Gene Cloning、Expression And Activity Analysis Of Chitinase HpChi From Holotrichia Parallela Motschulsky(Coleoptera:Scarabaeoidea)

Insect chitinase is within the hydrolytic enzymes, the ability to degrade chitin is a long chain oligomers. Chitinase is present in the insect midgut, molting fluid and venom of certain insects, and its precise regulation of chitin metabolism in the growth process insects. Currently, several studies chitinase focused application in pest control, disease control and biological waste chitin degradation.In this paper, Coleoptera Diablo Melolonthidae (Holotrichia parallela Motschulsky) as the research object, through which the bowel screening cDNA expression library immunization, access to full-length gene encoding chitinase hpChi’s. Gene consists of1591bp, the open reading frame (ORF)1443bp, encoding480amino acids, the predicted molecular weight of51.4kDa, chemical formula C2314H3539N603O714S20, isoelectric point (pI) of5.08. Half-life of30h, unstable factor of27.89, are unstable proteins. Amino acid sequence analysis shows it has a plurality of domains, respectively, the catalytic activity area (catalytic domain) N-terminal, C-terminal cysteine-rich chitin-binding domain (chitin-binding domain, CBD) and Fu connection area with serine and threonine (Linker).HpChi PDB protein using two sites of tertiary structure prediction results show HpChi family18chitinases typical "β/α drum type structure." Phylogenetic tree shows, Dark Melolonthidae chitinase HpChi Diablo Melolonthidae kinship with the same family with the most recent project by homology analysis showed95%of their genetic similarity. Second, with the Oriental migratory locust Orthoptera and Coleoptera red eyes castaneum closer relationship overall with Coleoptera Tenebrio, Mexican pine beetles, Phaedon system is a large class, and Hymenoptera and Lepidoptera insects distantly related. Lepidoptera from clustered together.Prokaryotic expression vector pET30a-hpChi, transformed into E. coli BL21. And optimize the expression condition induced by SDS-PAGE electrophoresis and Western Blot analysis, the molecular weight of51.4kDa successful expression of chitinase protein, consistent with the predicted results. Will be inclusion bodies after exogenous protein HpChi sonication, by Ni+-NTA resin affinity chromatography purified proteins, polyclonal antibodies.Construction of recombinant yeast expression vector pPICK9K-hpChi, transforming defective Pichia GS115. Methanol environmental screening, antibiotic selection, a single colony of yeast phenotypes showed recombinant expression vectors have been successfully transformed. But after induced expression, Western Blot not been able to successfully detect the expression of the target protein.Eukaryotic (baculovirus) expression vector pFast-hpChi, transformed into E. coli DH10Bac, using its own plasmid vectors seat will aim to further build baculovirus gene into Bacmid get Bacmid-hpChi. Transfected insect cells (Spodoptera frugiperda cell line sf9), respectively P1, P2, P3virus supernatant performed Western Blot test, proved to be successful HpChi sf9expressed in insect cells in. Chitin as substrate chitinase enzymatic activity was measured in cells expressing the principle of colorimetric detection of reducing sugars by DNS, results show that it has chitinase activity and more active in the acidic environment.This study successfully expressed in insect cells Diablo Melolonthidae larvae (grubs) chitinase, and its activity was measured, providing a theoretical basis for understanding Coleoptera chitinase biological functions, but also for its biological control provide data for reference.