Study On The Expressional Regulatory Mechanism Of Vbp Genes In Agrobacterium Tumefaciens

Agrobacterium tumefaciens is one of gram-negative plant pathogen that bear in the soil. In nature conditions it can infect injured plants, resulting in crown gall tumor disease. The T-DNA from Ti plasmid was transferred to the host cell in the form of protein-single-stranded-T-DNA (called T-complex) through the type IV secretion system (T4SS) witch located at both ends of the Agrobacterium cells. When the T-DNA from the natural transgenic plasmid was replaced by exogenous DNA, Agrobacterium tumefaciens is still able to transfer the exogenous T-DNA to the host plant and leads to genetic transformation in host cells. Thus, Agrobacterium-mediated T-DNA transfer technology has become the most widely used plant transgenic technology. The T-DNA transfer in Agrobacterium cells requires a lot of proteins to participate in. A recently identified protein that can bind to VirD2(VirD2-Binding Protein, VBP) is proved to play a role of recruiting T-complex to the T4SS. So they have been named as recruiting proteins of Agrobacterium T-complex. Agrobacterium contains three homologous genes, named vbp1, vbp2and vbp3; They encode three kinds of homologous proteins, named VBP1, VBP2and VBP3. Although the VBP has been compared to determine the biological function, its biochemical function is still not clear. In order to make the research of the recruit protein VBP form a complete system from the biological function of genes to biochemical function of the protein and expression regulation, this experiment is to research the expression regulation mechanism of VBP protein by looking for the binding proteins in the area of vbp gene regulation and studying the regulation of gene expression of these potential proteins. We analyze whether it can improve the efficiency of T-DNA by improving the quantity of VBP protein in Agrobacterium cells. At first, in this experiment, we use molecular biology methods to look for binding protein in the upstream of vbp gene fragment. Dynabeads(?) M-280Streptavidin has a streptavidin monolayer covalently coupled to the surface. We add the biotinylated vbp gene to the washed Dynabeads, let the complex combine with the supernatant fluid of induced Agrobacterium C58strains and elute the specific binding protein. Then we make SDS polyacrylamide gel electrophoresis to find the distinct band and cut down by comparing with the control. According to the protein components in the mass spectrometry results, we find there is no proteins that conform to the requirement of our experiment. Then, we turn out to study the regulation of gene expression by bioinformatics methods. On the web of Prokaryote Promoter Prediction, we preliminary search out some transcription factors which can regulate the expression of vbpl gene. These transcription factors are sigmaA, Fnr, tgntataat, sigma38and crp. Then we find out their binding sites and determine the length of the promoter. We will delete the promoter sequence of the pRSET-A plasmid, forming an empty carrier. Then we insert the vbpl promoter sequence that labeled gfp gene into the empty carrier, building a pRSET-avg recombinant expression plasmid. We can see green fluorescence from bacteria liquid by the fluorescence microscope, illustrating that in the recombinant expression plasmid, vbpl promoter can start the transcription of gfp gene under the regulation of these transcription factors. The following work is to analyze the base sequence of binding sites of these transcription factors, forming three mutational recombinant expression plasmids. They are pRSET-ml, pRSET-m2and pRSET-m3. After mutation, we will analyze whether the vbpl promoter can still start the transcription of gfp gene by seeing green fluorescence from bacteria liquid. We find out that if we delete the binding sites of sigmaA_breve, it will lower the expression of gfp gene; If we replace the conserved bases among these transcription factors, one mutation is that vbpl promoter can enhance the expression of gfp gene, the other mutation can stop the expression of gfp gene. Thus, in order to improve the Agrobacterium-mediated transgenic efficiency, we can enhance the expression quantity of VBP1protein by replacing some base sequence of vbpl promoter.