【作者】 顾杰；

【导师】 梁建生；

【作者基本信息】 扬州大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 本研究以拟南芥野生型Col-0、突变体rgs1-2、gpa1-4、aba3和abi1、过表达35S:RGS1为材料,通过转基因细胞系构建、差异显示分析和双突变体构建分析等技术,用分子生物学、生物化学、分子遗传学等方法研究了AtRGS1蛋白在拟南芥葡萄糖信号转导途径中的作用。取得了以下主要研究结果:1、建立了拟南芥悬浮培养细胞体系,并对AtRGS1蛋白及相关结构域进行了细胞定位根据AtRGS1蛋白的特有结构,将编码AtRGS1的两个结构域分别进行克隆表达,结合RGS1-GFP融合蛋白表达技术证明:AtRGS1蛋白定位于细胞膜,AtRGS1-N端结构域和AtRGS1-C端结构域分别定位于质膜和细胞核。D-葡萄糖和ABA处理可以改变AtRGS1全蛋白的亚细胞定位,而AtRGS1-N端结构域定位未发生改变。2、筛选了AtRGS1蛋白介导的葡萄糖信号转导途径中特异表达的基因以拟南芥野生型Col-0和突变体rgs1-2为材料,运用差异显示技术筛选AtRGS1蛋白介导的葡萄糖信号转导途径中特异表达的基因。经反向Northern验证,得到四个差异表达蛋白,分别是拟南芥UDP葡萄糖苷转移酶(UGTs)、拟南芥γ谷氨酰水解酶、拟南芥苏氨酸内切酶、拟南芥ADP-核糖基化因子(ARF)。它们很可能是AtRGS1蛋白介导的葡萄糖信号转导途径中重要的作用因子。其中ADP-核糖基化因子(ARF)可能和AtRGS1-C端结构域具有相互作用,参与了AtRGS1-C端结构域的迁移,而且可能将处于游离状态的AtRGS1的C端结构域通过胞内运输转运至细胞膜,使其和跨膜结构域重新融合。UDP葡萄糖苷转移酶(UGTs)是催化拟南芥内源脱落酸(ABA)糖基化的一种酶,在外源高浓度葡萄糖处理后,其表达量降低,这就表明,外源高浓度葡萄糖处理导致内源ABA分解的速度降低,从而促进ABA抑制的种子萌发。而且,UDP葡萄糖苷转移酶很可能是AtRGS1介导的葡萄糖信号转导途径中下游的效应分子。3、明确了拟南芥种子萌发对葡萄糖信号响应过程中AtRGS1的作用及其与ABA的关系(1)以拟南芥野生型Col-0为遗传背景的突变体为材料,研究了AtRGS1蛋白在葡萄糖介导的种子萌发过程中的作用。结果显示,与Col相比,AtRGS1过表达的35S:RGS1和Gα缺失突变体gpa1-4的种子萌发对D-葡萄糖的抑制作用表现为超敏感,而AtRGS1缺失突变体rgs1-2表现为低敏感。35S:RGS1、rgs1-2和Col-0种子萌发对葡萄糖类似物甘露糖的响应无明显差异。己糖激酶抑制剂NAG处理对葡萄糖抑制种子萌发的效应不明显,但显著降低了甘露糖对种子萌发的抑制作用。表明在种子萌发过程中,AtRGS1参与的葡萄糖信号转导途径是一特殊途径,且该途径不依赖于己糖激酶。(2)以AtRGS1缺失突变体rgs1-2、ABA合成突变体aba3、ABA响应不敏感突变体abi1和双突变体aba3rgs1-2、abi1rgs1-2,以及野生型Col-0为材料,研究了葡萄糖介导的拟南芥种子萌发过程中AtRGS1与ABA3和ABI1的相互关系。结果表明:双突变体aba3rgs1-2表现出比单突变体对高浓度葡萄糖的抑制作用更加不敏感。说明AtRGS1和ABA3都参与了拟南芥种子萌发过程的葡萄糖信号的转导。双突变体abi1rgs1-2表现出和单突变体rgs1-2相似的对高浓度葡萄糖的抑制作用不敏感,而单突变体abi1和野生型Col均表现为敏感。更多还原

【Abstract】 The regulator of G-protein signaling (RGS) protein, recently identified in Arabidopsis thaliana (named as AtRGS1) has a predicted seven-transmembrane structure as well as a RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 protein in glucose signaling of seed germination were investigated in the present study, using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1), the alpha subunit (GPA1) of the G protein, serine/threonine phosphatase and molybdenum cofactor sulfurase in Arabidopsis, named rgs1-2, gpa1-4, abi1 and aba3, respectively, with different approaches such as genetics, biochemistry and molecular biology. The main results were as follows:The 35S:RGS1 overexpression system was constructed by TOPO cloning. The transgenic suspension cell culture from Col was constructed. Protein localization analysis using RGS1-GFP fusion protein technique showed that, the RGS1 protein was localized in plasma membrane, and the N-terminal domain (7 TM domain) and C-terminal domain (RGS box) of AtRGS1 were localized in plasma membrane and nuclear, respectively, which was consistent with the predicated localization with software (TMHMM Server v. 2.0, http://www.cbs.dtu.dk/services/TMHMM/).We further investigated the expression of specific genes responding to glucose treatment using a differential display reverse transcription PCR (DDRT-PCR) approach, in combination with reverse northern blotting. Four different expressing genes were identified between Col-0 and rgs1-2, including ADP-ribosylation factor, Uridine diphosphate glycosyl transferases, threonine endopeptidase and gamma-glutamyl hydrolase. It was suggested that these four genes-encoded proteins may paly important roles in glucose signaling and their possible roles were also discussed.The roles of AtRGS1 in glucose-mediated seed germination were analyzed by comparing the different responses of seed germination of Col-0 and rgs1-2 mutant to glucose and the effects of glucose analog and a specific HXK inhibitor, N-acetyl-glucosamine (NAG). The results showed that, in contrast to wild type Col-0, rgs1-2 seed germination was insensitive to glucose, while those of gpa1-4 and 35S:RGS1 were hypersensitive to glucose. The germination of Col-0, 35S:RGS1 and rgs1-2 mutant seeds showed the same responses to mannose. The inhibition of seed germination by glucose treatment were not obviously affected by NAG, but significantly alleviated the inhibiting effect of mannose on seed germination. These data suggested that AtRGS1 protein most likely functions in an HXK-independent glucose signaling in Arabidopsis seed germination.The relationships between AtRGS1 with ABA3 and ABI1 in glucose signaling were also investigated using various single and double mutants. The seed germination of aba3rgs1-2 double mutant was less sensitive to high concentration glucose, as compared with those of single mutant, rgs1-2 and aba3, which implying that the hyposensitivity of rgs1-2 mutant seed germination to exogenous glucose might be the results of the impairment of ABA biosynthesis and the normal degradation of endogenous ABA during seed germination. The seed germination of abi1 mutant was hypersensitive to high concentration of glucose, and that of abi1rgs1-2 double mutant was insensitive to glucose.更多还原