Delta-8 desaturases and their use in making polyunsaturated fatty acids

Abstract

Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding delta-8 desaturases along with a method of making long-chain polyunsaturated fatty acids (PUFAs) and using these delta-8 desaturases in plants.

1 . An isolated polynucleotide comprising:
 (a) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24;   (b) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.   (c) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the nucleotide sequence hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20; or   (d) a complement of the nucleotide sequence of (a), (b) or (c), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.   
     
     
         2 - 38 . (canceled) 
     
     
         39 . The polynucleotide of  claim 1  wherein the nucleotide sequence comprises SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20. 
     
     
         40 . The polynucleotide of  claim 1 , wherein the amino acid sequence of the polypeptide comprises
 (a) SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24; or   (b) an amino acid sequence that differs from the amino acid sequences in (a) by at least one conservative amino acid substitution.   
     
     
         41 . A recombinant DNA construct comprising the polynucleotide of  claim 1  operably linked to at least one regulatory sequence. 
     
     
         42 . A plant cell comprising in its genome the recombinant DNA construct of  claim 41 . 
     
     
         43 . A method for producing a transformed plant comprising transforming a plant cell with the polynucleotide of  claim 1  and regenerating a plant from the transformed plant cell. 
     
     
         44 . A transgenic seed comprising in its genome the recombinant construct of  claim 41 . 
     
     
         45 . A transgenic seed obtained from the plant made by the method of  claim 43 . 
     
     
         46 . Oil or by-products obtained from the seed of  claim 44 . 
     
     
         47 . A method for making long-chain polyunsaturated fatty acids in a plant cell comprising:
 (a) transforming a cell with the recombinant construct of  claim 41 ; and   (b) selecting those transformed cells that make long-chain polyunsaturated fatty acids.   
     
     
         48 . A method for producing at least one polyunsaturated fatty acid in an oilseed plant cell comprising:
 (a) transforming an oilseed plant cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C 14/16  elongase, a C 16/18  elongase, a C 18/20  elongase and a C 20/22  elongase;   (b) regenerating an oilseed plant from the transformed cell of step (a); and   (c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed oilseed plant.   
     
     
         49 . The method of  claim 48  wherein the oilseed plant is selected from the group consisting of soybean,  Brassica  species, sunflower, maize, cotton, flax, and safflower. 
     
     
         50 . An oilseed plant comprising:
 (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and   (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C 14/16  elongase, a C 16/18  elongase, a C 18/20  elongase and a C 20/22  elongase.   
     
     
         51 . The oilseed plant of  claim 50 , wherein the oilseed plant is selected from the group consisting of soybean,  Brassica  species, sunflower, maize, cotton, flax and safflower. 
     
     
         52 . A transgenic seed obtained from the oilseed plant of  claim 50 . 
     
     
         53 . Oil or by-products obtained from the seed of  claim 52 . 
     
     
         54 . Oil obtained by the method of  claim 47 . 
     
     
         55 . Food or feed which incorporates the oil of  claim 53 . 
     
     
         56 . Food or feed which incorporates the oil of  claim 54 . 
     
     
         57 . Food or feed comprising the seed of  claim 52 . 
     
     
         58 . Food or feed comprising an ingredient derived from the processing of the seeds of  claim 52 . 
     
     
         59 . The oil or by-products of  claim 53  wherein the by-product is lecithin. 
     
     
         60 . A method for making long-chain polyunsaturated fatty acids in a plant cell having a reduced level of by-product fatty acids, said method comprising:
 (a) transforming a plant host cell with at least one recombinant DNA construct comprising an isolated polynucleotide encoding at least two delta-8 desaturases operably linked to at least one regulatory sequence; and   (b) selecting those transformed plant host cells obtained having a reduced level of by-product fatty acids, when compared to the level of such metabolic by-product fatty acids in a transformed host cell having at least one recombinant DNA construct comprising an isolated polynucleotide encoding one delta-8 desaturase operably linked to a regulatory sequence.   
     
     
         61 . Progeny plants obtained from the plant made by the method of  claim 43 . 
     
     
         62 . Progeny plants obtained from the oilseed plant of  claim 50 .