Abstract: 【Aim】 Insect chitinases are mainly involved in such important physiological processes as molting, degradation of peritrophic membranes and immune defense. This study aims to clone a group IV chitinase gene from Helicoverpa armigera and to analyze its expression profiles so as to provide the theoretical basis for the control of this insect using the gene as a molecular target. 【Methods】 A group IV chitinase gene was cloned from the midgut of H. armigera using RT-PCR and RACE techniques and subjected to multiple sequence alignment by DNAMAN, and the phylogenetic tree was constructed by MEGA. Its recombinant protein was expressed in Escherichia coli (DE3) and verified by Western-blot. The enzymatic properties of the recombinant protein were characterized after purification with Ni-NTA. The expression levels of this gene in different developmental stages and tissues of the 6th instar larvae of H. armigera were analyzed by qPCR. 【Results】 A chitinase gene was cloned from H. armigera and designated as HaCHT4 (GenBank accession no.: MH500771). Its full-length cDNA is 1 624 bp in length and contains an ORF of 1 527 bp that encodes 509 amino acids with a predicted molecular weight of 55.2 kD. Its encoded protein contains a signal peptide, one catalytic domain (CAD) and one chitin binding domain (CBD). Both multiple sequence alignment and phylogenetic analysis showed that HaCHT4 has the conserved domain of chitinase and belongs to group IV chitinases. The recombinant His-HaCHT4 was successfully expressed in E. coli. The purified recombinant protein exhibited the activity of digesting chitin, with the optimum reaction temperature of50℃and pH value of 7. The values of kinetic parameters K_m and V_max of the recombinant protein were 1.76±0.35 mg/mL and 0.0220±0.0012 μg/mL·s, respectively. qPCR results revealed that the relative expression levels of HaCHT4 inthe 1st and 2nd larval instars of H.armigera were significantly higher than those in other larval instars and the pre-pupal stage, and HaCHT4 was predominantly expressed in the midgut and fat body of the 6th instar larva, but lowly expressed in the integument and head. 【Conclusion】 The results suggest that HaCHT4 may participate in chitin degradation in the peritrophic matrix in H. arrmigera. These results lay a foundation for the further function study of HaCHT4 and provide useful information for pest control.

Key words: Helicoverpa armigera, chitin, group IV chitinase, enzyme characterization, gene expression analysis