| Agrobacterium tumefaciens is a gram-negative plant pathogens;it can infect the injured plant,causing plant crown gall disease.After sensing the signals from the injured plant,Agrobacterium tumefaciens can transport T-DNA to plant cells in the form of T-complex and integrate the T-DNA into the genome of plant cells to express,thereby interfering the normal division of plant cells and causing tumors.In the transport process of the T-complex,VBPs play the function of recruiting tne T-complex to T4SS by interacting with the VirD2 in the T-complex.Atu4860(vbp2),which is located in Agrobacterium tumefaciens linear DNA,is one of the three homologous genes encoding VBPs,and it affects the transformation efficiency of Agrobacterium-mediated method.We expect to explore the mechanism of Agrobacterium-mediated transformation from the perspective of mol-ecules by studying the expression regulation mechanism of vbp2 promoter,so that it can provide a theoretical basis for solving the problems of low transformation efficiency in plant transgenic technology,and improve the transformation efficiency of T-DNA.The research on the regulation mechanism of Agrobacterium tumefaciens vbp2 gene promoter ac-tivity mainly divides into two aspects:one side is the study on how the inducing factors induce the vbp2 gene promoter.To quantify the activity of the vbp2 gene promoter accurately,this experiment adopts the method of in situ replacement;this method uses the encoding sequence of the rfp coding frame to replace the vbp2 coding frame by using the principle of homologous recombination,so that the expression of rfp gene can be controlled by vbp2 promoter in situ.The constructed in situ substitution strain is named C58vbp2?rfP.We used acetyl syringone(AS),pH and L-rhamnose(Rha)to induce C58vbp2?rfp,and quantified the activity of vbp2 promoter with the fluorescence intensity of RFP in the total bacterial protein.The results show that AS,pH and Rha have a certain regulatory effect on the vbp2 promoter respectively.The optimum induction concentration of AS was 150 ?M.When the pH range is 5.5-7.5,the vbp2 promoter activity increases with the increase of pH.The optimal induction concentration of Rha is 100 ?M.The other side is the study on the active regu-lation region of the vbp2 gene promoter;the key point is to determine promoter's the active regu-latory region responding to the induction factor respectively.According to the predicting results of transcription factor binding point of vbp2 promoter,we shorten the vbp2 promoter in varying degrees,and then connect the shortened promoter to the reporter gene egfp.Finally,we built eight single-fluorescent carriers with different length promoters.We use AS and Rha to induce different length of promoters respectively at the optimum induction concentration.The outcome shows that the active regulation region of AS and Rha are on the upstream of the vbp2 promoter between 165 bp to 260 bp and between 126 bp to 165 bp,respectively.Comprehensive analysis of all results,AS,pH and Rha have a certain regulatory effect on the vbp2 promoter activity in Agrobacterium tumefaciens.By comparising the best inducement conditions of vbp2 promoter induced by AS and pH with the best inducement conditions for vir genes induced by AS and pH,we find that the two cases are not consistent.Therefore,we speculate that the function of vbp2 gene is not completely consistent with the function of other vir region genes,that is,VBP may have other functions besides participating in T-DNA transport with other Vir proteins.The results of inducing vbp2 promoter in Agrobacterium tumefaciens by Rha show that Rha is an inducer to regulate the transcriptional expression of vbp2,and it has a positive regulatory impact on the promoter of vbp2. |
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