花生小肽AhCEP1耐高盐、耐低氮偶联机制解析

High salinity and low nitrogen in saline-alkali land impart a plethora of harms to plants. Exploring the coupling mechanism of high salt and low nitrogen stress response is an important prerequisite for plants to withstand salt stress and survive in saline-alkali land. Peanuts as nitrogen fixing crops have higher salt stress tolerance than that of most other related plant varieties, which is more suitable crop in saline-alkali land for planting structure adjustment. Screening and breeding high salt and low nitrogen tolerant peanut varieties, and then exploring the coupling mechanism of high salt and low nitrogen stress response is an effective way to improve the peanut quality and utilize saline-alkali land. Using high salt and low nitrogen tolerant peanut varieties as materials, we identified AhCEP1, a gene dramatically induced by high salt and low nitrogen, encoding CEP small peptide. Through a variety of analyses, AhCEP1 and its receptor AhCEPR1 were proved to play a critical role in the salt and low nitrogen stress response. In our study, AhCEP1-AhCEPR1 protein interaction network will be built by using biomacromolecular interaction (Biacore). Moreover, AhCEPR1-mediated phosphorylation pathways and protein phosphorylation sites will be explored by phosphorylated protein chromatograms. Cell-free assay and xenopus oocyte expression system are used to investigate the stability and biological activity of phosphorylated protein regulated by AhCEPR1. All in all, the comprehensively study of AhCEP1-AhCEPR1 mediated coupling mechanism in salt and low nitrogen stress response provides effective gene resources and lays a solidly theoretical and technical foundation improving the quality of peanuts in saline-alkali land.

高盐和低氮胁迫是制约盐碱地植物生长的主要因素，解析其偶联机制是保障盐碱地作物高效生产的重要前提。花生固氮养地，且具备较强盐碱耐受能力，是盐碱地区较适宜种植的经济作物。培育耐高盐、耐低氮花生品种，解析其偶联机制，定向改良花生抗逆性状，是开发利用盐碱地的有效途径。我们以耐高盐、耐低氮花生品种为材料，通过转录组学分析，鉴定到同时响应高盐和低氮的基因AhCEP1，该基因编码CEP小肽。并证实，AhCEP1与其受体AhCEPR1共同作为高盐和低氮胁迫响应偶联关键因子。本项目以AhCEP1和AhCEPR1为研究对象，利用生物大分子互作等技术，构建蛋白互作网络；利用磷酸化蛋白质谱，探索AhCEPR1介导的磷酸化修饰及磷酸化位点；利用Cell-free和爪蟾卵母系统，研究磷酸化蛋白稳定性和生物学活性。全面解析AhCEP1介导花生耐盐、耐低氮偶联机制，为定向培育盐碱地高抗广适花生品种提供基因资源和技术支撑。

土壤盐碱化严重制约了粮食生产和农业可持续发展。高盐和低氮胁迫是限制盐碱地植物生长的主要因素，解析其偶联机制是保障盐碱地作物优质高产的重要前提。花生固氮养地，较耐盐碱，是盐碱地区较适宜种植的经济油料作物之一。培育耐高盐、耐低氮花生品种，解析其偶联机制，定向改良花生抗逆性状，是开发利用盐碱地的有效途径。为明确植物高盐、低氮胁迫偶联响应机制，我们以耐高盐、耐低氮花生品种为材料，通过转录组学分析，鉴定到同时响应高盐和低氮的小肽AhCEP1。并证实，AhCEP1与其受体AhCEPR1共同作为高盐和低氮胁迫响应偶联关键因子。利用酵母双杂交筛库调取到AhCEPR1互作蛋白为AhNRT1.2，MbSUS、LCI和pull-down实验进一步证实AhCEPR1与AhNRT1.2两者互作。遗传学实验表明AhCEPR1位于AhNRT1.2上游，两者协同调控高盐、低氮胁迫响应。体外磷酸化实验证实AhCEPR1能将AhNRT1.2磷酸化修饰。进一步Cell-free实验表明AhCEPR1将AhNRT1.2磷酸化可提高其蛋白稳定性。酵母和爪蟾卵母系统证实磷酸化的AhNRT1.2转运ABA和NO3-的活性显著提高，ABA调控的下游盐胁迫响应marker基因以及低氮胁迫响应marker基因显著上调，增强花生耐高盐、耐低氮能力。该项目全面解析AhCEP1介导花生耐盐、耐低氮偶联机制，为定向培育盐碱地高抗广适花生品种提供基因资源和技术支撑。