农杆菌vbp1基因表达调控分子机理的研究

Research on the Regulation of Molecular Mechanism by Agrobacterium tumefaciens vbp1 Gene Expression

为了提高农杆菌介导的T-DNA转化效率,本研究在实验中采用了生物信息学的方法,来研究农杆菌的vbp1基因表达调控的机理。利用TESS网站和Prokaryote Promoter Prediction网站,检索到tgntataat、fnrbsub、sigma A_breve、crp和sigma38这些调控因子可以调节vbp1基因,因此vbp1基因启动子的大小可以确定下来。然后通过插入绿色荧光基因(gfp)作为标记基因构建三个重组质粒PSET1、PSET2和PSET3来分析这些转录因子的调控序列,将调控序列进行突变,判断vbp1启动子是否能够启动荧光基因的表达。结果是sigmaA_breve的结合位点去除后会削弱荧光基因的表达,而tgntataat、fnrbsub、crp和sigma38这几个调控因子结合位点突变之后,一种可以增强荧光基因的表达,另一种不能表达荧光基因。因此,我们可以通过调节vbp1基因启动子的调控提高农杆菌的转运效率。

To improve the conversion efficiency of Agrobacterium tumefaciens-mediated T-DNA, we took bioinformatics methods to study the mechanism of vbp1 gene expression regulation in Agrobacterium. On the web of TESS and Prokaryote Promoter Prediction, we searched out that tgntataat, fnrbsub, sigma A_breve, crp and sigma38 could regulate the vbp1 gene expression, so we could determine the length of the vbp1 gene promoter. Then we formed three recombinant expression plasmids（PSET1, PSET2, PSET3） by inserting gfp gene as the marker gene to analyze the regulatory sequence of these transcription factors. After the mutation, we could judge whether the vbp1 promoter could start the expression of gfp gene by seeing green fluorescent from bacteria liquid. We found out that if we delete the binding sites of sigma A_breve, it would lower the expression of gfp gene;if the binding sites of these transcriptional regulatory factors（tgntataat, fnrbsub, crp, sigma38） were mutated, one mutation could enhance the expression of gfp gene, while the other mutation could stop the expression of gfp gene.Thus, we could improve the Agrobacterium-mediated transgenic efficiency by regulating some base sequence of vbp1 promoter.