Cloning And Prokaryotic Expression Of Trypsin From Mythimna Separata Midgut And Effects Of Perilocosides

Trypsin (EC3.4.21.4) is a kind of alkaline proteolytic enzyme exists in the midgut ofinsects, belong to the family of serine. Its precursor form is trypsin precursor. Previousresearch has shown that periplocoside T and P can activate the armyworm intestinal trypsin invivo, the activation rate is3.78times. But activate the effect is not obvious in vitro, and noconcentration-dependence. In order to confirm that mechanism of periplocoside T and P effecton intestinal trypsin of the armyworm in vitro, so carried out this study. In this thesis, wecloned the gene of trypsin exists in the midgut of M. separata and analyzed its nucleotidesequences, derived amino acids sequences and predicted senior protein structure. Meanwhile,the prokaryotic expression vector of trypsin was constructed and the activity of trypsinexpressed and purified was measured. At the last, the activity effect of periplocosidecompounds on trypsin expressed and purified from the midgut of M. separata were studied.The main result are as follows:1. cDNA sequences of the full length of trypsin gene from the midgut of M. separataBased on multiple alignment to trypsin genes of a variety of cutworm insects, threespecial middle segment primers were designed. Then middle segment sequences of trypsinfrom the midgut of M. separate were obtained with cDNA from the midgut of M.separata asamplification template. According to the middle segment sequences, four special3’-RACEprimers and5’-RACE primers were designed and3’-sequence and5’-sequence of trypsinfrom the midgut of M. separate were obtained. Finally the cDNA sequences of the full lengthof trypsin gene from the midgut of M. Separata was obtained. The full length of trypsin genesequence amplified according to the total RNA from the midgut of cDNA sequences of thefull length of trypsin gene from the midgut of M. separate is933bp, include the5’-untranslation region is81bp, protein translation region is765bp and3’-untranslationregion is88bp.2. The analysis of amino acid sequence deduced from the trypsinogen gene of the midgutof M. separata Open reading frame (ORF) of trypsin gene from the midgut of M. separate coding255amino acid, including16of signal peptide of amino acids. The obtained sequence possessesthe conservative sequence of serine protease:(1) it contains conservative active triplets of His(H96), Asp (D141), and Set (G265). Besides, Asp (D237), Gly (G255) and Gly (G265)located in the catalytic sites nearby are very conservative.(2) It contains the typical andconservative substrate binding sites-Asp (D232). The residue locates in the bottom of thesubstrate combination ditch and stabilizes the Lys or Arg residue of substrate cracking sitethrough charge effect. So it also determines the specificity of trypsin.(3) It contains the aminoacid sequence Lie-Val-Gly-Gly of N-terminal of trypsin.(4) it contains four disulfide bond. Itsgenetic structure is consistent with genetic structure of trypsin cloned from the other insects.3. Homology analysis of trypsin gene from the midgut of M. separataThe amino acid sequence have strong similarity between trypsin from the midgut of M.separata and the other insects, especially the substrate active site and active triplet iscompletely conservative. Besides, some of the amino acid residue locates in substrate activesites nearby have high conservative. Three disulfide bond six cysteine residues in the familyof serine are conservative in all eukaryotic animals. Vertebrates usually have four disulfidebond and the amino acid residues of these site are very conservative.4. Prokaryotic expression of trypsin from the midgut of M. separataConstruction of prokaryotic expression vector by placing the open reading code frame ofthe trypsin gene of armyworm cloned to the carrier of pET-28a. A about27kD molecularweight code protein of trypsin gene were expressed successful. More pure enzyme got afterpurification.5. The determination of activity of trypsin from prokaryotic expression and the activityeffect of periplocoside compounds on itTake BNpNA and TAME as the specificity substrate, the activity of trypsin ofprokaryotic expression after purification is measured and its activity is1.8375μmol/min·mgpro. There is no any effect of periplocoside E which is no insecticidal activity on purifiedtryptase of M. separata and there is no concentration dependent. Meanwhile, thePeriplocoside P and T with insecticidal activity is also no obvious activation to it and noconcentration dependent.These above results suggest that Periplocoside P and T is no obvious activation to trypsinof M separata in vitro. This result is same to the previous research results of our laboratory.So the mechanism of action periplocoside compounds to trypsin may be applied to thetranscription and translation of trypsin after armyworm fed and body metabolism. Futher thetrypsin showed high activity through influence trypsin expression and secretion. At the last the armyworm died by influence its normal metabolic function.