Identification of a New Chitinase Protein from Larval Holotrichia oblita (Coleoptera: Melolonthidae)

Chitinases are vital to moulting in insects, and may also affect gut physiology through their involvement in peritrophic membrane turnover. By screening Holotrichia oblita larva midgut cDNA expression library, using the polyclonal antiserum against Helicoverpa armigera, a full-length cDNA HoChi clone encoding chitinase was obtained. The HoChi cDNA was 1636bp in length (GenBank Accession No. HM596340). The open reading frame (ORF) encoded 486 amino acids, with the predicted MW of 51.8kDa and pi of 5.58, respectively, and flanked by a 5´ untranslated region(UTR) of 27bp and a 3´ untranslated region of 145bp. A potential polyadenylation signal sequence AAGAAA was located at 39bp upstream of the polyA tail. It shared the typical character of chitinase protein, including a signal peptide, a chitinase active site, a C-terminal threonine-rich region and a chitin-binding domain, belonging to the chitinases family 18. The deduced amino acid sequence showed a high identity to reported chitinases from other insect, especially to the Coleopteran insect Tribolium castaneum. The HoChi was recombined into pET28b vector, and transformed into E.coli. Western blot analysis demonstrated that HoChi protein was successfully expressed after IPTG induction.