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Gene cloning and expression of the NS3 gene of Rice grassy stunt virus and its antiserum preparation

Published online by Cambridge University Press:  12 February 2007

Lin Li-Ming ,
Wu Zu-Jian ,
Xie Lian-Hui  and
Lin Qi-Ying
Lin Li-Ming
Affiliation:
Institute of Plant Virology and Key Laboratory for Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Wu Zu-Jian
Affiliation:
Institute of Plant Virology and Key Laboratory for Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Xie Lian-Hui*
Affiliation:
Institute of Plant Virology and Key Laboratory for Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Lin Qi-Ying
Affiliation:
Institute of Plant Virology and Key Laboratory for Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
*Corresponding author. E-mal: xielh@fjau.edu.cn
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Abstract

Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, is composed of a single nucleocapsid protein and six genomic ssRNA segments. Based on the known RNA sequence of the RGSV-IR isolate, primers were designed and cDNA of the NS3 gene was obtained by reverse transcription of the virus sense (v) RNA3 and PCR amplification, with genomic RNAs of RGSV-SX isolate as template. The cDNA was then cloned and sequenced. The NS3 gene comprised 588 nt and the sequence identities were 99.1%, 96.2% at the nucleotide level and 98.4%, 96.4% at the amino acid level, compared with those of RGSV-IR and -SC isolates. The 22.9 kDa NS3 protein encoded by RGSV-SX vRNA3 showed 33.0% identity to the 21.6 kDa protein encoded by vRNA5 of RGSV over a span of 80 amino acids, and no other significant matches were found in the GenBank database. The NS3 gene was cloned in pGEX-2T to generate a prokaryotic expression plasmid pGTNS3, and a fusion protein of GST-NS3 at 49.0 kDa was induced in Escherichia coli. Antiserum prepared to this protein reacted with NS3 encoded by RGSV in Western blots. The protein was detected only in infected rice (Oryza sativa), not in purified virus or viruliferous planthopper vectors. Thus NS3 appears to be a protein encoded abundantly only while the virus infects the rice host plant and may be related to the virus pathogenesis.

Keywords

Rice grassy stunt virusNS3 geneantiserum
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 1 , Issue 1 , April 2004 , pp. 49 - 54
Copyright
Copyright © China Agricultural University and Cambridge University Press 2004

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