Brassica rod1 gene sequences and uses thereof
AssigneeBayer CropScience AG
InventorsPeter DENOLF, Michel VAN THOURNOUT, Stephane BOUROT
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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.
Claims21
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Dependencies are inferred from the claim text.