十字花科黑腐病菌致病调控因子HpaR的作用分子机制

The MarR family transcriptional regulators play important regulatory role in various cellular processes in bacteria; MarRs regulate gene expression by binding to their target promoters and then affecting the transcription of the target gene. In 2007, we found in Xanthomonas campestris pathovar campestris (Xcc) that the MarR family transcriptional regulator HpaR is essential for the virulence of the pathogen with an unknown mechanism. In 2010, under the financial support of the SNFC, the HpaR regulons were identified by using DNA microarray hybridization combined with semi-quantitative RT-PCR. The result showed that there are a total of 610 genes, including 32 known virulence genes, are regulated by HpaR. The aim of this project is to clarify the molecular baisis for the function of the virulence regulator HpaR through: (1) find out the potential HpaR-binding DNA sequences and candidate genes which are directly regulated by HpaR by analysis of the promoter sequences of the 610 genes; (2) determine the genes directly regulated by HpaR by using electrophoretic mobility shift assay (EMSA); (3) determine the exact HpaR-binding sequences in each target promoter by using DNaseI Footprinting analysis; (4) identify the nuclietides that is essential for HpaR binding by point mutation analysis of the HpaR-binding DNA sequences; (5) determine the effects of HpaR binding on the activity of the target promoter by using promoter-gus reporter and in vitro transcription analysis.

细菌的MarR家族转录调控蛋白调控基因表达的方式是和目标基因启动子结合从而影响基因的转录。2007年，我们发现十字花科黑腐病菌的一个MarR家族调控蛋白，HpaR，是该菌致病必须的，但机制不明。2010年，在自然科学基金短期项目资助下，我们鉴定了HpaR的调控元，发现有610个基因（包括32个致病基因）受HpaR调控。本项目计划：（1）通过分析这610个受HpaR调控的基因的启动子序列，预测HpaR结合的靶序列和HpaR直接调控的基因；（2）通过凝胶阻滞实验确定受HpaR直接调控的基因；（3）通过DNaseI Footprinting鉴定HpaR 在各个目标启动子上结合的确切位置和序列；（4）通过点突变分析确定HpaR识别序列中与HpaR结合必须的碱基；（5）通过体外转录实验确定HpaR的结合对目标基因转录的影响；以揭示HpaR在Xcc的致病以及其它生理过程中发挥作用的分子机制。