Thesis (M.Sc.) - Cairo University - Faculty of Science - Department of Biotechnology

Chitinase plays a highly important function in the growth of insects and its development by hydrolyzing chitin in the body of the insect. In this study we have cloned, expressed and characterized chitinase gene A (ChiA) from the cotton leafworm, Spodoptera littoralis nucleopolyhedrovirus, an Egyptian isolate. The Spli-ChiA gene shown to have an open reading frame of 1809 nucleotides, encoding a protein of 599 amino acid residues with a predicted weight of 66.8 kDa. The phylogenetic analysis revealed that the Spli-ChiA deduced protein sequence shared extensive similarities with other baculovirus chitinases to be found in the GenBank. The SDS-PAGE analysis of SpliNPV-infected larvae total protein followed by western blotting with anti-SpliNPV chitinase antibody identified a protein of about ~50 kDa corresponding to the mature chitinase of SpliNPV. Temporal transcriptional analyses using RT-PCR and qRT-PCR revealed that Spli-ChiA transcription might be detected in SpliNPV infect larvae at 12 hpi with a steady increase in the transcription levels up to 71.4-fold at 120 hpi, suggesting the late transcription of chitinase gene. In addition, the purified Spli-ChiA protein showed inhibition in vitro of the growth of two fungi species, Fusarium solani and Fusarium oxysporum, confirming the expressed protein activity. The results concluded that SpliNPV chitinase (Spli-ChiA) is a good applicant protein for significant contribution in pest control

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