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By using RACE (rapid amplification of cDNA ends) based homologous cloning strategy, we have successfully isolated the genomic and full-length cDNA sequences of a gene encoding typical DFR (dihydroflavonol-4-reductase) from black-seeded Brassica campestris L. var. oleifera DC.. The gene, designated BcDFR here, is 1 722bp in length and harbors 5 introns with typical splice sites of plant DFR genes. BcDFR cDNA is 1 311bp in length with a 1 158bp ORF as well as a 25bp 5’ UTR and a 128bp 3’ UTR. The encoded BcDFR protein is 385 aa with a calculated Mw of 42.85kD and a pI value of 5.55. The nucleotide and amino acid sequences of this gene share extensive homologies to plant DFR genes of wide origins especially high similarities to Cruciferous DFR genes. Sequence analyses such as phylogenetic analysis, conserved domain search and substrate specificity region detection all indicated that BcDFR gene is a quite potentially biofunctional gene. Its cloning enables us to further dissect the possible relatedness between DFR gene and Brassica seed coat color traits and to create transgenic novel yellow-seeded rapeseed germplasm through antisense- or RNAi-suppression of DFR gene expression in black-seeded elite cultivars.
By using RACE (rapid amplification of cDNA ends) based homologous cloning strategy, we have successfully isolated the genomic and full-length cDNA sequences of a gene encoding typical DFR (dihydroflavonol-4-reductase) from black-seeded Brassica campestris L. var. The gene, designated BcDFR here, is 1 722 bp in length and harbors 5 introns with typical splice sites of plant DFR genes. BcDFR cDNA is 1 311 bp in length with a 1 158 bp ORF as well as a 25 bp 5 ’UTR and a 128 bp 3 ’UTR The encoded BcDFR protein is 385 aa with a calculated Mw of 42.85 kD and a pI value of 5.55. The nucleotide and amino acid sequences of this gene share extensive extensiveologies to plant DFR genes of wide origins especially high similarities to Cruciferous DFR genes. Sequence analyzes such as phylogenetic analysis, conserved domain search and substrate specificity region detection all indicated that BcDFR gene is a potentially impossibly biofunctional gene. Its cloning enables us to further dissect the possibl e relatedness between DFR gene and Brassica seed coat color traits and to create transgenic novel yellow-seeded rapeseed germplasm through antisense- or RNAi-suppression of DFR gene expression in black-seeded elite cultivars.