反义CYP86MF基因植物表达载体的构建及其对白菜的转化

Construction of plant expression plasmid vector containning the antisense gene CYP86MF and its transformation to Chinese cabbage-pak-choi.

在获得一个与白菜核雄性不育相关的编码细胞色素P450(CYP450)的基因CYP86MF的基础上,采用PCR技术在该基因的内部第901个碱基至第1338个碱基之间设计引物,扩增出438bp序列作为反义基因片段.把该反义片段连接至pBI121双元载体质粒上得到CaMV35S组成型启动子的表达载体pBI35S-AMF,随后利用"三亲杂交"法将pBI35S-AMF质粒转入农杆菌LBA4404和EHA105两个菌株中.PCR扩增和酶切鉴定结果表明,所构建的反义CYP86MF基因植物表达载体pBI35S-AMF是正确的,并已成功导入了根癌农杆菌中.随后,按照已建立的遗传转化体系对'上海青'白菜进行了转化,并获得130了多株转化再生植株.

A male sterility related gene CYP86MF which encoded a plant cytochrome P450 was isolated in our former study, based on which an antisense gene fragment of 438 bp was amplified from 901^(st) to 1338^(th) base pairs. The fragment was introduced to pBI121 generating pBI35S-AMF which contained CaMV35S promoter, and then pBI35S-AMF was transferred into Agrobacterium tumefaciens strain LBA4404 and EHA105 through the assistant plasmid pRK2013/HB101 by triparental cross. Results of PCR amplification and restriction digestion showed that the fragment of antisense gene CYP86MF was introduced into pBI121 plasmid and the expression vector pBI35S-AMF was transferred into Agrobacterium successfully. Then, the antisense gene fragment was transferred into Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) cv. 'Shanghaiqing' and more than 130 Kan^R plantlets were obtained.