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Expression of β and γ Subunits of Arabidopsis G-protein in Pichia Pastoris and Identification of Interaction between the β Subunit and RACK1

Author: YangXueJiao
Tutor: LiangJianSheng
School: Yangzhou University
Course: Biochemistry and Molecular Biology
Keywords: Arabidopsis G protein Pichia pastoris GST pull down Cellular localization
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 102
Quote: 0
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Abstract


Heterotrimeric GTP-binding protein (heterotrimeric GTP-binding proteins, G proteins) are a class of proteins have important physiologic function in living cells. Since the early 1970s, the first to discover the existence of the G-protein in animal cells, the rapid development of the G protein. For the plants G protein research began in the late 1980s, is lagging behind the stage. Arabidopsis thaliana is an important model plant, Arabidopsis different trimeric G protein function and mechanism of action research to clarify the function and mechanism of action of other plants G protein has an important significance. Has been studied is the main function of heterotrimeric G protein subunit Gα, Gβγ the functions usually considered only as a negative regulator of the α subunit and fixed in the cytoplasmic membrane. Recent studies suggest that Gβγ dimer is one of the main link in the cell signaling system, the signal system in the regulation of metabolism and growth signaling functional subunit. This experiment is first isolated and cloned from Arabidopsis plants of the β and γ subunits of the G protein by transgenic means successfully cloned gene was transformed into Pichia pastoris. Successful expression of the Arabidopsis heterotrimeric G protein β and γ subunits using Pichia pastoris expression system. Culture conditions of this experiment, β, γ1 and γ2 subunit expression were 483.3μg/mL, 365μg/mL and 359.7μg/mL. In addition, wild type Arabidopsis Col. G protein β subunit function missing mutations on body agb1-2, of RACK1 lack of protein function mutant rack1 and the G protein β subunit and RACK1 protein functions are missing double mutant agb1-2 / rack1 seed for materials, research of AGB1, and RACK1 proteins in Arabidopsis seed germination process, and the results show that they participate in the Arabidopsis seed germination process of vernalization, sugar and hormone response. GST pull down technology research confirmed the interaction does not exist between the two, they exist each pathway. Finally, the use of the Topo cloning and Gateway subcloning technique transformed into Agrobacterium RACK1-GFP, and PEG-Ca solution and Agrobacterium-mediated transformation of recombinant plasmid to Col protoplast suspension culture cells by fluorescence microscopy RACK1 protein in protoplasts and cellular localization, and thus analysis of its function.

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