGAATG GTATATACTC

ATTGAACAAA ATTCAATAAA

TCTATGAGAA AACCACTGAA

CATATACCAT CTTCCATCTA

CCGTGGATAA TCATATTATT

AACAATACAT GTGATGTCGT

GGTTCGGGAC

ATCACTTTAT

TCCTGGTATT

GACCAAGTAT

TATCGTCTGG

AGAAGAAGTT

TCTTTTTGCC

CTACACCCCA

CAAGGACCAC

GCGGCACGAA

TACAGTGTTG

GTACGATGGA

GGTGTATACT

GGATGCTTTT

TCCAGCGAGG

ATCTGTCCCA

GAGTTGAGTG

GGCAT

(2) INFORMATION FOR SEQ ID
NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1693 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum (B) STRAIN: McGregor (xi) SEQUENCE
DESCRIPTION:
SEQ
ID NO:
3:

CTGTAACGTT

TGTTGGAGAA

CGGTGGCTCG

CGCAGGCCCC

ATGCCGTAGT

ATCGACCTGT

AGGATCAGGT

TATTTGTTGT

AACGCGATCA

TTCGGTTAAC

TATCAAGAAA

GATTTCGAGT

GCTCTTTGAT

TCAATGGCGA

AATGTCTTCT

TATATAGCTT

TGGCTACAAG

TGGTGACTCG

GTGTTTATTT

AAAGACATCT

AATGAAAAAA

AACATATTTA

AAAGCACATA GCAAAACATA

AAACCATGAA GCCCAAGTGA

CCGCTACCGG GTTAGGACAT

CAGTTAATGG AACAAATGAA

GAAAAAGTGA CCATTTAGAC

TAAACCGAAT GCAGGCGTTA

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1191 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Linum usitatissimum (B) STRAIN: McGregor (xi) SEQUENCE
DESCRIPTION:
SEQ ID NO:
4:

TGTAGTAACA

AACTGTTTCT

CCTGAAAAGC

CACAGAGCTG

AGTGGCGGTG

GATTGATCCA

ATGACAATGT TCAATCTCTGCAGATTTTTG TTAGCAGCAG~GTCATGATTC920 AACATCAATG

(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1371 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv} ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

AATGGCTCTC
AAGCTCAACC
CAGTCACCAC

CCTTTCTCAT

AAGTCACATG

GAAGCTGGGA

ACCTGAAGCC

ATGGGTTCGA

ATTTTGTTGT

TGCTCAACAC

TCTGGACAAG

TATACCTCTC

GCTCTGGAAT

TCCAAGAGAG

GGGACATGAA

CCGCATACAC

CACTGGCGGA

AAGACGACAA

AGGATTATGC

GGCTTTCCGG

GAAAGTTGGA

GCTGGATCTT

ATGATTGGAG

(2) INFORMATION
FOR
SEQ
ID
NO:
6:

(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE
TYPE:
cDNA

(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:

(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CCTTCAACAA CAATGGCTCT CAAGCTCAAC CCAGTCACCA CCTT gq (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

REFERENCES OF INTEREST TO THE PRESENT INVENTION
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2.Basiran N., Armitage, P., Scott, R. J., and Draper, J. 1987. Genetic transformation of flax (Linum usitatissimum) by Agrobacteria tumefaciens .
regeneration of transformed shoots via a callus phase. Plant Cell Reports 6:396-399.
3.Bennett, J. 1993. Genes for crop improvement. In Genetic Engineering, Vol. 15. J. K. Setlow (ed.).
Plenum Press, NY. Pp.165-189.
4.Chen ZL, Schuler MA, Beachy RN. 1986. Functional analysis of regulatory elements in a plant embryo-specific gene. Proc Natl Acad Sci U S A 1986 83:85~0-8564.
5.Datla, R.S.S., Hammerlindl, J.K., Panchuck, B., Pelcher, L.E., and Keller, W. 1992. Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 211:383-384.
6.Datla, R., Anderson, J. W., and Selvaraj, G. 1997.
Plant promoters for transgene expression. Biotech.
Ann. Rev. (In press).
7.Dehesh K, Jones A, Knutzon DS, Voelker TA. 1990.
Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana. Plant J
9:167-172.
8. Dellaporta, S.L., Wood, J., and Hicks J.B. 1983. A
plant DNA mini preparation: version II. Plant Mol.
Biol. Rep. 1:19-21.
9.de Silva, J., Robinson, S. J., and Safford, R. 1992.
The isolation and functional characterization of a B.
napus acyl carrier protein 5' flanking region involved in the regulation of seed storage lipid synthesis. Plant Mol. Biol. 18:1163-1172.
lO.Gait, M. J. 1989. Oligonucleotide,Synthesis-A
Practical Approach. IRL Press, Oxford.

ll.Hitz WD, Mauvis CJ, Ripp KG, Reiter RJ, DeBonte L, Chen, Z. 1995. The use of cloned rapeseed genes for cytoplasmic fatty acid desaturases and the plastid acyl-ACP thioesterase to alter relative levels of polyunsaturated and saturated fatty acids in rapeseed oil. Proc. 9th International Cambridge Rapeseed Congress, UK. Pp 47D-472.
12. Horsch, R. B. , Fry, J. E. , Hoffmann, N. L. , Eichholtz, D., Rogers S. G., and Fraley R. T. 1985. A
simple and general method for transferring genes into plants. Science 227:1229-1231.
13.Jefferson, R. A. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol.
Rep. 5:387-405.
l4.Jones A, Davies HM, Voelker TA. 1995. Palmitoyl-aryl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.
Plant Cell 7:359-371.
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Lem(Eds.), Physiology, Biochemistry, and Molecular Biology of Plant Lipids, Kluwer Academic Publ. Pp 298-300.
l6.Knutzon, D. S., Thompson, G. A., Radke, S. E., Johnson, W. B., Knauf, V. C., and Kridl, J. C. 1992.
Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene. Proc. Natl. Acad. Sci. USA. 89:2624-2628.
l7.Koncz, C., and Schell, J. 1986. The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet.
204:383-396.
l8.Mlynarova, L., Bauer, M., Nap, J.-P., and Pretova, A. 1994. High efficiency Agrobacterium-mediated gene transfer to flax. Plant Cell Reports 13:282-285.
l9.Moloney, M. M. and van Rooijen, G. J. H. (1996) Recombinant proteins via oleosin partitioning. Inform 7:107-113.

20.Moloney, M. M., Walker, J. M., and Sharma K. K.
1989. High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep.
8:238-242.
2l.Ochman, H., Ayala, F. J., and Hartl, D. L. 1993. Use of polymerase chain reaction to amplify segments outside boundaries of known sequences. Meth. Enzymol.
218:309-321.
22.Rowland, G. G., McHughen, A., Gusta, L. V., Bhatty, R. S., MacKenzie, S. L., and Taylor, D. C. 1995. The application of chemical mutagenesis and biotechnology to the modification of linseed. Euphytica 85:317-321.
23.Sanger, F., Nickler, S., and Coulson, A. R. 1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA. 74:5463-5467.
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Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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Sequence of a cDNA from Linum usitatissimum encoding the stearoyl-aryl carrier protein desaturase. Plant Physiol. 104:1075.
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Claims (17)

CLAIMS:

1. Isolated and purified deoxyribonucleic acid (DNA), characterized in that said DNA includes a sequence according to SEQ ID NO:1 or SEQ ID NO:2, or a sequence that has greater than or equal to 70% homology thereto.

2. Isolated and purified deoxyribonucleic acid (DNA), characterized in that said DNA includes a sequence according to SEQ ID NO:3 or SEQ ID NO:4, or a sequence that has greater than or equal to 70% homology thereto, or a that is a part of SEQ ID NO:3 or SEQ ID NO:4 and that contains any regulatory elements thereof.

3. A vector for introducing at least one gene into plant cells, characterized in that said vector contains a promoter having a sequence according to SEQ ID NO:3 or SEQ ID NO:4, or a sequence that has greater than or equal to 70% homology thereto, or a sequence that is a part of SEQ ID NO:3 or SEQ ID NO:4 and that contains any regulatory elements thereof.

4. A vector according to claim 3, characterized by further including at least one gene under expression control of said promoter.

5. Plasmid pCDC220 (ATCC 98192).

6. Plasmid pCDC214 (ATCC 98193).

7. An isolated and purified gene expression cassette characterized by containing a sequence selected from SEQ
ID NO:3 and SEQ ID NO:4, or a sequence that has greater than or equal to 70% homology thereto, or a sequence that is a part of SEQ ID NO:3 or SEQ ID NO:4 and that contains any regulatory elements thereof.

8. A plant characterized by having a genome containing an introduced nucleotide sequence of SEQ ID NO:3 or SEQ
ID NO:4, or a sequence that has greater than or equal to 70% homology thereto, or a sequence that is a part of SEQ ID NO:3 or SEQ ID NO:4 and that contains any regulatory elements thereof.

9. A plant seed characterized by having a genome containing an introduced nucleotide sequence of SEQ ID
NO:3 or SEQ ID NO:4, or a sequence that has greater than or equal to 70% homology thereto, or a sequence that is a part or SEQ ID NO:3 or SEQ ID NO:4 and that contains any regulatory elements thereof.

10. A plant as claimed in 8, characterized by exhibiting an alteration of an endogenous characteristic or an addition of a new characteristic compared to a genomically-unmodified plant of the same genotype.

11. A plant seed as claimed in 9, characterized by exhibiting an alteration of an endogenous characteristic or an addition of a new characteristic compared to a genomically-unmodified plant seed of the same genotype.

12. A method of producing transgenic plant by introducing a gene into a genome of said plant under control of a promoter, characterized in that said promoter is of SEQ ID NO:3 or SEQ ID NO:4, or a sequence that has greater than or equal to 70% homology thereto, or a sequence that is a part of SEQ ID NO:3 or SEQ ID
NO:4 and that contains any regulatory elements thereof.

13. A method according to claim 12, characterized in that said plant is selected from flax, canola and tobacco.

14. A method of producing a transgenic plant seed by introducing a gene into a genome of a plant under control of a promoter to produce a transgenic plant, growing said plant and obtaining transgenic seeds therefrom, characterized in that said promoter is of SEQ
ID NO:3 or SEQ ID NO:4, or is a sequence that is greater than or equal to 70% homologous thereto, or a sequence containing any regulatory elements that is a part of SEQ
ID NO:3 or SEQ ID NO:4.

15. A method of claim 14, characterized in that said plant is selected from flax, canola and tobacco.

16. A DNA sequence characterized in that the sequence has greater than or equal to 70% homology to SEQ ID NO:3 or SEQ ID NO:4, or a sequence that is a part of SEQ ID
NO:3 or SEQ ID NO:4 and that contains any regulatory elements thereof, and in that said sequence has been isolated or characterized using sequence information from SEQ ID NO:3 or SEQ ID NO:4.

17. A method of changing fatty acids of membrane and storage lipids of plants, characterized by introducing an antisense or sense construct comprising SEQ ID NO:3 or SEQ ID NO:4 into a plant transformation vector, using the vector to transform the genome of a plant or plant seed, and then growing the plant or plant seed and extracting membrane or storage lipids from the plants or plant seeds.