Expression Of β And γ Subunits Of Arabidopsis G-protein In Pichia Pastoris And Identification Of Interaction Between The β Subunit And RACK1

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.