Screening Of Arabidopsis CPK10 Interacting Proteins Based On The Tap Technology

In plant signal transduction pathways, Ca2+, as the basic of second messenger, plays an important role in plant growth and development, and response to adversity reaction. The further Ca2+ signal transfer finished is by Ca2+ receptor proteins and Calmodulin(Ca M) and calcium dependent protein kinase CDPKs(or CPKs). CDPKs named calcium dependent protein kinase, it is a kind of important calcium signal sensors, and plays an important role in the process of response to adversity stress. Arabidopsis thaliana genome encoding 34 CDPKs. Study of the interaction between the proteins is an important means of explanation various life activities in the cell, through the deep understanding of protein group to study the protein interaction network is to reveal the mysteries of life activities.By phenotype tests of the CDPK mutants, the cpk10 mutant, a T-DNA insertion mutant for Arabidopsis thaliana CPK10 gene, was found being much more sensitive to drought stress compared with wild-type plants. Existing research results showed that the CPK10 interact with HSP1(heat shock protein 20-like protein 1) directly, and regulate ion channels of guard cells indirectly, the guard cells showed the opening and closing movement in response to drought. So, CPK10 and downstream function protein-- ion channels, signal transmission is what members(or what)? This is we’ve been thinking about the problems in the process of research.In order to selected other CPK10-interacting proteins, further in-depth study CPK10 mechanism of drought resistance. This paper use TAP(Tandem Affinity Purification) technology,Screening interactions proteins in the body of physiological environment. Firstly, built a p CM1307-3FLAG-3HA-CPK10 binary expression vector which with two labels. Transformed it into Arabidopsis wild-type protoplast by PEG mediated method, and then detected the target protein of CPK10 expression. Secondly, on the basis of the target protein can be expression successful in protoplast, using the method of inflorescence impregnation, transformed the vector into Arabidopsis wild-type plant materials, screened for T3 generation positive strain on the Hygromycin medium, and detected the stability strain of expressed CPK10 protein fusion FLAG and HA labels by Western-blotting. Finally, the plants growth around 4 weeks of the stability strain of protein expression, selected the foliage of 3 and 4 rounds, extracted the proteins, using the TAP tag screening CPK10 protein complexes, further through the protein mass spectrometry analysis the information of protein complexes composition, and analysis of the possible gene CPK8、HSP70、IPGAM2、CAT3、TGG1、HSC70-1 by biological information and RT-PCR.The test results showed that the p CM1307-3FLAG-3HA-CPK10 vector plant successfully expressed proteins transformed into Arabidopsis wild-type protoplast, selected 2 stable positive strain of transformed wild type plant materials, using positive stable materials, screened 317 interacting protein signals by TAP-MS analysis. Bioinformatics analysis 6 interesting proteins. RT-PCR analysis of HSP70、CAT3、TGG1、CPK8 gene express raised induced by drought.