抗腐烂病苹果CrRLK1L基因鉴定及其调控机制研究

Valsa canker is a fungal disease of apple growing area in China even Asia. Receptor like kinase (RLK) takes crucial roles in plant recognition of the pathogen-drived signals and subsequent activation of host proper immune responses. Even though the subfamily CrRLK1L was found recently, several members have been confirmed to regulate plant immune responses against pathogens. In Apple, we demonstrated that 4 CrRLK1L genes showed significantly differential expression during the onset and progression of Valsa canker. In this project, we will focus on the functional analysis of the above 4 genes in ‘Royal Gala’. Taking advantage of over-expression analysis and RNA interference (RNAi) study to compare the resistance of wild types and transgenic lines, we expect to identify disease-resistant CrRLK1L (R-MdCrRLK1L). Furthermore, through comparing the resistant responses of wild types and transgenic lines by using Quantitative Proteomic Analysis, we will investigate the molecular networks regulated by R-MdCrRLK1L. Next, the potential interaction proteins of R-MdCrRLK1L will be screened by using a Yeast Two-Hybrid system and further confirmed by using Bimolecular Fluorescence Complementation (BiFC). Finally, through functional prediction of the interaction potential proteins, we looking forward to clarify the molecular pathway regulated by R-MdCrRLK1L.

腐烂病（Valsa canker）是我国乃至亚洲苹果产区的重大真菌性病害。类受体激酶（Receptor Like Kinase, RLK）在植物识别病原菌信号和激发寄主免疫反应中起重要的调控作用。其中，CrRLK1L亚家族的发现较晚，但部分成员对植物抗病反应的调控作用已得到证实。在苹果上，我们发现4个CrRLK1L基因的表达在腐烂病发生前后存在极为显著的差异。本项目拟以此4个基因为研究对象，在‘皇家嘎啦’中过表达和RNA干扰（RNAi），并比较野生型和转化株系的抗病性，筛选1个抗腐烂病CrRLK1L（R-MdCrRLK1L）基因。之后，基于定量蛋白组学分析，比较R-MdCrRLK1L野生型和转化株系抗病反应的差异，探讨其调控的分子网络。结合酵母双杂交筛选和双分子荧光互补验证，鉴定与其互作的蛋白。通过预测互作蛋白的功能，初步明确其调控的分子路径。

腐烂病（Valsa canker）是我国乃至亚洲苹果产区的重大真菌性病害。类受体激酶（Receptor Like Kinase, RLK）在植物识别病原菌信号和激发寄主免疫反应中起重要的调控作用。在苹果上，我们发现3个CrRLK1L基因的表达在腐烂病发生前后存在极为显著的差异。基于此，本项目按照预期计划，完成如下工作：基于生物信息学分析、基因表达分析、瞬时表达和稳定遗传转化，筛选获得1个负调控腐烂病抗性的成员，命名为MdFER-3；通过转录组分析和数据验证，发现防御反应、脂质代谢和细胞壁相关信号参与了MdFER-3对腐烂病抗性的调控过程；利用基因表达分析、瞬时表达分析和功能分析，筛选获得受MdFER-3影响的关键基因2个：富含半胱氨酸/跨膜结构域A样蛋白基因pbeCYSTM1与果胶甲酯酶基因pbePME1，如上基因都负调控腐烂病抗性；利用双分子荧光互补技术，发现MdFER-3与典型的模式识别受体MdBAK1、MdFLS2和MdBIK1存在直接的互作关系。通过本项目工作的开展，为腐烂病抗病分子育种提供了候选基因，并初步明确了该基因的调控机制。