Molecular Cloning And Prokaryotic Expression And Quantitative Analysis Of Heat Shock Protein70Gene From Aphis Glycine

Heat shock proteins is a group of highly conserved protein, it is also known as stress protein.Hsps usually can be divided according to molecular size: small Hsp, Hsp60, Hsp70, Hsp90andHsp110. Hsp70family is considered the most important and conservative Hsps. Hsp70expressionincreased significantly in response to stress factors, such as low oxygen, high temperature, heavymetal,pathogen invading, hunger. Heat shock protein of high expression can strengthen theenvironmental stresses mediated insect resistance, to the body of factor to maintain normalmetabolism and cellular structure integrity have very important role. Through the molecularbiology technology makes insect pests heat shock protein expression content decrease, to weakenthe harmful insects to outside adverse environmental defense ability, thus plays assist insecticidalrole. On insect Hsp70gene cloning and expression of cDNA sequence, a series of research frommolecular biology for insect resistance level study lays an important theoretic foundation forbiological control of pest, opening up new ways.Aphis glycines of Hemiptera Homoptera Aphididae Aphis, is an important pests of soybeanfields.At present, both at home and abroad has not been specifically for Hsp70protein study onprevention and treatment of soybean aphid. This paper obtained by rt-pcr and RACE cloningtechnology of soybean aphid heat shock protein70(Hsp70and Hsc70) gene, the successfulconstruction of expression vector, prokaryotic expression, for the soybean aphid Hsp70recombinant protein. And single head from the heat shock temperature under the condition ofsoybean aphid RNA, carries on the real-time fluorescence quantitative PCR analysis. For thefuture research of soybean aphid under heat stress after the expression of heat shock protein70and thermal control technology provides the basis for prevention and control of pests.This study of soybean aphid {Aphis glycines Matsumura) as materials, respectively toextract total RNA, using rt-pcr and RACE, amplification, respectively, to get over the span ofsoybean aphid Hsp70gene and Hsc70cDNA sequence. And use the pET21b e. coli expressionsystem for both the prokaryotic expression recombinant fusion protein.Over the span of this study cloned soybean aphid Hsp70cDNA sequence: the sequencecontains2281bases, and its interior contains an open reading frame of1965bases, and canencode a protein containing654amino acid residues, size71.41kDa, molecular weight andisoelectric point of pI:5.22. Soybean aphid Hsp70gene signature sequences containing twotypical Hsp70family: IDLGTTYS (11~18residues) residues, IFDLGGGTFDVSIL (199~212residues). Existing in the deduced amino acid sequence of6N glycosylation sites. Using SignalP4.0Server software analysis, the results show that the ORF18~19the nucleotide sequence of signal peptide cutting site. Login on GenBank soybean aphid Hsc70gene sequence, obtain loginid: JQ340173.Cloned soybean aphid Hsc70full length cDNA sequence: the sequence contains2268bases,including a1962bases of the open reading frame, can encode a protein containing653aminoacid residues, size71.22kDa, molecular weight and isoelectric point of pI:5.43. Soybean aphidHsc70gene signature sequences containing three Hsp70family: IDLGTTYS (10~17residues)IFDLGGGTFDVSIL (198~211residue), IVLVGGSTRIPK (335~349residues). Existing inthe deduced amino acid sequence of6N glycosylation sites. Using SignalP4.0Server softwareanalysis, the results show that the ORF of17~18the nucleotide sequence of signal peptidecutting site. Login on GenBank soybean aphid Hsc70gene sequence, obtain login id: KF052087.The research results show that the soybean aphid Hsp70and Hsc70gene contains20kindsof the same type of amino acids, and all kinds of amino acid content is basically the same, is richin glycine Gly, alanine (Ala, lysine Lys. Heat shock proteins (Hsp70as a kind of glycoprotein,glycosylation is one of the important factors enzyme activation. Using NetOGlyc2.0softwarefound in soybean aphid Hsp70in amino acid sequence and Hsc70amino acid sequence6glycosylation sites, in addition to the N-a glycosylation site, insect heat shock protein aminoacid sequence contains a lot of sulfating of Hsp70and phosphorylation sites, using molecularbiology Expasy Prosite software on the server to be able to find these sulphation andphosphorylation sites, the protein function by the locus of control.Consistency through the amino acid sequence comparison analysis, we can obviously seethat the soybean aphid Hsc70/Hsp70and other insects of amino acid sequence similarity ofHsp70gene are above90%.Reorganization of the soybean aphid respectively to pET21b Hsc70/Hsp70gene carrier,after polyacrylamide gel electrophoresis detection, fusion protein can detect the purpose. ByWestern blot analysis, the results showed that the recombinant plasmid pET/prokaryoticexpression of Hsp70results consistent with expected results, for sure the soybean aphid Hsp70gene expression.Using Real time PGR for Aphis glycines under different temperature stress Hsp70with Hsc7relative expression quantity analysis showed，in25~37℃range, expression quantity of Hsp7expression vary with temperature rising, peaked at37℃and was25℃when the expressioquantity of6.72times,35~39℃, Hsp70expression decreased; in25~34℃range, its Hsc7expression quantity varies with temperature rise, peaked when34℃, was25℃when thexpression quantity of1.79times,34~40℃and Hsp70decreased expression. So higtemperature stress can induce Hsc70and Hsp70of Aphis glycines expression.