Cloning, Expression Analysis And RNAi Vector Construction Of Seed Coat Color-Related Genes In Brassica Juncea

The yellow-seeded rapeseed has such advantages as thinner seed coat, higher oil content, clearer oil, higher protein and lower fiber content compared with the black-seeded one under the same genetic background. Thus, it will have a bright future to develop high quality rapeseed with yellow-seeded character. Development of high yielding, high quality and yellow-seeded rapeseed is a current major aim of breeding. Formation of a yellow seed coat is showed to be associated with the expression of the genes for flavonoid biosynthesis in Brassica juncea.RT-PCR analysis was used to study the gene expression in the 20-days-after-pollination (20-DAP) seed coat of 12 Brassica juncea accessions. The expression of the structural genes PAL, C4H, CHI, F3H, F3 'H, LAC, TT12 and GST and the regulatory genes 777, TT2, TT8, TTI6, TTG1 and TTG2 was detected in all the accessions studied. However, difference in level of expression of the four genes C4H, TT12, LAC and 777was found between the yellow and the black seed coat. The expression of the genes DFR and ANS was detected in all the five black-seeded accessions studied, while no expression of DFR and ANS was detected in the yellow-seeded accessions. The high expression of BAN was detected in all the five black-seeded accessions studied, but the weak expression in the three yellow-seeded accessions or no expression in the remaining yellow-seeded ones. It is suggested that DFR, ANS and BAN be the key genes responsible for differences in seed coat color.A Braasica juncea accession Purple Mustard(PM) was used for cloning of the gene ANS by RACE technology. 3'- and 5'-end fragments of the gene BjANS were cloned and joined with the conserved region using the software DNAMAN4.0. Full-length cDNA sequence of BjANS was obtained. Bioinformatics analysis showed that the gene BjANS encodes an open reading frame consisting of 359 amino acids.A plant RNA interference vector pRNAi-ANS under the promoter of a seed-specifically expressed gene napinA was constructed and subsequently introduced into the Agrobacterium tumefaciens strain EHA105 with freeze-thaw method, which provided foundations for analysis of the function of the gene.