CBL-CIPK1调控嫁接黄瓜RBOHD产生H2O2和耐盐性的机制

课题组前期研究发现，南瓜砧木通过根源RBOHD 基因介导的H2O2信号增强了根系Na+外排能力来提高嫁接黄瓜的耐盐性，CBL-CIPK途径是钙信号的重要通路，前期研究发现钙信号参与了耐盐性调控，已筛选出与RBOHD 互作的CIPK1，但南瓜RBOHD如何与CBL-CIPK1结合来调控H2O2尚不明晰。本项目拟通过CRISPR/Cas9创建南瓜RBOHD缺失突变体，遗传转化获得过表达RBOHD黄瓜，通过嫁接黄瓜实验验证南瓜ROBHD基因功能与耐盐性，利用酵母双杂、双分子荧光互补和免疫共沉淀鉴定与RBOHD-CIPK1互作的CBL蛋白，通过体外磷酸化实验和LC-MS/MS分析鉴定RBOHD蛋白的磷酸化位点，最后验证CBL-CIPK1调控RBOHD产生H2O2的机制。课题完成后可揭示CBL-CIPK1调控嫁接黄瓜RBOHD产生H2O2和耐盐性的机制，促进嫁接信号转导研究。

In our previous work, we found that pumpkin-grafted cucumber plants increase their salt tolerance via a mechanism involving the root-sourced respiratory burst oxidase homologue(RBOHD)-dependent H2O2 production, which enhances Na+ exclusion from the root， and we found that Ca2+ signal also involve in the regulation of salt resistance of grafted cucumber, CBL-CIPK is important Ca2+ signal, and we have already identified CIPK1, interacting with ROBHD, but how pumpkin RBOHD combine with CBL-CIPK1 and then control the salt resistance of grafted cucumber is not clear. In this study, we construct pumpkin RBOHD deficient mutant through CRISPR/Cas9 and obtain cucumber material overexpressed with pumpkin RBOHD, and then test the function of pumpkin RBOHD and salt resistance by grafting experiment under salt stress. Yeast two-hybrid analyses, BiFC and Co-IP analyses are used for screening CBL protein interact with RBOHD-CIPK1, and phosphorylation sites of pumpkin RBOH were obtained by in vitro phosphorylation and LC-MS/MS assays, finally ROS production generated by RBOH-CBL-CIPK were conducted by using HEK293T Cells. The implementation of this project will provide signal transduction mechanism for the interaction of pumpkin RBOHD and CBL- CIPK1 mediated salt tolerance of grafted cucumber and H2O2, and promote grafting signal transduction research.