根癌农杆菌Atu5117基因原核表达载体的构建

Construction of Prokaryotic Expression Vector of Agrobacterium Tumefaciens Atu5117 Gene

为进一步提高农杆菌在植物转基因中的效率,构建一个原核基因表达载体,为农杆菌T-复合物招募蛋白VBP1的编码基因Atu5117的表达调控机理研究奠定基础。首先,通过聚合酶链式反应(PCR)获得Atu5117基因5’端上游具有转录活性的基因片段。然后,用绿色荧光蛋白基因(gfp)和493bp Atu5117启动子构成嵌合基因,体外构建原核表达载体PRSET-Agfp1,并转化到宿主细胞(大肠杆菌DH5α)。通过对绿色荧光蛋白(GFP)的跟踪观察,最终确定有启动子转录活性的基因片段。结果表明,该原核表达载体PRSET-Agfp1构建成功,为Atu5117基因的表达调控机理研究奠定基础。

It is to further improve the efficiency of Agrobacterium in plant genetic transformation,construct a genetic transformation vector suitable for Agrobacterium tumefaciens,and lay the foundation for the study on the regulation of the expression of Atu5117,which encodes the T-complex recruitment protein VBP1 in Agrobacterium tumefaciens.At first,by polymerase chain reaction(PCR),the DNA fragment upstream of the 5′end of the gene which contains transcriptional activity can be obtained.Then,the chimeric gene is constructed with green fluorescent protein gene(gfp)and 493bp Atu5117 promoter,and the prokaryotic expression vector PRSET-Agfp1 is constructed in vitro and transformed into host cells(E.coli DH5α).Through the tracking and observation of green fluorescent protein(GFP),the gene fragment with promoter transcriptional activity is finally identified.The results show that the prokaryotic expression vector PRSET-Agfp1 is successfully constructed,which lays a foundation for the study of the expression regulation mechanism of Atu5117 gene.