无机焦磷酸化酶基因的克隆与植物表达载体的构建

Cloning of Inorganic Pyrophosphatase Gene and Construction of Its Plant Expression Vector

提取面包酵母基因组DNA,用无机焦磷酸化酶(PPase)基因特异引物通过PCR扩增出864bp的片段,并将该片段克隆至pMD18-T上。序列分析结果表明,该克隆序列与GeneBank上的PPase基因序列同源性达到100%。进一步将叶肉细胞特异性启动子rbcS和PPase基因克隆至植物表达载体pCBI上,构建了由rbcS启动子调控的PPase基因植物表达载体pCBIPR。通过冻融法将重组质粒导入根癌农杆菌EHA105中,为农杆菌介导的PPase基因对植物的遗传转化奠定基础。

An 864 bp DNA fragment was amplified by PCR using PPase gene special primer from Baker＇s yeast and then cloned into pMD18-T. The sequencing showed that the sequence of this fragment was 100 % homologous with that of PPase gene in GeneBank. The special promoter rbcS and PPase genes of mesophyll cells were cloned into plant expression vector pCBI, and a plant expression vector pCBIPR was constructed,in which PPase gene was controlled by the rbcS promoter.The recombination plasmids were then introduced into Agrobacterium tumefaciens EHA105 by the freezing-mehing transformation method, which facilitates Agrobacterium mediated transformation of PPase gene into plant.