【作者】 杨雪娇；

【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】 异三聚体GTP结合蛋白(heterotrimeric GTP-binding proteins, G蛋白)是活细胞内一类具有重要生理调节功能的蛋白质。自从上世纪70年代初在动物细胞中首先发现G蛋白的存在以来,有关G蛋白的研究发展迅速。对于植物G蛋白的研究于80年代后期才开始,处于相对滞后阶段。拟南芥是一种重要的模式植物,对拟南芥异三聚体G蛋白功能和作用机制的研究对于阐明其他植物中G蛋白的功能和作用机制有着重要的参考意义。而一直以来研究较多的是异三聚体G蛋白的主要功能亚基Gα,通常认为Gβγ的功能只是作为α亚基的负调节器并将其固定于细胞质膜上。最近的研究认为,Gβγ二聚体是细胞信号系统中的主要纽带之一,是调节代谢和生长信号传递的信号系统中的重要功能性亚基。本实验首先从拟南芥植物中分离克隆了G蛋白的β和γ亚基,通过转基因手段成功地将克隆的基因转化到巴斯德毕赤酵母中。利用巴斯德毕赤酵母表达体系成功表达了拟南芥异三聚体G蛋白的β和γ亚基。在本实验培养条件下,β、γ1和γ2亚基的表达量分别为483.3μg/mL、365μg/mL和359.7μg/mL。另外,以拟南芥野生型Col、G蛋白β亚基功能缺失突变体agb1-2, RACK1蛋白功能缺失突变体rack1以及G蛋白β亚基和RACK1蛋白功能都缺失的双突变体agb1-2/rack1种子为材料,研究AGB1和RACK1蛋白在拟南芥种子萌发过程中的作用,结果表明它们共同参与了拟南芥种子萌发过程中对春化作用、糖和激素的响应。经GST pull down技术研究证实两者之间不存在相互作用,说明它们存在各自的作用途径。最后,利用Topo克隆和Gateway亚克隆技术转化RACK1-GFP到农杆菌中,并分别用PEG-Ca溶液和农杆菌介导转化重组质粒到Col原生质体和悬浮培养细胞中,利用荧光显微镜观察RACK1蛋白在原生质体和细胞中的定位,进而分析其功能。更多还原

【Abstract】 Heterotrimeric GTP-binding protein is a kind of protein with important physiologically regulatory functions in living cells. Since its finding in 1970s in animal cells, researches on G proteins have developed rapidly; while researches on plant G proteins were started in the late 1980s, still at a relatively laggar phase. Arabidopsis thaliana is a vital model plant and studies for the functions and working pathways of the G proteins in it will provide very important references for explaining those in other plants. The main functional subunit Gαhas always been being studied much more and the Gβγdimer has been recognized as the negative regulator of Gαand its anchor on the cell membrane. Recent researches demonstrate that the Gβγdimer is one of the main taches among cellular signal systems as well as an important functional subunit in the signal systems for regulating metabolism and growth signal transduction.In this study,βandγsubunits of Arabidopsis G protein were firstly isolated from the seedling; then their genes were cloned; finally, the cloned genes were thansformed in the pichia pastoris yeast. Using the Pichia pastoris expression system, theβandγsubunits of Arabidopsis G protein were successfully expressed. Under the present culture conditions, the expression amounts ofβ,γ1 andγ2 subunits were respectively 483.3μg/mL、365μg/mL and 359.7μg/mL.Besides, with the seeds of Arabidopsis ecotype Col, theβsubunit deficient mutant agb1-2, RACK1 deficient mutant rack1 and the double mutant of agb1-2/rack1 as materials, I studied the effects of AGB1 and RACK1 proteins on Arabidopsis seeds germination,suggesting that both of them have taken part in the stratification, sugar and hormone responses during the Arabidopsis seed germination. However, each of them has its own action pathway as the result of that there is no interaction between them identified by the GST pull down technology. Finally, I transformed the reconstructed plasmid of RACK1-GFP into agrobacterium by Topo-cloning and Gateway sub-cloning technologies; then respectively through the PEG-Ca and agrobacterium-mediated transformation into Arabidopsis ecotype Columbia protoplasts and cells, the localization of RACK1 were observed by fluorescent microscope.更多还原