半胱氨酸蛋白酶基因RNA干扰表达载体的构建及油菜的遗传转化

Construction of a RNAi expression vector targeting the cysteine protease gene and the transformation of Brassica napus L.

将半胱氨酸蛋白酶基因(Bncp5)cDNA序列片段正反向插入表达载体pFGC5941中,构建了RNA干扰表达载体pFGC-Bncp5-RNAi.采用根癌农杆菌介导法,转化甘蓝型油菜下胚轴.在5mg/L BastaMS培养基中筛选,PCR鉴定结果显示,获得Bncp5转基因植株28株.半定量PCR分析结果显示,半胱氨酸蛋白酶基因在转基因油菜中表达受到一定程度的抑制,表明Bncp5的干扰载体在油菜中得到表达.

An inverted repeat DNA fragment of cysteine protease gene （Bncp5） was inserted into the binary plasmid pFGC5941 for constructing the RNA interference expression vector pFGC-Bnep5-RNAi. The RNAi fragment was introduced into hypocotyls of Brassica napus mediated by Agrobacterium tume- faciens. Twenty-eight transgenic plants with Bncp5 RNAi fragment were regenerated on the selective medium with 5mg/L PPT by PCR. The transgenic plants were identified semi-quantitative PCR. The results showed that cysteine protease gene expression was decreased in transgenic plants, indicating that the RNAi fragment affected expression of BncpS.