Rubisco活化酶基因反义表达载体的构建与水稻的遗传转化

Construction of Expression Vector with Antisense Rubisco Activase Gene and Its Genetic Transformation in Rice

利用pGEM Teasy、pBluescriptKS +获得了RCA基因两端可以引入与双元表达载体 pCAMBIA130 1多克隆位点相匹配的单酶切位点 ,将该片段连接到 pCAMBIA130 1载体 ,构建了RCA基因的反义植物表达载体 pCAMR0 2。通过电激法将该载体导入根癌农杆菌EHA10 5菌株中 ,以根癌农杆菌介导的方法 ,将反义载体 pCAMR0 2转化粳稻品种中花 11,获得了抗潮霉素的再生植株 ,经过GUS组织化学检测和PCR鉴定结果表明 ,目的基因确已经整合到这些转基因水稻基因组中。表型测定显示 ,大部分反义水稻不能在大气CO2 浓度条件下生长 ,存活下来转化株也是生长缓慢 ,植株瘦小 ,RCA和Rubisco含量发生了明显改变 。

In this research, Rubisco activase gene (rca) was amplified using specific primers and inserted into pGEM T-easy vector, and then cut with EcoRⅠ after confirming by sequencing. The fragment was subcloned into pBluescript KS+, digested with the enzyme BamHⅠ and inserted into the binary expression vector pCAMBIA1301, and the resulting construction with antisense rca was named pCAMR02.The pCAMR02 vector was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation and transformed to embryos of rice (Oryza. Sativa L.ssp.japonica) cultivar Zhonghua11 via Agrobacterium tumefaciens system.Plantlets were regenerated in vitro by resistance selection on medium containing various concentrations of hygromycin. Both GUS histochemical assays and PCR amplification demonstrated that antisense rca was integrated into T 0 genomes and inherited to T 1.The measurement of phenotypes of transgenic rice plants with antisense rca showed that most of them could hardly survive at ambient CO 2 concentration, even could not grow. The antisense plants that survived under natural condition were dwarf and grew slower than the wild-type controls, and their contents of RCA and Rubisco changed significantly. These plants generated in this experiment will be used to study the relationship between RCA and Rubisco and their regulation.