Brassica rod1 gene sequences and uses thereof

Abstract

The present invention relates to Brassica juncea ROD1 nucleic acid sequences and proteins and the use thereof to create plants with increased levels of C18:1 and reduced levels of saturated fatty acids in the seeds.

1 . A  Brassica juncea  plant, or a cell, part, seed or progeny thereof, comprising at least one ROD1 gene, characterized in that at least one ROD1 gene is a knock-out rod1 gene. 
     
     
         2 . The plant, or a cell, part, seed or progeny thereof, of  claim 1 , comprising two knock-out rod1 genes. 
     
     
         3 . The plant, or a cell, part, seed or progeny thereof, of  claim 1  or  2 , wherein said knock-out rod1 gene is a knock-out allele of the ROD1 gene encoding a protein having at least 90% sequence identity to SEQ ID No. 2 or SEQ ID No. 4. 
     
     
         4 . The plant of any one of  claims 1 - 3  which is homozygous for the knock-out ROD1 gene. 
     
     
         5 . A transgenic  Brassica juncea  plant, or a cell, part, seed or progeny thereof, comprising a chimeric gene, said chimeric gene comprising the following operably linked DNA fragments:
 a) a plant-expressible promoter;   b) a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one ROD1 gene; and optionally   c) a transcription termination and polyadenylation region functional in plant cells.   
     
     
         6 . The transgenic plant, or a cell, part, seed or progeny thereof, according to  claim 5  wherein said RNA molecule is inhibitory to a ROD1 gene encoding a protein having at least 90% sequence identity to SEQ ID No. 2 and to a ROD1 gene encoding a protein having at least 90% sequence identity to SEQ ID No. 4. 
     
     
         7 . Seeds from the plants of any one of  claims 1 - 6 . 
     
     
         8 . Oil from the seeds according to  claim 7 . 
     
     
         9 . A method for increasing the levels of C18:1 and/or decreasing the levels of saturated fatty acids in  Brassica juncea  seed oil, comprising modulating the expression of a ROD1 gene. 
     
     
         10 . The method according to  claim 9 , comprising the steps of:
 a) introducing or providing a chimeric gene to a  Brassica juncea  plant cell, to create transgenic cells, said chimeric gene comprising the following operably linked DNA fragments:
 a plant-expressible promoter 
 a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one ROD1 gene; and optionally 
 a transcription termination and polyadenylation region functional in plant cells; and 
   b) regenerating transgenic plants from said transgenic cells.   
     
     
         11 . The method according to  claim 9 , comprising the steps of:
 a) treating seeds or plant material with a mutagenic chemical substance or with ionizing radiation;   b) identifying plants with a mutated ROD1 gene, wherein the ROD1 gene, prior to being mutated, encodes a polypeptide having at least 90% sequence identity to SEQ ID No. 2, or to SEQ ID No. 4; and   c) selecting a plant with an increased level of C18:1 in the seeds compared to a plant in which the ROD1 gene is not mutated.   
     
     
         12 . A method for obtaining a  Brassica juncea  plant with increased levels of C18:1 and/or decreased levels of saturated fatty acids in the seeds comprising the step of introducing a knock-out allele of a ROD1 gene in said  Brassica juncea  plant, and selecting said  Brassica juncea  plant with increased levels of C18:1 in the seeds for the presence of said knock-out allele of a ROD1 gene by analyzing genomic DNA from said plant for the presence of at least one molecular marker, wherein said at least one molecular marker is linked to said knock-out allele of a ROD1 gene. 
     
     
         13 . A method to determine the presence or absence of a knock-out allele of a ROD1 gene in a biological sample, comprising providing genomic DNA from said biological sample, and analyzing said DNA for the presence of at least one molecular marker, wherein the at least one molecular marker is linked to said knock-out allele of a ROD1 gene. 
     
     
         14 . A kit for the detection of a knock-out allele of a ROD1 gene in  Brassica juncea  DNA samples, wherein said kit comprises one or more PCR primer pairs, which are able to amplify a DNA marker linked to said knock-out allele of a ROD1 gene. 
     
     
         15 . A method for determining the zygosity status of a mutant ROD1 allele in a  Brassica juncea  plant, or a cell, part, seed or progeny thereof, comprising determining the presence of a mutant and/or a corresponding wild type ROD1 specific region in the genomic DNA of said  Brassica juncea  plant, or a cell, part, seed or progeny thereof. 
     
     
         16 . A method for transferring at least one knock-out ROD1 allele from one  Brassica juncea  plant to another  Brassica juncea  plant comprising the steps of:
 (a) identifying a first  Brassica juncea  plant comprising at least one knock-out ROD1 allele,   (b) crossing the first  Brassica juncea  plant with a second  Brassica juncea  plant not comprising the at least one knock-out ROD1 allele and collecting F1 hybrid seeds from the cross,   (c) optionally, identifying F1  Brassica juncea  plants comprising the at least one knock-out ROD1 allele,   (d) backcrossing F1  Brassica juncea  plants comprising the at least one knock-out ROD1 allele with the second  Brassica juncea  plant not comprising the at least one knock-out ROD1 allele for at least one generation (x) and collecting BCx seeds from the crosses,   (e) identifying in every generation BCx  Brassica juncea  plants comprising the at least one knock-out ROD1 allele by analyzing genomic DNA of said BCx plants for the presence of at least one molecular marker, wherein the at least one molecular marker is linked to said knock-out ROD1 allele.   
     
     
         17 . A chimeric gene comprising the following operably linked elements:
 a) a plant-expressible promoter;   b) a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one ROD1 gene, said ROD1 gene encoding a protein having at least 90% sequence identity to SEQ ID No. 2 or SEQ ID No. 4; and optionally   c) a transcription termination and polyadenylation region functional in plant cells.   
     
     
         18 . A knock-out allele of a ROD1 gene, wherein the knock-out ROD1 allele is a mutated version of the native ROD1 gene selected from the group consisting of:
 (a) a nucleic acid molecule which comprises at least 90% sequence identity to SEQ ID No. 1 or SEQ ID No. 3;   (b) a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID No. 2 or SEQ ID No. 4,   wherein said mutant rod1 allele comprises a mutated DNA region consisting of one or more inserted, deleted or substituted nucleotides compared to a corresponding wild-type DNA region in the functional ROD1 gene and wherein said mutant rod1 allele encodes no functional ROD1 protein or encodes a ROD1 protein with reduced activity.   
     
     
         19 . Method for producing oil, comprising harvesting seeds from the  Brassica juncea  plants according to any one of  claims 1 - 6  and extracting the oil from said seeds. 
     
     
         20 . A method of producing food, feed, or an industrial product comprising
 (a) obtaining the plant or a part thereof, of any one of  claims 1 - 6 ; and   (b) preparing the food, feed or industrial product from the plant or part thereof   
     
     
         21 . The method of  claim 20  wherein
 (a) the food or feed is oil, meal, grain, starch, flour or protein; or 
 (b) the industrial product is biofuel, fiber, industrial chemicals, a pharmaceutical or a nutraceutical.