OsGPRP基因在水稻生长发育和逆境适应中的生物学功能研究

GPRP (Glycine- and Proline-rich Protein) genes play critical roles in plant growth as well as stress adaptation. After having confirmed the dramatic responses of 3 rice GPRP genes (OsGPRP genes) to drought, cold and abscisic acid (ABA) stress, we propose the following studies: (1) cloning of the 3 OsGPRP genes; (2) identification of the differential expression patterns and the subcellular localizations of the 3 OsGPRPs; (3) elucidation of the biological roles of the 3 OsGPRP genes during plant growth as well as the process of adaptation to stresses, by comparison of the physiological and phenotypic characteristics between wild type and homozygous transgenic rice plants under stress treated and untreated conditions. Homozygous transgenic rice lines will be obtained by genetic transformation of the japonica rice with gene overexpression plasmids, gene silencing constructs and loss-of-function constructs; (4) investigation of the putative metabolic pathways involving the 3 OsGPRP genes. Identification of relevant up-regulated or down-regulated genes in specific tissues of wild type and homozygous transgenic rice lines under stress treated and untreated conditions will be performed using small ncRNA and microRNA sequencing and Quantitative Real-time PCR. These studies will not only provide insight into the interactions among the 3 OsGPRP genes and their corresponding molecular networks/pathways, but also lay the theoretical foundations for the practice of breeding high-yield and stress-tolerant crops.

GPRP基因在植物生长发育与逆境适应中具有重要的作用。我们在确认3个水稻OsGPRP基因对干旱、低温和脱落酸（ABA）处理具有明显反应的基础上，1）克隆这3个OsGPRP基因；2）确定其在水稻不同组织中的表达特性，完成蛋白表达亚细胞定位；3）重点构建其过量、下调和缺失表达载体，转化粳稻，获得转基因纯合株系后，比较正常和逆境下野生型水稻与转基因纯合水稻在生理生化及表型性状上的差异，确定这3个OsGPRP基因在水稻生长发育和逆境适应过程中的生物学功能；4）通过小ncRNA和mRNA测序等方法，获得正常与逆境下野生型水稻和转基因纯合水稻特定组织中表达显著上调或下调的基因，初步确定3个水稻OsGPRP基因功能发挥可能涉及的代谢调控途径。为深入剖析这3个水稻OsGPRP基因在生物学功能方面的相互关系及其各自生物学功能发挥相关的分子网络途径奠定基础，也为高产、多抗的作物基因工程育种实践提供理论依据。

本结题项目主要完成了以下研究内容：1）克隆了3个水稻OsGPRP家族基因；2) OsGPRP蛋白家族基因的主要特征特性分析；3)该家族蛋白的亚细胞定位与原核表达分析；4)正常和逆境下该基因家族在水稻不同组织部位中的时空表达模式分析；5)该家族基因的 CRISPR敲除、RNAi干涉转基因水稻材料创制；6)转基因水稻材料的发育表型和逆境响应功能分析等。我们重点发现：与野生型相比，OsGPRP3的CRISPR敲除和RNAi干扰转基因水稻具有粒宽、粒厚和粒重减少的表型；这与正常情况下OsGPRP3特异性地在水稻P8期的穗子中较高表达和开花受精后第10天的种子中特高表达的结果基本相吻合；说明OsGPRP3在水稻籽粒发育过程中具有重要作用，但其分子机理有待进一步探究。本项目率先在植物中发现GPRP基因具有调控籽粒发育的功能。为深入揭示植物GPRP家族基因的功能及其作用机理奠定基础，为全面理解水稻种子发育的遗传机理提供了全新的知识，也为水稻种质改良提供了基因资源。