家稗Pdk基因叶片特异性表达载体的构建及农杆菌的导入

Construction of Plant Leaf-specific Expression Vector with PPDK of [Echinochloa crusgalli var frumentacea(Roxb.)W.F.Wight] and Transfer to Agrobacterium tumefaciens

【研究目的】构建含家稗Pdk基因的叶片特异性表达载体;【方法】通过PstI和SalI单酶切分别从质粒pRGN及pUCm-Pdk上获得Rubisco小亚基启动子和Pdk基因,将其连入表达载体pCAMBI1301,并通过冻融法将重组质粒导入根癌农杆菌EHA105;【结果】构建了由Rubisco小亚基启动子调控的Pdk基因植物表达载体p1301-Prbs-Pdk,选择标记基因为Hpt(潮霉素磷酸转移酶基因),经酶切和PCR鉴定,表达载体已成功导入农杆菌EHA105中;【结论】丙酮酸磷酸双激酶(PPDK)是C4光合途径中的关键光合酶,本实验中构建了含有rbcs启动子的适于单子叶植物转化的Pdk基因表达载体,为提高转基因植物光合效率奠定了基础。

[OBJECTIVE] The pyruvate-phosphate dikinase expressed effectively by utilizing leaf-specfic promoter of small subunits of rubisco; [METHOD]The small subunits of rubisco and the gene of Pdk were digested by PstI and SalI from pRGN and pUCm-Pdk,and inserted into expression vector pCAMBI1301. Then it was transformed into EHA105 by direct entering way; [RESULTS]The plant transformation vector p1301-Prbcs-PPDK was constructed in this experiment. The selective marker gene was Hpt（Hygomycin phosphate transferase）. By digestion and PCR identification, The vector p1301-Prbcs-PPDK was transferred to Agrobacterium tumefaciens EHA105 ; [CONCLUSION] The pyruvate-phosphate dikinase is the key enzyme of C4 photosynthesis.The experiment constructed a expression vector suit to monocotyledon,which will provide an important experimental base for study on PPDK function.