OsCML16在调控水稻耐冷性和细胞质钙信号解码中的分子互作机制

Calcium signal is an early event in the plant response to cold stress, so it is an effective way to identify the calcium signal transduction mechanism for rice genetic improvements with higher chilling tolerance. In our previous studies, transcript levels of OsCML16 gene, encoding a calmodulin-like protein in rice, was induced only by 12 degree but not by 4 degree, its overexpressor enhanced the chilling tolerance 56% higher than wild type did in the rice seedlings. Y2H (Yeast two hybrid) and BiFC (Bimolecular fluorescence complementation) assays confirmed that OsCML16 can interact with two proteins OsCaMBP1 and OsZOS3-18, respectively. In addition,OsZOS3-18 can transactivate the transcriptions of OsDREB1 and OsDREB2 genes in the yeast-one-hybrid assay. And thus, how do all these three interactive proteins function in the chilling tolerance and cytosoliic calcium signal decoding processes?. In this proposal, their overexpressor and repressors will be developed to assess their functions and downstream regulatory genes involved in chilling tolerance in rice seedlings; By the means of BiFC assays, their genuine real-time interactions in vivo will be detected in the rice protoplast exposed to cold stress and exogenous calcium signal antagonists; Regulatory proteins that can interact with OsCML16, will be screened out via the Co-immnue precipitation (Co-IP) followed by Mass spectrometry (MS) techniques, and the effects of phosphorylation and oxidation sites will be assayed on the binding property of OsCML16 with calcium ion in vitro and in vivo; Finally, all above three overexpressors will be used to explore the cytosolic calcium signal dynamics via the calcium signal fluorescence probe yellow chameleon 3.6 under the chilling stress treatments and exogenous calcium signal chemical agents.. The accomplishment of this proposal will clarify the interaction mechanism of OsCML16 and two target proteins involved in the chilling tolerance and cytosolic calcium signal decoding and transduction pathways, and provide the basis for the genetic breedings and resistance cultivation in the rice chilling stress in the future.

钙信号是植物感受冷胁迫的最初信号之一，鉴定钙信号传导机制是水稻耐冷性改良的有效途径。申请人研究表明，OsCML16基因表达受12℃明显诱导，超表达幼苗耐冷性提高56%。Y2H和BiFC证明OsCML16与OsCaMBP1和OsZOS3-18互作。OsZOS3-18调控OsDREB1和OsDREB2基因的转录。这些互作蛋白在水稻耐冷性和钙信号解码中的功能如何呢？为此，本项目将创建上述3个基因的功能突变体，鉴定耐冷性及其下游调控基因；利用BiFC验证它们在水稻幼苗原生质体中的互作；利用Co-IP/MS技术初筛与OsCML16互作的调控蛋白；探究磷酸化和氧化对OsCML16在离体和活体下与Ca2+结合的影响；利用钙信号荧光蛋白YC3.6检测3个超表达幼苗冷胁迫下钙信号变化特征。阐明OsCML16及其2个互作靶蛋白在调控水稻耐冷性和Ca2+信号解码中的机制，为水稻耐冷性遗传育种和抗逆栽培提供依据。

本项目研究首先揭示出OsCML16的磷酸化和氧化过程与其在体外的Ca2+结合是2个独立的生化过程。首先，本项目鉴定了OsCML16正调控水稻苗期的耐冷性，靶蛋白基因OsZOS3-18负调控水稻的耐冷性。Y2H和BiFC鉴定了OsCML16以依赖和不依赖CaMBD（1-10motif）方式与靶蛋白CaMBP1互作，该蛋白是一个抗稻瘟病相关的蛋白OsPi304。暗示该基因在水稻耐冷性和耐稻瘟病中的交谈（cross-talk）机制。其次，本项目以OsCML16为诱饵（bait）蛋白，大规模筛选了本实验室构建的水稻幼苗冷胁迫酵母双杂交文库，初步获得了32个与OsCML16互作的靶蛋白。成对Y2H和BiFC进一步鉴定了6个与OsCML16互作的靶蛋白。利用WGCNA方法，获得了57个与6个靶基因共表达的水稻非生物逆境响应相关的基因，构建了OsCML16基因调控水稻冷害等非生物逆境的基因互作网络，阐明了OsCML16调控水稻耐冷性等非生物逆境的分子机制。为研究水稻钙调素类似蛋白OsCMLs在调控水稻耐冷性等非生物逆境和Ca2+信号解码中的分子互作机制提供了借鉴。