花生CLAVATA1-like基因AhRLK1调控青枯病抗性的分子机理研究

青枯病是影响花生品质和产量的重要细菌性病害，其抗病机理尚未阐明。前期研究表明ClAVATA1负调控拟南芥对青枯菌的抗性，但花生CLAVATA1-like基因AhRLK1超表达可提高烟草青枯病抗性。本项目拟通过超表达和基因编辑载体分别转化花生感病和抗病品种，通过细胞学、生理生化和qPCR等技术分析AhRLK1对花生青枯病抗性的影响。通过构建该基因上游启动子系列截短载体分析响应青枯菌胁迫的顺式元件，利用酵母单杂交鉴定其上游调控因子，通过ChIP和EMSA等方法研究识别的motif；采用酵母双杂交筛选AhRLK1互作蛋白，利用BiFC和Co-IP等方法予以验证。分析调控因子的亚细胞定位及编码基因对青枯菌侵染的表达特征。通过遗传互补转化花生，研究AhRLK1与其他调控因子的互作在花生青枯病抗性中调控机制。研究将阐明AhRLK1调控花生青枯病抗性的分子机制，为花生抗病遗传改良提供理论依据和基因资源。

Bacterial wilt is an important disease caused reduction in yield and quality severely in peanut. Up to now, resistant underlying mechanism of this disease is also not well understood. Previous researches showed that ClAVATA1 overexpression of peanut AhRLK1 enhanced resistance to bacterial wilt significantly in tobacco. In the present project, the complementation of Arabidopsis mutant will be studied by AhRLK1 overexpression transformation. Overexpression and gene editing vectors of AhRLK1 will be transformed into susceptible and resistant peanut cultivars, respectively. The effect of AhRLK1 in peanut bacterial wilt resistance will be analyzed by cytology, physiology, biochemistry and qPCR techniques. The cis-elements involved in Ralstonia solanacearum infection will be identified by analyzing the activities of serial truncated mutants of promoters. Based on the identified cis-elements, upstream regulatory transcription factors will be identified by Yeast one-hybrid (Y1H). Chromatin Immunoprecipitation (ChIP) and EMSA is employed in studying the motifs binded by AhRLK1. Yeast two-hybrid (Y2H) technique will be employed to identify the interacting proteins of AhRLK1. Y2H results will be confirmed in planta by Bimolecular Fluorescence Complementation (BiFC) and co-immunoprecipitation (Co-IP). The putative Y1H and Y2H interactors will be characterized by subcellular localization analysis and the expression pattern of encoded genes responded to Ralstonia solanacearum infection will be investigated. Using overexpression and CRISPR/Cas9 silencing analysis in peanut plants, the function of potential Y1H and Y2H interactors in transcriptional regulation of AhRLK1 and interacting protein, respectively, will be studied in detail. The results will help to reveal the underlying mechanism of the resistance to the bacterial wilt. It will benefit the theoretical foundation but also can provide gene resources for efficient genetic improvement of peanut disease resistance.