花生NBS-LRR类抗病基因AhRRS5抗青枯病机理研究

由根部侵染引起的青枯病是影响花生产量和品质的土传性细菌病害，迄今尚未获得花生抗青枯病的关键基因，也缺乏有效的化学防治手段。绝大多数抗病基因编码NBS-LRR结构蛋白，阐明抗病分子机理是花生抗青枯病分子育种的重要基础。前期通过基因芯片筛选获得花生抗青枯病新基因AhRRS5，超表达转化烟草能显著提高青枯病抗性。基于此，本项目拟通过发根农杆菌介导将超表达和Cas9沉默载体分别转化花生感和抗青枯病品种产生发状根，通过细胞和分子生物学研究方法比较分析转基因发状根接种青枯菌前后差异，明确该基因在花生上的抗青枯病功能。通过酵母双杂交筛选AhRRS5的互作蛋白，通过免疫共沉淀和双分子荧光互补验证候选互作蛋白，将互作基因遗传互补转化花生产生发状根，并通过细胞分子生物学手段研究其互作蛋白在花生青枯病抗性中的作用，分析其上下游关系。研究结果有利于揭示花生AhRRS5基因抗病机理，为花生抗青枯病遗传改良提供基础。

Bacterial wilt is an important soil-borne bacterial disease which infects peanut roots and cause reduction in yield and quality severely. Up to now, no key resistance gene has been identified in peanut, and no effective chemical methods can be used. Most cloned disease-resistant genes are NBS-LRR encoding proteins, a better understanding of NBS-LRR disease resistance can benefit genetic improvement of peanut disease resistance to solve the problems caused by Rastonia solanacerum in peanut production. In our previous research, a novel NBS-LRR resistance gene AhRRS5, associated with resistance to bacterial wilt, was screened from microarray analysis and transformed into tobacco. The transgenic tobacco showed enhanced resistance to bacterial wilt significantly. In the present project, Overexpression and CRISPER/Cas9 silencing of AhRRS5 will be transformed into susceptible and resistant peanut cultivar mediated by Agrobacterium rhizogenes, hairy roots were harvested respectively. The resistant function of AhRRS5 will be identified by comparing the difference of cytological and molecular analysis between susceptible and resistant peanut transformed hairy root after inoculated by Rastonia solanacerum. The resistent function will be elucidated in peanut. Interacting proteins of AhRRS5 will be screened by yeast two-hybrid system. The potential interaction proteins will be confirmed by co-immunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC). The potential interaction genes will be transformed into peanuts by complement genetics. The resistant function of AhRRS5-interaction proteins will be identified through cytological and molecular analysis. The relationship of upstream and downstream between and AhRRS5 and interaction proteins will be clarified. The results will help to reveal the underlying mechanism of the resistance to bacterial wilt and benefit the genetic improvement of peanut disease resistance.