拟南芥异三聚体G蛋白β和γ亚基在毕赤酵母中的表达及β亚基和RACK1相互作用研究
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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。
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.
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。
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.
引文
陈云(2005)拟南芥G蛋白信号转导调节蛋白(AtRGS1蛋白)功能研究.博士学位论文
杜中军,朱水芳,黄文胜,翟衡(2002)毕赤酵母外源基因表达系统研究进展.生物技术通报, 4: 7-9
郝岗平,吴忠义,陈茂盛,曹鸣庆,黄丛林,杨清(2004) ATHK1基因调节拟南芥渗透胁迫信号转导过程.植物生理与分子生物学学报30(5): 553-560
季芳芳(2006) ATRGS1蛋白的定位及其在拟南芥种子萌发和幼苗生长过程中的作用研究.硕士学位论文
李生茂(2004)应用GST Pull Down实验验证NF-κB相互作用多肽.硕士学位论文
尚忠林,马力耕,王学臣,孙大业(2003)异三聚体G蛋白参与调节花粉细胞内钙离子浓度.自然科学进展, 13(7): 746-749
孙大业,郭艳林,马力耕(2001)细胞信号转导.北京:科学出版社, 1-9
王谨,李天增,才学鹏(2003)甲醇酵母表达外源基因的研究近况.中国兽医科技, 33 (7): 34-38
武维华,赵云云(1996)植物细胞G蛋白研究进展.植物学报, 38(5): 406-413
闫诚(2005)拟南芥WD40重复蛋白AtARCA和AtAGB1对干旱胁迫信号响应机理的研究.
姚斌,张春义(1998)高效表达具有生物学活性的植酸酶的毕赤酵母.中国科学(C辑), 28(3): 237-243
赵翔,霍克克,李育阳(2000)毕赤酵母的密码子用法分析.生物工程学报, 16(3): 308-311
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杜中军,朱水芳,黄文胜,翟衡(2002)毕赤酵母外源基因表达系统研究进展.生物技术通报, 4: 7-9
郝岗平,吴忠义,陈茂盛,曹鸣庆,黄丛林,杨清(2004) ATHK1基因调节拟南芥渗透胁迫信号转导过程.植物生理与分子生物学学报30(5): 553-560
季芳芳(2006) ATRGS1蛋白的定位及其在拟南芥种子萌发和幼苗生长过程中的作用研究.硕士学位论文
李生茂(2004)应用GST Pull Down实验验证NF-κB相互作用多肽.硕士学位论文
尚忠林,马力耕,王学臣,孙大业(2003)异三聚体G蛋白参与调节花粉细胞内钙离子浓度.自然科学进展, 13(7): 746-749
孙大业,郭艳林,马力耕(2001)细胞信号转导.北京:科学出版社, 1-9
王谨,李天增,才学鹏(2003)甲醇酵母表达外源基因的研究近况.中国兽医科技, 33 (7): 34-38
武维华,赵云云(1996)植物细胞G蛋白研究进展.植物学报, 38(5): 406-413
闫诚(2005)拟南芥WD40重复蛋白AtARCA和AtAGB1对干旱胁迫信号响应机理的研究.
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