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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。
The pUCEM-4zf plasmids containing the CryIA (a) and CryIA (c) genes, respectively, were digested with restriction endonuclease HindIII to give the CryIA (a) and CryIA (c) gene expression cassettes driven by the Ubiquitin And inserted into the vector pCAMBIA3300 (containing the bar gene encoding the herbicide glufosinate) opened with HindIII, respectively, to form the intermediate vector p3300-bt. The pta gene expression cassette containing the CaMV35S promoter and the NOS terminator was amplified from plasmid pBI121-pta by PCR using Pfu high-fidelity DNA polymerase and then inserted into an intermediate vector p3300-bt cut with SmaI to obtain Bivalent insect-resistant gene with a resistance gene expression vector p3300-bt-pta. Agrobacterium tumefaciens-mediated leaf disc method was used to transform tobacco variety Hundred Days Red, and a number of herbicide-resistant transgenic tobacco plants were obtained.