Preparation Of Tobacco Protein Kinase NtGCN2 Antibody And Screening Its Binding Proteins Using CoIP Approach

Under the stress environment,the organism can response to the stress environment by regulation of the protein synthesis.A protein kinase called GCN2(General Control Non-derepressible 2)plays an important role in the connection between the stress and metabolic signaling networks,which could phosphorate ? subunit of eukaryotic translation initiation factor 2(e IF2?)and involved in the regulation of protein synthesis in response to stress response.At present,the research on GCN2 has made great progress in animal and microbial cells.However,there are less studies in plants and mainly in Arabidopsis thaliana.In this study,we cloned the protein kinases catalytic domain of Nt GCN2(Gen Bank accession number:KJ706220).Also,We constructed the recombinant plasmid p ET15b-PKc and successfully expressed PKc protein in E.coli BL21-Codon Plus-(DE3)-RIPL.Then the obtained proteins were purified using the Ni2+-NTA column,anion exchange chromatography and gel filtration,respectively.Moreover,We prepared the polyclonal antibody of PKc protein and detected the expression of Nt GCN2 in tobacco.Finally,we preliminary explored these proteins which interacted with GCN2 by using prepared antibody by Co-IP method.This study lay a foundation for further studying the function of GCN2 and its roles in regulation of protein synthesis in plant.The main results of the study were shown as follows:Firstly,according to the full-length sequence of Nt GCN2,we predicted the sequence of kinase domain and designed its primers.We cloned the PKc from Nt GCN2 and obtained the fragment of kinase domain.The fragment was connected with T vector and sequenced,and the PKc functional domain was successfully inserted into the T vector.Secondly,We constructed the recombinant plasmid p ET15b-PKc and transformed the plasmid into Escherichia coli BL21-Codon Plus-(DE3)-RIPL strains for expression of PKc protein.Meanwhile,we optimized the expression conditions of PKc protein.The PKc protein was expressed under induction of different concentrations IPTG at 16 ?,28 ?,37 ?,respectively.The results showed that the optimized expression condition were under induction of 0.5 m M IPTG for 13 h at 16?.The obtained proteins were then purified using the Ni2+-NTA column,anion exchange chromatography and gel filtration,respectively.The purified protein was analyzed by SDS-PAGE.Thirdly,we prepared polyclonal antibodie of Nt GCN2 and PKc by using purified p KC protein and Nt GCN2.It was showed that the titers of two antibodies were both 1:520K.We detected the expression of Nt GCN2 in tobacco by Western-blotting using anti-Nt GCN2 or anti-PKc antibody.The results showed that there was no hybrid band by using anti-Nt GCN2 antibody.While,single hybrid band was obtained by using anti-PKc antibody.We hypothesized that the immunogenicity of the antibody obtained from rabbit immunized with full-length GCN2 was weaker,since the antigen of recombinant Nt GCN2 was recovered from SDS-PAGE.Finally,We extracted the whole protein from tobacco and explore the proteins that would interreact with GCN2 using anti-PKc antibody by Co-IP method.The results showed that proteins such as,EF1 A X4(related to protein synthesis),RPS4(related to ribosome formation),UEP(related to stress resistance),Ru Bis CO activase(involved in carbon cycle),CAB8(related to photosynthesis),atp A1(involved in energy metabolism),LTCP(related to seed germination)etc,might be able to interact with GCN2 directly or indirectly.The results indicate that Nt GCN2 might be related to these pathways,which remains to be confirmed in the future.