钙依赖性蛋白激酶基因CsCDPK2调控茶树低温响应的机制研究

Low temperature is one of the most important environmental factors affecting the growth and economic benefits of tea plant. How to improve cold resistance becomes more and more important in tea production. In our previous studies, we found that calcium signaling pathways play an important role during cold stress responding in tea plant based on transcriptomic and physiological studies. As a key node in signal transduction, calcium-dependent protein kinases (CDPKs) play important roles in plant abiotic stress response. Based on our gene expression profile under long- or short-term cold stress, a CDPKs gene, CsCDPK2, was identified in tea plant. In order to illustrate the mechanism of CsCDPK2 in regulating the cold stress response of tea plant, we propose to over-express CsCDPK2 in poplar and treat the transgenic plants with long- or short-term cold, analyze the changes of physiological parameters, gene expressions, transcriptome and quantitative phosphoproteome, and measure the kinase activity of CsCDPK2. It is expected to identify the function of CsCDPK2 and its down-stream regulated genes or networks, including the substrates of CsCDPK2 kinase. The results will greatly improve our understanding of the downstream genes or metabolites of CsCDPK2 and its regulation mechanism in cold resistance. This study will benefit tea breeders in cold resistance germplasm rapid screening and provide important theoretical basis for the tea cultivation in cold regions.

低温是影响茶树生长发育和经济效益的最重要环境因子之一，如何提高茶树的抗寒性是茶学研究的热点问题。申请人利用生理学和转录组学等方法开展的前期研究发现，钙离子信号通路在调控茶树低温胁迫响应中发挥重要的作用，并鉴定出一个在茶树长期和短期低温胁迫下都上调表达的钙依赖性蛋白激酶基因CsCDPK2。为进一步解析CsCDPK2调控茶树低温响应的机制，本项目在分析CsCDPK2基因基本信息的基础上，构建CsCDPK2的杨树转基因株系，进行长期与短期低温胁迫处理，然后开展生理学、转录组学、磷酸化蛋白组学以及激酶酶活验证等方面的研究，鉴定出CsCDPK2激酶的靶物质及下游调控基因，阐明其潜在调控网络，提出CsCDPK2调控茶树低温响应的可能机制，为后续不同低温抗性品种间差异基因、代谢物挖掘和利用奠定基础，也为茶树抗寒品种的早期鉴定及抗寒栽培提供理论依据。

低温是影响茶树生长发育和经济效益的最重要环境因子之一，如何提高茶树的抗寒性是茶学研究的热点问题。钙离子作为重要的“第二信使”，在植物响应逆境胁迫的信号传导中起着重要的作用。申请人前期研究发现，钙离子信号通路在调控茶树低温胁迫响应中发挥重要的作用，发现了一个在茶树长期和短期低温胁迫下都上调表达的钙依赖性蛋白激酶基因CsCDPK2（后修定名称为CsCPK9），但其调控机制并不清楚。本项目在此基础上，对茶树CPK基因家族在全基因组层面进行了鉴定，鉴定出26个茶树CPK基因，利用低温胁迫和Ca2+抑制剂处理鉴定出响应低温胁迫的CsCPKs，并重点对目标基因CsCPK9响应低温胁迫的调控机制进行了研究。研究结果发现CsCPK9通过激活ICE-CBF-COR信号通路正调控植物的低温胁迫；过表达CsCPK9改变了植物“碳水化合物的运输”、“磷光信号转导系统”、“微管骨架”、“细胞壁的结构成分”等代谢通路的基因表达，从而提高抗寒性。通过酵母双杂交等手段鉴定了9个潜在的磷酸化底物，并对其中的CsFIB1a与CsCPK9的互作进行了研究，结果表明CsCPK9和CsFIB1a能够在细胞质膜发生互作，在茶树中共同参与低温调控。研究结果为下一步通过钙离子信号途径提高茶树抗寒性、进而提出茶树低温防治技术提供了理论基础。