病程相关蛋白激酶（GhPR5K）调控棉花抗黄萎病的分子机制

培育抗病品种是棉花黄萎病防治最经济有效的措施，棉花的抗病机制是抗病育种关键及核心问题。前期通过抗、感病品种根部磷酸化蛋白质组学分析，筛选到一个类受体蛋白激酶Gh_D04G1868（命名为GhPR5K），研究表明GhPR5K基因提高了棉花对黄萎病的抗性。本项目拟分析GhPR5K激活的相关次生代谢产物变化及细胞信号传导途径上标记基因的表达，解析GhPR5K调控的棉花抗病反应途径；利用qRT-PCR和平行反应监测（PRM）的方法分析GhPR5K基因和磷酸化GhPR5K在不同抗性品种根部的应激表达，解析GhPR5K磷酸化水平与抗病性的关系；将GhPR5K转化棉花原生质体，分析GhPR5K的亚细胞定位；通过酵母双杂交等筛选与GhPR5K互作的靶蛋白，分析GhPR5K磷酸化作用及关键位点，解析GhPR5K与其互作蛋白的作用机理，阐明GhPR5K调控棉花抗黄萎病的分子机制，为棉花抗病育种提供理论依据。

The use of resistant varieties is an economical and effective way of controlling Verticillium wilt disease. Previously, a receptor like protein kinase (RLKs) Gh_D04G1868 (named as GhPR5K) was detected in cotton root phosphorylated proteins, its phosphorylation level was significant higher in Zhongzhimian 2 inoculated than that in control (12.69 folds), while it was not significant in Jimian 11. The gene knockout (VIGS) and functional complementation assay showed that the GhPR5K could improves cotton resistance to Verticillium wilt. In this study, the gene and phosphorylated protein expression level will be analysied by qRT-PCR and Quadrupole-MS in different resistant varieties. The secondary metabolites (lignin, callose), signal pathways of Salicylic, Ethylene, Jasmonic, H2O2/NO, and the gene expression in this metabolic pathways will be detected. Moreover, to prove the mechanism of GhPR5K regulate cotton resistant to Verticillium wilt, the protein in cotton specific targeting by GhPR5K will be detected by Yeast-two hybrid (Y2H), Bimolecular fluorescence complementation (BiFC), Co-Immunoprecipitation (Co-IP) and pull down. Furtherly, we plan to examine the phosphorylation of interaction, and the conserved phosphorylation sites, to explore how the GhPR5K regulate plant immunity signal pathways. This project will provide base for more effective and targeted cotton resistant breeding.