การศึกษาอณูพันธุศาสตร์ของยีนไคติเนสของแบคคิวโลไวรัส Helicoverpa armigera nucleopolyhedrovirus สายพันธุ์ไทย

A molecular study of chitinase gene of Thai isolated baculovirus Helicoverpa armigera nucleopolyhedrovirus

Name: นวพร โปษยะพิสิษฐ์

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Name: Navaporn Posayapisit

keyword: Chitinase

; HaNPV

; gfp

; Baculovirus transfer vector

Abstract: A chitinase gene of a local strain Helicoverpa armigera nucleopolyhedrovirus (HaNPV-TH), isolated from cotton bollworm, was cloned, sequenced and analyzed. PCR primers for amplification of chitinase gene were designed from conserved regions of chitinase amino acid sequences of several baculoviruses. An open reading frame (ORF) of 1,725 bp was obtained and designated chiA. The chiA ORF encoded a polypeptide of 575 amino acids with a molecular mass of 64 kDa. A consensus late transcription motif, TAAG, was found at 24 bases upstream of the translational start codon. Comparison of the deduced amino acid sequences of HaNPV-TH chitinase with other chitinase sequences from various organisms showed a significant similarity to a number of chitinases. The most similarity sequence was the chitinase gene of HaSNPV-G4 strain giving 98% identity. A phylogenetic tree was constructed for chitinases from baculoviruses and other species. The baculoviruschitinases were monophyletic with HaNPV-TH chitinase clustered within a group ofHaSNPV-G4 strain and HzNPV-Elcar strain. To demonstrate and confirm the ORF1,725 bp of HaNPV-TH was the chitinase gene, the chitinase activity wasinvestigated by cloning into an E. coli expression vector, pTrcHis. Immunoblotanalysis showed that the recombinant chitinase was approximately 64 kDa, whichwas corresponding to the protein, deduced from the nucleotide sequences. However,the recombinant protein had no chitinase activity against fluorescent chitin oligomeranalogues. A transfer vector pNVBGFP was constructed and use to insert gfp intothe chitinase gene of HaNPV-TH. A recombinant HaNPV-TH showing bright greenanalogues. A transfer vector pNVBGFP was constructed and use to insert gfp intothe chitinase gene of HaNPV-TH. A recombinant HaNPV-TH showing bright greenfluorescent was obtained. No chitinase activity of the recombinant was detectedusing SDS-PAGE and fluorescent chitin oligomer analogue, confirming that ORF1,725 bp encoded a functional chitinase.

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร (Bangkok)

Email: info.lib@mail.kmutt.ac.th

Name: Dr. Somkiet Techkarnjanaruk

Role: Supervisor

Created: 2545

Issued: 2005-10-03

Modified: 2006-05-10

วิทยานิพนธ์/Thesis

ISBN: 9749550978

CallNumber: BIT374

tha

DegreeName: Master of Science

Level: Master's Degree

Descipline: Biotechnology

Grantor: King Mongkut's University of Technology Thonburi

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