细菌第二信使环二鸟苷酸(c-di-GMP)分子对芽胞杆菌防病活性、游动性、生物膜起重要调控作用，但目前对c-di-GMP代谢调控研究较少。枯草芽胞杆菌GLB191对葡萄霜霉病防效显著，前期发现GLB191唯一的c-di-GMP降解酶PdeH具有自我负调控功能，但PdeH不能与自身启动子结合，与已报道的E. coli PdeL自调控机制不同，推测PdeH具有自我调控的新机制。本研究拟通过转座子突变体库的筛选、构建单/双突变体和功能回补等，确定PdeH通过中间调控因子对自身转录进行负调控；采用EMSA和位点突变等方法，检测中间调控因子与pdeH启动子的结合；通过报告质粒、qPCR、Western blot和ITC等方法，确定PdeH对中间调控因子的调控机制；最终明确PdeH自我负调控新机制。研究结果有利于解析细菌c-di-GMP代谢通路的调控机制，而且为生防芽胞杆菌的筛选及防效提高提供参考。

Cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger in bacteria and plays an important role in regulating biocontrol activity, swarming and biofilm in Bacillus. However, the regulation of c-di-GMP metabolism is not known much. B. subtilis GLB191 is an endophyte which showed high control efficacy against grape downy mildew. Our previous results showed that the only phosphodiesterase (PDE) PdeH in B. subtilis regulates its own promoter activity but not via directly binding its own promoter region, which is different from the known mechanism of PdeL auto-regulation in E. coli, indicating a new auto-regulation mechanism for PdeH in Bacillus. To interpret the new mechanism, screening the transposon library in combination with construction of single/double deletion mutants and complementation were used to determine the autoregulation of PdeH via the regulatory gene. Then EMSA will be used to determine the direct binding of regulatory protein to pdeH promoter or not. GFP reporter system, qPCR, Western blot and ITC et al. will be further used to determine the regulatory mechanism of PdeH on regulatory gene or its protein, and finally interpret the auto-regulation mechanism of PdeH. This project will contribute not only to exploring the regulatory mechanisms of c-di-GMP signaling metabolic pathway, but also to screening and increasing the biocontrol efficacy of Bacillus.