花生NBS-LRR类抗病基因AhRRS5抗青枯病机理研究

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.

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。

花生是我国重要的油料和经济作物，由青枯雷尔氏菌引起的青枯病严重危害花生的产量和品质，迄今尚未获得花生抗青枯病的关键基因。前期研究表明在烟草中异源超表达花生NBS-LRR类新基因AhRRS5能稳定增强对青枯菌侵染的抗性，但其抗病分子机制尚不清楚。本项目通过发根农杆菌侵染法获得AhRRS5基因超表达和基因编辑的转基因发状根，通过伤根接种青枯菌抗病鉴定表明该基因在花生上同样具有抗青枯病功能。在青枯菌处理后，超表达转基因发状根抗氧化酶类和R基因信号通路的标记基因的表达都表现持续升高，而基因编辑材料则表现不同程度的降低，表明AhRRS5在花生上具有抗青枯病功能，且通过R基因信号通路介导。基于同源重组方法构建了受青枯菌诱导的花生根部酵母双杂交初级和次级文库，构建pGBKT7-AhRRS5诱饵载体，经验证无自激活和毒性活性，与酵母双杂交文库共转酵母Y2H Gold菌株后，经多次筛库和回转验证，最终获得12个候选互作蛋白，这些蛋白涉及到植物生长发育、能量代谢、激素信号转导、胁迫响应等多个方面。通过酵母双杂交筛选获得AhRRS5互作靶蛋白AhRD21A，该蛋白属半胱氨酸蛋白酶类，其mRNA受青枯菌和低温胁迫诱导上调表达，通过Y2H、Co-IP和BIFC验证了二者在体内和体外都能发生互作，DAB、台盼蓝染色和电导率测定显示瞬间共表达AhRRS5/AhRD21A能够增强AhRRS5引起的过敏性反应，防御反应信号通路的标记基因显著上调表达，表明AhRD21A可以正调控AhRRS5引起的抗病反应。异源超表达AhRD21A基因能够增强烟草对青枯菌侵染的抗性，且防御反应信号通路的标记基因显著上调表达，表明该基因能正调控植物对青枯病侵染的抗性反应。异源超表达花生AhRD21A基因可以提高拟南芥对冷害胁迫的耐受反应，qPCR结果显示冷害胁迫信号通路的标记基因明显上调表达，表明该基因能正调控植物对冷害胁迫的响应，而且是依赖于CBFs的信号通路介导的耐低温响应机制。本研究为系统解析花生抗青枯病信号转导机制提供了理论支持，为花生抗逆分子育种提供新的基因资源。