The Study Of Chitin Degradation By Insect Group ? Chitinase

Chitin is one of the most abundant biomass resources in nature.It is a major structural component in insect cuticle that protects insects from external threats.However,its rigid structure restricts the growth and development of the insect,so the cuticular chitin is periodically degraded by an efficient and complex chitin degradation system during the development of insect.The insect chitin degradation system includes three chitinases(ChtI,Cht? and Chi-h).Among the three chitinases,Cht? is a very large and complicated chitinase containing several catalytic domains and chitin-binding domains.It has been proved to be an essential chitinase for insect molting.However,Cht?' s enzymatic activity and its role in chitin degradation during insect development remain poorly understood.This thesis mainly focused on OfCht? from Ostrinia furnacalis and carried out the following work:(1)A truncated protein named OfCht?-B4C1 was recombinantly expressed in the yeast strain Pichia pastoris and purified to homogeneity by metal chelate chromatography.The purity of OfChtII-B4C1 was verified by SDS-PAGE and Western Blot analyses.The practical molecular mass of OfChtII-B4C1 was about 120 kDa and the yield was about 5 mg/L.(2)The enzymatic properties of OfCht?-B4C1 were detected using a-chitin.OfCht?-B4C1 could hydrolyze the substrate.The compact chitin particles was dramatically loosened and collapsed after the digestion by OfCht?-B4C1 as evidenced by scanning electron microscopy.When cuticular chitin was used as substrate,big holes and cut chitin fibrils were observed on the cuticle surface after hydrolysis by OfCht?-B4C1.(3)The stage-specific expression patterns of OfCht?,OfCht?,and OfChi-h were investigated at both the gene and protein levels as evidenced by Real-Time PCR and Western Blot analyses respectively.The results demonstrated that OfCht? was expressed earlier than the other two chitinases.OfChtd? also responded to 20E more rapidly than the other two chitinase genes,which was consistent with OfCht? being expressed before the other chitinases.(4)OfCht? and OfChi-h were expressed and purified to homogeneity.When using a-chitin as substrate,electron microscopy imaging demonstrated that OfCht? could break the compact chitin particles after hydrolysis,while OfCht?-treated particles were still compact and the effect of OfChi-h was somewhere in between.Synergistic effect was proved between either of the two chitinases.The chitinase systems from O.furnacalis and Serratia marcescens were compared and the results showed that the chitinase system from O.furnacalis was very effective toward ?-chitin,however,it was slower than that from S.marcescens when using?-chitin as substrate.Our work will contribute to understanding of the chitin degradation mechanism in insects.It will also provide useful information for efficient utilization of chitin biomass and the development of specific biological pesticide targeting chitin degradation of insect.