含绿色荧光蛋白(gfp)基因的植物重组表达载体pBI121-GFP-62390和pBI121-GFP-51780的构建

[目的]构建含绿色荧光蛋白(gfp)基因的植物重组表达载体pBI121-GFP-62390和pBI121-GFP-51780。[方法]用PCR方法扩增psmGFP的GFP片段,BamHI和SacI双酶切PCR产物,同时用BamHI和SacI双酶切pBI121。从琼脂糖凝胶中回收纯化pBI121的大片段,经连接、转化、鉴定出改造后的质粒。[结果]用PCR方法扩增得到长度为740 bp的增强型的绿色荧光蛋白基因片段,克隆入pBI121表达载体后,获得了新的重组质粒pBI121-GFP。分别将编码拟南芥BAG7、BAG4蛋白的基因At5g62390和At3g51780通过PCR方法扩增后,克隆入pBI121-GFP,构建了用于超量表达BAG蛋白基因的GFP融合蛋白双元表达载体pBI121-GFP-62390和pBI121-GFP-51780。利用细胞感受态法将该植物表达载体分别导入根癌农杆菌GV3101中。[结论]为进一步研究BAG蛋白在拟南芥抗性胁迫中的功能及其在细胞内的动态分布奠定了基础。

Construction of the Recombinant Expression Plasmid pB1121-GFP-62390 and pB1121-GFP-51780 Contained Green Fluorescent Protein Gene

[Objective] The research aimed to construct the recombinant expression plasmid pBI121-GFP-62390 and pBI121-GFP-51780 contained green fluorescent protein gene.[Method] The GFP fragment was amplified by PCR.PCR production and pBI121 were double digested using BamH I SacI.Big pBI121 fragment was purified from agarose gels.The plasmid was appraised through joint and transformation.[Result] A 740 bp fragment of green fluorescent protein gene was amplified by PCR.The fragment was cloned into pBI121 vector,and a new recombined vector was obtained.Arabidopsis' calmodulin genes At5g62390 and At3g51780 were cloned into the recombined vector pBI121-GFP.The new plant expression vector named pBI121-GFP-62390 and pBI121-GFP-51780 were constructed.The recombined plant expression vector were transferred into Agrobacterium tumefaciens strains of GV3101.[Conclusion] The study is a good start for the further research on the functions and location of genes At5g62390 and At3g51780.