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 nucleic acid fragment encoding a plant fatty acid modifying enzyme involved in desaturation of fatty acids resulting in conjugated double bond formation and is involved in the formation of α-eleostearic and/or α-parinaric acid, wherein said fragment:
 a) remains hybridized to the complement of the nucleotide sequence set forth in SEQ ID NO: 1, 3, 19, 23, or 29 under conditions of moderate stringency, or 
 b) is at least 45% identical to any of the nucleotide sequences set forth in SEQ ID NO: 1, 3, 19, 23, or 29 as determined by a comparison method designed to detect homologous sequences, wherein the comparison method is the Clustal method of alignment with parameters KTUPLE=2, GAPPENALTY+5, WINDOW=4 and DIAGONALS SAVED=4, or 
 c) encodes a protein comprising any one of the amino acid sequences set forth in SEQ ID NO: 2, 4, 20, 24, or 30. 
 
     
     
         64 . An isolated nucleic acid fragment as claimed in  claim 63 , wherein the sequence identity referred to in part b) is at least 50%. 
     
     
         65 . An isolated nucleic acid fragment as claimed in  claim 63 , wherein the sequence identity referred to in part b) is at least 60%. 
     
     
         66 . The isolated nucleic acid fragment of  claim 63 ,  64 , or  65  wherein said fragment is isolated from  Impatiens balsamina, Momordica charantia, Chrysobalanus icaco, Licania michauxii,  or  Aleurites fordii.    
     
     
         67 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 63 ,  64 , or  65  or a complement thereof operably linked to suitable regulatory sequences. 
     
     
         68 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 66 , or a complement thereof operably linked to suitable regulatory sequences. 
     
     
         69 . A transformed host cell comprising the chimeric gene of  claim 67 . 
     
     
         70 . The host cell of  claim 69 , wherein said cell is a plant or a microorganism. 
     
     
         71 . The host cell of 70, wherein the said cell is soybean, oilseed Brassica species, corn, peanut, rice, wheat, sunflower, safflower, cotton, flax, palm, or cocoa. 
     
     
         72 . A method of altering the level of fatty acids with conjugated double bonds in a cell comprising:
 a) transforming a host cell with the chimeric gene of  claim 67 ;   b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and   c) selecting those transformed host cells having altered levels of eleostearic and/or parinaric acid compared with corresponding cells that have not been transformed with the chimeric gene of  claim 67 .   
     
     
         73 . The method of  claim 72  wherein the host cell is a plant or microorganism. 
     
     
         74 . A method for producing seed oil containing fatty acids with conjugated double bonds in the seeds of plants comprising:
 a) transforming a plant cell with the chimeric gene of  claim 67 ;   b) growing a fertile mature plant from the transformed plant cell of step a);   c) screening progeny seeds from the fertile plants of step b) for altered levels of eleostearic and/or parinaric acid; and   d) processing the progeny seed of step c) to obtain seed oil containing altered levels of eleostearic and/or parinaric acid compared with corresponding seed that have not been transformed with the chimeric gene of  claim 67 .   
     
     
         75 . The method  claim 74  wherein the plant is soybean, oilseed Brassica species, corn peanut, rice, wheat, sunflower, safflower, cotton, or cocoa. 
     
     
         76 . A method for producing fatty acid modifying enzymes involved in desaturation of fatty acids resulting in conjugated double bond formation and is associated with the formation of α-eleostearic acid and/or α-parinaric acid comprising:
 a) transforming a microbial host cell with the chimeric gene of  claim 67 ; 
 b) growing the transformed host cell under conditions suitable for the expression of the chimeric gene; and 
 c) selecting those transformed host cells containing altered levels of protein encoded by the chimeric gene compared with corresponding host cells not transformed with the chimeric gene of  claim 67 . 
 
     
     
         77 . A method to isolate nucleic acid fragments encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation comprising:
 a) comparing SEQ ID NOs: 2,4,20, 24, or 30′   b) identifying conserved sequences of 4 or more amino acids obtained in step a);   c) designing degenerate oligomers based on the conserved sequences identified in step (b);   d) using the degenerate oligomers of step c) to isolate nucleic acid fragments suspected of encoding a plant fatty acid modifying enzyme or a portion thereof associated with conjugated double bond formation by sequence dependent protocols; and   e) testing the nucleic acid fragments by the method of any one of  claims 74 - 76 .   
     
     
         78 . A method of preparing animal feed comprising:
 a) obtaining seeds from a transgenic plant derived from plant cells transformed with the chimeric gene of  claim 67 ;   b) processing the seeds to obtain a feed ingredient; and   c) incorporating the feed ingredient into the animal feed.   
     
     
         79 . Animal feed prepared using the method of  claim 78 . 
     
     
         80 . Animal feed comprising at least one conjugated fatty acid derived from the processing of seeds obtained from a transgenic plant derived from plant cells transformed with the chimeric gene of  claim 67 . 
     
     
         81 . A method of feeding an animal comprising feeding the animal a grain, at least one conjugated fatty acid or oil of said grain obtained from a transgenic plant comprising in its genome the chimeric gene of  claim 67  and further wherein expression of the chimeric gene results in an altered conjugated fatty acid oil phenotype compared to the oil phenotype of a corresponding grain not transformed with the chimeric gene of  claim 67 . 
     
     
         82 . The complement of the isolated nucleic acid fragment of any one of  claims 63 - 66 . 
     
     
         83 . Use of the nucleic acid fragment of any one of  claims 63 - 66  to transform a host cell, wherein said host cell produces an oil comprising altered levels of α-eleostearic acid and/or α-parinaric acid compared to a corresponding host cell not transformed with said nucleic acid fragment. 
     
     
         84 . Use of the nucleic acid fragment of any one of  claims 63 - 66  to produce a transformed plant, wherein said plant produces a seed oil comprising altered levels of α-eleostearic acid and/or α-parinaric acid compared to a corresponding seed oil from a plant not transformed with said nucleic acid fragment. 
     
     
         85 . Use of the nucleic acid fragment of any one of  claims 63 - 66  to produce a transformed plant, wherein said plant produces an animal feed comprising altered levels of α-eleostearic acid and/or α-parinaric acid compared to a corresponding animal feed from a plant not transformed with said nucleic acid fragment. 
     
     
         86 . The use of  claim 84  wherein said plant is soybean, oilseed  brassica  species, corn, peanut, rice, wheat, sunflower, safflower, cotton, or cocoa. 
     
     
         87 . The use of  claim 85  wherein said plant is soybean, oilseed  brassica  species, corn, peanut, rice, wheat, sunflower, safflower, cotton, or cocoa.