Nucleotide sequence and in vitro translation of the coat protein gene of cymbidium mosaic virus

Abstract

The nucleotide sequence of the coat protein gene of cymbidium mosaic virus (CyMV) has been determined from a cDNA clone that encompassed the 3'-region of the virus genome. The 0.75 kb coat protein open reading frame (ORF) codes for a 24kD polypeptide. The coat protein coding region was separated from the 3'-poly(A) terminal of the viral genome by an 80 base noncoding region. The derived amino acid sequence of this ORF showed considerable homology with other potexvirus coat protein sequences, notably with narcissus mosaic virus (NMV), potato virus X (PVX), and white clover mosaic virus (WClMV). In vitro expression of the CyMV coat protein gene was carried out by T7 RNA polymerase on the linearized cDNA clone template, followed by in vitro translation of the purified transcript in wheat germ extract. One of the translated products was found to comigrate with the coat protein of the virus at about 24 kD, as determined by NaDodSO4-PAGE. This translated product was also found to crossreact with antibodies raised against CyMV.

Transgenic resistance to Cymbidium mosaic virus in Dendrobium expressing the viral capsid protein gene

“…This CP sequence and other reported CP isolates were aligned using the Genetics Computer Group (GCG) BestFit Program (Wisconsin Package Version 10.3, USA). The CymMV-CS shared 96.5-98.7% nucleotide homology with Singapore isolates (Chia et al, 1992;Neo et al, 1993;Wong et al, 1997) and 92.4-97.6% nucleotide homology with Korean isolates (Ryu et al, 1995;Youn et al, 1997;Kim et al, 1998). At the amino acid level, the Cym-MV-CS CP shared a 92.7-99.1% identity with the other isolates.…”

“…The primer pairs included the forward primer (5¢-CGCGGATCCACCACAATAA-TTTGAAAT-3¢, based on reported CymMV CP gene with a BamHI site (in bold) at the 5¢ end) and a reversed primer (5¢-GTCGACTCTAGA-T (15) with an XbaI (in bold) at the 5¢ end) (Chia et al, 1992;Neo et al, 1993;Ryu et al, 1995). The RT-PCR products were subsequently ligated into the pGEM-T easy vector.…”

Coat Protein Gene ofCymbidiummosaic virus fromVanilla fragransin Reunion Island

“…The RNA of 12 Reunion isolates of CyMV (CyMV‐R) from V. fragrans was sequenced. Sequences were compared with each other and with that of two Korean strains, CyMV‐K1 (Ryu et al., 1995) and CyMV‐K2 (Youn et al., 1997) and two Singapore strains, CyMV‐S1 (Chia et al., 1992) and CyMV‐S2 (Neo et al., 1993).…”

Elimination of cymbidium mosaic virus and odontoglossum ringspot virus from orchids by meristem culture and thin section culture with chemotherapy

“…Primer 1 was a 20-mer oligonucleotide, 5'-TTTGCCGCCTTTGACTTCTT-3', corresponding to the Cterminal of the coat protein region of CyMV, which possesses conserved amino acid sequence in several potexviruses (Harbison, Forster, Guildford & Gardner, 1988;Jelkmann et af., 1990). Primer 2 was also a 20-mer oligonucleotide, 5'-ATTTAAGCTGGCTAAGT-ATA-3', corresponding to the conserved CyMV 3' non-coding region (Neo, Wong & Wu, 1993). Prior to the PCR assay, first strand cDNA synthesis was carried out using 1 pl of CyMV-infected orchid leaf or PLB homogenate in ELISA coating buffer, 16 pmoles primer 2, 50 mM Tris-HC1, pH 8.3, 75 mM KCI, 3 mM MgC1,lO mM DTT, 0.5 mM dNTPs (Perkin Elmer Cetus), one unit rRNasin@ ribonuclease inhibitor (Promega) and three units Moloney Murine Leukaemia Virus RNase Hreverse transcriptase (M-MLV H-RT; Gibco-BRL), added to a final volume of 20 pl and incubated at 37°C for 1 h. Subsequently, 80 p1 of PCR buffer (50 mM KCI, 10 mM Tris-HC1, pH 8.3, 2.5 mM MgCl,), 50 pmoles primer 1, 25 pmoles primer 2 and 2.5 units Taq DNA Polymerase (Perkin Elmer Cetus) were added to each reaction tube, and covered with one drop of mineral oil.…”