Prokaryotic Expression And Purification,Antibody Preparation Of NtGCN2 From Tobacco And Its Characterization In Vitro

In the face of adverse environments such as drought,cold damage,salt,etc.,plants usually regulate their metabolism to cope with stress and maintain their own growth,and protein as a material basis of life plays a vital role in this regulation.As an eIF2a kinase,GCN2 phosphorylates eIF2a for the purpose of regulating protein synthesis in vivo.In studies of yeast and animal GCN2,it was found that either alone or in combination with other eIF2a kinases involved in a variety of stress regulation.The eIF2a kinase found in plants only has GCN2,so it should play an important role in the metabolic pathways of plants to cope with stress.However,there are still many unknown mysteries in the study of plant GCN2.In this study,we cloned the N-terminal 1-437aa gene sequence of NtGCN2(GenBank accession number:KJ706220),constructed the recombinant plasmid pCznl-NtGCN2,and successfully expressed NtGCN2 protein in E.coli BL21(DE3)Plys.Also,the protein was purified by inclusion body renaturation and Ni2+-NTA column chromatography to obtain pure NtGCN2 protein,and polyclonal antibodies capable of detecting recombinant NtGCN2 protein were prepared by using these proteins.Moreover,we performed an in vitro characterization of NtGCN2,demonstrating that it can phosphorylate eif2.Finally,we have initially verified the interaction gene of NtGCN2 to lay a foundation for further study of the function of plant GCN2 and its role in dealing with stress.The main results of the study are as follows:Firstly,based on the full-length sequence of NtGCN2,we selected the 1-437aa sequence of N-terminal and designed its primers.We obtained a N-terminal 1-437aa fragment from the pMD-NtGCN2 clone and inserted the fragment into the pCznl vector to construct the recombinant plasmid pCznl-NtGCN2(1-437aa).This plasmid was transformed into Escherichia coliBL21(DE3)Plys strain,and expression was induced by 0.5 mM IPTG at 37 °C for 4 hours.The target protein NtGCN2(1-437aa)was mainly found in the protein precipitate by SDS-PAGE analysis.Secondly,we purified the expressed protein by using inclusion body renaturation and Ni-NTA agarose affinity chromatography column to obtain a higher purity NtGCN2 protein.The obtained protein was immunized with New Zealand white rabbits to prepare polyclonal antibodies to NtGCN2(1-437aa)and NtGCN2.Indirect ELISA results showed that the titers of both antibodies were 1:520K.Western blot analysis showed that the prepared NtGCN2(1-437aa)and NtGCN2 antibodies could detect the expression of recombinant NtGCN2,but did not detect the expression of NtGCN2 in tobacco.We hypothesize that the prokaryotic expression system lacks methylation,glycosylation and other modification processes,resulting in the antigenic binding site not being presented normally.Thirdly,we characterized the kinase activity of NtGCN2 by isotope labeling in vitro.The entire identification system consists of the kinase domain protein of NtGCN2,NteIF2aprotein,[?-32P]ATP and buffer.The concentration gradient of NtGCN2 showed that NteIF2awas only phosphorylated in the presence of NtGCN2,and the phosphorylated NteIF2?content increased with the increase of NtGCN2 content,which showed a positive correlation.In the study of the reaction time of the NtGCN2 and NteIF2a,it was found that the phosphorylated NteIF2acontent increased with time in a certain range,but the longer the time,the smaller the phosphorylated NteIF2aincrease.It is indicated that the amount of phosphorylation of eIFadepends on the amount of NtGCN2 and NteIF2a.Finally,we performed a preliminary validation of the interaction gene of NtGCN2.Tobacco K326 and NtGCN2 overexpressing plants were subjected to normal treatment and drought treatment,and then GCN2,eIF2(related to protein synthesis),HIPP(related to ion transport),PHAO(related to photorespiration),NQOl(related to ROS)and CCR4(related to mRNA stability)were quantified in real time.The results showed that the expression levels of eIF2,HIPP and PHAO of NtGCN2 overexpressing lines increased,and the increase range was similar to that of GCN2.The expression of CCR4 decreased slightly,but the change in the expression of NQO1 was not very obvious.This indicates that the overexpressing line has a stronger ability to scavenge reactive oxygen species and cope with stress than K326.Under drought treatment,the expression levels of NtGCN2,eIF2,HIPP and NQO1 genes in overexpressing lines increased significantly,and the expression level of PHAO increased by about six times.In addition,the decline in CCR4 has become larger.The results indicated that drought induced the changes of NtGCN2,eIF2,HIPP,NQO1 and CCR4.From the level of gene expression,it was verified that the overexpressing lines had stronger ability to cope with stress,and that NtGCN2 could regulate protein synthesis and mRNA content in plants.The ion transport and redox states respond to stress,which lays a foundation for combing the stress regulation network involved in plant GCN2.