| Agrobacterium tumefaciens is a phytopathogenic Gram-negative soil bacterium that is able to genetically transform many plant cells with the production of tumors. Agrobacterium contains Agrobacterium tumefaciens and Agrobactrium rhizogenes, in which Agrobacterium tumefaciens is the best studied. Agrobacterium tumefaciens can cause typical crown gall disease, which is caused by Agrobacterial Ti plasmid (tumor-inducing plasmid). Agrobacterium tumefaciens can transfer the tumorigenic segment of Ti-plasmid (T-DNA) to the plant cells, and T-DNA can exist in plant chromosome stably. If T-DNA is replaced by a target gene, the target gene can express in plant cells. Although the Agrobacterium mediated genetic transformation is widely used, the problems of low efficiency and the unpredictable transformation effects are still existed.VBPs (VirD2-binding protein, VBPs) can specifically bind to VirD2 and are involved in the tumorigesis. VBPs are involed in the recruitment of T-complex to the VirB/VirD4 type IV secretion system (T4SS) transport site. Meanwhile, VBPs are important in the recruitment of a conjugative plasmid to a different transfer system from the VirB/VirD4 T4SS. Therefore, VBPs were defined as ’recruiting proteins’. VBPs are highly conserved; there are three copies of homologous genes in Agrobacterium. atu5117 is located on the plasmid pAtC58, both atu4856 and atu4860 are located on the linear chromosome and very close to each other, and atu5117 is located only 3 kb away from the conjugative transfer genes. By researching the mechanism of atu4860 promoter, we expect to explore the function of VBPs, improving the efficiency of transformation.The key to reasearching the regulatory mechanism of Agrobacterium atu4860 (vbp2) promoter activity, is to quantitatively determine atu4860 promoter activity. Thus, we constructed a dual-fluorescent report carrier, and established a method for quantitatively detection the promoter activity. In addition to the lacZ’ promoter of pUCA19 plasmid was removed, two DNA fragments were inserted reversely in this carrier. A DNA fragment is atu3617 promoter with rfp, another fragment is atu4860 promoter with egfp. These dual-fluorescent report carriers meet four requirements. They can copy and express in both E. coli and Agrobacterium, no other promoter will affect the results of the experiment, the influence of copy number of the plasmid can be eliminated, and atu4860 promoter regulatory function can be quantitatived. On the basis of several constructed dual-fluorescent report carriers, we found that both atu3617 and atu4860 promoters can initiate the reporter gene expression in Escherichia coli DH5a and Agrobacterium GMI9017△vbp2 by fluorescence microscopy. Also, it is found that a method in which fluorescence intensity of total protein represent the amount of fluorescent protein is more reliable than another method in which bacterial fluorescence intensity represent the amount of fluorescent protein by fluorescence spectrophotometer. Red fluorescence intensity as a reference, the ratio of green fluorescence intensity and red fluorescence intensity can represent atu4860 promoter activity.By the growth curve, we found that the expression of fluorescent protein in bacteria will not affect the growth of bacteria. Tumor-inducing factors including AS and pH have effects on atu4860 (vbp2) promoter. And in the experiments induced by AS and pH, we found that the optimal AS concentration and pH for atu4860 promoter regulation is between 100μM and 200 μM, between pH 5.0 and pH 6.5, respectively. The result is consistent with the effective use of AS concentration in the range of 50～200μM and is consistent with the range of pH 5.2～6.0. Therefore, AS and pH can affect atu4860 promoter activity, and atu4860 may have some connection with vir gene expression. |
PDF Full Text Request