Study On The Expressional Regulatory Mechanism Of Atu5117 In Agrobacterium Tumefaciens

Agrobacterium is a plant pathogen that can survive in the soiLAgrobacterium can invade a plant cell, its genes into plant genes, resulting in disease.Genetic transformation techniques are the best plant transgenic technology, this technology was developed by Agrobacterium-mediated, but the low efficiency of this technology, its results unpredictable.This process is very complex and linked to the transfer of T-DNA and VBPs and a plurality of complex processes. We try to understand the principles and mechanisms of this process, to improve the efficiency of this technology. Such efforts are important.In the research of the expression regulation mechanism of Agrobacterium tumefaciens’ promoter, most researchers are used to choose single fluorescent gene like gfp as the promoter’s reporter gene. However, the expression product is affected not only by promoter but also by the copy number of the carrier. So this method cannot be ruled out the interference of vector’s copy number, so it has a big error. To solve the problem, this experiment is to build a dual fluorescence indicated carrier pUCA19-GFP/RFP. We use fluorescent protein gene rjp as the reporter gene of carrier’s copy number. First, connect the target Agrobacterium promoter atu5117 and gfp fragments. Second, connect it with Agrobacterium expression vector pUCA19 through the technologies of enzyme digestion, enzyme link and others. Then we get a kind of recombinant plasmid pUCA19-GFP. Third, connect pUCA19-GFP with promoter atu3617 and rfp. In this way dual fluorescent indicated carrier pUCA19-GFP/RFP can be constructed. We use fluorescence microscopy to detect whether the recombinant vector pUCA19-GFP/RFP emit red and green fluorescence. We use genome sequencing to detect whether the dual fluorescence reporter genes are in the recombinant vector pUCA19-GFP/RFP. The test results show that the dual fluorescence indicated Agrobacterium carrier pUCA19-GFP/RFP is successfully constructed.Heterologous expression in E. coli experiments regulation, the expression in E. coli atu5117 promoter activity was significantly higher than the expression and activity in Agrobacterium tumefaciens GMI9017A2 and GMI9017, atu5117 promoter activity as the growing changes in the logarithmic of highest activity, presumably to start express atu5117 promoter has certain chronological. Under different growth conditions E. coli Agrobacterium atu5117 start against AS affect promoter activity concentrations, probably because there is no response mechanism in E. coli and other VirA-VirG of AS. In the alkaline pH environment, atu5117 Promoters expression irregular activity at acidic pH environment activity rise significantly, possibly with Agrobacterium plant during chemotaxis acidic substance related. Promoter activity was significantly affected by temperature, the promoter activity as the temperature increases, the cells may start to resist high temperatures mechanism atu5117 relevant.Expression control experiments in Agrobacterium, atu5117 promoter activity changes with growth promoter activity in log phase maximized. In the absence of homologous genes atu4860 of GMI9017A2 in Agrobacterium atu5117 start expression activity was higher than in sub-expression activity in GMI9017, atu5117 start there is some regulation of the relationship between the child and the homologous gene atu4860 promoter. At different temperatures and pH conditions, it is similar promoter activity in E. coli. AS concentration on experiments, atu5117 promoter activity did not change the law, the mechanism needs further study.