油菜BnCr4基因RNAi载体构建及植株转化

该实验构建了含甘蓝型油菜黄化相关基因BnCr4特异片段反向重复结构的RNA干扰(RNAi)载体pFGC5941-Cr4,通过根癌农杆菌介导转化油菜,获得47株抗Basta的抗性再生油菜植株,其中10株经PCR鉴定为阳性转基因植株.随机选取3株经鉴定的转基因阳性油菜植株进行半定量RT-PCR分析,结果显示,相对于非转基因的野生型油菜,3株转基因植株中BnCr4基因的表达量分别降低了78.5%、8.5%、11.8%,表明该干扰载体转入油菜能特异引起植株BnCr4基因表达量下降.

Construction of the RNAi Vector Carrying BnCr4 Gene and Transformation of Brassica napus L.

BnCr4 was related with chlorophyll reduced seedling mutation.In this study,the plant RNA interference(RNAi)expression vector pFGC5941-Cr4,which contains BnCr4 gene-specific sequence in the sense and antisense orientations,was constructed and transferred into B.napus mediated by Agrobacterium tumefacien.Forty seven regenerated plants resistant to Basta (5.0 mg·L-1) were obtained.Ten transgenic plants were confirmed by PCR.Three transgenic plants were selected for expression analysis.Semi-quantity RT-PCR analysis result showed the expression levels of BnCr4 in the three transgenic plants were down-regulated 78.5%,8.5% and 11.8% compared with that of wild type,respectively.The results revealed that the pFGC5941-Cr4 vector has been inserted the genome of B.napus and induced the decrease of BnCr4 gene expression.