LRR型类受体蛋白激酶OsRPK1负调控水稻耐盐性的分子机制研究

Soil salinity is a major abiotic stress that decreases plant growth and productivity. Receptor-like kinases play major roles in plant development and defense responses against stresses. In our previous study, a putative leucine-rich repeat-RLK gene named OsRPK1 was identified as a salt stress-responding protein by proteomics. OsRPK1 was expressed mainly in root and leaf ,localized to the plasma membrane. In vitro phosphorylation assay showed that the protein could be phosphorylated, but must be incubated with the total protein extracted of the roots treated with high salt. Transgenic plants under-expressing OsRPK1 enhanced salt stress compared to the control. In contrast, over-expressing OsRPK1 resulted in decreased salt stress.These results suggested that OsRPK1 may be a negative regulator in salt stress signal transduction pathway . Building on our recent results, we will (a) determine the phosphorylation site of the OsRPK1, and then confirm whether or not phosphorylation of key site contributes to the function of OsRPK1 in response to salt by using nonphosphorylatable and phosphomimic forms of overexpressing OsRPK1 rice;(b) screen and find the intracellular kinase domain interacting protein and downstream regulatory genes of OsRPK1 using yeast two hybrid system and high throughput transcriptome sequencing to investigate the OsRPK1 function;(c) determine the relationship between OsRPK1 and ion stress / osmotic stress.The study also may provide the evidences in investigation of regulatory mechanisms for salt signaling pathway.

土壤盐渍化是目前影响农作物产量和质量的主要环境因子之一。植物类受体蛋白激酶RLKs在植物生长发育和抗逆过程中发挥了重要作用。本课题前期通过蛋白质组学的方法鉴定一个盐响应、定位于细胞质膜的富亮氨酸重复片断类受体蛋白激酶OsRPK1，体外磷酸化实验证明在高盐处理后的根部总蛋白孵育前提下，该蛋白才能发生磷酸化，进一步发现OsRPK1抑制表达转基因水稻耐盐性增强，提示OsRPK1可能负调控盐胁迫信号转导途径。因此本项目首先拟确定其激酶磷酸化位点，利用磷酸化位点突变的转基因水稻验证该激酶磷酸化前后响应盐胁迫的变化。其次通过酵母双杂交实验和转录组测序技术筛选和挖掘与OsRPK1胞内激酶区互作蛋白和下游调控基因，明确OsRPK1及激酶活性与离子胁迫和渗透胁迫的关系，阐明OsRPK1参与高盐胁迫信号转导途径的分子机制，为进一步研究植物抗逆机制奠定理论和实验基础。

植物LRR型类受体蛋白激酶是受体蛋白激酶中最大的亚族，大量研究表明其在植物生长发育和逆境胁迫中发挥重要作用。本课题前期通过蛋白质组学的方法鉴定一个盐响应、定位于细胞质膜的富亮氨酸重复片断类受体蛋白激酶OsRPK1，OsRPK1抑制表达能增强水稻的耐盐性。本研究首先分析OsRPK1基因响应非生物胁迫的表达模式，表明OsRPK1表达量主要受高盐和渗透胁迫诱导表达。以OsRPK1转基因水稻材料，在盐胁迫过程中，抑制表达OsRPK1能提高水稻提内的叶绿素、可溶性蛋白、可溶性糖、脯氨酸、抗氧化酶活性等含量，从而增强水稻耐盐性，以上结果都表明OsRPK1主要与高盐引起的渗透胁迫有关。进一步我们对关键磷酸化位点进行质谱鉴定，定点突变关键磷酸化位点并在水稻中遗传转化，通过耐盐性分析确定了2个关键磷酸化位点，此项工作为基因编辑技术改良水稻耐盐性提供试验依据。针对OsRPK1如何负调控盐胁迫信号转导途径这一关键科学问题，我们构建水稻高盐胁迫下的酵母双杂交文库，筛选并验证了一个胁迫响应蛋白AKP1与OsRPK1相互作用。通过上述研究，明确了OsRPK1与离子胁迫和渗透胁迫的关系；通过磷酸化位点的确定，探讨提高水稻耐盐的有效途径；初步阐明OsRPK1参与高盐胁迫信号转导途径的分子机制，为耐盐水稻的分子设计育种提供重要的试验依据。