棉蚜Catb5基因RNAi载体的构建及其对烟草的转化

Construction of RNAi Expression Vector for Aphis gossipy Catb5 Gene and Transformation of Tobacco

依据细胞色素氧化酶亚基Ⅰ序列的多态性设计特异性引物,通过PCR技术扩增出的特异性条带鉴定为棉蚜。提取棉蚜总RNA,利用RT-PCR技术从棉蚜中扩增出大小为515 bp的Catb5目的基因,进一步将两段Catb5基因分别正向和反向连接到植物表达载体pBi35SG12上,构建了pBi35SC5 RNAi表达载体,并通过农杆菌介导法导入烟草中,PCR检测表明已得到转基因烟草再生苗。

Aphis gossipy was identified by PCR with primers according to the sequencing length polymorphism method based on the cytochrome oxidase subunit I gene.The total RNA was extracted from A.gossipy and the 515 bp length product of Catb5 gene was cloned by RT-PCR.RNAi vector pBi35SC5 was constructed by inserting the two Catb5 genes into plant expression vector pBi35SG12 forward and reversely.The vector is transformed into tobacco mediated by Agrobacterium tumefaciens GV3101.Transgenic plants were screened by kanamycin resistant and verified by PCR analysis,which provides a foundation for further experiment of resisting A.gossipy.