水稻冷应答类受体激酶OsCI-RLK1耐冷功能鉴定及其作用机制

Developing cold tolerance (CT) rice varieties based on mining of the CT genetic resources in rice is regarded as the most cost-effective strategy to reduce the cold injury losses, which is vital for food security in China. We have identified seedling CT QTLs using CT introgression line (IL) populations in an elite japonica restorer line C418 background. Furthermore, genome-wide gene expression profiling of a CT IL K354 with introgressed alleles from indica rice variety BG300 was conducted to explore the possible CT enhancement mechanisms. We screened a K354-specific cold-induced receptor-like protein kinase gene (OsCI-RLK1) located in the QTL region for CT on the chromosome 8. Although the molecular mechanism that cold-signal transduction mediated by RLK genes in rice is not yet clear, the bioinformatic analysis showed the promoter region of OsCI-RLK1 (upstream 2kb sequence) contained the core motif for calmodulin or calmodulin binding transcription activators recognition, suggesting OsCI-RLK1 may be involved in the cold-signaling pathway. Based on the pre-existing OsCI-RLK1 transgenic (knockout and overexpression) rice plants, we will confirm whether OsCI-RLK1 confers tolerance to cold stress and provide a better understanding of molecular and physiological mechanisms of CT in rice. Yeast two-hybrid system, bimolecular fluorescence complementary, and CoIP technology, etc. will be used to identify proteins which interact with OsCI-RLK1. The goal of this research is to decipher the regulation mechanisms underlying OsCI-RLK1 genotype-specific induced by cold in rice, and provide new genetic resources and the theoretical basis for the CT improvement in rice breeding.

挖掘水稻耐冷基因资源，培育耐冷性强的水稻品种，是降低水稻冷害损失最经济有效的方式，对我国粮食安全生产至关重要。本研究前期利用粳稻恢复系C418背景耐冷导入系群体定位了耐冷QTL，并对供体为籼稻BG300的耐冷导入系K354进行了全基因组表达谱分析，在第8染色体的耐冷QTL区域内筛选到一个K354特异的受冷胁迫诱导的类受体激酶基因OsCI-RLK1。尽管水稻RLK基因是否参与冷信号转导尚不明确，但是生物信息学分析发现OsCI-RLK1启动子区域含有钙调素识别的核心基序，推测其可能参与冷信号通路。本研究拟在OsCI-RLK1敲除和过表达转基因水稻基础上，明确OsCI-RLK1耐冷功能，探讨水稻耐冷相关分子和生理机制。应用酵母双杂交、双分子荧光互补、CoIP等技术，鉴定OsCI-RLK1的互作蛋白，以期探明OsCI-RLK1参与冷胁迫反应的调控机制，为水稻耐冷育种提供新的基因资源和理论基础。

挖掘水稻耐冷基因资源，培育耐冷水稻品种，是降低水稻冷害损失最经济有效的方式。本研究前期利用粳稻恢复系C418 背景耐冷导入系群体定位了耐冷QTL，结合全基因组表达谱分析，筛选到一个受冷胁迫诱导的类受体激酶基因OsCI-RLK1。本研究利用遗传群体发掘了与OsCI-RLK1相关的苗期耐冷多位点遗传结构。通过抑制OsCI-RLK1基因表达可以降低水稻苗期耐冷性，从而验证了OsCI-RLK1的耐冷功能。冷胁迫条件下对野生型与RNAi转基因植株进行转录组比较分析，发现差异基因在分子功能上富集在转录因子活性类，并有多个钙离子/钙调蛋白相关基因差异表达。利用非损伤微测技术，发现冷胁迫导致野生型的叶肉组织和根部的钙离子均内流，而在RNAi转基因植株中均外排，表明OsCI-RLK1可能与钙离子信号有关。同时，发现抑制OsCI-RLK1基因表达能够降低种子在萌发过程中对ABA的敏感性。OsCI-RLK1亚细胞定位于细胞核和细胞质中，利用诱饵载体对野生型在冷胁迫下建立的酵母双杂交文库进行筛选，获得了18个互作蛋白。通过酵母体内验证和双分子荧光互补验证了其中一个亚硫酸盐氧化酶与OsCI-RLK1存在互作。此外，在正常栽培条件下抑制OsCI-RLK1基因表达，会减少苗期总根长，缩短第3节间长，从而降低株高，这些结果表明 OsCI-RLK1基因可能影响水稻发育。本研究的结果一方面为进一步深入研究冷胁迫信号的分子机理奠定基础，另一方面为培育耐冷水稻品种提供新的基因资源和理论依据。