刺芹侧耳抗软腐病相关基因的挖掘及功能解析

Recently, soft rot disease occurring to Pleurotus eryngii has become an emerging disease, epidemically out broken in Korea and many parts of China, causing the production of P. eryngii decreased by 15-20%. According to previous study, pathogen to the soft rot disease of P. eryngii epidemic in China was isolated and identified as Panteoa beijingensis. So far there has been few reports about resistance associated gene against soft rot disease in P.eryngii. Our preceding study indicated that P. beijingensis had 364 type-III effector proteins (T3Es). Interactions between T3Es and resistance proteins were the most important recognizing ways, providing with new ideas about discovery of resistance genes against soft rot disease in P.eryngii. Therefore, this study initially protein interaction network between P. beijingensis type-III effector proteins (T3Es) and Pleurotus eryngii based on the two kinds of methods of interolog protein and domain-based interacting. Thus analyses and focuses on interaction characteristics between type-III effector proteins (T3Es) and proteins possessing resistance conserved domain. Then verifying the protein interacting characteristics by bacterial two-hybrid system and GST pull-down technology, screen and acquire the target genes against soft rot disease in P.eryngii, and the functional analysis of resistance associated gene was investigated in vivo and in vitro. Not only can this study probe the interactions mechanism between pathogen and host, but also provide a theoretical basis for disease resistant breeding of P. eryngii. It is significant for safe, economical and efficient prevention of soft rot disease epidemic in P. eryngii.

刺芹侧耳（Pleurotus eryngii）软腐病是近年来发生的新病害，已发现在韩国和我国多地爆发，导致刺芹侧耳减产15～20%，国内流行的病原菌为北京泛菌（Pantoea beijingensis），迄今尚无刺芹侧耳抗软腐病相关基因的报道。前期研究发现北京泛菌具有364个Ⅲ型效应蛋白，鉴于抗病蛋白与Ⅲ型效应蛋白的相互作用是二者最重要的直接识别方式，为刺芹侧耳抗软腐病相关基因挖掘提供了新思路。因此，本申请拟首先采用基于同源蛋白映射和结构域互作两种方法构建北京泛菌Ⅲ型效应蛋白与刺芹侧耳的蛋白互作网络，由此分析并锁定Ⅲ型效应蛋白与具有抗病蛋白保守结构域的蛋白的互作特征，通过细菌双杂交和pull-down验证蛋白互作特性，从而筛选并获得刺芹侧耳抗软腐病相关基因，并对其进行体内、体外功能验证。本研究不仅可探究该病原菌与宿主的相互作用机制，也可为刺芹侧耳的抗病育种和病原菌防控提供理论依据。

刺芹侧耳（杏鲍菇）软腐病是近年来发生的新病害，已发现在韩国和我国多地爆发，导致刺芹侧耳减产15～20%。本项目组前期研究发现，国内流行的病原菌为北京泛菌，迄今尚无刺芹侧耳软腐病抗病相关基因的报道。以抗病相关基因为靶标，与优异的农艺性状基因聚合，培育优良的刺芹侧耳抗病品种，是安全、高效的绿色防控方法。本课题采用“有向的贝叶斯算法”构建刺芹侧耳与北京泛菌III型效应蛋白的互作网络，数据训练采用所有已知的病原-宿主蛋白互作对，并通过实验验证蛋白互作关系。从微观结构和转录组两个方面分析刺芹侧耳对软腐病病原菌-北京泛菌的响应机制，扫描电镜和透射电镜观察显示，侵染9天后，菌丝出现大面积的破裂，细胞壁结构变形，边缘模糊，细胞结构完全被破坏，且细胞内的活性氧自由基升高，参与氧化还原、过氧化物酶体、自噬、细胞壁降解、丝氨酸苏氨酸（STK）蛋白激酶等基因在感染的组织中表达发生显著改变。筛选出抗病菌株1个，与易感菌株进行变异检测，结合转录组以及III型效应蛋白与刺芹侧耳的蛋白互作网络综合分析，筛选出6个STK蛋白激酶和1个含有FAD-binding蛋白与刺芹侧耳抗病相关，并明确了抗病相关基因的功能和结构特征。本研究不仅可探究该病原菌与宿主的相互作用机制，也可为刺芹侧耳的抗病育种和建立有效的病原防控措施提供理论依据和相关思路。