Genes for plant fatty acid modifying enzymes associated with conjugated double bond formation

Abstract

The preparation and use of nucleic acid fragments encoding plant fatty acid modifying enzymes associated with conjugated double bond formation or functionally equivalent subfragments thereof are disclosed. Chimeric genes incorporating such nucleic acid fragments or functionally equivalent subfragments thereof or complement thereof and suitable regulatory sequences can be used to create transgenic plants having altered lipid profiles.

1 - 62 . (canceled) 
     
     
         63 . An isolated polynucleotide encoding a plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation wherein said isolated polynucleotide (a) hybridizes to the nucleotide sequence set forth in SEQ ID NO:3 under conditions of moderate stringency or (b) is at least 45% identical to a polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 3 as determined by a comparison method designed to detect homologous sequences.
 further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:   (i) eight conserved histidine residues; and   (ii) at least one modification selected from the group of:
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) there is a proline at what corresponds to position 312 of SEQ ID NO:4 as determined by the Clustal method of alignment. 
   
     
     
         64 . An isolated polynucleotide encoding a plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation wherein said isolated polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 4. 
     
     
         65 . An isolated polynucleotide encoding a plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation wherein said isolated polynucleotide (a) hybridizes to the isolated nucleic acid fragment of  claim 64  under conditions of moderate stringency or (b) is at least 45% identical to a polypeptide encoded by the isolated nucleic acid fragment of  claim 64  as determined by a comparison method designed to detect homologous sequences, further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:
 (i) eight conserved histidine residues; and   (ii) at least one modification selected from the group of:
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) there is a proline at what corresponds to position 312 of SEQ ID NO:4 as determined by the Clustal method of alignment. 
   
     
     
         66 . An isolated polynucleotide comprising:
 (a) a nucleotide sequence encoding a plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation, wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has an amino acid sequence of at least 57% identity, when compared to SEQ ID NO: 4, based on the Clustal method of alignment, further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:
 (i) eight conserved histidine residues; and 
 (ii) at least one modification selected from the group of: 
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) there is a proline at what corresponds to position 312 of SEQ ID NO:4 as determined by the Clustal method of alignment; 
   (b) all or part of the isolated polynucleotide comprising (a) for use in co-suppression or antisense suppression of endogenous nucleic acid sequences encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation, or   (c) a complement of the nucleotide sequence of (a) or (b) wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.   
     
     
         67 . The polynucleotide of  claim 66 , wherein the amino acid sequence of the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation and the amino acid sequence of SEQ ID NO: 4 have at least 60% identity based on the Clustal alignment method, further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:
 (i) eight conserved histidine residues; and   (ii) at least one modification selected from the group of:
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) there is a proline at what corresponds to position 312 of SEQ ID NO:4 as determined by the Clustal method of alignment. 
   
     
     
         68 . The polynucleotide of  claim 66 , wherein the amino acid sequence of the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation and the amino acid sequence of SEQ ID NO: 4 have at least 65% identity based on the Clustal alignment method, further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:
 (i) eight conserved histidine residues; and   (ii) at least one modification selected from the group of:
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) there is a proline at what corresponds to position 312 of SEQ ID NO:4 as determined by the Clustal method of alignment. 
   
     
     
         69 . The polynucleotide of  claim 66 , wherein the amino acid sequence of the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation and the amino acid sequence of SEQ ID NO: 4 have at least 70% identity based on the Clustal alignment method, further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:
 (i) eight conserved histidine residues; and   (ii) at least one modification selected from the group of:
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) there is a proline at what corresponds to position 312 of SEQ ID NO:4 as determined by the Clustal method of alignment. 
   
     
     
         70 . The polynucleotide of  claim 66 , wherein the amino acid sequence of the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation and the amino acid sequence of SEQ ID NO: 4 have at least 75% identity based on the Clustal alignment method, further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:
 (i) eight conserved histidine residues; and   (ii) at least one modification selected from the group of:
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) the amino acid at position 312 is proline. 
   
     
     
         71 . The polynucleotide of  claim 66 , wherein the amino acid sequence of the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation and the amino acid sequence of SEQ ID NO: 4 have at least 80% identity based on the Clustal alignment method, further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:
 (i) eight conserved histidine residues; and   (ii) at least one modification selected from the group of:
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) there is a proline at what corresponds to position 312 of SEQ ID NO:4 as determined by the Clustal method of alignment. 
   
     
     
         72 . The polynucleotide of  claim 66 , wherein the amino acid sequence of the plant fatty acid modifying involved in desaturation of fatty acids resulting in conjugated double bond formation and the amino acid sequence of SEQ ID NO: 4 have at least 85% identity based on the Clustal alignment method, further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:
 (i) eight conserved histidine residues; and   (ii) at least one modification selected from the group of:
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) there is a proline at what corresponds to position 312 of SEQ ID NO:4 as determined by the Clustal method of alignment. 
   
     
     
         73 . The polynucleotide of  claim 66 , wherein the amino acid sequence of the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation and the amino acid sequence of SEQ ID NO: 4 have at least 90% identity based on the Clustal alignment method, further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:
 (i) eight conserved histidine residues; and   (ii) at least one modification selected from the group of:
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) there is a proline at what corresponds to position 312 of SEQ ID NO:4 as determined by the Clustal method of alignment. 
   
     
     
         74 . The polynucleotide of  claim 66 , wherein the amino acid sequence of the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation and the amino acid sequence of SEQ ID NO: 4 have at least 95% identity based on the Clustal alignment method, further wherein the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation has the following structural features:
 (i) eight conserved histidine residues; and   (ii) at least one modification selected from the group of:
 (a) an amino acid immediately preceding a first conserved histidine box is glycine instead of alanine; 
 (b) the amino acid immediately following the first histidine in the first histidine box is glutamate instead of aspartate; and 
 (c) there is a proline at what corresponds to position 312 of SEQ ID NO:4 as determined by the Clustal method of alignment. 
   
     
     
         75 . The polynucleotide of  claim 66 , wherein the amino acid sequence of the plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation comprises SEQ ID NO:4. 
     
     
         76 . The polynucleotide of  claim 66 , wherein the nucleotide sequence comprises SEQ ID NO:3. 
     
     
         77 . The polynucleotide of any one of  claims 63 - 76  wherein the plant fatty acid modifying enzyme activity results in the formation of eleostearic acid. 
     
     
         78 . The polynucleotide of any one of  claims 63 - 76  wherein the plant is selected from the group consisting of soybean, oilseed  Brassica  species, corn, peanut, rice, wheat, sunflower, safflower, cotton, flax, palm, and cocoa. 
     
     
         79 . The polynucleotide of any one of  claims 63 - 76  wherein the plant fatty acid modifying enzyme results in the formation of eleostearic acid and wherein the plant is selected from the group consisting of soybean, oilseed  Brassica  species, corn, peanut, rice, wheat, sunflower, safflower, cotton, flax, palm, and cocoa. 
     
     
         80 - 97 . (canceled)