Clone,prokaryotic Expression Of NtGCN2 And Its Responses To Stresses In Tobacco

In the initiation of eukaryotic translation,GCN2 can regulate the translation of protein by phosphorylation eukaryotic initiation factor 2(eIF2).Which plays a key role in the stress responding.In animals and yeast cells,the research on GCN2 has made great progress.While,in plant,GCN2 has been studied in the arabidopsis thaliana.However,this gene has not been studied in the Nicotiana tabacum so far.In this study,we clone the gene of GCN2 and heterogeneously expressed the NtGCN2 and NteIF2a in BL21-DE3-Codenplus-RIPL of E.coli.The obtained proteins were preliminary purified using the anion exchange column or Ni2+-NTA column,respectively.Also,the gene expression of GCN2 was detected under biotic(TMV)and abiotic(SA,AZA signal substance)by Real-Time PCR.The results of the study were shown as follows:we cloned the GCN2 from Nicotina tobaccum L.K326 by rapid amplification of cDN A ends(RACE),named NtGCN2,which includs a 3759 bp open reading frame encoding 1252 amino acid residues.The molecular weight of the predicted amino acid sequence of the NtGCN2 protein was 141.4 kDa with a theoretical pI of 5.58.Sequence alignment by BLASTP showed NtGCN2 shared the high similarity to Solanum tuberosum GCN2(90%)and Solanum lycopersicum GCN2(88%)and contained the a typical kinase catalytic domain and a His-tRNA synthetase-related domain.Real-time PCR was porfermed to reveal that NtGCN2 was expressed in roots,stems,leaves and flowers,with higher expression in leaves and lower in root.Secondly,in order to study the function of NtGCN2 protein,We constructed the recombinant plasmid pET15b-NtGCN2 and transformed the plasmid into Escherichia coli BL21-CodonPlus-(DE3)-RIPL strainsfor expression of NtGCN2 protein This recombinant protein was successfully expressed under the induction of IPTG in 0.5 mM and 1 mM and was confirmed by Western-Blotting.Then the obtained proteins were further purified by the anion exchange column Hitrap.Thirdly,we conducted the pET15b-NteIF2a vector to express the recombinant NteIF2α,which was prepared for the biological activity assay of the NtGCN2.NteIF2αprotein was also produced in the Escherichia coli BL21-CodonPlus-(DE3)-RIPL strains under the induction of 0.5 mM IPTG and confirmed by Western-Blotting.The protein was further purified by Ni2+-NTA agarose column.Finally,For exploring the response of NtGCN2 to biotic and abiotic,the gene expression of NtGCN2 was detected by Real-Time PCR under biotic(TMV)and abiotic(SA,AZA signal substance).We found that NtGCN2 was activated under biotic(TMV)and abiotic(SA,AZA signal substance).The specific results were that the gene expression of GCN2 increased significantly under the treatment of 10 mg/mL TMV,0.2 mM SA,0.1 mM AZA and 1 mM AZA,12 hours later;but only 0.2 mM SA,and 1 mM AZA,24 hours later.During seeding stage and resettling stage of tobacco we found the similar regulations under the treatment of AZA,but the difference of treatment between 0.1 mM AZA and 1 mM AZA was more obvious during the resettling stage.The GCN2 from Nicotina tobaccum L.K326 was cloned and NtGCN2 protein was expressed and preliminarily purified.The research laied the foundation for the exploration of GCN2.At the same time its responses to stresses in tobacco was explored preliminary,which provided a theoretical basis for the mechanism of stress response with GCN2.