含VHb基因和GFMcryIA基因的双价基因植物高效表达载体的构建

将人工合成的、密码子优化后的透明颤菌血红蛋白基因与人工合成的GFMcryIA基因构建成双价基因植物高效表达载体PGBI4ASVHBBt,vgbM基因表达盒中含2个增强子的35S启动子、Ω前导序列、Kozak序列、多联终止密码子及Nos终止子;Bt基因表达盒中,除含有以上提高转录和翻译的调控元件外,还包含有正确切割、加工序列、Poly(A)信号序列。利用根癌农杆菌介导转化烟草,获得了转基因植株;PCR及Southern blot检测,证实了双价基因在烟草基因组中的整合;Western blot检测证实了vgbM基因在转基因烟草中的表达;杀虫实验表明GFMcryIA基因也表达出活性毒蛋白。

Construction of Plant Expression Vector Harbouring Bivalent Genes Composed of Synthetic VHb Gene and GFMcryIA Gene

The highly-efficient plant expression vector harbouring bivalent genes was constructed by applying the artificially-modified-synthesized VHb gene(vgbM gene)and the modified Bt gene(GFMcryIA gene),vgbM gene experession cassettes contained such cis-acting elements as 35S promoter,two enhancers,Ω and Kozak sequence,poly-terminator and Nos terminator,besides these elements,Bt gene expression cassettes contained splicing sequence,processing sequence and Poly(A)sequence.Transgenic tobacco plants were obtained through the transformation with Agrobacterium tumefaiens carrying the expression vector PGBI4ASVHBBt,by PCR amplification and Southern blot analysis,it was proved that the bivalent genes were inserted into the tobacco plant genome;through western blot analysis it was also verified that vgbM gene expressed in the transgenic plants.Toxicity assay indicated that transgenic plants expressed pesticidal toxin protein.