Cloning And Prokaryotic Expression Of Hsp70and Hsc70Genes From Hypena Tristalis

It will produce a class of highly conserved heat shock proteins (HSPs) after the living organisms are stimulated by heat, pathogens, and other physical or chemical factors. HSPs usually can be divided according to molecular size:small HSP, HSP60, HSP70, HSP90and HSP110. HSP70family is considered the most important and conservative HSPs.HSP70as a molecular chaperon, can help the nascent peptide chain to fold and participate in its transporter in the cell. The cell of insects are stimulated by extmal or internal physiological changes to stretch the protein peptide chain, lose the original state of hovering and folding, thus changing the spatial configuration of the molecule, and some oligomeric protein complexs gradually degradation, and ultimately loss the function of protein. At this point, Hsp70quickly gathered in the chromatin and nuclear matter, it plays a role in chromatin after depolymerization in a special way, to prevent the degradation of chromatin in the cells, to maintain homeostasis of insect cells, to increase tolerance of the insect cells against external stimulation, and to prevent apoptosis of insect cells, and to accelerat the repair of damage in the insect cells. The expression of heat shock protein is one of the most important changes in the performance of organisms to adapt to changes in ambient temperature at the molecular level.The study of HSP70, as a highly conserved protein, can reveal the insect evolutionary relationship and determine the proximity of the phylogenetic relationship between insect species. According to the insect subjected to temperature stress, HSP70expression changes of body also can reveal its protective effect on the organism. Regulation the expression of insect’s HSP70by molecular biology techniques to reduce the adverse the environmental defense capabilities, and ultimately achieve the purpose of prevention and control of pests. The adaptability and the role of the resistance mechanism of HSP70in organisms, provides a new idea about new insecticide target sites.Hypena tristalis of Lepidoptera Noctuidae, is an important leaf-eating pests of soybean fields. According to the results for many years field survey of soybean pest of our laboratory that the occurrence of Hypena tristalis is increasing year by year in the Northeast region, and has a certain explosive occurrence. Hypena tristalis reported only limited pest occurrence investigation, and has not been specifically research on HSP70of Hypena tristalis at home and abroad. In this study, I have obtained HSP70gene of Hypena tristalis by RT-PCR and successfully constructed the prokaryotic expression vector. And obtain the HSP70recombinant protein of Hypena tristalis to provide the basis for the future research of HSP70expression by heat stress and thermal control technology of pest control.In this paper, Hsp70full-length cDNA sequences of Hypena tristalis was cloned:this cDNA contain2226nucleotide and include an ORF of1905nucleotide which encodes a protein of635amino acid residues, molecular weight is about69.6kDa and p15.36. Hsp70of Hypena tristalis has three typical signature sequence of the HSP70family:IDLGTTYS(11-18), IPDLGGGTFDVSVL(199-212), VVLVGGSTRIPKIQS(337-351). C-terminal consensus sequence:GPTVEEVD(629-635). There were5N-glycosylation sites in the Hsp70amino acid sequences from Hypena tristalis.The analysis show that no signal peptide cleavages sites in ORF by SignalP4.0Server. The Hsp70cDNA sequence has been deposited at GenBank with accession number JQ316541.Hsc70full-length cDNA sequences of Hypena tristalis was cloned:this cDNA contain2198nucleotide and include an ORF of1959nucleotide which encodes a protein of653amino acid residues, molecular weight is about71.5kDa and p15.32. Hsc70of Hypena tristalis has three typical signature sequence of the HSP70family:IDLGTTYS(10-17), IFDLGGGTFDVSIL(198-211), IVLVGGSTRIPK(335-346). C-terminal consensus sequence: GPTIEEVD(646-653). There were6N-glycosylation sites in the Hsc70amino acid sequences of Hypena tristalis. The analysis results show that no signal peptide cleavages sites in ORF by SignalP4.0Server. The Hsp70cDNA sequence has been deposited at GenBank with accession number JQ340172.Analysed the consistency of amino acid sequence show that Hsp70of Hypena tristalis and other insects similarity about79%. Analysis of amino acid sequence consistency show that Hsc70of Hypena tristalis and other insects similarity about90%, and the difference was significantly less than the Hsp70. Homology between the two amino acids and other biological Hsc70and Hsp70is very high, and the homology of the Hsc70gene is significantly higher than the Hsp70gene.Application of the E. coli expression system, Hsp70/Hsc70gene of Hypena tristalis was respectively restructuring into the pET21b vector. The anlysis of SDS-PAGE show we have got the fusion protein. Western blot show that the prokaryotic expression of pET/HtriHsp70and pET/HtriHsc70is consistent with the expected results, indeed is expression of Hsp70and Hsc70of Hypena tristalis.