Expression of a Variant Chitinase Transcript of Helicoverpa armigera in E. coli

Abstract

Gene encoding for chitinase was isolated and sequenced from the cotton bollworm Helicoverpa armigera, using the polymerase chain reaction technique directed by primers designed from the chitinase cDNA of the same insect reported by Ahmad et al. (2003). The nucleotide sequence analysis suggested that the gene is of 1737 bp showing homolgy of 96% with the reference chitinase sequence (AY325496) and 80% homology with that of Manduca sexta, the first cloned insect chitinase gene (U02270). The cDNA encoded a polypeptide of 578 amino acids, short of 10 amino acid residues compared to reference sequence. The major differences in the protein sequence to the one reported earlier was in proline threonine linker region where four threonine residues were replaced by alanine and one threonine by serine and further, condensation of protein towards the C-terminal region. Surprisingly the protein sequence of chitin binding domain shows 98% homolgy to Lymantria dispar chitinase (KP337328.1) and 96% homology to Phyllonorycter ringoniella chitinase (JN607321.1) and 100% homology to the reference protein(AAQ91786.1). Expression of the gene in bacterial system resulted in the formation of inclusion bodies retaining enzymatic activity. Bioassay for insecticidal activity against Helicoverpa armigera showed 60% mortality for injection assay, reduction in larval weight gain in case of oral application and 40% mortality for topical application.