Study On The Expressional Regulatory Mechanism Of Atu4860in Agrobacterium Tumefaciens

Agrobacterium species are known as the only organism capable of interkingdom gene transfer. The phytopathogenic bacterium has the ability to cause crown gall disease of dicotyledonous plants. Agrobacterium tumefaciens mediated T-DNA transfer technology has become the most widely used plant transgenic method, but its efficiency is so low and the unpredictability of the results are severe problems facing transgenic technology. More research should be done to further understand molecular mechanism of Agrobacterium-mediated T-DNA transfer, and to discuss whether we can improve VBP expressing quantity in Agrobacterium cells to improve the efficiency. The research is of great significance on its application in transgenic plants. The expressional regulatory mechanism of Atu4860encoding Agrobacterium T-complex recruiting proteins VBP2is studied.Contents:1. DNA fragment (500bp) that may have promoter transcriptional activity of Atu4860were acquired by PCR. Construct promoter detection carrier containing a modified green fluorescent protein as the reporter gene, and convert the recombinant plasmid into E.coli DH5a. Through antibiotics screening and the tracking observation of EGFP, preliminarily determine the promoter fragments with transcriptional activity.2. Predict transcription factors that may in combination with the promoter, and localize binding sites in the sequence. Construct recombinant plasmids containing shorter upper sequences of Atu4860(468bp、248bp、175bp、130bp) to identify the core region with promoter activity.3. To drive the transcription of rfp by promoter of Atu3617encoding constitutive expressional50S ribosomal protein, the gene fragment was inserted into multiple cloning site of pRSET-AGFP2S, providing a basis for the quantitative analysis of green fluorescent protein. 4. Use different induction conditions (culture, temperature, pH) to cultivate E.coli containing pRSET-AGFP2S to validate how environmental factors affect the activity of Atu4860.5. Use online software to predict the position weight matrix of transcription factor binding sites, design primers, and do site-directed mutagenesis for several important bases with the method of overlap-PCR. Analysis expression of fluorescence protein qualitatively and quantitatively.6. The above experiments were based on predictions, so we should use different induction conditions to verify whether transcription factor fadR binding site, rhaS binding site and CAP/CRP binding sites are really existed.Conclusions:(1) The promoter probe vector was transformed into E. coli after the successful construction.(2) The175bp upstream sequences of Atu4860is the core region that having promoter activity.(3) Red fluorescent protein was properly expressed in host E. coli.(4) The CAP/CRP binding sites predicted by bioinformatics software are important for the promoter activity.(5) When E. coli were cultured at different temperatures and pH conditions, different expression of EGFP regulated by Atu4860promoter indicated that the Atu4860promoter may be regulated by temperature and pH.(6) The base A in-125mutation point is not the critical regulatory site. The base G in-140mutation point has a negative regulatory effect for the expression of protein. The base G in-107mutation point has a positive regulatory effect for the expression of protein. Agrobacterium T-complex recruiting protein VBP2begins to express at a early time, decreases after middle time and remains at a low level.(7) We deduce that their are fadR binding site and CAP/CRP binding site in Atu4860promoter sequences, but rhaS binding site not existed.