茶树CsSnRK3.9/CIPK12调控低温响应的机制研究

Cold adaptability of the tea plant affects its geographical distribution, growth and development and the production economic benefits. The CBL-SnRK3s/CIPK regulatory network is involved in Ca2+ signal transduction and abiotic stress response in plant. According to the analysis based on the previous transcriptome data, we found that the expression level of CsSnRK3.9/CIPK12 was induced by long- and short- term cold stress. Moreover, CsSnRK3.9/CIPK12 was coexpressed with the known cold stress signaling regulatory genes. We proposed that CsSnRK3.9/CIPK12 transferred Ca2+ signal which induced by low temperature by binding to CsCBL proteins and therefore regulated the cold stress response of tea plant. To prove the hypothesis, we will develop genetic materials such as overexpression lines in poplar, determine the physiological function of CsSnRK3.9/CIPK12 gene, identify its interacting CsCBL proteins and the target-phosphorylated proteins by combining of technologies related to physiology, yeast two-hybrid library screening, interaction protein verification, phosphoproteome analysis and phosphorylation verification. It is helpful to clarify the mechanism of CsSnRK3.9/CIPK12 in regulating cold response in tea plant and provide theoretical basis for developing tea cultivars with improved cold resistance.

茶树的低温适应性影响其地理分布、生长发育和生产经济效益。CBL蛋白与SnRK3s/CIPK蛋白激酶互作(CBL-SnRK3s/CIPK)调控网络参与植物钙离子信号传导和非生物胁迫响应。根据前期的转录组数据分析，课题组发现CsSnRK3.9/CsCIPK12基因受长期低温和短期低温诱导表达，并且该基因与已知的低温信号调控基因共表达，推测CsSnRK3.9/CsCIPK12通过结合CsCBL蛋白来传递低温胁迫诱发的钙离子信号，从而调控茶树的低温胁迫响应。本项目拟在前期研究的基础上，创制该基因的杨树超表达等遗传材料，结合生理学、酵母双杂交文库筛选、互作蛋白验证、磷酸化蛋白组及磷酸化作用验证等技术手段，明确茶树CsSnRK3.9/CsCIPK12基因的生理功能，鉴定与其互作的CsCBL蛋白及目标磷酸化蛋白，阐明其在茶树低温响应信号中的调控机制，为抗寒茶树品种的培育提供理论基础。

茶树的低温适应性影响其地理分布、生长发育和生产经济效益。钙离子（Ca2+）是植物响应环境刺激信号转导网络中的第二信使，当受到低温刺激后，植物细胞内的Ca2+浓度在短时间内迅速增加，从而激发下游耐寒防御反应。CBL蛋白与CIPK蛋白激酶互作网络参与植物Ca2+信号传导和非生物胁迫响应。本研究在茶树中鉴定了分属4个进化分支的8个CsCBLs和分属5个进化分支的25个CsCIPKs。明确了它们在茶树各组织中的表达模式，对非生物胁迫（成熟叶经4℃、盐和干旱处理；新梢一芽二叶经4℃处理）的响应模式。通过酵母双杂交，证明了低温诱导的CsCBLs和CsCIPKs之间的互作关系。构建了CsCBL-CsCIPK模块调控茶树非生物胁迫响应的分子模型。.通过异源转化拟南芥和杨树，进一步对CsCIPK12进行功能鉴定。研究发现CsCIPK12受低温诱导，定位于细胞膜、细胞质和细胞核中。CsCIPK12与CsCBL1/9在细胞膜上互作。生理实验表明，过表达CsCIPK12的拟南芥株系在种子发芽阶段对ABA和NaCl敏感；过表达CsCIPK12的拟南芥和杨树株系的低温抗性降低，表现为在低温处理下，与野生型相比，过表达株系的相对电导率和丙二醛含量升高，AtCBF1/2、AtICE1/2和AtCOR15a/47等低温标记基因的表达显著降低。通过酵母文库筛选及蛋白互作鉴定发现，CsCIPK12与CsKIN10、CsFIB、CsWRKYx互作。因此推测当茶树遭受低温和盐胁迫时，细胞中的Ca2+浓度发生变化，CsCBL1/9蛋白结合Ca2+并通过与CsCIPK12互作，激活CsCIPK12将Ca2+信号传递下去，激活的CsCIPK12可能通过与CsKIN10、CsFIB和CsWRKYx互作来负调控ABA信号依赖的低温和盐胁迫响应。项目共发表标注论文7篇，其中JCR Q1区SCI论文6篇。研究结果为茶树抗寒育种提供基因资源，也为阐明茶树抗寒分子机理提供理论基础。