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反义CYP86MF基因植物表达载体的构建及其对白菜的转化
引用本文:余小林,曹家树.反义CYP86MF基因植物表达载体的构建及其对白菜的转化[J].浙江大学学报(农业与生命科学版),2004,30(2):179-184.
作者姓名:余小林  曹家树
作者单位:浙江大学,蔬菜研究所,浙江,杭州,310029
基金项目:国家自然科学基金,浙江省科研项目
摘    要: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438 bp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.

关 键 词:CYP86MF基因  表达载体  反义RNA技术  遗传转化
文章编号:1008-9209(2004)02-0179-06
修稿时间:2003年2月24日

Construction of plant expression plasmid vector containning the antisense gene CYP86MF and its transformation to Chinese cabbage-pak-choi
YU Xiao-lin,CAO Jia-shu.Construction of plant expression plasmid vector containning the antisense gene CYP86MF and its transformation to Chinese cabbage-pak-choi[J].Journal of Zhejiang University(Agriculture & Life Sciences),2004,30(2):179-184.
Authors:YU Xiao-lin  CAO Jia-shu
Abstract: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.
Keywords:CYP86MF  expression vector  the antisense RNA technology  genetic transformation
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