羊草和小麦特有组氨酸富集钙结合蛋白HRC的生理功能研究

Sheepgrass (Leymus chinensis (Trin.) Tzvel.) is a perennial forage and ecological grass in China, with strong resistance to abiotic stress, therefore, exploring novel genes conferring abiotic stress resistance from sheepgrass has great theoretical significance and practical value. Calcium as second messenger is a vital signaling molecule in plants for maintaining cellular stability and sensing interior and exterior environment. The abiotic stress were sensed by diverse intracellular Ca2+ binding sensor proteins. However, so far no Ca2+ binding proteins was identified in the sheepgrass. In our previous study, a homologous gene of animal histidine-rich Ca2+ binding protein gene (HRC) was first identified in the sheepgrass and the bioinformatics analysis results show it was only found in the sheepgrass, wheat and its closed related species. Preliminary results show that the sheepgrass HRC (LcHRC) showed a Ca2+ binding activity and sensitivity to ABA in the plant. On the basis of these results, methods of Virus Induced Gene Silencing (VIGS), CRISPR/Cas9 mediated genome editing technology as well as overexpression of LcHRC in Arabidopsis will be used to identify the function of LcHRC in plants. The aims of this project are to identify LcHRC in sheepgrass and its potential functions in the responses to drought and ABA sensitivity, which will also provide effective theory basis for the use of HRC gene in wheat.

羊草（Leymus chinensis (Trin.) Tzvel.）是我国重要的禾本科牧草和生态草，具有较强的抗逆特性，相关基因资源的发掘和利用具有重要的意义。钙做为细胞内第二信使直接参与介导植物对多种逆境的应答，而钙信号主要通过钙结合蛋白进行感受和转导，目前关于羊草钙结合蛋白的相关研究还尚未见报道。项目组前期研究中获得羊草和小麦特有的组氨酸富集钙结合蛋白(HRC)，初步研究表明羊草HRC（LcHRC）具有钙离子结合特性并与ABA敏感性相关，本项目拟在此基础上，进一步分析LcHRC基因的分子特征，通过过表达、病毒介导的基因沉默及基因组编辑等技术验证LcHRC在钙离子结合、ABA敏感性及抗旱性中的作用，解析LcHRC的功能、作用机制及其调控网络。该项目的开展可望获得羊草和小麦特有的钙结合蛋白新基因，并为解析羊草和小麦等重要植物钙结合蛋白参与逆境调控的分子和生理基础提供依据。

羊草（Leymus chinensis (Trin.) Tzvel.）是我国重要的禾本科牧草和生态草，具有较强的抗逆特性，相关基因资源的发掘和利用具有重要的意义。钙做为细胞内第二信使直接参与介导植物对多种逆境的应答，而钙信号主要通过钙结合蛋白进行感受和转导，然而关于羊草钙结合蛋白的相关研究还尚未见报道，本研究在羊草中克隆了组氨酸富集钙结合蛋白LcHRC基因，明确了其在羊草中的表达特征，利用项目组开发的羊草瞬时表达方法明确了LcHRC定位于细胞核，体外实验证明LcHRC可以和Ca2+、Zn2+特异性结合，同Mg2+、Mn2+没有结合特性。转LcHRC基因拟南芥生长对ABA敏感且抵抗干旱的能力提高，ABA处理拟南芥植株叶片，转LcHRC基因拟南芥细胞质内Ca2+浓度升高倍数低于野生型拟南芥中升高倍数且ABA信号应答相关基因表达量下调。酵母双杂交筛选拟南芥cDNA酵母文库，发现LcHRC可以同拟南芥中含Tudor/PWWP/MBT结构域蛋白AtPWWP3相互作用，该蛋白组可能在组蛋白去乙酰化中具有重要作用。拟南芥AtPWWP3基因突变体表现出对ABA不敏感，进一步分析发现AtPWWP3与羊草中LcTPM基因同源，酵母双杂交实验证明LcHRC和LcTPM可以相互作用，亚细胞定位表明LcTPM同样定位于细胞核。最新研究报道表明LcHRC高度同源基因TaHRC基因在小麦抗赤霉病中发挥着重要作用，鉴于此，项目组收集来自内蒙古自治区的羊草种质资源227份，通过对LcHRC基因测序发现LcHRC基因的自然突变体，该突变体对ABA不敏感。综上，本项目不仅证明LcHRC可能通过调节细胞质内Ca2+浓度的变化参与ABA依赖信号通路对干旱胁迫的应答，还筛选获得可被进一步应用的LcHRC基因资源。