| The beet armyworm, Spodoptera exigua(Lepidoptera: Noctuidae), is a serious pest of numerous agricultural crops worldwide, and causes severe losses in agricultural production including vegetables, ornamentals, industrial crop, oil crops, peanut and cotton. Chitin deacetylases(CDAs)are members of an extracellular chitin-modifying enzyme family, carbohydrate esterase family 4(CE-4)of the carbohydrate active enzymes(CAZY)database, which deacetylate chitin to chitosan, a polymer of β-1,4-linked D-glucosamine residues. CDA is a key enzyme in insect metabolism and plays an important role in a variety of physiological activities of insects.In this study, midgut from fifth instar larvae of S. exigua was extracted for isolating total RNA. c DNA sequence encoding chitin deacetylase 1 of S. exigua(secda1)was cloned by RT-PCR and rapid amplification of c DNA ends(RACE)methods. The full length of secda1 c DNA was 1659bp(Gen Bank Accession No. KJ621414)and contained an ORF of 1554 bp. The deduced protein sequence showed that Se CDA1 was synthesized as a preprotein of 518 amino acid residues with the predicted molecular weight of 61.5 k Da, p I of 4.83 and a 23-amino acid signal peptide predicted by the software Signa IP. BLAST analysis shows that the protein has a low-density lipoprotein receptor class A domain( LDLa), a chitin-binding peritrophin-A domain(Ch BD) and a polysaccharide deacetylase-like domain. Three predicted N–glycosylation sites and five O-glycosylation sites were found based on Ex PASy Proteomics Server.The secda1 was successfully expressed in E.coli and was purified for preparation of a specific antibodies against Se CDA1. Western blot analysis showed that the secda1 was successfully secreted 80 k Da protein in yeast Pichia pastoris. As well as, the secda1 was successfully expressed in insect cells BTI-Tn-5B1-4(High Five)as a secreted protein using recombinant baculoviruses(Bac-to-Bac system).Enzyme activity assays demonstrated that Se CDA1 expressed by both yeast and insect cells exhibited chitin deacetylase activity. Enzyme activity of Se CDA1 secreted by insect cells BTI-Tn-5B1-4(High Five)and pichia yeast is 1.88U/m L and 1.35U/m L.Tissue immunolocalization showed that Se CDA1 was commonly present in egg, larvae and pupae, especially high in egg. Se CDA1 was also detectable in head, midgut, malphabian tube, fatbody and epidermic of larvae. secda1 exhibited high expression in egg and epidermic by q RT–PCR approach, which was hypothesized that Se CDA1 protein might be responsible for incubation and metamorphosis process.At the aim of making a further study of the function of Se CDA1, a sequence specific to secda1 at the length of 536 bp was cloned into vector p GEM-T, and ds RNA-secda1 was synthesized successfully using linearized p GEM-T-secda1 as template. |
