水稻丝氨酸羧肽酶基因参与干旱耐受的分子机理

The use of a biotechnological approach to develop new rice varieties with enhanced drought tolerance is one of the most efficient ways to improve rice production due to the increasing scarcity of water resources. The discovery and use of excellent genes from germplasm resources is the key to achieve breakthrough breeding. Preliminary research indicated that drought tolerance was improved in transgenic rice plants of over-expression of serine carboxypeptidase (OsSCP) gene but reduced in the mutant. In this proposal, we are planning to investigate the subcellular localization of OsSCP by the green fluorescent protein (GFP); analyze rice OsSCP interacting proteins in response to drought by yeast two-hybrid and verify by GST Pull Down.In addition, differentially expressed proteins will be detected by proteomics technology in the over-expression of OsSCP plants, mutants and wild-type in response to drought stress,the differences in the levels of RNA by real-time PCR and protein expression by western blot will be confirmed. These work will elucidate the molecular mechanisms of drought tolerance of rice OsSCP, explore some new drought tolerant proteins and provide a theoretical basis for further enrichment and improvement of drought stress signaling pathways and network.

水资源的日益匮乏使得耐旱水稻品种的培育成为粮食安全的重要目标之一，而从种质资源中发掘和利用优异基因是实现水稻突破性育种的关键。本课题组前期的研究结果表明水稻丝氨酸羧肽酶（OsSCP）基因过表达植株提高干旱耐受性，而突变株的干旱耐受性降低。本项目拟在现有研究基础上，采用绿色荧光蛋白GFP进行OsSCP的亚细胞定位；通过酵母双杂交和GST Pull Down技术分析和验证OsSCP在水稻干旱响应中的相互作用蛋白；利用蛋白质组学技术分析OsSCP过表达植株、突变体及其野生型响应干旱胁迫时的差异表达蛋白质,并通过real-time PCR和western blot验证RNA和蛋白质水平的差异表达。研究结果将初步阐明OsSCP参与水稻干旱耐受的分子机制，有望发掘与耐旱相关的新蛋白质，为进一步丰富和完善干旱胁迫信号途径及其网络提供理论基础。

干旱胁迫已成为限制水稻生产非常重要的环境因素。前期的研究结果表明水稻丝氨酸羧肽酶（OsSCP）基因过表达植株提高干旱耐受性，而突变株的干旱耐受性降低。本项目采用酵母双杂交、双分子荧光互补和免疫共沉淀技术筛选和验证水稻OsSCP在干旱响应中的互作蛋白；通过蛋白质组学技术分析OsSCP突变体及野生型响应干旱胁迫的差异表达蛋白质，探讨OsSCP介导水稻干旱耐受的分子机制。.研究表明，OsSCP定位在细胞膜上；利用酵母双杂交系统筛选9个在干旱胁迫处理下与OsSCP相互作用的蛋白质，分别为OsHSP40、Ospoll-L、Os21980、OsMADS1、Osp59-Y1、OsUPF0195-L、OsRPL14E、OsS59-L和OsCaLB-1，并采用双分子荧光互补和免疫共沉淀分析技术验证了OsUPF0195-L和OsHSP40与OsSCP蛋白之间的互作，同时鉴定OsSCP与蛋白互作结合的功能结构域为蛋白水解酶肽酶S28。进一步的研究发现，OsSCP与OsHSP40在细胞膜上发生互作且受Mg2+的调控，推测定位在细胞质的OsHSP40和OsSCP发生互作时被切除信号肽而迁移至细胞膜，参与调控水稻植株的耐旱性。iTRAQ分析表明干旱胁迫下差异表达蛋白最显著富集的是钙离子结合蛋白，KEGG Pathway功能富集分析发现差异蛋白与碳代谢、糖酵解/糖异生和氨基酸的生物合成以及不饱和脂肪酸代谢途径相关。此外，利用转基因技术获得了OsUPF0195-L、OsHSP40互作基因的过量表达和CRISPR/Cas9突变植株，进一步验证OsSCP互作基因参与水稻的干旱响应。上述研究结果从蛋白质水平阐明OsSCP参与水稻对干旱耐受的机理，为培育耐旱水稻新材料提供理论和材料基础；与此同时，发掘一些与耐旱相关的蛋白质，为进一步丰富和完善干旱胁迫信号途径及其网络提供重要信息。