Diacylglycerol acyltransferases from flax

Abstract

The invention relates to isolated diacylglycerol acyltransferases and polynucleotide sequences encoding the DGAT enzymes; polynucleotide constructs, vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same. Also provided are transformed cells and transgenic plants, with enhanced oil accumulation and quality.

Description

PRIORITY

[0001] This application claims priority of U.S. provisional application No. 61/034,787 filed on Mar. 7, 2008

FIELD OF THE INVENTION

[0002] The present invention relates to isolated diacylglycerol acyltransferases and polynucleotide sequences encoding the DGAT enzymes; polynucleotide constructs, vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same.

BACKGROUND

[0003] Oils obtained from plant seeds are important sources of fatty acids for human consumption and for use as chemical feedstocks. These fatty acids include essential fatty acids, saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids. In plant seed oils, fatty acids are stored predominantly as triacylglycerols (TAGs). TAGs represent the most efficient form of stored energy in eukaryotic cells.

[0004] TAG biosynthesis occurs mainly in the endoplasmic reticulum (ER) of the cell using acyl-CoA and sn-glycerol-3-phosphate as primary substrates. Biosynthesis of TAG is effected through a biochemical process generally known as the Kennedy pathway (Kennedy, 1961) which involves the sequential transfer of fatty acids from acyl-CoAs to the glycerol backbone (acyl-CoA-dependent acylation). The pathway starts with the acylation of sn-glycerol-3-phosphate to form lysophosphatidic acid through the action of sn-glycerol-3-phosphate acyltransferase. The second acylation is catalyzed by lysophosphatidic acid acyltransferase, leading to the formation of phosphatidic acid which is dephosphorylated by phosphatidate phosphatase 1 to form sn-1,2-diacylglycerol. The final acylation is catalyzed by diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) to form TAG. The DGAT enzyme catalyzes the transference of the acyl group from acyl-coenzymeA (acyl-CoA) donor to a sn-1,2-diacylglycerol, producing CoA and TAG. Previous research results suggest that the level of DGAT activity may have a substantial effect in the flow of carbon into seed oil (Ichihara and Noda, 1988; Perry and Harwood, 1993; Stobart et al., 1986; Settlage et al., 1998).

[0005] Two types of DGAT (DGAT1 and DGAT2) have been identified in animals and plants (Cases et al., 2001; Hobbs et al., 1999; Lardizabal et al., 2001; Kroon et al., 2006; Shockey et al., 2006). DGAT1 has been most studied and displays broad substrate specificity. DGAT1 null mutants in plants and animals have been shown to have substantially reduced levels of TAG (Routaboul et al., 1999; Smith et al., 2000). Furthermore, over-expression of DGAT1 in seeds of Arabidopsis thaliana results in increased seed weight and oil content (Jako et al., 2001). These results suggest that DGAT1 is the predominant type, although some studies indicate that DGAT2 might be more important for TAG biosynthesis in plants like castor bean (Kroon et al., 2006).

[0006] Flax is an oilseed that substantially accumulates .alpha.-linolenic acid (.alpha.-18:3) which is an omega-3 fatty acid. Other omega-3 fatty acids include eicosapentaenoic acid (EPA) and docosahexaneoic acid (DHA) which produce beneficial health effects in humans (Simopoulos, 2002). Flaxseed oil displays chemical attributes which are advantageous for industrial applications including, for example, the production of linoleum, preservation of concrete and as an ingredient in paints and varnishes. The enzymatic activity of DGAT has been studied in isolated ER of flax developing seeds (Sorensen et al., 2005). DGAT is able to incorporate polyunsaturated fatty acids (C18:3 n-3) at higher rates compared to monounsaturated (C18:1) fatty acids. In addition, flax microsomes incorporate EPA and DHA into TAGs (Sorensen et al., 2005), highlighting the usefulness of TAG biosynthetic enzymes such as DGAT as genetic tools for engineering vegetable oils. Over-expression of DGAT in oilseed plants could potentially increase TAG production or enhance seed oil content in plants. However, since numerous enzymatic activities occur within microsomes, it is difficult to evaluate the effect of DGAT in flax using a microsome-based system. Genetically modified organisms have not achieved widespread public acceptance; however, use of native flax DGAT genes for improving the oil content through biotechnology may more readily meet stringent controls.

SUMMARY OF THE INVENTION

[0007] The present invention relates to isolated diacylglycerol acyltransferases and polynucleotide sequences encoding the DGAT enzymes; nucleic acid constructs, vectors and host cells incorporating the polynucleotide sequences; and methods of producing and using same.

[0008] In one aspect, the invention provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from:

[0009] at least 300, at least 400 or at least 500 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 2 or of an amino acid sequence having at least 85% sequence identity therewith;

[0010] at least 300 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 4 or of an amino acid sequence having at least 85% sequence identity therewith; or

[0011] at least 300 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 6 or of an amino acid sequence having at least 85% sequence identity therewith.

[0012] In one embodiment, the invention provides an isolated polynucleotide, wherein the encoded polypeptide comprises the amino acid sequence depicted in SEQ ID NO: 2.

[0013] In one embodiment, the encoded polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO: 1 from nucleotide 57 to nucleotide 1580.

[0014] In one embodiment, the encoded polypeptide comprises the amino acid sequence depicted in SEQ ID NO: 4.

[0015] In one embodiment, the polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO: 3 from nucleotide 1 to nucleotide 1029.

[0016] In one embodiment, the encoded polypeptide comprises the amino acid sequence depicted in SEQ ID NO: 6.

[0017] In one embodiment, the polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO. 5 from nucleotide 1 to nucleotide 1048.

[0018] In one embodiment, the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.

[0019] In one embodiment, the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4.

[0020] In one embodiment, the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6.

[0021] In a further aspect, the invention provides a polynucleotide construct comprising any of the above polynucleotides operably linked to a promoter expressible in bacterial, yeast, fungal, mammalian or plant cells.

[0022] In a further aspect, the invention provides a vector comprising any of the above polynucleotides. In one embodiment, the invention provides a microbial cell comprising any of the above polynucleotides. In one embodiment, the microbial cell is Saccharomyces cerevisiae.

[0023] In a further aspect, the invention provides a transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore, comprising any of the above polynucleotides. In one embodiment, the transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore is selected from a flax, canola, soybean, mouse-ear cress, castor, sunflower, linola, oat, wheat, triticale, barley, corn or Brachypodium distachyon plant, plant cell, plant seed, plant embryo, or microspore.

[0024] In another aspect, the invention provides a method for producing an oil, comprising the steps of growing the above transgenic plant and recovering oil which is produced by the plant. In one embodiment, the plant is selected from a flax, canola, soybean, mouse-ear cress, castor, sunflower, linola, oat, wheat, triticale, barley, corn or Brachypodium distachyon plant.

[0025] In yet another aspect, the invention provides a method for producing a transgenic plant comprising the steps of introducing into a plant cell or a plant tissue any of the above polynucleotides to produce a transformed cell or plant tissue, and cultivating the transformed plant cell or transformed plant tissue to produce the transgenic plant. In one embodiment, the plant is selected from a flax, canola, soybean, mouse-ear cress, castor, sunflower, linola, oat, wheat, triticale, barley, corn or Brachypodium distachyon plant.

[0026] Additional aspects and advantages of the present invention will be apparent in view of the description, which follows. 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 from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The invention will now be described in relation to the drawings in which:

[0028] FIGS. 1A, 1B and 1C show a contig of LuDGAT1 obtained by assembling six isolated fragments (amplicon1 (SEQ ID NO: 28), amplicon2 (SEQ ID NO:29), RT-PCR (SEQ ID NO:30), 3' RACEm (SEQ ID NO:33), 5' RACE (SEQ ID NO:31) and 5' RACEB (SEQ ID NO:32)).

[0029] FIG. 2 is a schematic drawing of the LuDGAT1 cDNA contig. The fragments obtained by PCR are represented by the rectangles. The 5' and 3' untranslated regions are designated by lines. The annealing position and orientation of the oligonucleotides are described on the top.

[0030] FIG. 3 shows the cDNA sequence of LuDGAT1 (SEQ ID NO:1) and the predicted polypeptide sequence (SEQ ID NO:2).

[0031] FIGS. 4A, 4B, 4C and 4D show an amino acid alignment of plant DGAT1 with the accession numbers and species indicated for each sequence, black highlight indicating identical residues and grey highlight indicating blocks of conserved residues.

[0032] FIG. 5 shows a phylogenetic tree of plant DGAT1 with the accession numbers indicated for each plant species and oilseed members of the Cruciferae family highlighted in grey.

[0033] FIG. 6 shows a hydropathy plot of LuDGAT1 polypeptide using the Kyte and Doolittle scale (Kyte and Doolittle, 1982).

[0034] FIG. 7 is a graph showing the specific activity of type-1 DGAT in microsomes from yeast expressing plant type-1 DGAT.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0035] As will be apparent to those skilled in the art, various modifications, adaptations and variations of the foregoing specific disclosure can be made without departing from the scope of the invention claimed herein. The various features and elements of the described invention may be combined in a manner different from the combinations described or claimed herein, without departing from the scope of the invention.

[0036] To facilitate understanding of the invention, the following definitions are provided.

[0037] "Isolated" means that a substance or a group of substances is removed from the coexisting materials of its natural state.

[0038] A "polynucleotide" is a linear sequence of ribonucleotides (RNA) or deoxyribonucleotides (DNA) in which the 3' carbon of the pentose sugar of one nucleotide is linked to the 5' carbon of the pentose sugar of another nucleotide. The deoxyribonucleotide bases are abbreviated as "A" deoxyadenine; "C" deoxycytidine; "G" deoxyguanine; "T" deoxythymidine; "I" deoxyinosine. Some oligonucleotides described herein are produced synthetically and contain different deoxyribonucleotides occupying the same position in the sequence. The blends of deoxyribonucleotides are abbreviated as "W" A or T; "Y" C or T; "H" A, C or T; "K" G or T; "D" A, G or T; "B" C, G or T; "N" A, C, G or T.

[0039] A "polypeptide" is a linear sequence of amino acids linked by peptide bonds. The amino acids are abbreviated as "A" alanine; "R" arginine; "N" asparagine; "D" aspartic acid; "C" cysteine; "Q" glutamine; "E" glutamic acid; "G" glycine; "H" histidine; "I" isoleucine; "L" leucine; "K" lysine; "M" methionine; "F" phenylalanine; "P" proline; "S" serine; "T" threonine; "W" tryptophan; "Y" tyrosine and "V" valine.

[0040] "Downstream" means on the 3' side of a polynucleotide while "upstream" means on the 5' side of a polynucleotide.

[0041] "Expression" refers to the transcription of a gene into RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein.

[0042] A "promoter" is a polynucleotide usually located within 20 to 5000 nucleotides upstream of the initiation of translation site of a gene. The "promoter" determines the first step of expression by providing a binding site to DNA polymerase to initiate the transcription of a gene. The promoter is said to be "inducible" when the initiation of transcription occurs only when a specific agent or chemical substance is presented to the cell. For instance, the GAL "promoter" from yeast is "inducible by galactose," meaning that this GAL promoter allows initiation of transcription and subsequent expression only when galactose is presented to yeast cells.

[0043] A "coding sequence" or "coding region" or "open reading frame (ORF)" is part of a gene that codes for an amino acid sequence of a polypeptide.

[0044] A "complementary sequence" is a sequence of nucleotides which forms a duplex with another sequence of nucleotides according to Watson-Crick base pairing rules where "A" pairs with "T" and "C" pairs with "G." For example, for the polynucleotide 5'-AATGCCTA-3' the complementary sequence is 5'-TAGGCATT-3'.

[0045] A "cDNA" is a polynucleotide which is complementary to a molecule of messenger RNA mRNA. The "cDNA" is formed of a coding sequence flanked by 5' and 3' untranslated sequences.

[0046] "DGAT" is an enzyme of the class EC 2.3.1.20 which catalyzes the reaction: acyl-CoA+sn-1,2-diacylglycerol.fwdarw.CoA+triacylglycerol. Alternative names include: diacylglycerol O-acyltransferase, diacylglycerol acyltransferase, diglyceride acyltransferase and acylCoA:diacylglycerol acyltransferase.

[0047] A polypeptide having "DGAT activity" is a polypeptide that has, to a greater or lesser degree, the enzymatic activity of DGAT.

[0048] A "recombinant" polynucleotide is a novel polynucleotide sequence formed in vitro through the ligation of two DNA molecules.

[0049] A "construct" is a polynucleotide which is formed by polynucleotide segments isolated from a naturally occurring gene or which is chemically synthesized. The "construct" which is combined in a manner that otherwise would not exist in nature, is usually made to achieve certain purposes. For instance, the coding region from "gene A" can be combined with an inducible promoter from "gene B" so the expression of the recombinant construct can be induced.

[0050] "Transformation" means the directed modification of the genome of a cell by external application of a polynucleotide, for instance, a construct. The inserted polynucleotide may or may not integrate with the host cell chromosome. For example, in bacteria, the inserted polynucleotide usually does not integrate with the bacterial genome and might replicate autonomously. In plants, the inserted polynucleotide integrates with the plant chromosome and replicates together with the plant chromatin.

[0051] A "transgenic" organism is the organism that was transformed with an external polynucleotide. The "transgenic" organism encompasses all descendants, hybrids and crosses thereof, whether reproduced sexually or asexually and which continue to harbor the foreign polynucleotide.

[0052] A "vector" is a polynucleotide that is able to replicate autonomously in a host cell and is able to accept other polynucleotides. For autonomous replication, the vector contains an "origin of replication." The vector usually contains a "selectable marker" that confers the host cell resistance to certain environment and growth conditions. For instance, a vector that is used to transform bacteria usually contains a certain antibiotic "selectable marker" which confers the transformed bacteria resistance to such antibiotic.

[0053] Two polynucleotides or polypeptides are "identical" if the sequence of nucleotides or amino acids, respectively, in the two sequences is the same when aligned for maximum correspondence as described here. Sequence comparisons between two or more polynucleotides or polypeptides can be generally performed by comparing portions of the two sequences over a comparison window which can be from about 20 to about 200 nucleotides or amino acids, or more. The "percentage of sequence identity" may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of a polynucleotide or a polypeptide sequence may include additions (i.e., insertions) or deletions (i.e., gaps) as compared to the reference sequence. The percentage is calculated by determining the positions at which identical nucleotides or identical amino acids are present, dividing by the number of positions in the window and multiplying the result by 100 to yield the percentage of sequence identity. Polynucleotide and polypeptide sequence alignment may be performed by implementing specialized algorithms or by inspection. Examples of sequence comparison and multiple sequence alignment algorithms are: BLAST and ClustalW softwares. Identity between nucleotide sequences can also be determined by DNA hybridization analysis, wherein the stability of the double-stranded DNA hybrid is dependent on the extent of base pairing that occurs. Conditions of high temperature and/or low salt content reduce the stability of the hybrid, and can be varied to prevent annealing of sequences having less than a selected degree of homology. Hybridization methods are described in Ausubel et al. (1995).

[0054] The invention provides isolated DGAT1 and DGAT2 polynucleotides and polypeptides. DGAT1 and DGAT2 polynucleotides include, without limitation (1) single- or double-stranded DNA, such as cDNA or genomic DNA including sense and antisense strands; and (2) RNA, such as mRNA. DGAT1 and DGAT2 polynucleotides include at least a coding sequence which codes for the amino acid sequence of the specified DGAT polypeptide, but may also include 5' and 3' untranslated regions and transcriptional regulatory elements such as promoters and enhancers found upstream or downstream from the transcribed region.

[0055] In one embodiment, the invention provides a DGAT1 polynucleotide which is a cDNA comprising the nucleotide sequence depicted in SEQ ID NO: 1, and which was isolated from Linum usitatissimum. The cDNA is 1778 base pairs in length including a coding region of 1524 base pairs (SEQ ID NO: 1 from nucleotide 57 to nucleotide 1580) and untranslated 5' and 3' regions of 56 and 198 base pairs, respectively. The DGAT1 encoded by the coding region (designated as LuDGAT1, SEQ ID NO: 2) is a 507 amino acid polypeptide with a predicted molecular weight of 58,012 Daltons and an isoelectric point of 8.74.

[0056] In one embodiment, the invention provides a DGAT2 polynucleotide which is a coding region comprising the nucleotide sequence depicted in SEQ ID NO: 3, which was also isolated from Linum usitatissimum. The coding region is 1029 base pairs in length and the DGAT2 encoded by the coding region (designated as LuDGAT2A, SEQ ID NO: 4) is a 343 amino acid polypeptide with a predicted molecular weight of 38,201 Daltons and an isoelectric point of 9.28.

[0057] In one embodiment, the invention provides a DGAT2 coding region comprising the nucleotide sequence depicted in SEQ ID NO: 5 and which was isolated from Linum usitatissimum. The coding region is 1048 base pairs in length and the DGAT2 encoded by the coding region (designated here by LuDGAT2B, SEQ ID NO: 6) is a 349 amino acid polypeptide with a predicted molecular weight of 38,737 Daltons and an isoelectric point of 9.18.

[0058] Those skilled in the art will recognize that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for identical polypeptides. Accordingly, the invention includes polynucleotides of SEQ ID NOS: 1, 3 and 5, and variants of polynucleotides encoding polypeptides of SEQ ID NOS: 2, 4 and 6. In one embodiment, polynucleotides having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequences depicted in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 are included in the invention. Methods for isolation of such polynucleotides are well known in the art (see for example, Ausubel et al., 1995).

[0059] In one embodiment, the invention provides isolated polynucleotides which encode polypeptides having DGAT activity and which comprise amino acid sequences having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequences depicted in SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 6.

[0060] In one embodiment, the invention provides isolated polynucleotides which encode polypeptides having DGAT activity and which comprise amino acid sequences having a length of at least 300, at least 400 or at least 500 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 2. In one embodiment, the invention provides isolated polynucleotides which encode polypeptides having DGAT activity and which comprise amino acid sequences having a length of at least 300 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 4. In one embodiment, the invention provides isolated polynucleotides which encode polypeptides having DGAT activity and which comprise amino acid sequences having a length of at least 300 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 6.

[0061] The above described polynucleotides of the invention may be used to express polypeptides in recombinantly engineered cells including, for example, bacterial, yeast, fungal, mammalian or plant cells. In one embodiment, the invention provides polynucleotide constructs, vectors and cells comprising DGAT polynucleotides. Those skilled in the art are knowledgeable in the numerous systems available for expression of a polynucleotide. All systems employ a similar approach, whereby an expression construct is assembled to include the protein coding sequence of interest and control sequences such as promoters, enhancers, and terminators, with signal sequences and selectable markers included if desired. Briefly, the expression of isolated polynucleotides encoding polypeptides is typically achieved by operably linking, for example, the DNA or cDNA to a constitutive or inducible promoter, followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA. High level expression of a cloned gene is obtained by constructing expression vectors which contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. Vectors may further comprise transit and targeting sequences, selectable markers, enhancers or operators. Means for preparing vectors are well known in the art. Typical vectors useful for expression of polynucleotides in plants include for example, vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaM-VCN transfer control vector. Promoters suitable for plant cells include for example, the nopaline synthase, octopine synthase, and mannopine synthase promoters, and the caulimovirus promoters.

[0062] Those skilled in the art will appreciate that modifications (i.e., amino acid substitutions, additions, deletions and post-translational modifications) can be made to a polypeptide of the invention without eliminating or diminishing its biological activity. Conservative amino acid substitutions (i.e., substitution of one amino acid for another amino acid of similar size, charge, polarity and conformation) or substitution of one amino acid for another within the same group (i.e., nonpolar group, polar group, positively charged group, negatively charged group) are unlikely to alter protein function adversely. Some modifications may be made to facilitate the cloning, expression or purification. Variant DGAT polypeptides may be obtained by mutagenesis of the polynucleotides depicted in SEQ ID NOS: 1, 3 and 5 using techniques known in the art including, for example, oligonucleotide-directed mutagenesis, region-specific mutagenesis, linker-scanning mutagenesis, and site-directed mutagenesis by PCR (Ausubel et al., 1995). Variant DGAT polypeptides can be tested for DGAT activity by the assay described in Example 4.

[0063] Various methods for transformation or transfection of cells are available. For prokaryotes, lower eukaryotes and animal cells, such methods include for example, calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation, biolistics and microinjection. The transfected cells are cultured, and the produced DGAT polypeptides may be isolated and purified from the cells using standard techniques known in the art. Accordingly, in one embodiment, the invention provides methods for producing DGAT in yeast as described in Example 4. Various industrial strains of microorganisms including for example, Aspergillus, Pichia pastoris, Saccharomyces cerevisiae, E. coli, Bacillus subtilis) may be used to produce DGAT polypeptides. In one embodiment, the microbial cell is Saccharomyces cerevisiae.

[0064] Methods for transformation of plant cells include for example, electroporation, PEG poration, particle bombardment, Agrobacterium tumefaciens- or Agrobacterium rhizogenes-mediated transformation, and microinjection. The transformed plant cells, seeds, callus, embryos, microspore-derived embryos, microspores, organs or explants are cultured or cultivated using standard plant tissue culture techniques and growth media to regenerate a whole transgenic plant which possesses the transformed genotype. Transgenic plants may pass polynucleotides encoding DGAT polypeptides to their progeny, or can be further crossbred with other species. Accordingly, in one embodiment, the invention provides methods for producing transgenic plants, plant cells, callus, seeds, plant embryos, microspore-derived embryos, and microspores comprising DGAT polynucleotides.

[0065] In one embodiment, the invention provides transgenic plants, plant cells, callus, seeds, plant embryos, microspore-derived embryos, and microspores comprising DGAT polynucleotides. Plant species of interest for transformation include, without limitation, crops used for commercial oil production such as, for example, flax (Linum spp.), canola, soybean (Glycine and Soja spp.), mouse-ear cress (Arabidopsis thaliana), castor, sunflower and linola. In one embodiment, the plant is a flax plant. In one embodiment, the plant is a canola plant. It will be appreciated by those skilled in the art that the plant species for transformation are not limited to crops grown for commercial oil production. Such additional plant species include, without limitation, oats, wheat, triticale, barley, corn and Brachypodium distachyon.

[0066] The DGAT polynucleotides, polypeptides, and methods of the invention are useful in a wide range of agricultural, industrial and nutritional applications. Transgenic plants with increased seed oil content can be developed; for example, transgenic plants which have the unique preference of incorporating omega-3 fatty acids into TAGs. Co-expression of LuDGAT1 and LuDGAT2 with a delta-15 desaturase gene to feed LuDGAT with a polyunsaturated fatty acid may be conducted in plant species which do not normally produce C18:3 fatty acid. Recombinant expression of DGAT may be achieved in plants which are typically not grown for commercial oil production, resulting in development of new cultivars which produce oil having a similar composition to flaxseed oil.

[0067] Further, the DGAT polynucleotides and polypeptides may be used in the industrial production and recovery of oil products using recombinant technology such as transformed bacterial, yeast or fungal cells. Transformed cells may be engineered to accumulate omega-3 fatty acids in TAGs.

[0068] The DGAT polynucleotides and polypeptides may be incorporated into human food and animal feed applications to provide healthier products or to improve the fat quality of products. For example, a healthier dietary oil having a fatty acid profile which reduces the risk of coronary heart disease and decreases plasma cholesterol may be developed for humans. Livestock are unable to convert n-6 fatty acids into n-3 fatty acids since they lack an n-3 fatty acid desaturase gene. However, co-expression of LuDGAT1 and/or LuDGAT2 with fat-1 desaturase gene in livestock may increase the amount of n-3 fatty acids.

[0069] The Examples provided below are not intended to be limited to these examples alone, but are intended only to illustrate and describe the invention rather than limit the claims that follow.

EXAMPLES

Example 1

Isolation of RNA from Flax Embryos

[0070] Flax plants (Linum usitatissimum L. cv A C Emerson) were grown in greenhouse conditions, irrigated at 2-3 day intervals and fertilized weekly with 1% Peters 20-20-20 general purpose fertilizer (Scotts, Marysville, Ohio). Flax embryos were isolated from developing seeds and RNA was obtained using 350 mg of embryos frozen in liquid nitrogen and ground with mortar and pestle. Ground embryos were transferred to a 2 ml tube and 500 .mu.l of extraction buffer (50 mM TrisHCl pH 9.0, 200 mM NaCl, 1% Sarkosyl, 20 mM EDTA, and 5 mM DTT) was added, mixing with a vortex. 500 .mu.l of phenol chloroform mixture (Sigma-Aldrich Ltd, Oakville, ON) was added and mixed with a vortex. The sample was centrifuged for 5 minutes at 12000 g at 4.degree. C. The aqueous upper phase was transferred to a new tube and 1 ml of Trizol reagent (Gibco, Burlington, ON, Canada) was added, followed by addition of 250 .mu.l of chloroform. The sample was mixed with a vortex and centrifuged for 5 minutes at 12000 g at 4.degree. C. The aqueous upper phase (750 .mu.l) was transferred to a new tube and 500 .mu.l of chloroform was added and mixed in a vortex. The sample was centrifuged for 5 minutes at 12000 g at 4.degree. C. The upper phase (600 .mu.l) was transferred to a new tube and the RNA was precipitated with addition of 60 .mu.l of sodium acetate (3M) and 1.2 ml of ethanol. The sample was incubated at -80.degree. C. for 1 hour and centrifuged for 20 minutes at 14000 g at 4.degree. C. The RNA pellet was washed with 70% ethanol, followed by brief centrifugation (2 minutes at 14000 g at 4.degree. C.) and dried with a vacufuge (Ependorf, Westbury, N.Y., U.S.). The RNA pellet was diluted in 50 .mu.l of water and centrifuged for 20 minutes at 14000 g at 4.degree. C. Total RNA was quantified by using a Nanodrop.TM. spectrophotometer (NanoDrop Technologies, Wilmington, Del., U.S.).

Example 2

Isolation of LuDGAT1 cDNA

[0071] Recombinant DNA techniques such as digestion by restriction endonucleases, ligation and plasmid preparation were performed as described by Ausubel et al. (1995). First strand synthesis of complementary DNA (cDNA) was produced by reverse transcription. Five micrograms of flax embryo RNA were mixed with 50 pmoles of oligonucleotide 5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTTVN-3' (oligodT adaptor; SEQ ID NO: 7) and 1 mM of dNTP in a total volume of 10 .mu.l. The mixture was incubated at 65.degree. C. for 5 minutes and immediately cooled on ice for 2 minutes. A total volume of 10 .mu.l of cDNA synthesis mix was added. This mix consisted of 2.times. transcriptase buffer, 10 mM of MgCl.sub.2, 20 mM of DTT and 200 units of Superscript II (Invitrogen, Burlington, ON). The reaction was incubated at 50.degree. C. for 50 minutes followed by enzyme inactivation at 85.degree. C. for 5 minutes. The reaction was cooled to 37.degree. C. and incubated at this temperature for 20 minutes in the presence of 4 units of RNAseH (Invitrogen) and 1 unit of RNAseT1 (Ambion, Austin, Tex., U.S.) to remove the mRNA strand from RNA-DNA duplexes and single-stranded RNA, respectively. The synthesized cDNA was stored at -20.degree. C.

[0072] Polymerase chain reaction (PCR) was performed using 200 .mu.M of each dNTP, 0.1 volumes of PCR reaction buffer, and varying amounts of oligonucleotide, polymerases, DNA template and MgSO.sub.4 or MgCl.sub.2, according to the application in a final volume of 50 .mu.l. The general PCR thermal cycling conditions were: 2 minutes preheat at 94.degree. C. followed by 30 cycles of 94.degree. C. denaturing for 30 seconds, 55.degree. C. annealing for 30 seconds and 72.degree. C. or 68.degree. C. extension for 1 to 2 minutes. After the final cycle, the PCR reactions were incubated for 10 minutes at 72.degree. C. or 68.degree. C. for further extension and cooled to 10.degree. C. until used for analysis.

[0073] The degenerate oligonucleotides 5'-GARTTYTAYCANGAYTGGTGG-3' (RS-007; SEQ ID NO: 8), 5'-GGNACNGCNATRCANARYTCRTG-3' (RS-008; SEQ ID NO: 9), 5'-GARAANYTNATGAARTAYGG-3' (RS-009; SEQ ID NO: 10) and 5'-TANTGYTCNATDATRAANCCCAT-3' (RS-010; SEQ ID NO: 11) were designed based on several plant DGAT1 sequences available in public databases. Such oligonucleotides, which provide limited specificity to the template, were used on PCR amplification of two different segments of DGAT1 using cDNA previously described as a template. These two DNA segments were sequenced using BigDye Version 3.1 dye terminator cycle sequencing kit (Applied Biosystems, Streetsville, ON, Canada). Samples were analyzed in an automatic sequencer (Applied Biosystems 373A Sequencer) at the University of Alberta Molecular Biology Service Unit (MBSU). Sequencing chromatograms were trimmed and assembled using Contig Express and "BLASTed" (Altschul et al., 1995; www.ncbi.nlm.nih.gov/BLAST) against public DNA databases.

[0074] Sequences from these two DNA segments, here named amplicon1 and amplicon2 (FIGS. 1 and 2), were used to design specific oligonucleotides to LuDGAT1 cDNA. These specific oligonucleotides were used in rapid amplification of cDNA ends (RACE) and reverse transcription-PCR (RT-PCR) amplification reactions to obtain the full nucleotide sequence of DGAT1.

[0075] 3' RACE was obtained using the oligonucleotides 5'-GCCATATCTATTTCCCATGTCTGCGG-3' (RS027; SEQ ID NO: 12) and 5'-GGCCACGCGTCGACTAGTAC-3' (adaptor; SEQ ID NO: 13) using cDNA previously described as template. For 5' RACE flax cDNA was produced using the oligonucleotide 5'-CACTGGAAGTGTTAGACAG-3' (RS-039; SEQ ID NO: 14) using the same conditions described before. The cDNA and the 3' end were tailed with dCTP using Terminal deoxynucleotidyl Transferase (TdT). 5'-RACE was amplified using the oligonucleotides 5'-GGCCACGCGTCGACTAGTACGGGGGGGGGGGGGGGGGN-3' (oligo dG adaptor; SEQ ID NO: 15) and 5'-ACTGAACCAGAAGCCTGTC-3' (RS-038; SEQ ID NO: 16). This reaction yielded a truncated 5' RACE product. A second 5'-RACE (here called 5'RACEB) was performed by producing flax embryo cDNA with the oligonucleotide 5'-CGGAACTAAGCGGACTCTC-3' (RS-057; SEQ ID NO: 17) and tailing with dCTP. 5'-RACEB was performed using 5'-GGCACGGAAGGGCGGTAAG-3' (RS-056; SEQ ID NO: 18) and oligo dG adaptor. Sequences obtained from 5' and 3' RACE products were aligned with amplicon1 and amplicon2 sequences (FIG. 2).

[0076] RT-PCR of the DNA segment between amplicon1 and amplicon2 (FIG. 1) was performed using the oligonucleotides 5'-CAAGTTAGTAATATTTACAGGC-3', (RS-054; SEQ ID NO: 19) and 5'-TCCACATTCTCCAGTATTCTTC-3' (RS-055; SEQ ID NO: 20) (FIGS. 1 and 2) and a cDNA from flax embryos previously described. The RT-PCR product was sequenced and aligned with sequences from other LuDGAT1 segments previously obtained (FIG. 2).

[0077] The coding region of LuDGAT1 was obtained through RT-PCR using the oligonucleotides 5'-ATTAGGATCCGACCATGGGCGTGCTCGACACTCCTGACAATC-3' (RS-100; SEQ ID NO: 21) and 5'-TTTAAGCTTGATTCCATCTTTCCCATTCCTG-3' (RS-101; SEQ ID NO: 22) and flax embryo cDNA as template. The RT-PCR product of LuDGAT1 coding region was sequenced and analyzed.

Example 3

Analysis of LuDGAT1 Sequence

[0078] DNA sequences were analyzed using Vector NTi.TM. Advance 10.1.1 (Invitrogen) software package. Amino acid and DNA alignments were performed with AlignX, and phylogenetic trees were visualized using Tree View.TM. version 1.6.6.

[0079] The full sequence of DGAT1 cDNA has 1778 base pairs with an open reading frame of 1521 base pairs comprising 507 amino acids with a molecular weight of 58.02 kDa. The predicted LuDGAT1 polypeptide, obtained by analysis of the ORF of LuDGAT1 cDNA (FIG. 3), was compared to other DGAT polypeptides from plants available in public databases. This comparison showed that LuDGAT1 is 74% identical with Vernicia fordii (tung tree), 75% with Jatropha curcas, 73% with Euonymus alatus (burning bush) and 65% with Brassica napus but only 40% with Mus musculus and 39% with Homo sapiens. An alignment of LuDGAT1 with several other plant DGAT1 (FIG. 4) showed many similarities and also some unique features. When compared to DGAT1 from other plants, LuDGAT1 presents the polypeptide "APSAALNV" (SEQ ID NO: 23) in the region between positions 253 and 259, which is absent in DGAT1 from cruciferaceae (Arabidopsis and Brassica sp.). A phylogenetic tree obtained with the previous alignment (FIG. 5) shows higher similarity between LuDGAT1 and Vernicia fordii, Jatropha curcas and Ricinus communis, compared to Oryza sativa, Brassica napus and Arabidopsis thaliana. LuDGAT presents unique features such as the substitution of the aspartic acid with glycine at position 103 in the conserved motif "ESPLSSD" (SEQ ID NO: 24). In position 271, the motif "LAYF" (SEQ ID NO: 25) is modified to "LVYF" (SEQ ID NO: 26). In the conserved motif "MWNMPVH" (SEQ ID NO: 27) present in other plant DGATs, the conserved asparagine in position 395 is substituted by a serine. These variations could reflect unique characteristics of LuDGAT1 enzymatic activity and specificity.

[0080] LuDGAT presents a hydrophilic N-terminus and several hydrophobic regions (FIG. 6), which is typical in other DGAT1 proteins. The first 80 residues present much higher variability compared to the rest of the protein, which is a characteristic found in other DGATs. Nine transmembrane regions were predicted in LuDGAT1 using TMPRED (http://www.ch.embnet.org/) (TM1 from 127 to 148, TM2 from 172 to 191, TM3 from 204 to 225, TM4 from 230 to 252, TM5 from 311 to 331, TM6 from 360 to 380, TM7 from 432 to 457, TM8 from 461 to 477 and TM9 from 493 to 511). Motif searches revealed that LuDGAT1 has a membrane bound O-acyl transferase (MBOAT) motif (pfam03062.12) which is present in a variety of acyltransferase enzymes such as DGAT1.

Example 4

Expression of LuDGAT1 in Yeast

[0081] The LuDGAT1 coding region was subcloned into pYES2.1/V5-HIS vector under control of GAL1 promoter which is inducible by galactose. Ai yeast consensus sequence for initiation of translation, composed of 5-(`g/a) nnatgg-3`, was introduced and the second amino acid codon was changed from gcg (A) to ggg (G). The translation stop sequence 5'-tga-3' was removed in order to fuse LuDGAT1 in frame with V5 and HIS tags. The recombinant plasmid, called pYES LuDGAT1, was introduced into Saccharomyces cerevisiae strain H1246. A single colony containing pYES LuDGAT1 was inoculated in medium containing 2% glucose and grown overnight. The expression of LuDGAT1 was induced with medium containing 2% galactose. The same procedure was performed for pYES BnDGAT1 which contains the cDNA encoding DGAT1 from Brassica napus (Nykiforuk et al., 2002). pYES BnDGAT1 was used to compare the activity of another plant DGAT to LuDGAT1. Microsomes were extracted from induced yeast cells as described by Urban et al. (1994) and DGAT activity was determined by measuring the incorporation of .sup.14C-oleyl-CoA into TAG. As S. cerevisiae strain H1246 is deficient in TAG biosynthesis (Sandager et al., 2002), the DGAT activity observed results only from the recombinant DGAT expressed.

[0082] DGAT assays were performed according to Byers et al. (1999). The standard reaction mixture (60 .mu.L) consisted of 0.2 M Hepes-NaOH buffer (pH 7.4) containing 0.15 mg BSA/mL, 20 mM MgSO.sub.4, 330 .mu.M sn-1,2-diolein in 0.2% (wt/vol) Tween.TM. 20, 15 .mu.M [1-.sup.14C]oleoyl-CoA (56 mCi/mmo) and microsomal protein (80-120 .mu.g). The reaction was performed for 15 min at 30.degree. C. Each reaction mixture was spotted directly onto a silica gel thin layer chromatography plate, which was developed in hexane/ether (80:20, vol/vol).

[0083] Sections of silica containing TAG were scraped into scintillation vials, combined with 5 mL Ecolite(+) and assayed for radioactivity in a liquid scintillation counter. As observed in FIG. 7, LuDGAT1 has comparable DGAT specific activity to BnDGAT1.

REFERENCES

[0084] Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215: 403-10. [0085] Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Deidman, J. G. and Tatham, A. S. (1995) Current Protocols in Molecular Biology. New York. [0086] Byers, S. D., Laroche, A., Smith, K. C. and Weselake, R. J. (1999) Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape. Lipids 34:1143-1149. [0087] Cases, S., Stone, S. J., Zhou, P., Yen, E., Tow, B., Lardizabal, K. D., Voelker, T. and Farese, R. V. (2001) Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members. J. Biol. Chem. 276:38870-38876. [0088] Hobbs, D. H., Lu, C. F., and Hills, M. J. (1999) Cloning of a cDNA encoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression. Febs Letters 452:145-149. [0089] Hong, H., Datla, N., Reed, D. W., Covello, P. S., MacKenzie, S. L., and Qiu, X. (2002) High-level production of gamma-linolenic acid in Brassica juncea using a Delta 6 desaturase from Pythium irregulare. Plant Physiology 129:354-362. [0090] Ichihara, K., Takahashi, T. and Fujii, S. (1988) Diacylglycerol acyltransferase in maturing safflower seeds--its influences on the fatty-acid composition of triacylglycerol and on the rate of triacylglycerol synthesis. Biochimica et Biophysica Acta 958:125-129. [0091] Jako, C., Kumar, A., Wei, Y. D., Zou, J. T., Barton, D. L., Giblin, E. M., Covello, P. S. and Taylor, D. C. (2001) Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiology 126:861-874. [0092] Kennedy, E. P. (1961) Biosynthesis of Complex Lipids. Federation Proceedings 20:934-940. Kroon, J. T., Wei, Y. D., Simon, W. J. and Slabas, A. R. (2006) Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals. Phytochemistry 67:2541-2549. [0093] Lai, L. X., Kang, J. X., Li, R. F., Wang, J. D., Witt, W. T., Yong, H. Y., Hao, Y. H., Wax, D. M., Murphy, C. N., Rieke, A., Samuel, M., Linville, M. L., Korte, S. W., Evans, R. W., Starzl, T. E., Prather, R. S. and Dai, Y. F. (2006) Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nature Biotechnology 24:435-436. [0094] Lardizabal, K. D., Mai, J. T., Wagner, N. W., Wyrick, A., Voelker, T. and Hawkins, D. J. (2001) DGAT2 is a new diacylglycerol acyltransferase gene family--purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity. J. Biol. Chem. 276:38862-38869. [0095] Nykiforuk, C. L., Furukawa-Stoffer, T. L., Huff, P. W., Sarna, M., Laroche, A., Moloney, M. M. and Weselake, R. J. (2002) Characterization of cDNAs encoding diacylglycerol acyltransferase from cultures of Brassica napus and sucrose-mediated induction of enzyme biosynthesis. Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids 1580:95-109. [0096] Perry, H. J. and Harwood, J. L. (1993) Changes in the Lipid-Content of Developing Seeds of Brassica-Napus. Phytochemistry 32:1411-1415. [0097] Routaboul, J. M., Benning, C., Bechtold, N., Caboche, M. and Lepiniec, L. (1999) The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase. Plant Physiology and Biochemistry 37:831-840. [0098] Sandager, L., Gustavsson, M. H., Stahl, U., Dahlqvist, A., Wiberg, E., Banas, A., Lenman, M., Ronne, H. and Stymne, S. (2002) Storage lipid synthesis is non-essential in yeast. J. Biol. Chem. 277:6478-6482. [0099] Settlage, S. B., Kwanyuen, P., and Wilson, R. F. (1998) Relation between diacylglycerol acyltransferase activity and oil concentration in soybean. Journal of the American Oil Chemists Society 75:775-781. [0100] Shockey, J. M., Gidda, S. K., Chapital, D. C., Kuan, J. C., Dhanoa, P. K., Bland, J. M., Rothstein, S. J., Mullen, R. T. and Dyer, J. M. (2006) Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum. Plant Cell 18:2294-2313. [0101] Simopoulos, A. P. (2002) The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomedicine & Pharmacotherapy 56:365-379. [0102] Smith, S. J., Cases, S., Jensen, D. R., Chen, H. C., Sande, E., Tow, B., Sanan, D. A., Raber, J., Eckel, R. H., and Farese, R. V. (2000) Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat. Nature Genetics 25:87-90. [0103] Sorensen, B. M., Furukawa-Stoffer, T. L., Marshall, K. S., Page, E. K., Mir, Z., Forster, R. J. and Weselake, R. J. (2005) Storage lipid accumulation and acyltransferase action in developing flaxseed. Lipids 40:1043-1049. [0104] Stobart, A. K., Stymne, S. and Hoglund, S. (1986) Safflower Microsomes Catalyze Oil Accumulation Invitro--A Model System. Planta 169:33-37. [0105] Urban, P., Werckreichhart, D., Teutsch, H. G., Durst, F., Regnier, S., Kazmaier, M. and Pompon, D. (1994) Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast--kinetic and spectral properties of the major plant P450 of the phenylpropanoid pathway. European Journal of Biochemistry 222:843-850. [0106] Wu, G. H., Truksa, M., Datla, N., Vrinten, P., Bauer, J., Zank, T., Cirpus, P., Heinz, E. and Qiu, X. (2005) Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants. Nature Biotechnology 23:1013-1017.

[0107] All publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

Sequence CWU 1

1

3311778DNALinum usitatissimumCDS(57)..(1580)LuDGAT1 1ctttcggtgt gttatcatcg ttctttctgc gactgcttcc cccctctcct cttcca atg 59Met1gcg gtg ctc gac acc cct gac aat cac ata aac ccc tct ccc tcc acc 107Ala Val Leu Asp Thr Pro Asp Asn His Ile Asn Pro Ser Pro Ser Thr 5 10 15tct gct att gac tcc tcc gat ctt aac ggt ctc tcc ctt cga cgt cgt 155Ser Ala Ile Asp Ser Ser Asp Leu Asn Gly Leu Ser Leu Arg Arg Arg 20 25 30tca gtt gcc act aac tcc gac caa ggt act tct tcc acc gct gta gaa 203Ser Val Ala Thr Asn Ser Asp Gln Gly Thr Ser Ser Thr Ala Val Glu 35 40 45tca ctc cac gcg gat cgg cca gcc gat tct gat ggg gcg aac cgc gag 251Ser Leu His Ala Asp Arg Pro Ala Asp Ser Asp Gly Ala Asn Arg Glu50 55 60 65gat aag aag att gac aat cgg gac ggt caa gtt gcg aga tcg gat atc 299Asp Lys Lys Ile Asp Asn Arg Asp Gly Gln Val Ala Arg Ser Asp Ile 70 75 80aaa ttc act tac cgc cct tcc gtg ccc gct cac gtc aag gtt aaa gag 347Lys Phe Thr Tyr Arg Pro Ser Val Pro Ala His Val Lys Val Lys Glu 85 90 95agt ccg ctt agt tcc ggc gcc att ttt aag cag agc cat gca ggc ctc 395Ser Pro Leu Ser Ser Gly Ala Ile Phe Lys Gln Ser His Ala Gly Leu 100 105 110ttc aat ctc tgt att gta gtc cta gtt gca gtc aac agc agg ctt att 443Phe Asn Leu Cys Ile Val Val Leu Val Ala Val Asn Ser Arg Leu Ile 115 120 125atc gag aat atc atg aag tat ggt tgg tta att agg aca ggc ttc tgg 491Ile Glu Asn Ile Met Lys Tyr Gly Trp Leu Ile Arg Thr Gly Phe Trp130 135 140 145ttc agt tca aaa tcg ttg aga gat tgg cct ctt ttc atg tgc tgt cta 539Phe Ser Ser Lys Ser Leu Arg Asp Trp Pro Leu Phe Met Cys Cys Leu 150 155 160aca ctt cca gtg ttc gcg ctt gct gca tat cta gtt gag aaa ttg gcg 587Thr Leu Pro Val Phe Ala Leu Ala Ala Tyr Leu Val Glu Lys Leu Ala 165 170 175tac cgg aaa tat ctc tct gaa cct ata gtc gtt tcc ctc cac ata atc 635Tyr Arg Lys Tyr Leu Ser Glu Pro Ile Val Val Ser Leu His Ile Ile 180 185 190atc tcc gtg gta gca gtt gtg tac cct gtt tca gtg att ctc agc tgc 683Ile Ser Val Val Ala Val Val Tyr Pro Val Ser Val Ile Leu Ser Cys 195 200 205gac tct gca ttt gta tct ggt gtg acg ttg atg ctt ttt gct tgc att 731Asp Ser Ala Phe Val Ser Gly Val Thr Leu Met Leu Phe Ala Cys Ile210 215 220 225gtg tgg tta aaa ttg gtc tca tat gct cat acg aac tat gat atg aga 779Val Trp Leu Lys Leu Val Ser Tyr Ala His Thr Asn Tyr Asp Met Arg 230 235 240gcg gtt gcc aag tca gtt gaa aag gga gaa gca cct tct gct gct ttg 827Ala Val Ala Lys Ser Val Glu Lys Gly Glu Ala Pro Ser Ala Ala Leu 245 250 255aat gtt gat tac tct tat gac gtt aac ttc aag agc ttg gtg tat ttt 875Asn Val Asp Tyr Ser Tyr Asp Val Asn Phe Lys Ser Leu Val Tyr Phe 260 265 270atg att gct cca aca ctg tgc tat cag cca agc tat cca cgc act cca 923Met Ile Ala Pro Thr Leu Cys Tyr Gln Pro Ser Tyr Pro Arg Thr Pro 275 280 285tgt atc cga aag ggt tgg ctg gtt cat cag ttc atc aag tta gta ata 971Cys Ile Arg Lys Gly Trp Leu Val His Gln Phe Ile Lys Leu Val Ile290 295 300 305ttt aca ggc ttg atg gga ttc att ata gag caa tat atc aat cca att 1019Phe Thr Gly Leu Met Gly Phe Ile Ile Glu Gln Tyr Ile Asn Pro Ile 310 315 320atc cag aac tct cag cat cct ttt aaa ggg aat cta tta tat gga att 1067Ile Gln Asn Ser Gln His Pro Phe Lys Gly Asn Leu Leu Tyr Gly Ile 325 330 335gag agg gtt ttg aaa ctt tcg gtc cca aac ttg tat gtg tgg ctg tgc 1115Glu Arg Val Leu Lys Leu Ser Val Pro Asn Leu Tyr Val Trp Leu Cys 340 345 350atg ttc tac tgc ttc ttt cat cta tgg tta aat ata ctt gca gag ctc 1163Met Phe Tyr Cys Phe Phe His Leu Trp Leu Asn Ile Leu Ala Glu Leu 355 360 365cta cga ttt ggt gat aga gaa ttc tac aaa gat tgg tgg aat gca aaa 1211Leu Arg Phe Gly Asp Arg Glu Phe Tyr Lys Asp Trp Trp Asn Ala Lys370 375 380 385act gtt gaa gaa tac tgg aga atg tgg agc atg cca gtt cat aaa tgg 1259Thr Val Glu Glu Tyr Trp Arg Met Trp Ser Met Pro Val His Lys Trp 390 395 400atg gtt cgc cat atc tat ttc cca tgt ctg cgg cac aac att cct aag 1307Met Val Arg His Ile Tyr Phe Pro Cys Leu Arg His Asn Ile Pro Lys 405 410 415gga gta gca ata ctt att gcc ttc ttt gtt tct gca gca ttt cat gag 1355Gly Val Ala Ile Leu Ile Ala Phe Phe Val Ser Ala Ala Phe His Glu 420 425 430ttg tgt atc gca gtt cct tgc cac ata ttc aag ctg tgg gct ttt ctt 1403Leu Cys Ile Ala Val Pro Cys His Ile Phe Lys Leu Trp Ala Phe Leu 435 440 445ggg att atg ttc cag att cca ctg gtg tgg ata aca aac gtt cta cag 1451Gly Ile Met Phe Gln Ile Pro Leu Val Trp Ile Thr Asn Val Leu Gln450 455 460 465cag aag ttc aag agc tca atg gtg ggg aac atg ata ttc tgg tca atg 1499Gln Lys Phe Lys Ser Ser Met Val Gly Asn Met Ile Phe Trp Ser Met 470 475 480ttc tgc ata ttt ggt caa cca atg tgt gtg ctt cta tac tat cat gac 1547Phe Cys Ile Phe Gly Gln Pro Met Cys Val Leu Leu Tyr Tyr His Asp 485 490 495ttg atg aac agg aat ggg aaa gat gga atc tga aaagggaaac aaaaaacaac 1600Leu Met Asn Arg Asn Gly Lys Asp Gly Ile 500 505taattcttac ttggttcatt tcattagtgt tgttgttgcc ttggaaatgg agtgcatgct 1660tggttgcttt agaaaagagg agaaaaccaa agatacattg aggcgttgtc tgcaatgtaa 1720tggtaatgtt ggcgagaatg taagaaaaga agccatttat tcgaaaaaaa aaaaaaaa 17782507PRTLinum usitatissimum 2Met Ala Val Leu Asp Thr Pro Asp Asn His Ile Asn Pro Ser Pro Ser1 5 10 15Thr Ser Ala Ile Asp Ser Ser Asp Leu Asn Gly Leu Ser Leu Arg Arg 20 25 30Arg Ser Val Ala Thr Asn Ser Asp Gln Gly Thr Ser Ser Thr Ala Val 35 40 45Glu Ser Leu His Ala Asp Arg Pro Ala Asp Ser Asp Gly Ala Asn Arg 50 55 60Glu Asp Lys Lys Ile Asp Asn Arg Asp Gly Gln Val Ala Arg Ser Asp65 70 75 80Ile Lys Phe Thr Tyr Arg Pro Ser Val Pro Ala His Val Lys Val Lys 85 90 95Glu Ser Pro Leu Ser Ser Gly Ala Ile Phe Lys Gln Ser His Ala Gly 100 105 110Leu Phe Asn Leu Cys Ile Val Val Leu Val Ala Val Asn Ser Arg Leu 115 120 125Ile Ile Glu Asn Ile Met Lys Tyr Gly Trp Leu Ile Arg Thr Gly Phe 130 135 140Trp Phe Ser Ser Lys Ser Leu Arg Asp Trp Pro Leu Phe Met Cys Cys145 150 155 160Leu Thr Leu Pro Val Phe Ala Leu Ala Ala Tyr Leu Val Glu Lys Leu 165 170 175Ala Tyr Arg Lys Tyr Leu Ser Glu Pro Ile Val Val Ser Leu His Ile 180 185 190Ile Ile Ser Val Val Ala Val Val Tyr Pro Val Ser Val Ile Leu Ser 195 200 205Cys Asp Ser Ala Phe Val Ser Gly Val Thr Leu Met Leu Phe Ala Cys 210 215 220Ile Val Trp Leu Lys Leu Val Ser Tyr Ala His Thr Asn Tyr Asp Met225 230 235 240Arg Ala Val Ala Lys Ser Val Glu Lys Gly Glu Ala Pro Ser Ala Ala 245 250 255Leu Asn Val Asp Tyr Ser Tyr Asp Val Asn Phe Lys Ser Leu Val Tyr 260 265 270Phe Met Ile Ala Pro Thr Leu Cys Tyr Gln Pro Ser Tyr Pro Arg Thr 275 280 285Pro Cys Ile Arg Lys Gly Trp Leu Val His Gln Phe Ile Lys Leu Val 290 295 300Ile Phe Thr Gly Leu Met Gly Phe Ile Ile Glu Gln Tyr Ile Asn Pro305 310 315 320Ile Ile Gln Asn Ser Gln His Pro Phe Lys Gly Asn Leu Leu Tyr Gly 325 330 335Ile Glu Arg Val Leu Lys Leu Ser Val Pro Asn Leu Tyr Val Trp Leu 340 345 350Cys Met Phe Tyr Cys Phe Phe His Leu Trp Leu Asn Ile Leu Ala Glu 355 360 365Leu Leu Arg Phe Gly Asp Arg Glu Phe Tyr Lys Asp Trp Trp Asn Ala 370 375 380Lys Thr Val Glu Glu Tyr Trp Arg Met Trp Ser Met Pro Val His Lys385 390 395 400Trp Met Val Arg His Ile Tyr Phe Pro Cys Leu Arg His Asn Ile Pro 405 410 415Lys Gly Val Ala Ile Leu Ile Ala Phe Phe Val Ser Ala Ala Phe His 420 425 430Glu Leu Cys Ile Ala Val Pro Cys His Ile Phe Lys Leu Trp Ala Phe 435 440 445Leu Gly Ile Met Phe Gln Ile Pro Leu Val Trp Ile Thr Asn Val Leu 450 455 460Gln Gln Lys Phe Lys Ser Ser Met Val Gly Asn Met Ile Phe Trp Ser465 470 475 480Met Phe Cys Ile Phe Gly Gln Pro Met Cys Val Leu Leu Tyr Tyr His 485 490 495Asp Leu Met Asn Arg Asn Gly Lys Asp Gly Ile 500 50531029DNALinum usitatissimumCDS(1)..(1029)LuDGAT2B 3atg ggc cag aaa gta gag gag gaa aac cgg ctc gcc gga gga act gct 48Met Gly Gln Lys Val Glu Glu Glu Asn Arg Leu Ala Gly Gly Thr Ala1 5 10 15tct aat agg tgg cag gag aat gta aac ggt aat agc atc aac ggc ggc 96Ser Asn Arg Trp Gln Glu Asn Val Asn Gly Asn Ser Ile Asn Gly Gly 20 25 30gga gta gct acg att ttg aga tcc agt gat gtc gtc tct gga agc aaa 144Gly Val Ala Thr Ile Leu Arg Ser Ser Asp Val Val Ser Gly Ser Lys 35 40 45ttg aag tct tta ttg tcg ctt ggg ata tgg ctg ggg gct atc cat ttc 192Leu Lys Ser Leu Leu Ser Leu Gly Ile Trp Leu Gly Ala Ile His Phe 50 55 60aac gtc gcc tta ggt gtt gta tcc ttc gtc ttc ctc cct ttc tcc tat 240Asn Val Ala Leu Gly Val Val Ser Phe Val Phe Leu Pro Phe Ser Tyr65 70 75 80ttc ctg atg cta ttg gga ttt ctt ttg atg ttg atg ttc gtc cca atc 288Phe Leu Met Leu Leu Gly Phe Leu Leu Met Leu Met Phe Val Pro Ile 85 90 95aac gat tcc agc tac ttg ggc cgc cga ttc tgc aga tac gtt tgc aga 336Asn Asp Ser Ser Tyr Leu Gly Arg Arg Phe Cys Arg Tyr Val Cys Arg 100 105 110cat gcg tgt agt tac ttt ccg atc act ctt cac gtc gag gat atg aac 384His Ala Cys Ser Tyr Phe Pro Ile Thr Leu His Val Glu Asp Met Asn 115 120 125gcc ttc cgt tct gat cgt tct tac gtt ttc ggg tat gag ccg cat tct 432Ala Phe Arg Ser Asp Arg Ser Tyr Val Phe Gly Tyr Glu Pro His Ser 130 135 140gtt ctt ccc att ggc gtt gtt gct cta tcg gat cat gtg ggt ttt cta 480Val Leu Pro Ile Gly Val Val Ala Leu Ser Asp His Val Gly Phe Leu145 150 155 160cct cta ccg aaa att aaa gtt ctt gct ggc aca gct gtg ttc tac acc 528Pro Leu Pro Lys Ile Lys Val Leu Ala Gly Thr Ala Val Phe Tyr Thr 165 170 175cct ttc ctt aga cat ata tgg acg tgg tgt ggt ctt gcc ccg gca acc 576Pro Phe Leu Arg His Ile Trp Thr Trp Cys Gly Leu Ala Pro Ala Thr 180 185 190aag aag aat ttt acg tct ctc ttg gca tcc ggt tat agt tgc att gtg 624Lys Lys Asn Phe Thr Ser Leu Leu Ala Ser Gly Tyr Ser Cys Ile Val 195 200 205gtt cct ggt ggc gtt caa gaa gca ttt cac atg gaa cat gga gca gag 672Val Pro Gly Gly Val Gln Glu Ala Phe His Met Glu His Gly Ala Glu 210 215 220gtc gct ttc ctg aac aag cga aaa gga ttc gtt cgg tta gcc ata gag 720Val Ala Phe Leu Asn Lys Arg Lys Gly Phe Val Arg Leu Ala Ile Glu225 230 235 240atg ggt agc ccc ttg gtt cca gtt ttc tcc ttc ggt cag tca gat gtg 768Met Gly Ser Pro Leu Val Pro Val Phe Ser Phe Gly Gln Ser Asp Val 245 250 255tac aag tgg tgg aaa cct agg gga aag tgg ttc ttg gca ttt gcg aga 816Tyr Lys Trp Trp Lys Pro Arg Gly Lys Trp Phe Leu Ala Phe Ala Arg 260 265 270gct ata agg ttc acc cct att atc ttt tgg ggt ata ctc gga act cca 864Ala Ile Arg Phe Thr Pro Ile Ile Phe Trp Gly Ile Leu Gly Thr Pro 275 280 285ttg cca ttc cag caa ccg atg cat gtt gtg att ggc cga ccc atc gaa 912Leu Pro Phe Gln Gln Pro Met His Val Val Ile Gly Arg Pro Ile Glu 290 295 300ttt aag aaa aac gca cag cct agc atg gaa gag gtg gct gaa gtt cat 960Phe Lys Lys Asn Ala Gln Pro Ser Met Glu Glu Val Ala Glu Val His305 310 315 320ggg aag ttt gtt gca gca ctg aaa gac ctc ttt gat agg cac aaa gtg 1008Gly Lys Phe Val Ala Ala Leu Lys Asp Leu Phe Asp Arg His Lys Val 325 330 335gag gct ggt tgt gct gat ctt 1029Glu Ala Gly Cys Ala Asp Leu 3404343PRTLinum usitatissimum 4Met Gly Gln Lys Val Glu Glu Glu Asn Arg Leu Ala Gly Gly Thr Ala1 5 10 15Ser Asn Arg Trp Gln Glu Asn Val Asn Gly Asn Ser Ile Asn Gly Gly 20 25 30Gly Val Ala Thr Ile Leu Arg Ser Ser Asp Val Val Ser Gly Ser Lys 35 40 45Leu Lys Ser Leu Leu Ser Leu Gly Ile Trp Leu Gly Ala Ile His Phe 50 55 60Asn Val Ala Leu Gly Val Val Ser Phe Val Phe Leu Pro Phe Ser Tyr65 70 75 80Phe Leu Met Leu Leu Gly Phe Leu Leu Met Leu Met Phe Val Pro Ile 85 90 95Asn Asp Ser Ser Tyr Leu Gly Arg Arg Phe Cys Arg Tyr Val Cys Arg 100 105 110His Ala Cys Ser Tyr Phe Pro Ile Thr Leu His Val Glu Asp Met Asn 115 120 125Ala Phe Arg Ser Asp Arg Ser Tyr Val Phe Gly Tyr Glu Pro His Ser 130 135 140Val Leu Pro Ile Gly Val Val Ala Leu Ser Asp His Val Gly Phe Leu145 150 155 160Pro Leu Pro Lys Ile Lys Val Leu Ala Gly Thr Ala Val Phe Tyr Thr 165 170 175Pro Phe Leu Arg His Ile Trp Thr Trp Cys Gly Leu Ala Pro Ala Thr 180 185 190Lys Lys Asn Phe Thr Ser Leu Leu Ala Ser Gly Tyr Ser Cys Ile Val 195 200 205Val Pro Gly Gly Val Gln Glu Ala Phe His Met Glu His Gly Ala Glu 210 215 220Val Ala Phe Leu Asn Lys Arg Lys Gly Phe Val Arg Leu Ala Ile Glu225 230 235 240Met Gly Ser Pro Leu Val Pro Val Phe Ser Phe Gly Gln Ser Asp Val 245 250 255Tyr Lys Trp Trp Lys Pro Arg Gly Lys Trp Phe Leu Ala Phe Ala Arg 260 265 270Ala Ile Arg Phe Thr Pro Ile Ile Phe Trp Gly Ile Leu Gly Thr Pro 275 280 285Leu Pro Phe Gln Gln Pro Met His Val Val Ile Gly Arg Pro Ile Glu 290 295 300Phe Lys Lys Asn Ala Gln Pro Ser Met Glu Glu Val Ala Glu Val His305 310 315 320Gly Lys Phe Val Ala Ala Leu Lys Asp Leu Phe Asp Arg His Lys Val 325 330 335Glu Ala Gly Cys Ala Asp Leu 34051047DNALinum usitatissimumCDS(1)..(1047)LuDGT2B 5atg ggc cag aaa gta gag gag gaa gac cgg ctc gcc gga gga act gct 48Met Gly Gln Lys Val Glu Glu Glu Asp Arg Leu Ala Gly Gly Thr Ala1 5 10 15tct aat agc tgg cag gag aac gta aac ggt aat agc att aac ggt ggc 96Ser Asn Ser Trp Gln Glu Asn Val Asn Gly Asn Ser Ile Asn Gly Gly 20 25 30ggc ggc ggt gga gga gga gga gtt gct acg att ttg aga tcc act gat 144Gly Gly Gly Gly Gly Gly Gly Val Ala Thr Ile Leu Arg Ser Thr Asp 35 40 45gtc gtc tct aga agc aaa ttg aag tct tta ttg tcg ctt ggg ata tgg 192Val Val Ser Arg Ser Lys Leu Lys Ser Leu Leu Ser Leu Gly Ile Trp 50 55 60ctg ggg gct atc cat ttc aac gtc gcc tta gtt gtt gta tcc ttc gtc 240Leu Gly Ala Ile His Phe Asn Val Ala Leu Val Val Val Ser Phe Val65 70 75 80ttc ctc cct ttc tcc tat ttc ctg atg cta ttg gga ttt ctt ttg atg 288Phe Leu Pro Phe Ser Tyr Phe Leu Met Leu Leu Gly Phe Leu Leu Met 85 90 95ttg gtg ttc att cca atc aac gat tcc agc tac ttg ggc cgc cga ttc 336Leu Val Phe Ile Pro Ile Asn Asp Ser Ser Tyr Leu Gly Arg Arg Phe 100 105 110 tgc aga tac gtt tgc aga cat gcg tgt agt tac ttt ccg atc act ctt

384Cys Arg Tyr Val Cys Arg His Ala Cys Ser Tyr Phe Pro Ile Thr Leu 115 120 125cat gtc gag gat atc aac gcc ttc cgt tct gat cgt tct tac gtt ttc 432His Val Glu Asp Ile Asn Ala Phe Arg Ser Asp Arg Ser Tyr Val Phe 130 135 140ggg tac gag ccg cat tct gtt ctt ccc att ggc gtt gtt gtt cta tcg 480Gly Tyr Glu Pro His Ser Val Leu Pro Ile Gly Val Val Val Leu Ser145 150 155 160gat cat gtg ggt ttt ctg cct tta ccg aag ata aaa gtt ctt gct agc 528Asp His Val Gly Phe Leu Pro Leu Pro Lys Ile Lys Val Leu Ala Ser 165 170 175aca gct gtg ttc tat acc cct ttc ctt aga cat ata tgg acg tgg tgt 576Thr Ala Val Phe Tyr Thr Pro Phe Leu Arg His Ile Trp Thr Trp Cys 180 185 190 ggt ctt gcc ccg gca acc aag aag aat ttc acg tct ctc ttg gca tcc 624Gly Leu Ala Pro Ala Thr Lys Lys Asn Phe Thr Ser Leu Leu Ala Ser 195 200 205ggt tat agt tgc att gtg gtt ccc ggt ggc gtt caa gaa gca ttt cac 672Gly Tyr Ser Cys Ile Val Val Pro Gly Gly Val Gln Glu Ala Phe His 210 215 220atg gaa cat gga gta gag gtc gct ttc ctg aac aag cga aaa gga ttc 720Met Glu His Gly Val Glu Val Ala Phe Leu Asn Lys Arg Lys Gly Phe225 230 235 240gtt cgg tta gcc ata gag atg ggt agc ccc ttg gtt cct gtt ttc tcc 768Val Arg Leu Ala Ile Glu Met Gly Ser Pro Leu Val Pro Val Phe Ser 245 250 255ttc ggt cag tcg gat gtg tac aag tgg tgg aaa cct agg gga aag tgg 816Phe Gly Gln Ser Asp Val Tyr Lys Trp Trp Lys Pro Arg Gly Lys Trp 260 265 270 ttc ttg gca ttt gcg aga gtg att agg ttc acc cct att atc ttt tgg 864Phe Leu Ala Phe Ala Arg Val Ile Arg Phe Thr Pro Ile Ile Phe Trp 275 280 285ggt gta ctc gga act cca ttg cca ttc cgg caa cca atg cac gtt gtg 912Gly Val Leu Gly Thr Pro Leu Pro Phe Arg Gln Pro Met His Val Val 290 295 300atc ggc cga ccc atc gaa ttt aag aaa aac gca cag cct acc atg gaa 960Ile Gly Arg Pro Ile Glu Phe Lys Lys Asn Ala Gln Pro Thr Met Glu305 310 315 320gag gtg gct gaa gtt cat ggg cag ttt gtt gca gca ctg aaa gac ctc 1008Glu Val Ala Glu Val His Gly Gln Phe Val Ala Ala Leu Lys Asp Leu 325 330 335ttt gat agg cac aaa gtg gag gct ggc tgt gct gat ctt 1047Phe Asp Arg His Lys Val Glu Ala Gly Cys Ala Asp Leu 340 3456349PRTLinum usitatissimum 6Met Gly Gln Lys Val Glu Glu Glu Asp Arg Leu Ala Gly Gly Thr Ala1 5 10 15Ser Asn Ser Trp Gln Glu Asn Val Asn Gly Asn Ser Ile Asn Gly Gly 20 25 30Gly Gly Gly Gly Gly Gly Gly Val Ala Thr Ile Leu Arg Ser Thr Asp 35 40 45Val Val Ser Arg Ser Lys Leu Lys Ser Leu Leu Ser Leu Gly Ile Trp 50 55 60Leu Gly Ala Ile His Phe Asn Val Ala Leu Val Val Val Ser Phe Val65 70 75 80Phe Leu Pro Phe Ser Tyr Phe Leu Met Leu Leu Gly Phe Leu Leu Met 85 90 95Leu Val Phe Ile Pro Ile Asn Asp Ser Ser Tyr Leu Gly Arg Arg Phe 100 105 110Cys Arg Tyr Val Cys Arg His Ala Cys Ser Tyr Phe Pro Ile Thr Leu 115 120 125His Val Glu Asp Ile Asn Ala Phe Arg Ser Asp Arg Ser Tyr Val Phe 130 135 140Gly Tyr Glu Pro His Ser Val Leu Pro Ile Gly Val Val Val Leu Ser145 150 155 160Asp His Val Gly Phe Leu Pro Leu Pro Lys Ile Lys Val Leu Ala Ser 165 170 175Thr Ala Val Phe Tyr Thr Pro Phe Leu Arg His Ile Trp Thr Trp Cys 180 185 190Gly Leu Ala Pro Ala Thr Lys Lys Asn Phe Thr Ser Leu Leu Ala Ser 195 200 205Gly Tyr Ser Cys Ile Val Val Pro Gly Gly Val Gln Glu Ala Phe His 210 215 220Met Glu His Gly Val Glu Val Ala Phe Leu Asn Lys Arg Lys Gly Phe225 230 235 240Val Arg Leu Ala Ile Glu Met Gly Ser Pro Leu Val Pro Val Phe Ser 245 250 255Phe Gly Gln Ser Asp Val Tyr Lys Trp Trp Lys Pro Arg Gly Lys Trp 260 265 270Phe Leu Ala Phe Ala Arg Val Ile Arg Phe Thr Pro Ile Ile Phe Trp 275 280 285Gly Val Leu Gly Thr Pro Leu Pro Phe Arg Gln Pro Met His Val Val 290 295 300Ile Gly Arg Pro Ile Glu Phe Lys Lys Asn Ala Gln Pro Thr Met Glu305 310 315 320Glu Val Ala Glu Val His Gly Gln Phe Val Ala Ala Leu Lys Asp Leu 325 330 335Phe Asp Arg His Lys Val Glu Ala Gly Cys Ala Asp Leu 340 345739DNAArtificial sequencechemically synthetized 7ggccacgcgt cgactagtac tttttttttt tttttttvn 39821DNAartificial sequencechemically synthetized 8garttytayc angaytggtg g 21923DNAartifical sequencemisc_feature(1)..(23)oligonucleotide RS-008 9ggnacngcna trcanarytc rtg 231020DNAartificial sequencechemically synthetized 10garaanytna tgaartaygg 201123DNAartificial sequencechemically synthetized 11tantgytcna tdatraancc cat 231226DNAartificial sequencechemically synthetized 12gccatatcta tttcccatgt ctgcgg 261320DNAartificial sequencechemically synthetized 13ggccacgcgt cgactagtac 201419DNAartificial sequencechemically syntetized 14cactggaagt gttagacag 191538DNAartificial sequencechemically syntehtized 15ggccacgcgt cgactagtac gggggggggg gggggggn 381619DNAartificial sequencechemically sytnetized 16actgaaccag aagcctgtc 191719DNAartificial sequencechemically syntehtized 17cggaactaag cggactctc 191819DNAartificial sequencechemically synthetized 18ggcacggaag ggcggtaag 191922DNAartificial sequencechemically synthtetized 19caagttagta atatttacag gc 222022DNAartificial sequencechemically synthetized 20tccacattct ccagtattct tc 222142DNAartificial sequencechemically synthetized 21attaggatcc gaccatgggc gtgctcgaca ctcctgacaa tc 422231DNAartificial sequencechemically synthetized 22tttaagcttg attccatctt tcccattcct g 31238PRTLinum usitatissimumPEPTIDE(1)..(8)LuDGAT1 residues from 253 to 259 23Ala Pro Ser Ala Ala Leu Asn Val1 5247PRTLinum usitatissimum 24Glu Ser Pro Leu Ser Ser Asp1 5254PRTBrassicaPEPTIDE(1)..(4)amino acid sequences in DGAT position 301-307 25Leu Ala Tyr Phe1264PRTLinum usitatissimumPEPTIDE(1)..(4)amino acid sequence in LuDGAT in positions 311-314 26Leu Val Tyr Phe1277PRTBrassica napusPEPTIDE(1)..(7)amino acid sequence of DGAT in positions 234-241 27Met Trp Asn Met Pro Val His1 528176DNALinum usitatissimummisc_feature(1)..(176)isolated nucleotide fragment amplicon 1 28tggtggaatg caaaaactgt tgaagaatac tggagaatgt ggagcatgcc agttcataaa 60tggatggttc gccatatcta tttcccatgt ctgcggcaca acattcctaa gggagtagca 120atacttattg ccttctttgt ttctgcagca tttcatgagt tgtgtatcgc agttcc 17629539DNAlinum usitatissimummisc_feature(1)..(539)isolated polynucleotide frgment amplicon 2. 29atgaagtatg gttggttaat taggacaggc ttctggttca gttcaaaatc gttgagattg 60gcctcttttc atgtgctgtc taacacttcc agtgttcgcg cttgctgcat atctagttga 120ggaaattggc gtaccggaaa tatctctctg aacctatagt cgtttccctc cacataatca 180tctccgtggg tagcagttgt gtaccctgtt tcagtgattc tcagctgcga ctctgcattt 240gtatctggtg tgacgttgat gctttttgct tgcattgtgt ggttaaaatt ggtctcatat 300gctcatacga actatgatat gagagcggtt gccaagtcag ttgaaaaggg agaagcacct 360tctgctgctt tgaatgttga ttactcttat gacgttaact tcaagagctt ggtgtatttt 420atgattgctc caacactgtg ctatcagcca agctatccac gcactccatg tatccgaaag 480ggttggctgg ttcatcagtt catcaagtta gtaatattta caggcttgat gggcttcat 53930275DNALinum usitatissimummisc_feature(1)..(275)isolated RT-PCR polynucleotide fragment 30ttagtaatat ttacaggctt gatgggattc attatagagc aatatatcaa tccaattatc 60cagaactctc agcatccttt taaagggaat ctattatatg gaattgagag ggttttgaaa 120ctttcggtcc caaacttgta tgtgtggctg tgcatgttct actgcttctt tcatctatgg 180taaatatact tgcagagctc ctacgatttg gtgatagaga attctacaaa gattggtgga 240atgcaaaaac tgttgaagaa tactggagaa tgtgg 27531438DNALinum usitatissimummisc_feature(1)..(438)isolated 5'RACE polynucleotide fragment 31cggtgctcga cacccctgac aatcacataa acccctctcc ctccacctct gctattgact 60cctccgatct taacggtctc tcccttcgac gtcgttcagt tgccactaac tcccgaccaa 120ggtacttctt ccaccgctgt agaatcactc cacgcggatc ggccagccga ttctgatggg 180gcgaaccgcg aggataagaa gattgacaat cgggacggtc aagttgcgag atcggatatc 240aaattcactt accgcccttc cgtgcccgct cacgtcaagg ttaaagagag tccgcttagt 300tccggcgcca tttttaagca gagccatgca ggcctcttca atctctgtat tgtagtccta 360gttgcagtca acagcaggct tattatcgag aatatcatga agtatggttg gttaattagg 420acaggcttct ggttcagt 43832327DNALinum usitatissimummisc_feature(1)..(327)isolated 5'RACEB polynucleotide fragment 32ctttcggtgt gttatcatcg ttctttctgc gactgcttcc cccctctcct cttccaatgg 60cggtgctcga cacccctgac aatcacataa acccctctcc ctccacctct gctattgact 120ccctccgatc ttaacggtct ctcccttcga cgtcgttcag ttgccactaa ctccgaccaa 180ggtacttctt ccaaccgctg tagaatcact ccacgcggat cggccagccg attctgatgg 240ggcgaaccgc gaggataaga agattgacaa tcgggacggt caagttgcga gatcggatat 300caaattcact taccgccctt ccgtgcc 32733511DNALinum usitatissimummisc_feature(1)..(511)isolated 3'RACE polynucleotide fragment 33gccatatcta tttcccatgt ctgcggcaca acattcctaa gggagtagca atacttattg 60ccttctttgt ttctgcagca tttcatgagt tgtgtatcga gttccttgcc acatattcaa 120gctgtgggct tttcttggga ttatgttcca gattccactg gtgtggataa caaacgttct 180acagcagaag ttcaagagct caatggtggg gaacatgata ttctggtcaa tgttctgcat 240atttggtcaa ccaatgtgtg tgcttctata ctatcatgac ttgatgaaca ggaatgggaa 300agatggaatc tgaaaaggga aacaaaaaac aactaattct tacttggttc atttcattag 360tgttgttgtt gccttggaaa tggagtgcat gcttggttgt tttagaaaag aggagaaaac 420caaagataca ttgaggcgtt gtctgcaatg taatggtaat gttggcgaga atgtaagaaa 480agaagccatt tattcgaaaa aaaaaaaaaa a 511

Claims

1. An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from: at least 300, at least 400 or at least 500 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 2 or of an amino acid sequence having at least 85% sequence identity therewith; at least 300 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 4 or of an amino acid sequence having at least 85% sequence identity therewith; or at least 300 contiguous residues of the amino acid sequence depicted in SEQ ID NO: 6 or of an amino acid sequence having at least 85% sequence identity therewith.

2. The isolated polynucleotide of claim 1, wherein the encoded polypeptide comprises the amino acid sequence depicted in SEQ ID NO: 2.

3. The isolated polynucleotide of claim 1, wherein the encoded polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO: 1 from nucleotide 57 to nucleotide 1580.

4. The isolated polynucleotide of claim 1, wherein the encoded polypeptide comprises the amino acid sequence depicted in SEQ ID NO: 4.

5. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO: 3 from nucleotide 1 to nucleotide 1029.

6. The isolated polynucleotide of claim 1, wherein the encoded polypeptide comprises the amino acid sequence depicted in SEQ ID NO: 6.

7. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO: 5 from nucleotide 1 to nucleotide 1048.

8. The isolated polynucleotide of claim 1, wherein the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2.

9. The isolated polynucleotide of claim 1, wherein the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4.

10. The isolated polynucleotide of claim 1, wherein the encoded polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6.

11. A polynucleotide construct comprising a polynucleotide of claim 1 operably linked to a promoter expressible in bacterial, yeast, fungal, mammalian or plant cells.

12. A vector comprising a polynucleotide of claim 1.

13. A microbial cell comprising a polynucleotide of claim 1.

14. The microbial cell of claim 13, wherein the cell is Saccharomyces cerevisiae.

15. A transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore, comprising a polynucleotide of claim 1.

16. The transgenic plant, plant cell, plant seed, callus, plant embryo, microspore-derived embryo, or microspore of claim 17, which is selected from flax, canola, soybean, mouse-ear cress, castor, sunflower, linola, oats, wheat, triticale, barley, corn or Brachypodium distachyon plant, plant cell, plant seed, plant embryo, or microspore.

17. A method for producing oil, comprising the steps of: growing a transgenic plant according to claim 15; and recovering oil which is produced by the plant.

18. The method according to claim 17, wherein the plant is selected from flax, canola, soybean, mouse-ear cress, castor, sunflower, linola, oats, wheat, triticale, barley, corn or Brachypodium distachyon plant.

19. A method for producing a transgenic plant comprising the steps of: introducing into a plant cell or a plant tissue a polynucleotide of claim 1 to produce a transformed cell or plant tissue; and cultivating the transformed plant cell or transformed plant tissue to produce the transgenic plant.

20. The method of claim 19, wherein the plant is selected from flax, canola, soybean, mouse-ear cress, castor, sunflower, linola, oats, wheat, triticale, barley, corn or Brachypodium distachyon plant.