Molecular cloning, nucleotide sequencing and detection of coat protein gene of cymbidium mosaic and odontoglossum ringspot virus
1995
Nanyawan Rungroj
Genomic RNAs of cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV), isolated from three different species of orchids: Cattleya, Mokara and Oncidium, were used as a template for reverse transcription to complementary DNAs (cDNAs) synthesis in polymerase chain reaction (PCR) to amplify coat protein (CP) genes region. The complete nucleotide sequences of CyMV-CP gene were obtained from a number of potential clones. CyMV-CP gene isolated from Mokara is 669 base pair and deduced protein consists of 223 amino acids with Mr 23,761.43. The amino acid sequence of CyMV-CP isolated from Mokara has 73.18 and 96.86 percent homology to one isolated in Singapore and Oncidium isolated in Thailand, respectively. In addition, sequence comparison of CyMV-CP to the CP sequences of other potexvirus such as papaya mosaic virus (PMV), potato virus X (PVX) and white clover mosaic virus (WClMV) revealed 24.65, 31.39 and 39.47 percent homology, respectively. The nucleotide sequences of ORSV-CP gene isolated from Oncidium is 474 base pair. The ORF predicts a polypeptide chain of Mr 17,725.89. Amino acid sequence comparison this ORF showed 96.83 and 99.37 percent homology to the CP sequences reported in Korea and USA, respectively. More, the CP sequences of ORSV shared 32.28 to 72.15 percent to those of eight other tobamovirus: cucumber green mottle mosaic virus (CGMMV), sunn-hemp mosaic virus (SHMV), pepper mild mottle virus (PMMV) and four strains of tobacco mosaic virus (TMV). A reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization technique has been developed for CyMV and ORSV detection. The results clearly demonstrate the effectiveness of RT-PCR as a sensitive method for detecting virus in infected orchids.
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