CHX-GFP融合基因的构建及在胡萝卜愈伤组织中瞬时表达

摘要

In order to construct CHX-GFP fusion gene and expressed it in the calli of carota,fusion PCR was used because it needed not analysis restriction enzyme site of DNA fragments and took less labor and time. The plant expression vector of p1301-35S-CHX-GFP-Nos was constructed by recombinant technology. Using 1/2MS+AS 100μmol/L and MS+6-BA 0 . 5 mg/L+2 ,4-D 0 . 5 mg/L+agar 6 g/L+sucrose 30 g/L+glucose 10 g/L+AS 50μmol/L as infection and co-cultivation medium respectively the vector was transformed into the cells of calli after 4 duration co-cultivation with agro-bacterium ( EHA105 ) . The CHX-GFP was expressed transiently in the calli with frequence on average was up to 75%. These results would provide foundation for further investigation of CHX sub-cell localization and gene function.%为构建CHX-GFP融合基因并将其在胡萝卜愈伤组织中瞬时表达。采用融合PCR方法,将属于CAP2家族的Na+/H+反向转运蛋白基因CHX和GFP基因相融合,利用中间载体,采用DNA重组技术将CHX-GFP融合基因插入到p1301植物表达载体中,采用冻融法将重组质粒导入到农杆菌中,遗传转化时所用侵染液为1/2MS+AS 100μmol/L,共培养基为MS+6-BA 0.5 mg/L+2,4-D 0.5 mg/L+琼脂6 g/L+蔗糖30 g/L+葡萄糖10 g/L+AS 50μmol/L,共培养时间为4 d。成功构建了p1301-35S-CHX-GFP-Nos植物表达载体,CHX-GFP融合基因在胡萝卜愈伤组织中得到瞬时表达,表达效率为75%。采用融合PCR获得CHX-GFP融合基因的方法比较简单,无须知道DNA序列的酶切位点,省时省力,CHX-GFP融合基因及其表达载体的成功构建将为进一步探查CHX亚细胞定位及CHX基因的功能奠定了基础。