大豆疫霉菌无毒效应因子Avr3a逃避抗病蛋白识别的分子机制研究

Race-specific resistance, induced by the interaction between avirulence effectors and resistance proteins, is a very important form of plant resistance. However, avirulence effectors often evade the recognition from resistance proteins and plant resistance has been overcome. The purpose of this research is to elucidate the molecular mechanism by which avirulence effectors evade the recognition from resistance proteins. Phytophthora sojae, a very important soybean pathogen, is taken as the research object. The critical amino acid sites related to avirulent function of avirulence effector Avr3a has been analyzed preliminarily. On the basis of the result, the transient expression assay would be used to study the influence of mutation of critical amino acids on avirulent function of Avr3a. Furthermore, the nuclear magnetic resonance method would be used to study the influence of mutation of critical amino acids on tertiary structure of Avr3a. Then the relation between avirulent function and tertiary structure would be analyzed in depth with the combination of the two aspects. These research results would contribute to elucidate the molecular mechanism by which avirulence effectors evade the recognition from resistance proteins. It has great significance to prevent loss of resistance and prolong use of varieties.

无毒效应因子与抗病蛋白互作产生的小种专化抗性是植物的一种十分重要的抗病形式，而无毒效应因子常常逃避抗病蛋白的识别导致植物抗性丧失。为阐明无毒效应因子逃避抗病蛋白识别的分子机制，本项目拟以大豆的一种重要病原菌大豆疫霉菌为研究对象，在初步分析出无毒效应因子Avr3a无毒功能关键氨基酸位点的基础上，采用植物瞬时表达技术研究关键位点氨基酸突变对Avr3a无毒功能的影响，进一步采用核磁共振技术研究关键位点氨基酸突变对Avr3a三维结构的影响，然后将二者结合深入分析Avr3a无毒功能与三维结构的关系。本项目将揭示大豆疫霉菌无毒效应因子逃避抗病蛋白识别的分子机制，对于防止品种抗性的快速丧失，延长抗病基因的使用年限具有十分重要的意义。

本项目通过分析无毒效应因子Avr3a在自然群体中的多态性及其对Rps3a的毒性，初步分析出Avr3a逃避Rps3a识别的关键位点，第64和65位的氨基酸位点是关键位点，并进一步利用植物瞬时表达技术验证这两个氨基酸位点是关键识别位点；同时根据毒性序列与无毒序列的差异设计CAPS标记成功区分Avr3a的毒性序列与无毒序列，建立Avr3a毒性序列的分子检测方法；利用原核表达系统表达Avr3a蛋白，进一步通过Ni柱亲和层析获得高纯度的Avr3a蛋白，并采用同源建模法构建Avr3a蛋白的三维结构。通过以上研究阐明了大豆疫霉菌无毒效应因子Avr3a逃避抗病蛋白识别的分子机制，对于防止大豆品种抗性的快速丧失，延长抗病基因的使用年限具有十分重要的意义，同时大豆疫霉菌无毒基因Avr3a毒性序列分子检测方法的建立，为大量、快速和准确鉴定大豆疫霉菌的致病型提供有力支持。