双价抗虫植物表达载体p3300-bt-pta的构建及转基因烟草的初步检测  

Construction of Plant Expression Vectors Containing Binary Insect Resistance Genes Bt CryIA(a)-pta or Bt CryIA(c)-pta and their Expression in Transgenic Tobaccos

作　　者：范媛媛[1] 庞永珍[2] 吴为胜[2] 姚剑虹[2] 唐克轩[1,3] 武天龙[1] 

机构地区：[1]上海交通大学农业与生物学院 [2]复旦大学生命科学学院遗传工程国家重点实验室 [3]复旦大学生命科学学院遗传工程国家重点实验室,上海200433

出　　处：《上海交通大学学报（农业科学版）》2004年第1期1-6,共6页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：国家863计划资助项目(2001AA212141)

摘　　要：用限制性内切酶HindⅢ酶切分别含有CryIA(a)和CryIA(c)基因的pGEM-4zf质粒得到Ubiquitin(玉米泛素)基因启动子驱动的CryIA(a)和CryIA(c)基因表达盒,将它们分别插入到用HindⅢ开环的pCAMBIA3300(含编码抗除草剂草丁膦的bar基因)载体上形成中间载体p3300-bt。采用Pfu高保真DNA聚合酶用PCR的方法从质粒pBI121-pta上扩增得到含CaMV35S启动子和NOS终止子的pta基因表达盒,然后插入到用SmaⅠ切开的中间载体p3300-bt中,获得带有抗性基因的双价抗虫基因表达载体p3300-bt-pta。通过根癌农杆菌介导的叶盘法转化烟草品种百日红,获得一批抗除草剂的转基因烟草植株。Plant expression vectors p3300 CryIA(a) pta and p3300 CryIA(c) pta with binary insect resistance genes were constructed by inserting expression cassettes of CryIA(a) gene or CryIA(c) which were cut from pGEM 4zf plasmids with restriction enzyme HindⅢand pta gene expression cassette obtained by PCR method into HindⅢand SmaⅠsites of pCAMBIA3300 respectively, which also contains bar gene cassette encoding phosphinothricin acetyltransferase (PAT). The CryIA(a) or CryIA(c) genes were under the control of Ubiquitin promoter and NOS terminator and the pta gene was under the control of CaMV 35S promoter and NOS terminator respectively. Leaf discs of Nicotiana tobacco were infected by Agrobacterium tumefaciens strain LBA4404 containing these plant expression vectors. Phosphinothricin resistant plants from individual transformation events were obtained and PCR checking confirmed the integration of foreign genes.

关 键 词：转基因植株 表达载体 限制性内切酶 质粒 DNA聚合酶 PCR 双价抗虫基因 转基因技术 

分 类 号：S433[农业科学—农业昆虫与害虫防治] Q785[农业科学—植物保护]