【作者】 彭威风；

【导师】 梁卫红；

【作者基本信息】 河南师范大学 ， 细胞生物学， 2011， 硕士

【摘要】 OsRacD是水稻小GTP结合蛋白Rho家族的一个新成员,已有的研究结果显示,OsRacD的功能之一是通过调控花粉管的延伸生长,参与水稻的育性控制,是一个重要的发育调控基因和信号通路中的开关分子。为进一步研究OsRacD相关信号通路及互作蛋白,梁卫红等以OsRacD为诱饵,采用酵母双杂交技术筛选了水稻幼穗cDNA文库,获得了若干潜在互作蛋白编码基因的cDNA序列。基于本实验室对OsRacD及其互作蛋白的研究,本论文对已经鉴定的一个负调控OsRacD活性的鸟苷酸解离抑制因子的编码基因—OsRhoGDI2进行了系列研究,对该基因在水稻育性控制的功能进行了初步鉴定。采用生物信息学分析,预测和比较了OsRhoGDI2和OsRacD蛋白的理化特点、修饰位点和亚细胞定位,进而构建了受控于CaMV35S启动子的与绿色荧光蛋白融合表达的OsRhoGDI2基因的双元植物表达载体pCAMBIA1302-OsRhoGDI2- GFP,采用农杆菌介导法转化洋葱表皮细胞,通过荧光显微镜观察了融合蛋白在活细胞内分布特点,证实OsRhoGDI2和OsRacD具有一些相似的理化特性和翻译后修饰位点,OsRhoGDI2融合蛋白主要分布在细胞质、细胞膜和细胞核。OsRhoGDI2与OsRacD在蛋白理化特性和胞内分布上存在的相关性提示,OsRhoGDI2蛋白可能在调控OsRacD的胞内分布和活性中发挥重要作用。为确定OsRhoGDI2基因的功能,借助农杆菌介导法将双元植物表达载体pCAMBIA1302-OsRhoGDI2-GFP转化水稻愈伤,经过浸染、筛选和分化,获得了12株分化苗,经基因组DNA的PCR检测,获得7株阳性转基因水稻。半定量RT-PCR检测结果显示,转基因水稻中OsRhoGDI2基因转录水平显著高于对照,说明OsRhoGDI2基因在CaMV35S启动子下,能够在水稻中过表达。表型分析显示,与对照水稻相比,转基因水稻的分蘖数和总稻粒数变化不大,而授粉粒数和结实率明显降低。统计分析显示,转基因水稻的授粉率和结实率显著降低,大多不足10%,而对照水稻结实率则达到96%,表明OsRhoGDI2基因的过表达和水稻育性存在负相关。为确认OsRhoGDI2基因在水稻育性控制中的功能,构建了OsRhoGDI2基因的干扰载体pART27-OsRhoGDI2,以期探讨OsRhoGDI2基因沉默对水稻的生长发育的影响,确定OsRhoGDI2与OsRacD蛋白的相互作用在水稻育性控制中的作用机制。更多还原

【Abstract】 OsRacD belonging to the rice Rho family of small GTP-binding protein, previous results showed one of its function involved in rice photoperiod fertility conversion of photoperiod sensitive genic male sterile rice Nongken 58S, which influences rice fertility via controlling the pollen tube growth. To further investigate OsRacD-related signaling pathway and partners, using OsRacD as bait, the rice panicle cDNA library were screened by yeast two-hybrid system, and some cDNA sequences encoding putative targets were obtained. In this study, the functions of the gene OsRhoGDI2, a putative partner of OsRacD, which encoding a GDP dissociation inhibitor in rice fertility control had been identified by transgenic methods.Using bioinformatics analysis, the physicochemical characteristics, modification site and subcellular localization of OsRhoGDI2 and OsRacD protein had been predicted and analyzed. The GFP-fused binary plant expression vector pCAMBIA1302-OsRhoGDI2-GFP which controlled by the CaMV35S promoter was constructed. By Agrobacterium-mediated method, the recombinant vector pCAMBIA1302-OsRhoGDI2-GFP was transformed into onion epidermal cells, the subcellular localization of the fusion protein were observed under the fluorescence microscopy. The results showed that OsRhoGDI2 and OsRacD shared some similarities in physical and chemical properties, post-translational modification sites, and OsRhoGDI2 mainly distributed in the cytoplasm, nucleus and cell membrane in onion epidermal cells, suggest that OsRhoGDI2 may be an important partner of OsRacD which regulated its subcellular localization and activities.In order to identify the function of OsRhoGDI2 gene, the binary plant expression vector pCAMBIA1302-OsRhoGDI2-GFP was transformed into rice callus by Agrobacterium-mediated method, after infection, screening and re-differentiation, twelve transgenic rice were obtained, and seven of them were further verified by PCR amplification. RT-PCR detection showed the expression level of the gene OsRhoGDI2 were higher than that of control, suggest that the gene over-expressed in transgenic rice. The phenotype analysis showed that the tiller numbers and total rice grain numbers were similar between transgenic rice and control, but the pollination grain number and maturing rate of transgenic rice were decreased significantly compared with that of control. The statistic analysis data showed the pollination grain number and maturing rate of the transformation rice was less than 10%, while the control was up to 96%, suggested that function of OsRhoGDI2 may related with rice fertility. In order the study the relationships between OsRhoGDI2 and OsRacD in rice fertility control, the RNA interference binary vector pART27-OsRhoGDI2 was constructed, help the further study on the effects of gene silencing of OsRhoGDI2 and OsRhoGDI2-OsRacD interaction in the growth and development of rice panicle, which participated in the process of rice fertility regulation.更多还原