| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 09.99 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 14.09.99 |
| PATENT TITLE |
Fatty acid desaturase genes from plants |
| PATENT ABSTRACT | The preparation and use of nucleic acid fragments encoding fatty acid desaturase enzymes are described. The invention permits alteration of plant lipid composition. Chimeric genes incorporating such nucleic acid fragments with suitable regulatory sequences may be used to create transgenic plants with altered levels of unsaturated fatty acids. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 26.08.94 |
| PATENT CT FILE DATE | 03.12.92 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | 10.06.93 |
| PATENT REFERENCES CITED |
Walter, M.H. et al, Mol. Gen. Genet, 222, 353-360 (1990). Browse, J. et al, Plant Physiol., 81, 859-864 (1986). Wada, H. et al, Nature, 347, 200-203 (1990). Lemieux, B. et al, Theor. Appl. Genet, 80, 234-240 (1990). Brockman, J. et al, Biological Abstracts, 89(12), Abstract No. 131194 (Jun. 1990). Thompson, G.A. et al, Proc. Natl. Acad. Sci. USA, 88, 2578-2582 (1991). Browse, J., Annu. Rev. Plant Physiol. Plant Mol. Biol., 42, 467-506 (1991). Mattson, F.H. et al, Journal of Lipid Research, 26, 194-202 (1985). Gailliard, T. In: Stumpf, P.K. Ed., The Biochemistry of Plants, 4, 85-116, Academic Press, NY (1980). Mensink, R.P. et al, New England J. Med., N323, 439-445 (1990). Ohlrogge, J.G. et al, Biochim. Biophys. Acta., 1082, 1-26 (1991). Knowles, P.F. In, Applewhite, T.H. Ed., World Conf. on Biotechnology for the Fats and Oils Industry Proceedings, American Oil Chemists' Society, pp. 35-38 (1980). Wang, X.M. et al, Plant Physiol. Biochem., 26, 777-792 (1988). Gunstone et al, Eds. The Lipds Handbook, Chapman and Hall, Ltd., Cambridge (1986) pp. 55-112. Shanklin, J. et al, Proc. natl. Acad. Sci, USA, 88, 2510-2514 (1991). Stukey, J.E. et al, J. Biol. Chem., 265, 20144-20149 (1990). Thiede, et al, J. Biol. Chem., 261, 13230-13235 (1986). Kaestner, K.H. et al, J. Biol. Chem., 264, 14755-14756 (1989). Browse, J. et al, UCLA Symp. Mol. Cell. Biol.; New Ser., Plant Gene Transfer, 129, 301-309 (1990). Somerville, C. et al, Science, 252, 80-87 (1991). Arondel, V. et al, Science, 258, 1353-1355 (1992). |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. An isolated nucleic acid fragment encoding a plant plastid or microsomal delta-15 fatty acid desaturase enzyme, which catalyzes a reaction at C15-C16 of a fatty acyl chain and further wherein said isolated nucleic acid fragment hybridizes to one of the nucleotide sequences set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, and 15 under one of the following sets of conditions: (a) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt's, 0.5% sodium dodecyl sulfate (SDS), 100 .mu.g denatured calf thymus DNA at 50.degree. C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, repeat with 2X SSC, 0.5% SDS at 45.degree. C. for 30 min, then repeat twice with 0.2X SSC, 0.5% SDS at 50.degree. C. for 30 min each; (b) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt's, 0.5% sodium dodecyl sulfate (SDS), 100 .mu.g denatured calf thymus DNA at 50.degree. C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, repeat with 2X SSC, 0.5% SDS at 45.degree. C. for 30 min, then repeat twice with 0.2X SSC, 0.5% SDS at 60.degree. C. for 30 min each; (c) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50.degree. C. for eighteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, then washing for 5 min at 50.degree. C. in 0.2X SSPE, 1% SDS; (d) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50.degree. C. for sixteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, then wash with fresh solution for 10 min, then wash for 5 min at 50.degree. C. in 0.5X SSPE, 1% SDS; (e) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt's, 0.5% sodium dodecyl sulfate (SDS), 100 .mu.g denatured calf thymus DNA at 50.degree. C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, then wash twice with 2X SSC, 0.5% SDS at 45.degree. C. for 30 min each and then wash twice with 0.2X SSC, 0.5% SDS at 60.degree. C. for 30 min each; or (f) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50.degree. C. for eighteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, followed by washing for 5 min at 50.degree. C. in 0.2X SSPE, 1% SDS. 2. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a plant plastid or microsomal enzyme which catalyzes the formation of a double bond between carbon positions 3 and 4 numbered from the methyl end of a fatty acyl chain, and further wherein the amino acid sequence comprising said enzyme contains at least one of the following amino acid sequences selected from the group consisting of FVLGHDCGHGSF, GHDCGH, HDIGTHVIHHLFP, HDIGTH, or HVIHHL. 3. An isolated nucleic acid fragment encoding a plant plastid or microsomal enzyme which catalyzes the formation of a double bond between carbon positions 3 and 4 numbered from the methyl end of a fatty acyl chain, wherein said isolated nucleic acid fragment encodes a protein comprising any one of the amino acid sequences set forth in SEQ ID NOS:2, 5, 7, 9, 11, 12, 15 or 17. 4. An isolated nucleic acid fragment encoding an enzyme which catalyzes a reaction at C15-C16 of a fatty acyl chain wherein said isolated nucleic acid fragment hybridizes to the isolated nucleic acid fragment of claim 3 under one of the following sets of conditions: (a) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt's, 0.5% sodium dodecyl sulfate (SDS), 100 .mu.g denatured calf thymus DNA at 50.degree. C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, repeat with 2X SSC, 0.5% SDS at 45.degree. C. for 30 min, then repeat twice with 0.2X SSC, 0.5% SDS at 50.degree. C. for 30 min each; (b) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt's, 0.5% sodium dodecyl sulfate (SDS), 100 .mu.g denatured calf thymus DNA at 50.degree. C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, repeat with 2X SSC, 0.5% SDS at 45.degree. C. for 30 min, then repeat twice with 0.2X SSC, 0.5% SDS at 60.degree. C. for 30 min each; (c) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50.degree. C. for eighteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, then washing for 5 min at 50.degree. C. in 0.2X SSPE, 1% SDS; (d) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50.degree. C. for sixteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, then wash with fresh solution for 10 min, then wash for 5 min at 50.degree. C. in 0.5X SSPE, 1% SDS; (e) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt's, 0.5% sodium dodecyl sulfate (SDS), 100 .mu.g denatured calf thymus DNA at 50.degree. C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, then wash twice with 2X SSC, 0.5% SDS at 45.degree. C. for 30 min each and then wash twice with 0.2X SSC, 0.5% SDS at 60.degree. C. for 30 min each; or (f) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50.degree. C. for eighteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, followed by washing for 5 min at 50.degree. C. in 0.2X SSPE, 1% SDS. 5. An isolated nucleic acid fragment of claim 1, claim 2, claim claim 3 or claim 4 wherein said fragment is isolated from a plant selected from the group consisting of soybean, oilseed Brassica species, Arabidopsis thaliana and corn. 6. A chimeric gene capable of causing altered levels of linolenic acid in a transformed plant cell, the gene comprising a nucleic acid fragment of any of claims 1, claim 2, claim 3 or claim 4 the fragment operably linked to suitable regulatory sequences. 7. Plants containing the chimeric gene of claim 6. 8. A method of producing seed oil containing altered levels of linolenic (18:3) acid comprising: (a) transforming a plant cell of an oil-producing species with a chimeric gene of claim 5; (b) growing fertile plants from the transformed plant cells of step (a); (c) screening progeny seeds from the fertile plants of step (b) for the desired levels of linolenic (18:3) acid; and (d) processing the progeny seed of step (c) to obtain seed oil containing altered levels of linolenic (18:3) acid. 9. A method of claim 8 wherein said plant cell of an oil-producing species is selected from the group consisting of Arabidopsis thaliana, soybean, oilseed Brassica napus, sunflower, cotton, peanut, and corn. 10. The isolated genomic DNA of Arabidopsis thaliana comprising the microsomal delta-15 desaturase identified by accession number ATCC 75167. 11. A plasmid designated pXF1 and bearing accession number ATCC 68874 comprising the DNA sequence of SEQ ID NO:10 which encodes a soybean delta-15 desaturase enzyme. 12. A plasmid designated pBNSF3 and bearing accession number ATCC 68854 comprising the DNA sequence of SEQ ID NO:6 which encodes an oilseed Brassica species delta-15 desaturase enzyme. 13. An isolated Polymerase Chain Reaction Product designated PCR20 comprising the DNA sequence of SEQ ID NO:14 which encodes a portion of the Zea mays delta-15 desaturase enzyme. 14. The method of claim 8 wherein said plant cell of an oil-producing species is selected from the group consisting of Arabidopsis thaliana, soybean, oilseed Brassica napus, sunflower, cotton and corn. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The invention relates to the preparation and use of nucleic acid fragments encoding fatty acid desaturase enzymes to modify plant lipid composition. BACKGROUND OF THE INVENTION Plant lipids have a variety of industrial and nutritional uses and are central to plant membrane function and climatic adaptation. These lipids represent a vast array of chemical structures, and these structures determine the physiological and industrial properties of the lipid. Many of these structures result either directly or indirectly from metabolic processes that alter the degree of unsaturation of the lipid. Different metabolic regimes in different plants produce these altered lipids, and either domestication of exotic plant species or modification of agronomically adapted species is usually required to economically produce large amounts of the desired lipid. Plant lipids find their major use as edible oils in the form of triacylglycerols. The specific performance and health attributes of edible oils are determined largely by their fatty acid composition. Most vegetable oils derived from commercial plant varieties are composed primarily of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) acids. Palmitic and stearic acids are, respectively, 16- and 18-carbon-long, saturated fatty acids. Oleic, linoleic, and linolenic acids are 18-carbon-long, unsaturated fatty acids containing one, two, and three double bonds, respectively. Oleic acid is referred to as a mono-unsaturated fatty acid, while linoleic and linolenic acids are referred to as poly-unsaturated fatty acids. The relative amounts of saturated and unsaturated fatty acids in commonly used, edible vegetable oils are summarized below (Table 1): TABLE 1 ______________________________________ Percentages of Saturated and Unsaturated Fatty Acids in the Oils of Selected Oil Crops Mono- Poly- Saturated unsaturated unsaturated ______________________________________ Canola 6% 58% 36% Soybean 15% 24% 61% Corn 13% 25% 62% Peanut 18% 48% 34% Safflower 9% 13% 78% Sunflower 9% 41% 51% Cotton 30% 19% 51% ______________________________________ Many recent research efforts have examined the role that saturated and unsaturated fatty acids play in reducing the risk of coronary heart disease. In the past, it was believed that mono-unsaturates, in contrast to saturates and poly-unsaturates, had no effect on serum cholesterol and coronary heart disease risk. Several recent human clinical studies suggest that diets high in mono-unsaturated fat and low in saturated fat may reduce the "bad" (low-density lipoprotein) cholesterol while maintaining the "good" (high-density lipoprotein) cholesterol (Mattson et al., Journal of Lipid Research (1985) 26:194-202). A vegetable oil low in total saturates and high in mono-unsaturates would provide significant health benefits to consumers as well as economic benefits to oil processors. As an example, canola oil is considered a very healthy oil. However, in use, the high level of poly-unsaturated fatty acids in canola oil renders the oil unstable, easily oxidized, and susceptible to development of disagreeable odors and flavors (Gailliard, 1980, Vol.4, pp. 85-116 In: Stumpf, P. K., Ed., The Biochemistry of Plants, Academic Press, New York). The levels of poly-unsaturates may be reduced by hydrogenation, but the expense of this process and the concomitant production of nutritionally questionable trans isomers of the remaining unsaturated fatty acids reduces the overall desirability of the hydrogenated oil (Mensink et al., New England J. Medicine (1990) N323: 439-445). Similar problems exist with soybean and corn oils. For specialized uses, high levels of poly-unsaturates can be desirable. Linoleate and linolenate are essential fatty acids in human diets, and an edible oil high in these fatty acids can be used for nutritional supplements, for example in baby foods. Linseed oil, derived from the Flax plant (Linum usitatissimum), contains over 50% linolenic acid and has widespread use in domestic and industrial coatings since the double bonds of the fatty acids react rapidly with oxygen to polymerize into a soft and flexible film. Although the oil content of flax is comparable to canola (around 40% dry weight of seed), high yields are only obtained in warm temperatures or subtropical climates. In the USA flax is highly susceptible to rust infection. It will be commercially useful if a crop such as soybean or canola could be genetically transformed by the appropriate desaturase gene(s) to synthesize oils with a high linolenic acid content. Mutation-breeding programs have met with some success in altering the levels of poly-unsaturated fatty acid levels found in the edible oils of agronomic species. Examples of commercially grown varieties are high (85%) oleic sunflower and low (2%) linolenic flax (Knowles, (1980) pp. 35-38 In: Applewhite, T. E., Ed., World Conference on Biotechnology for the Fats and Oils Industry Proceedings, American Oil Chemists' Society). Similar commercial progress with the other plants shown in Table 1 has been largely elusive due to the difficult nature of the procedure and the pleiotropic effects of the mutational regime on plant hardiness and yield potential. The biosynthesis of the major plant lipids has been the focus of much research (Browse et al., Ann. Rev. Plant Physiol. Mol. Biol. (1991) 42:467-506). These studies show that, with the notable exception of the soluble stearoyl-acyl carrier protein desaturase, the controlling steps in the production of unsaturated fatty acids are largely catalyzed by membrane-associated fatty acid desaturases. Desaturation reactions occur in plastids and in the endoplasmic reticulum using a variety of substrates including galactolipids, sulfolipids, and phospholipids. Genetic and physiological analyses of Arabidopsis thaliana nuclear mutants defective in various fatty acid desaturation reactions indicates that most of these reactions are catalyzed by enzymes encoded at single genetic loci in the plant. The analyses show further that the different defects in fatty acid desaturation can have profound and different effects on the ultra-structural morphology, cold sensitivity, and photosynthetic capacity of the plants (Ohlrogge, et al., Biochim. Biophys. Acta (1991) 1082:1-26). However, biochemical characterization of the desaturase reactions has been meager. The instability of the enzymes and the intractability of their proper assay has largely limited researchers to investigations of enzyme activities in crude membrane preparations. These investigations have, however, demonstrated the role of delta-12 desaturase and delta-15 desaturase activities in the production of linoleate and linolenate from 2-oleoyl-phosphatidylcholine and 2-linoleoyl-phosphatidylcholine, respectively (Wang et al., Plant Physiol. Biochem. (1988) 26:777-792). Thus, modification of the activities of these enzymes represents an attractive target for altering the levels of lipid unsaturation by genetic engineering. Genes from plants for stearoyl-acyl carrier protein desaturase, the only soluble fatty acid desaturase known, have been described (Thompson, et al., Proc. Natl. Acad. Sci. U.S.A. (1991) 88:2578-2582; Shanklin et al., Proc. Natl. Acad. Sci. USA (1991) 88:2510-2514). Stearoyl-coenzyme-A desaturase genes from yeast, rat, and mice have also been described (Stukey, et al., J. Biol. Chem.(1990) 265:20144-20149; Thiede, et al., J. Biol. Chem. (1986) 261:13230-13235; Kaestner, et al., J. Biol. Chem. (1989) 264:14755-1476). No evidence exists in the public art that describes the isolation of fatty acid desaturases other than stearoyl-ACP desaturases from higher plants or their corresponding genes. A fatty acid desaturase gene from the cyanobacterium, Synechocystis PCC 6803, has also been described (Wada, et al., Nature (1990) 347:200-203). This gene encodes a fatty acid desaturase, designated des A, that catalyzes the conversion of oleic acid at the 1 position of galactolipids to linoleic acid. However, these genes have not proven useful for isolating plant fatty acid desaturases other than stearoyl-ACP desaturase via sequence-dependent protocols, and the present art does not indicate how to obtain plant fatty acid desaturases other than stearoyl-ACP desaturases or how to obtain fatty acid desaturase-related enzymes. Thus, the present art does not teach how to obtain glycerolipid desaturases from plants. Furthermore, there is no evidence that a method to control the nature and levels of unsaturated fatty acids in plants using nucleic acids encoding fatty acid desaturases other than stearoyl-ACP desaturase is known in the art. The biosynthesis of the minor plant lipids has been less well studied. While hundreds of different fatty acids have been found, many from the plant kingdom, only a tiny fraction of all plants have been surveyed for their lipid content (Gunstone, et al., Eds., (1986) The Lipids Handbook, Chapman and Hall Ltd., Cambridge). Accordingly, little is known about the biosynthesis of these unusual fatty acids and fatty acid derivatives. Interesting chemical features found in such fatty acids include, for example, allenic and conjugated double bonds, acetylenic bonds, trans double bonds, multiple double bonds, and single double bonds in a wide number of positions and configurations along the fatty acid chain. Similarly, many of the structural modifications found in unusual lipids (e.g., hydroxylation, epoxidation, cyclization, etc.) are probably produced via further metabolism following chemical activation of the fatty acid by desaturation or they involve a chemical reaction that is mechanistically similar to desaturation. For example, evidence for the mechanism of hydroxylation of fatty acids being part of a general mechanism of enzyme-catalyzed desaturation in eukaryotes has been obtained by substituting a sulfur atom in the place of carbon at the delta-9 position of stearic acid. When incubated with yeast cell extracts the thiostearate was converted to a 9-sulfoxide (Buist et al. (1987) Tetrahedron Letters 28:857-860). This sulfoxidation was specific for sulfur at the delta-9 position and did not occur in a yeast delta-9-desaturase deficient mutant (Buist & Marecak (1991) Tetrahedron Letters 32:891-894). The 9-sulfoxide is the sulfur analogue of 9-hydroxyoctadecastearate, the proposed intermediate of stearate desaturation. Thus fatty-acid desaturase cDNAs may serve as useful probes for cDNAs encoding fatty-acid hydroxylases and other cDNAs which encode enzymes with reaction mechanisms similar to fatty-acid desaturation. Many of these fatty acids and derivatives having such features within their structure could prove commercially useful if an agronomically viable species could be induced to synthesize them by introduction of a gene encoding the appropriate desaturase. SUMMARY OF THE INVENTION Applicants have discovered a means to control the nature and levels of unsaturated fatty acids in plants. Nucleic acid fragments from glycerolipid desaturase cDNAs or genes are used to create chimeric genes. The chimeric genes may be used to transform various plants to modify the fatty acid composition of the plant or the oil produced by the plant. More specifically, one embodiment of the invention is an isolated nucleic acid fragment comprising a nucleotide sequence encoding a plant delta-15 fatty acid desaturase or a fatty acid desaturase-related enzyme with an amino acid identity of 50%, 65%, 90% or greater to the polypeptide encoded by SEQ ID NOS:1, 4, 6, 8, 10, 12, 14, or 16. The isolated fragment in these embodiments is isolated from a plant selected from the group consisting of soybean, oilseed Brassica species, Arabidopsis thaliana and corn. Another embodiment of this invention involves the use of these nucleic acid fragments in sequence-dependent protocols. Examples include use of the fragments as hybridization probes to isolate other glycerolipid desaturase cDNAs or genes. A related embodiment involves using the disclosed sequences for amplification of DNA fragments encoding other glycerolipid desaturases. Another aspect of this invention involves chimeric genes capable of causing altered levels of the linolenic acid in a transformed plant cell, the gene comprising nucleic acid fragments encoding encoding a plant delta-15 fatty acid desaturase or a fatty acid desaturase-related enzyme with an amino acid identity of 50%, 65%, 90% or greater to the polypeptide encoded by SEQ ID NOS:1, 4, 6, 8, 10, 12, 14, or 16 operably linked in suitable orientation to suitable regulatory sequences. Preferred are those chimeric genes which incorporate nucleic acid fragments encoding delta-15 fatty acid desaturase cDNAs or genes. Plants and oil from seeds of plants containing the chimeric genes described are also claimed. Yet another embodiment of the invention involves a method of producing seed oil containing altered levels of linolenic (18:3) acid comprising: (a) transforming a plant cell with a chimeric gene described above; (b) growing fertile plants from the transformed plant cells of step (a); (c) screening progeny seeds from the fertile plants of step (b) for the desired levels of linolenic (18:3) acid, and (d) processing the progeny seed of step (c) to obtain seed oil containing altered levels of the unsaturated fatty acids. Preferred plant cells and oils are derived from soybean, rapeseed, sunflower, cotton, cocoa, peanut, safflower, coconut, flax, oil palm, and corn. Preferred methods of transforming such plant cells would include the use of Ti and Ri plasmids of Agrobacterium, electroporation, and high-velocity ballistic bombardment. The invention also is embodied in a method of breeding plant species to obtain altered levels of poly-unsaturated fatty acids, specifically linolenic (18:3) acid in seed oil of oil-producing plants. This method involves (a) making a cross between two varieties of an oilseed plant differing in the linolenic acid trait; (b) making a Southern blot of restriction enzyme digested genomic DNA isolated from several progeny plants resulting from the cross of step (a); and (c) hybridizing the Southern blot with the radiolabeled nucleic acid fragments encoding the claimed glycerolipid desaturases. The invention is also embodied in a method of RFLP mapping that uses the isolated Arabidopsis thaliana delta-15 desaturase sequences described herein. The invention is also embodied in plants capable of producing altered levels of glycerolipid desaturase by virtue of containing the chimeric genes described herein. Further, the invention is embodied by seed oil obtained from such plants. The invention is also embodied in a method of RFLP mapping ina genomic RFLP marker comprising (a) making a cross between two varieties of plants; (b) making a Southern blot of restriction enzyme digested genomic DNA isolated from several progeny plants resulting from the cross of step (a); and (c) hybridizing the Southern blot with a radiolabelled nucleic acid fragments of the claimed fragments. The invention is also embodied in a method to isolate nucleic acid fragments encoding fatty acid desaturases and fatty acid desaturase-related enzymes, comprising (a)comparing SEQ ID NOS:2, 5, 7, 9, 11, 13, 15 and 17 with other fatty acid desaturase polypeptide sequences; (b) identifying the conserved sequence(s) of 4 or more amino acids obtained in step a; (c) making region-specific nucleotide probe(s) or oligomer(s) based on the conserved sequences identified in step b; and d) using the nucleotide probe(s) or oligomers(s) of step c to isolate sequences encoding fatty acid desaturases and fatty-acid desaturase-related enzymes by sequence-dependent protocols. The product of the method of isolation method described is also part of the invention. BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS The invention can be more fully understood from the following detailed description and the Sequence Descriptions which form a part of this application. The Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter code for amino acids in conformity with the IUPAC-IUB standard described in Nucleic Acids Research 13:3021-3030 (19085) and 37 C.F.R. 1.822 which are incorporated herein by reference. SEQ ID NO:1 shows the complete 5' to 3' nucleotide sequence of 1350 base pairs of the Arabidopsis cDNA which encodes delta-15 desaturase in plasmid pCF3. Nucleotides 46 to 48 are the putative initiation codon of the open reading frame (nucleotides 46 to 1206). Nucleotides 1204 to 1206 are the termination codon. Nucleotides 1 to 45 and 1207 to 1350 are the 5' and 3' untranslated nucleotides, respectively. The 386 amino acid protein sequence in SEQ ID NO:1 is that deduced from the open reading frame. SEQ ID NO:2 is the deduced peptide of the open-reading frame of SEQ ID NO:1. SEQ ID NO:3 is a partial nucleotide sequence of the Arabidopsis genomic DNA insert in plasmid pF1 which shows the genomic sequence in the region of the Arabidopsis genome that encodes delta-15 desaturase. Nucleotides 68-255 are identical to nucleotides 1-188 of SEQ ID NO:1. Nucleotides 47 to 49 and 56 to 58 are termination codons in the same reading frame as the open reading frame in SEQ ID NO:1. SEQ ID NO:4 shows the 5' to 3' nucleotide sequence of the insert in plasmid pACF2-2 of 1525 base pairs of the Arabidopsis thaliana cDNA that encodes a plastid delta-15 fatty acid desaturase. Nucleotides 10-12 and nucleotides 1348 to 1350 are, respectively, the putative initiation codon and the termination codon of the open reading frame (nucleotides 10 to 1350). Nucleotides 1 to 9 and 1351 to 1525 are, respectively, the 5' and 3' untranslated nucleotides. SEQ ID NO:5 is the deduced peptide of the open reading frame of SEQ ID NO:4. SEQ ID NO:6 shows the complete 5' to 3' nucleotide sequence of 1336 base pairs of the Brassica napus seed cDNA, found in plasmid pBNSF3-2, which encodes a microsomal delta-15 glycerolipid desaturase. Nucleotides 79 to 82 are the putative initiation codon of the open reading frame (nucleotides 79 to 1212). Nucleotides 1210 to 1212 are the termination codon. Nucleotides 1 to 78 and 1213 to 1336 are the 5' and 3' unstranslated nucleotides respectively. SEQ ID NO:7 is the deduced peptide of the open reading frame of SEQ ID NO:6. SEQ ID NO:8 is the complete 5' to 3' nucleotide sequence of 1416 base pairs of the Brassica napus seed cDNA found in plasmid pBNSFd-2 which encodes a plastid delta-15 glycerolipid desaturase. Nucleotides 1 to 1215 correspond to a continuous open reading frame of 404 amino acids. Nucleotides 1213 to 1215 are the termination codon. Nucleotides 1215 to 1416 are the 3' untranslated nucleotides. SEQ ID NO:9 is the deduced peptide of the open reading frame of SEQ ID NO:8. SEQ ID NO:10 is the complete nucleotide sequence of the soybean (glycine max) microsomal delta-15 desaturase cDNA, found in plasmid pXF1, which the 2184 nucleotides of this sequence contain-both the coding sequence and the 5' and 3' non-translated regions of the cDNA. Nucleotides 855 to 857 are the putative initiation codon of the open reading frame (nucleotides 855 to 2000). Nucleotides 1995 to 1997 are the termination codon. Nucleotides 1 to 854 and 1998 to 2184 are the 5' and 3' unstranslated nucleotides respectively. The 380 amino acid protein sequence in SEQ ID NO:7 is that deduced from the open reading frame. SEQ ID NO:11 is the deduced peptide of the open reading frame in SEQ ID NO:10. SEQ ID NO:12 is the complete 5' to 3' nucleotide sequence of 1676 base pairs of the soybean (Glycine max) seed cDNA found in plasmid pSFD-118bwp which encodes a soybean plastid delta-15 desaturase. Nucleotides 169 to 1530 correspond to a continuous open reading frame of 453 amino acids. Nucleotides 169 to 171 are the putative initiation codon of the open reading frame. Nucleotides 1528 to 1530 are the termination codon. Nucleotides 1531 to 1676 are the 3' untranslated nucleotides. Nucleotides 169 to 382 encode the putative plastid transit peptide, based on comparison of the deduced peptide with the soybean microsomal delta-15 peptide. SEQ ID NO:13 is the deduced peptide of the open reading frame in SEQ ID NO:12. SEQ ID NO:14 is the complete nucleotide sequence of a 396 bp polymerase chain reaction product derived from corn seed mRNA that is found in the insert of plasmid pPCR20. Nucleotides 1 to 31 and 364 to 396 correspond to the amplification primers described in SEQ ID NO:18 and SEQ ID NO:19, respectively. Nucleotides 31 to 363 encode an internal region of a corn seed delta-15 desaturase that is 61.9% identical to the region between amino acids 137 and 249 of the Brassica napus delta-15 desaturase peptide sequence shown in SEQ ID NO:7. SEQ ID NO:15 is the deduced amino acid sequence of SEQ ID NO:14. SEQ ID NO:16 shows the partial composite 5' to 3' nucleotide sequence of 472 bp derived from the inserts in plasmids pFadx-2 and pYacp7 for Arabidopsis thaliana cDNA that encodes a plastid delta-15 fatty acid desaturase. Nucleotides 2-4 and nucleotides 468 to 470 are, respectively, the first and the last codons in the open reading frame. SEQ ID NO:17 is deduced partial peptide sequence of the open reading frame in SEQ ID NO:16. SEQ ID NO:18 One hundred and twenty eight fold degenerate sense 31-mer PCR primer. Nucleotides 1 to 8 correspond to the Bam H1 restriction enzyme recognition sequence. Nucleotides 9 to 137 correspond to amino acid residues 130 to 137 of SEQ ID NO:6 with a deoxyinosine base at nucleotide 11. SEQ ID NO:19 Two thousand and forty eight-fold degenerate antisense 35-mer PCR primer. Nucleotides 1 to 8 correspond to the Bam H1 restriction enzyme recognition sequence. Nucleotides 9 to 35 correspond to amino acid residues 249 to 256 of SEQ ID NO:6 with a deoxyinosine base at nucleotide 15. SEQ ID NO:20 Sixteen-fold degenerate sense 36-mers made to amino acid residues 97-108 in SEQ ID NO:2. SEQ ID NO:21 Sixteen-fold degenerate sense 36-mers made to amino acid residues 97-108 in SEQ ID NO:2. SEQ ID NO:22 Seventy two-fold degenerate sense 18-mers made to amino acid residues 100-105 in SEQ ID NO:2. SEQ ID NO:23 Seventy two-fold degenerate sense 18-mers made to amino acid residues 100-105 in SEQ ID NO:2. SEQ ID NO:24 Seventy two-fold degenerate antisense 18-mers made to amino acid residues 299-304 in SEQ ID NO:2. SEQ ID NO:25 Seventy two-fold degenerate antisense 18-mers made to amino acid residues 299-304 in SEQ ID NO:2. SEQ ID NO:26 Seventy two-fold degenerate antisense 18-mers made to amino acid residues 304-309 in SEQ ID NO:2. SEQ ID NO:27 Seventy two-fold degenerate antisense 18-mers made to amino acid residues 304-309 in SEQ ID NO:2. SEQ ID NO:28 Sixteen-fold degenerate sense 36-mers made to amino acid residues 97-108 in SEQ ID NO:2. SEQ ID NO:29 Sixteen-fold degenerate sense 36-mers made to amino acid residues 97-108 in SEQ ID NO:2. SEQ ID NO:30 Sixty four-fold degenerate antisense 38-mers made to amino acid residues 299-311 in SEQ ID NO:2. SEQ ID NO:31 Sixty four-fold degenerate antisense 38-mers made to amino acid residues 299-311 in SEQ ID NO:2. SEQ ID NO:32 A 135-mer made as an antisense strand to amino acid residues 97-141 in SEQ ID NO:2. |
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