核桃细菌性黑斑病菌环二鸟苷酸降解酶在DSF信号传导中的功能研究

核桃细菌性疫病是世界性核桃病害，严重影响了核桃的产量和品质，同时危害森林健康。环二鸟苷酸（c-di-GMP）是普遍存在于细菌中的第二信使，能调控病原菌的致病相关过程。本项目以核桃黑斑病菌为研究对象，鉴定其信号受体蛋白的功能。研究内容包括：（1）通过酶活性的测定及基因敲除、过表达等方法，研究RpfR在调控胞内c-di-GMP水平中的功能；（2）采用等温滴定量热法以及酶促反应速率的测定，研究RpfR与DSF信号分子的结合，同时通过基因敲除的方法研究RpfR对DSF信号传导的影响；（3）对RpfR突变株进行毒力分析、植物侵染试验、毒力因子的表达检测，鉴定RpfR对病原菌致病性的影响。研究目标是揭示参与c-di-GMP代谢的酶对细菌DSF信号分子的传导调控功能。本项目的实施，将为深入研究核桃细菌性病害的致病机理提供新的思路，以控制和减少核桃病害的发生。

Bacterial blight of walnut is worldwide disease，it affected the yield and quality of walnut seriously，and it is harmful to forest health. Cyclic di-GMP has emerged as one of the most common and important bacterial second messengers, c-di-GMP has been shown to regulate pathogenicity related processes. This study takes walnut bacterial blight pathogen as object，to identify the function of its signal receptor. The detailed research contents including: (1) to study the function of RpfR in modulating intracellular c-di-GMP level through phosphodiesterase activity determination, gene knockout and overexpression; (2) to investigate the interaction of RpfR and the DSF signal by the aid of isothermal titration calorimetry (ITC) analysis, furthermore, through the method of gene knockout to research the impact on DSF signal transduction; (3) to characterize the function of RpfR in the influence on bacterial pathogenicity through analysis of the virulence, plant infection test, and to detection expression of virulence factor in RpfR mutant strain by quantitative RT-PCR. The goal of this project is to reveal the function of enzyme participating in c-di-GMP metabolic in DSF signal transduction. The implementation of this project will definitely promote our further study of walnut bacterial disease pathogenic mechanism, and provide a strong basis in theory for effective and new technology.