钙依赖型蛋白激酶OsCPK9介导的水稻耐旱性分子机理研究

Calcium-dependent protein kinases (CDPKs) are involved in plants’ response to various environmental stresses. Our previous study showed that drought, high salt and ABA treatment could significantly up-regulate OsCPK9 expression in rice. Transgenic rice over-expressing OsCPK9 (OsCPK9-OX) exhibited increased tolerance to drought and osmotic stress and became more sensitive to ABA treatment; by contrast, OsCPK9-RNAi transgenic lines was intolerant to drought stress, indicating that OsCPK9 plays a positive role in drought stress tolerance. While, the detailed molecular mechanisms of drought tolerance mediated by OsCPK9 remains unclear. In the present study, Pull-down and in-gel kinase assay will be used to investigate OsCPK9 activation mechanism in response to drought stress; Yeast two-hybrid, co-immunoprecipitation, subcellular localization combined with comparative phosphoproteome analyses are to be employed to screen and identify OsCPK9 interacting proteins or its kinase substrates; Genome-wide gene expression analysis will be performed in OsCPK9-OX and OsCPK9-RNAi transgenic rice under drought condition to clarify those genes that may be regulated by OsCPK9. This investigation will reveal the underlying molecular mechanisms of drought tolerance mediated by OsCPK9 in rice.

钙依赖型蛋白激酶（CDPK）参与植物对多种逆境胁迫响应。我们前期研究工作发现：干旱、高盐和ABA处理能显著上调水稻OsCPK9基因的表达；OsCPK9基因过表达（OsCPK9-OX）的转基因水稻对干旱和渗透胁迫的耐受性显著提高、对ABA处理的敏感增强，而OsCPK9 基因RNA干扰（OsCPK9-RNAi）的转基因水稻对干旱胁迫耐受性显著降低，表明OsCPK9作为一种正调控因子在水稻对干旱胁迫响应中发挥重要作用。本研究以野生型和转基因水稻为材料，采用Pull-down结合激酶活性检测技术，分析OsCPK9激活机制；采用酵母双杂交、免疫共沉淀及亚细胞共定位技术，并参考比较磷酸化蛋白质组数据，筛选、鉴定OsCPK9在干旱胁迫下的作用位点，即其激酶底物；利用基因芯片技术，在基因组水平上分析受OsCPK9调控的基因。通过上述研究工作，揭示OsCPK9介导的水稻耐旱性分子调控机理。

本项目的目的是阐明OsCPK9介导的水稻耐旱性的分子调控机制。首先成功实现了OsCPK9在大肠杆菌中的表达，通过分离纯化和体外激酶活性实验，证明了OsCPK9的钙依赖性蛋白激酶特性。综合运用免疫共沉淀技术，结合经干旱、渗透胁迫和ABA处理后水稻幼苗提取mRNA构建的cDNA文库的筛选以及生物信息学分析，最终确定了过氧化氢酶OsCatB等为OsCPK9高置信度的互作蛋白。在前期工作的基础上发现OsCPK9可以提高水稻小穗育性以及增加穗重；OsCPK9基因可以响应稻瘟病病原菌侵染，OsCPK9过表达的转基因水稻可增强对稻瘟病的抗性。OsCPK9可能通过提高OsCatB的活性减轻水稻细胞在生物和非生物逆境胁迫下的损伤。此外，利用CRISPR基因编辑技术、定点突变技术获得了组成型激活、失活和棕榈酰化或/和豆蔻酰化位点突变的OsCPK9突变体，为进一步研究OsCPK9的作用机理提供了材料。以上结果为全面揭示水稻钙依赖性蛋白激酶家族的生物学功能以及OsCPK9基因在水稻抗逆育种上的应用提供了理论依据。