title: Molecular and immunohistochemical characterization of the chitinase gene from Pieris rapae granulovirus.
abstract: 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.
authors.affiliation: Division of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National University, Gwangju, 500-757, Korea.
authors.forename: Seunghan
authors.initials: S
authors.lastname: Oh
authors.forename: Dong Hyun
authors.initials: DH
authors.lastname: Kim
authors.forename: Bharat Bhusan
authors.initials: BB
authors.lastname: Patnaik
authors.forename: Yong Hun
authors.initials: YH
authors.lastname: Jo
authors.forename: Mi Young
authors.initials: MY
authors.lastname: Noh
authors.forename: Hyo Jeong
authors.initials: HJ
authors.lastname: Lee
authors.forename: Kwang Ho
authors.initials: KH
authors.lastname: Lee
authors.forename: Kwang Ho
authors.initials: KH
authors.lastname: Yoon
authors.forename: Wan-Jong
authors.initials: WJ
authors.lastname: Kim
authors.forename: Ju Young
authors.initials: JY
authors.lastname: Noh
authors.forename: Heon Cheon
authors.initials: HC
authors.lastname: Jeong
authors.forename: Yong Seok
authors.initials: YS
authors.lastname: Lee
authors.forename: Chuan-Xi
authors.initials: CX
authors.lastname: Zhang
authors.forename: Yong-Su
authors.initials: YS
authors.lastname: Song
authors.forename: Woo-Jin
authors.initials: WJ
authors.lastname: Jung
authors.forename: Kisung
authors.initials: K
authors.lastname: Ko
authors.forename: Yeon Soo
authors.initials: YS
authors.lastname: Han
mesh_terms: D000825:Animal Structures / Q000201:enzymology / Q000821:virology; D000818:Animals; D015153:Blotting, Western; D002688:Chitinases / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D016679:Genome, Viral; D025722:Granulovirus / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D007915:Lepidoptera / Q000821:virology*; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
chemical_list: D004279:DNA, Viral; D021382:Protein Sorting Signals; D002688:Chitinases