利用MLO缺失突变体解析ROR在甜瓜白粉病广谱抗性中的作用

Resistance breeding about powdery mildew was difficult since of physiological race fast differentiation in melon. The MLO protein was considered a negative regulator to plant defense mechanism. Our previous study showed that CmMLO2 mutants in wild melon has resistant to powdery mildew. After inhibition of CmMLO2 the expression by RNAi technology, susceptible melon germplasm also obtained broad spectrum resistance to powdery mildew.So we made speculated the role is likely to rely on a synaptic fusion protein which named ROR. The project is proposed on the basis of ROR genes have been cloned, constructed the RNAi expression vector, respectively validation susceptible cultivated strains and the MLO mutations resistant strains missing ROR powdery mildew resistance performance, Using the yeast two-hybrid (Y2H) system and co-immunoprecipitation (Co-Ip) technology to investigate the interaction of the the ROR and MLO in powdery mildew pathogenesis. Combination of the ROR fluorescent cellular localization and expression in different tissues at the same time, clarify the link between the MLO and ROR in broad spectrum of powdery mildew resistance performance, definitive the protein interaction mechanism for the create broad-spectrum resistance to the melon germplasm materials .

甜瓜白粉病生理小种众多，分化快，是抗性育种面临的主要困难。MLO蛋白被认为是一种植物防卫机制的负调控因子，前期研究发现具有CmMLO2缺失突变的野生甜瓜对白粉菌具有抗性，通过RNAi技术抑制CmMLO2的表达后，感病的甜瓜种质也获得了对白粉病的广谱抗性，进一步研究发现，甜瓜的这种广谱抗性机制很有可能要依赖一种突触融合蛋白ROR。本项目拟在已经克隆ROR基因的基础上，通过构建ROR基因的RNAi表达载体，分别验证易感栽培株和MLO突变抗性株在缺失ROR后对白粉病菌的抗性表现，利用酵母双杂交（Y2H）系统和免疫共沉淀（Co-Ip）技术探讨ROR和MLO两个蛋白在白粉病发病过程中的相互作用。同时结合ROR的荧光细胞定位和在不同组织中的表达情况，阐明甜瓜白粉病的广谱抗性表现中MLO和ROR两个蛋白之间的联系，最终明确ROR和MLO蛋白的互作机制，为创制广谱抗性甜瓜种质材料提供理论基础。

本项目通过现代生物技术方法，克隆、分离了甜瓜的ROR2基因，将其与绿色荧光蛋白（GFP）构建融合表达载体，进行细胞定位研究，结果表明，CmROR2蛋白定位在质膜上。进一步通过Real-time PCR 定量分析白粉病发病过程中ROR基因的表达模式，在白粉病发病过程中，抗性材料的ROR表达量高于感病品种，ROR属于甜瓜白粉病的正向调控蛋白。采用生物信息学预测和酵母双杂交系统验证的方法，研究了ROR和MLO两个蛋白在白粉病发病过程中的互作机制。两个蛋白以互作方式参与了甜瓜白粉病菌丝的入侵或者延迟进程。利用MLO缺失突变的甜瓜野生种质，通过RNA干扰技术，构建干扰载体将改造后的ROR分别转入具有MLO缺失突变的甜瓜植株和MLO正常栽培甜瓜种质，通过抑制内源ROR基因的表达，从而验证了ROR在甜瓜白粉病广谱抗性中的作用，这一研究结果明晰甜瓜白粉病野生种质的广谱抗性机理，为下一步创制优良的广谱抗性育种材料提供了坚实的理论基础。