Molecular characterization of chitinase from polyphagous pest Helicoverpa armigera

https://doi.org/10.1016/j.bbrc.2003.08.136Get rights and content

Abstract

Chitinase from a polyphagous pest, Helicoverpa armigera, has been cloned and expressed. The Helicoverpa chitinase cDNA is 2870 bp in length and contains an open reading frame of 1767 bp. The cDNA encodes a polypeptide of 588 residues with a predicted molecular weight of 66 kDa and a pI of 5.99. The polypeptide has distinct catalytic and substrate binding domains at the N- and the C termini, respectively. The two domains are held together by a proline, threonine rich linker region. The catalytic and the substrate binding domains shared a high level of homology with other lepidopteran chitinases, but the proline and threonine rich region is longer in H. armigera chitinase than in other lepidopteran chitinases. The transcription of chitinase at different developmental stages and in different tissues was analysed by RT-PCR. Chitinase transcript was found in the integument, gut, and fat bodies but was absent in the haemocytes. The levels of chitinase mRNA were abundant at the moulting stages and a basal level of transcript was maintained throughout the development of the insect. Interestingly, Western blot analysis of total proteins from the integument and the gut showed the presence of chitinase in the moulting stages but was absent in the intermoult periods, suggesting post-transcriptional control. The chitinase cDNA was expressed in bacteria and in insect cells. The insect cell expressed chitinase was glycosylated and catalytically active against the simple and complex substrates. The chitinase gene spans about 6.8 kb of genomic DNA and is organized into 10 exons and 9 introns. The 6.8 kb genomic clone of chitinase revealed a high degree of conservation in the position and size of the exons with other lepidopteran insects.

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Materials and methods

Insect rearing and tissue collection. The culture of H. armigera was maintained in the laboratory under controlled conditions of temperature 25 °C, 70% relative humidity, and a photoperiod of 12 h light:12 h dark. The larvae were reared on a semi-synthetic diet. The larvae were dissected under insect physiological saline and integuments, midguts, and fat bodies were collected. Haemocytes were separated from the haemolymph by centrifuging at 7500g. Moulting fluid was extracted from the prepharate

Cloning and sequence analysis of chitinase cDNA

A 1 kb fragment was amplified when degenerate primers, synthesized from the conserved regions ADSKRI and VEIKMNW of lepidopteran chitinases, were used with pupal cDNA as template. The fragment was cloned into the PCR cloning vector and the sequence was subjected to the BLAST application at the NCBI server. It showed extensive homology with S. litura chitinase and other lepidopteran chitinases. To amplify the flanking 5 and 3 regions of the chitinase cDNA, 3 and 5 RACE was conducted and a 1.6

Chitinase cDNA and its genomic organization

The cloned chitinase cDNA showed a high level of homology with other lepidopteran chitinases from M. sexta[8], B. mori, Hy. cunea[9], S. litura[10], and C. fumiferana[11]. As in other species, the H. armigera chitinase cDNA showed a modular structure consisting of a catalytic domain, a chitin-binding region, and a connecting linker. The presence of conserved motif in the catalytic domain that has the consensus, [LIVMFY]-[DN]-G-[LIVMF]-[DN]-[LIVMF]-[DN]-x-E motif, where E is the catalytic

Acknowledgements

This work was supported by a grant from the INDO-SWISS programme.

References (28)

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