Cloning And Expression Analysis Of NteIF2? And The Preparation Of Antibody

Eukaryotic translation initiation factor eIF2??? subunit of eukaryotic translation initiation factor 2?plays an important role in the regulation of protein synthesis of eukaryotes.In animals and yeast cells,the research on eIF2? has made great progress.While,in plant,eIF2? has been studied in the arabidopsis thaliana.However,this gene has not been studied in the Nicotiana tabacum so far.In this study,we cloned the gene of NteIF2? and analyzed the sequence of it.The m RNA expression level in different organs of different tissues were analyzed by q PCR method and the gene expression of NteIF2? was detected under biotic?TMV?and abiotic?SA,AZA,Me JA signal substance?by Real-Time PCR.In addition,NteIF2? was successfully produced by E.coli BL21-Codon Plus-?DE3?-RIPL strain and this protein was purified by Ni²⁺-NTA agarose colunm and gelfiltration column,respectively.The results of the study were shown as follows :Firstly,to investigate the function of eIF2? and explore its roles in plant protein synthesis,the ORF?Opening Read Frame?of Nte IF2 a was cloned from Nicotiana tabacum L.K326 and the analysis software is adopted to analysis the sequence of bioinformatics.Our results showed that there were 2 copies of NteIF2? in N.tabacum K326?NteIF2?-1and NteIF2?-2?,and the protein sequences show 99.7% similarity to the other.Also,NteIF2? contains the conserved domain of eIF2? which includes kinase interaction site,phosphorylation site and e IF2 B interaction site.Further,phylogenetic analysis revealed that NteIF2?-1 and NteIF2?-2 might be derived from its progenitor Nicotiana sylvestris and Nicotiana tomentosiformis,respectively.In addition,The potential phosphorylation sites were predicted in the protein sequence.Among them,the Ser56 which was the phosphorolated site of protein kinases showed highly conservation from animals to plants and participated in the regulation of phosphorylation maybe.Secondly,we extractded the RNA of leaf,root,stem,flower in the middle of Nicotaina tobaccum L.K326.Then the m RNA expression level in different tissues were analyzed by q PCR method with the c DNA of reverse transcription and the expression pattern was detected in different organs of N.tabacum K326.The results showed NteIF2?-1 and NteIF2?-2 were expressed in all organs examined and the expression profiles in N.Tabacum displayed differently.Because of the expression of NteIF2?-2 showed higher than that of NteIF2?-1,the functions of eIF2? family members is different in different organs.For exploring the response of NteIF2? to biotic and abiotic,the gene expression of NteIF2? was detected by Real-Time PCR under biotic?TMV?and abiotic?SA,AZA,Me JA?.Results showde that the AZA,SA and Me JA could activate the expression of NteIF2?.The expression of NteIF2? gene was the highest under the treatment of AZA 3 hours later,but SA 48 hours later and Me JA 24 hours later.The expression of NteIF2? gene was the highest under the treatment of TMV 12 hours later.TMV inhibitded the expression of NteIF2? first,then gradually increased after 24 h and 48 h.Thirdly,to investigate the function of eIF2? and explore its roles in the plant protein synthesis,the recombinant vector p ET15b-NteIF2? was constructed for prokaryotic expression of NteIF2?.The results showed that NteIF2? was successfully produced by E.coli BL21-Codon Plus-?DE3?-RIPL strain under induction of 0.2 m M IPTG for 4 h at 16 ?.Also,this protein was purified by Ni²⁺-NTA agarose colunm and anion exchange column.The protein was further purified by gelfiltration column.A single protein band was obtained.Further,the obtained protein was identified by Western-blotting using anti-His antibody.Lastly,the polyclonal antibody of NteIF2? was prepared which showed high specificity and sensitivity.Results showed that the NteIF2? antibody titer was 1:400000.Also,adopting the antibodies detected the expression in tobacco K326 alpha in tobacco K326 of different organizations.As a result,NteIF2? only expressed in leaves,and expressed in root,stem and flower with low or no expression.The NteIF2? from Nicotiana tobacum L.K326 was cloned and NteIF2? protein was expressed and preliminarily purified.This study lay a foundation for further study the function of eIF2? and its roles in regulation of protein synthesis in plant.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 NteIF2?.