钙调磷酸酶B类蛋白GmCBL1在大豆低温胁迫应答中的作用机制

Low-temperature damage is one of the main constraints in soybean(Glycine max) cultivation and production. Calcineurin B-like proteins were crucially involved in relaying plant responses to calcium signal transduction pathway and in regulating ion fluxes. Researches had shown that negative regulation of CBL1 might be involed in the signal transduction pathway of cold response. CBL1 researches were mainly concentrated in dicotyledonous model plant Arabidopsis thaliana,while few researches in soybean CBL1.Soybean germplasm DAS7,salt-resistant but cold-sensitive, was selected in this research. Then DAS7 gene expression profiles were established under salt and low temperature stresses against the control by RNA-Seq. According to the obtained information, low-temperature-sensitive GmCBL1 was prepared to be the candidate gene for cloning from soybean. This research would be focused on expression and subcellular localization of GmCBL1,protein binding capacity with calcium ions, plant responses to abiotic (salt, low temperature, drought) and hormonal stresses. After 35S:: GmCBL1vector was constructed, it was transferred into wild type A. thaliana and AtCBL1 gene deletion mutant to validate the function of GmCBL1. Under low temperature and hormones treatments, it was analysed not only the changes of A. thaliana growth and physiology,but also relationships of genes in calcium signaling networks. According to the cDNA library of soybean yeast two-hybrid, GmCBL1-interacting proteins would be obtained. Mechanism of GmCBL1 would be illuminated under low temperature stress. This work should lay a certain foundation for the molecular breeding of cold resistant soybean.

低温寒害是制约大豆栽培及生产的主要原因之一。钙调磷酸酶B类蛋白CBLs在植物钙离子信号传导途径中起重要调节作用，CBL1可能对低温胁迫应答基因的表达起负调控作用。CBL1的研究主要集中在拟南芥，而大豆GmCBL1的研究却很少。本研究选用本实验室收集筛选出的抗盐、低温敏感型大豆种质DAS7，建立了盐、低温胁迫及对照的基因表达谱，比较了它们的基因表达差异，筛选出对低温敏感的候选基因GmCBL1，拟研究其表达特异性、钙离子结合能力、亚细胞定位、非生物胁迫（盐、低温、干旱）及激素应答反应。将35S::GmCBL1分别转入拟南芥野生型及其同源基因缺失突变型，分析低温、激素处理下转基因拟南芥的生长及生理变化、低温胁迫应答相关基因表达变化，初步确定该基因功能；通过构建大豆酵母双杂交cDNA文库，筛选与GmCBL1互作的蛋白。初步阐述GmCBL1在低温胁迫应答中的作用机制，为耐寒大豆的分子育种奠定基础。

钙调磷酸酶B类蛋白CBLs在大豆低温诱导的钙离子信号传导途径中起重要调节作用。本研究项目利用前期工作中收集筛选出的抗盐、低温敏感型大豆种质DAS7，构建了其酵母cDNA文库，克隆出大豆低温敏感蛋白钙调磷酸酶B类蛋白GmCBL3并获得其基因全长cDNA（根据该基因全长cDNA的序列特征及比对分析结果，大豆GmCBL1基因最终命名为GmCBL3）。. 在大豆GmCBL3的亚细胞定位研究中，我们发现GmCBL3较多的存在于大豆茎中，次之为叶和根。进一步对GmCBL3在非生物胁迫（盐、低温）究中发现，GmCBL3能够对盐和低温进行应答，伴随着的GmCBL3表达量上升，植株的耐寒、耐盐性也随之增加。在对激素应答反应中发现，GmCBL3对ABA、IAA和干旱信号都有所响应。. 通过酵母双杂交发现了GmCBL3的互作蛋白激酶GmCIPK2（CBL-interacting protein kinases, CIPKs），及GmCIPK2的互作蛋白真核转录起始因子1亚基基因2 GmSUI2(Eukaryotic translation initiation factor 1 Subunit 2)。经研究发现，当大豆种质DAS7受到低温胁迫时，胞内Ca2+浓度增加引起了GmCBL3表达量的增高，后者通过磷酸化激活了GmCIPK2的蛋白活性，从而促进了大豆GmCIPK2的互作蛋白GmSUI2和耐寒相关基因的表达，最终提高了植株的耐寒性。在其后的拟南芥转基因实验中对GmCBL3进行了功能验证，进一步证实了以上结论，发现GmCBL3的过表达明显提高了转基因拟南芥对低温的耐受性。本研究初步揭示了GmCBL3大豆在低温胁迫下对钙离子信号传导途径的调控作用，为阐明其在的调控机制奠定基础。