大麦钙调蛋白HvCaM1及其结合蛋白HvCBP60耐盐调控机理研究

Understanding salt-tolerant mechanism in plants and identification of relevant germplasm are the essential basis for developing crop cultivars with high salinity tolerance, whereas Ca2+/CaM-dependent signal regulation pathway is an important key for the establishment of salt stress response and tolerance. In the previous studies, one specific salt-associated calmodulin (HvCaM1) has been identified in one Tibetan annual wild barley accession XZ26 with high salt-tolerance. The preliminary results proved HvCaM1 can negatively regulate salt tolerance by using genetic transformation of RNA interference and over-expression in barley. Meanwhile, we found that one CaM-binding protein HvCBP60 can interact with HvCaM1 in the experiment of yeast two-hybrid screening. Accordingly, the salt-tolerant regulation mechanism of HvCaM1 and HvCBP60 will be deeply addressed in this study. The main objectives are as follows: (1) to obtain loss-of-function barley mutants of HvCaM1 and HvCBP60, respectively, so as to determine their physiological and ionic responses under salt stress; (2) to verify cell localization, tissue localization and salt-responded expression pattern of HvCaM1 and HvCBP60 by using BiFC, in situ PCR and qRT-PCR techniques; (3) to clarify the association pattern of Ca2+ in HvCaM1 and HvCBP60 pathway, along with ROS, NO and other signal molecules by using ratio imaging technique; (4) to find key genes and their roles in salt regulation pathway of HvCaM1 and HvCBP60 by using RNA-Seq and WGCNA. As a result, we may have a deep understanding of salt-tolerant mechanisms underlying HvCaM1 and HvCBP60 in barley, thus enlarging the knowledge of salinity tolerance in crop and providing elite genotypes or genes useful for improving salt tolerance。

解析植物耐盐机理与获得耐盐种质及基因是培育耐盐作物品种的基础，Ca2+/CaM信号调控通路是植物盐胁迫反应及耐性体系建成的重要途径。在前期研究中，申请人从西藏野生大麦耐盐种质XZ26中鉴定到一个盐响应特异钙调蛋白（HvCaM1），经基因沉默和过表达研究表明其负调控大麦耐盐性，并用酵母双杂鉴定到其互作蛋白HvCBP60。本项目拟深入研究HvCaM1与HvCBP60的耐盐调控机理，主要内容有：（1）利用大麦遗传转化获得两者的功能缺失突变体，明确其耐盐表型和离子响应特征；（2）利用BiFC、原位PCR和qRT-PCR明确其细胞和组织定位及盐响应模式；（3）利用比值成像技术明确该途径Ca2+与ROS、NO等信号关联模式；（4）利用转录组测序和WGCNA明确该途径耐盐调控的关键基因及作用机制。研究结果可望阐明大麦HvCaM1与HvCBP60耐盐机理，丰富和创新作物耐盐理论，并提供优异耐盐种质及基因。

Ca2+信号及其结合蛋白转导途径，现已成为植物耐盐调控机理研究的热点。前期从西藏野生大麦根中首次鉴定到一个盐响应特异的钙调蛋白HvCaM1，经酵母双杂鉴定到其互作结合蛋白HvCBP60b，但它们的功能未知。本项目研究了大麦钙调蛋白HvCaM1及其互作结合蛋白HvCBP60b负调控大麦耐盐性的生物学功能。研究发现核定位转录因子HvCBP60b通过转录调控下游钠钾转运蛋白基因KUP12和NHX1的表达，调节根往地上部的Na+转运及根对K+吸收的生理特性，由此揭示了HvCaM1-HvCBP60b-KUP12/NHX1负调控耐盐性的分子路径，丰富了作物耐盐理论。本研究还鉴定到一批受HvCaM1和HvCBP60b共同调控的盐响应差异表达候选基因，同时创制的大麦HvCBP60b基因编辑和HvCaM1基因敲除材料均具有显著的耐盐表型，可为作物耐盐育种提供新的基因及种质。项目实施期间，负责人以第一或通讯作者在Plant Physiology等国际知名期刊上正式发表SCI收录的学术论文6篇，申请发明专利6件，其中授权2件、实质审查4件、转化1件。培养硕士研究生4名、协助培养博士研究生2名、指导本科毕业生3名、联合培养博士后1名。多次受邀在国内学术交流会议上作学术报告，获行业专家指导与研究学者好评。