Biochemical and Biophysical Research Communications
Molecular characterization of chitinase from polyphagous pest Helicoverpa armigera
Section snippets
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.
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