Molecular and immunohistochemical characterization of the chitinase gene from Pieris rapae granulovirus
The chitinase gene of baculoviruses is expressed in the late phase of virus replication in insects and possesses high exo- and endochitinase activity, which can hydrolyze chitin in the body of the insect, thus promoting terminal host liquefaction. Alphabaculovirus viral chitinases (vChitA) have been well analyzed, but information regarding viral chitinases from betabaculoviruses is limited. Whole-genome sequencing of a Korean isolate of Pieris rapae GV (PiraGV-K) predicted a putative chitinase gene corresponding to ORF10. The PiraGV-K chitinase gene had a coding sequence of 1,761 bp, encoding a protein of 586 amino acid (aa) residues, including an 18-aa putative signal peptide. Time course induction pattern observed by SDS-PAGE and subsequent Western blot with anti-PiraGV-K chitinase antibody revealed the cleavage of the signal peptide from the intact chitinase. Edman sequencing analysis was further conducted to confirm the exact nature of the mature chitinase, and the N-terminal amino acid sequence (KPGAP) exactly matched the sequence following the signal peptide sequence. The transcriptomics of PiraGV-K chitinase in infected P. rapae larvae, examined by real-time PCR, revealed a significant 75-fold increase after four days of feeding with PiraGV-K-treated leaves, with a subsequent decline at the later stages of infection. Confocal microscopic analysis showed that PiraGV-K chitinase possibly exists as a secreted protein, with strong chitinase-specific signals in fat body cells and integument at four days postinfection. Furthermore, immunogold labeling and electron microscopy studies localized the PiraGV-K chitinase in the cytoplasm and sparsely within vacuolar structures in the fat body apart from the extensive aggregation in the cuticular lining of the integument.
KeywordsChitinase Chitinase Gene Immunogold Label Infected Larva Signal Peptide Sequence
This work was supported by the Mid-Career Researcher Program through NRF grant funded by the MEST (No. 2010-0027694).
- 4.Brurberg MB, Synstad B, Klemsdal SS, van Aalten DMF, Sundheim L, Eijsink VGH (2001) Chitinases from Serratia marcescens. Recent Res Dev Microbiol 5:187–204Google Scholar
- 18.Henrissat B (1999) Classification of chitinase modules. In. Chitin and Chitinases. Muzzarelli RAA (ed) Birkhauser-Verlag, Basel, Switzerland, pp137-159Google Scholar
- 32.Maeda S (1989) Gene transfer vectors of a baculovirus, Bombyx mori nucleopolyhedrovirus, and their use for expression of foreign genes in insect cells. In: Mitsuhasi J (ed) Invertebrate cell system application, vol 1. CRC Press, Boca Raton, pp 167–181Google Scholar
- 43.Su J, Fu CS, You CB (1990) Characterization of inclusion body protein of granulosis virus from Pieris rapae. Acta Agriculturae Nucleatae Sinica 4:151–156Google Scholar
- 48.Watanabe T, Kono M, Aida K, Nagasawa H (1996) Isolation of cDNA encoding a putative chitinase precursor in the Kuruma prawn Penaeus japonicas. MolMar Biol Biotechnol 5:299–303Google Scholar