根癌农杆菌GW4中磷酸盐转运调控因子PhoB介导的砷氧化调控机制

项目名称： 根癌农杆菌GW4中磷酸盐转运调控因子PhoB介导的砷氧化调控机制

项目编号： No.31500089

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 黄静

作者单位： 华中农业大学

项目金额： 18万元

中文摘要： 砷氧化细菌能将高毒性的As(III)氧化成低毒的(AsV)。砷磷具有很多相似的性质，细菌砷磷代谢及其调控之间可能关系密切。前期研究发现，根癌农杆菌GW4的砷氧化产能，氧化产物As(V)进入细胞并取代部分磷。我们近期发现磷酸盐转运调控基因phoB的缺失使砷氧化速率加快，砷抗性增强，且胞外多聚物减少。生物信息学分析表明砷氧化基因启动子区存在PhoB结合域，预示phoB可能参与了砷氧化的调控，胞外多聚物的屏障作用减弱可能加快砷氧化进而增强对砷的抗性。本研究在此基础上，将通过报告基因融合、qRT-PCR、蛋白质与DNA互作、DNase I足迹法、蛋白质磷酸化等技术研究phoB在砷氧化酶的表达调控及胞外多聚物合成中的作用，建立phoB砷氧化调控及胞外多聚物影响砷氧化的模型。该研究对于认识砷氧化调控及砷-磷共代谢具有重要作用，为理解胞外多糖的生物学意义提供新见解，为砷污染环境的微生物修复提供理论依据。

中文关键词： 基因表达调控；功能基因；互作机制；调控因子

英文摘要： Some microorganisms could oxidize the more toxic As(III) to the less toxic As(V) by arsenite oxidase AioAB to relieve toxic effect. Since many of the similar physic-chemical properties between arsenic and phosphorus, there may be close relationship between metabolism of phosphorus and arsenic, and the co-regulation of arsenite oxidation and phosphorus metabolism is complex. Our previous studies found that Agrobacterium tumefaciens GW4 could obtain energy by oxidizing As(III), and the oxidation product As(V) was incorporated into cells by replacing partial phosphorus. In this study, we focused on the Agrobacterium tumefaciens GW4 which could oxidize arsenite to arsenate. By gene in frame deletion and Atomic fluorescence analysis form, it is found that the missing of phosphate transport system regulator phoB accelerated the arsenite oxidation, and resulted in improvement of arsenite resistance and significantly reduction of exopolysaccharides. The bioinformatic analysis suggested there were some potential PhoB-binding domain in the promoter of arsenic oxidation gene operon. That means phoB may be involved in the regulation of arsenite oxidation. The reduction of exopolysaccharides may allow much more arsenite to enter bacterial cells, and arsenite resistance was enhanced because of the more effective arsenic oxidation. To confirm our hypothesis, we will study the arsenite oxidation regulation mechanism of phoB by introducing comprehensive techniques, which including gene complementation, report gene fusion, qRT-PCR, in vivo/vitro interactions between proteins and DNA, DNase I footprinting, in vivo protein phosphorylation and its detection. The aim of this research is to clarify the function of phoB in arsenic resistance, arsenic oxidase expression regulation and biosynthesis of exopolysaccharides, establish the regulation model of arsenite oxidation mediated by phoB and the influences of exopolysaccharides to arsenite oxidation. The results of this project will give us a significant perspective to better understanding the regulation of arsenite oxidation and the co-regulation of arsenic-phosphorus metabolism, to provide new insights into the biological significances of bacterial exopolysaccharides, and to provide theoretical basis for microbial remediation of arsenic-pollution.

英文关键词： Regulation of gene expression;Functional gene;Interaction mechanism;Regulation factor