Molecular Characterization of the Radish mosaic virus Taiwan isolate (RaMV-TW)

• Main Authors: Ching-Chu Wang, 王淨竹
• Other Authors: 陳煜焜
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/91413894661651903569

碩士 === 國立中興大學 === 植物病理學系所 === 98 === Cruciferous vegetables are one of the important crops in Taiwan and generally suffer from virus infections in the field. Radish plants (Raphanus sativus) with severe mosaic symptoms on leaves were observed in the fields of Dar-Hu, Miaoli in 2007. Spherical virus-like particles (ca. 30 nm in diameter) were observed in the preparations of infected crude saps by electron microscopic examination . Pure virus isolate obtained from the single lesion induced by mechanical inoculation on Chenopodium quinoa successively and was back-inoculated to original host to confirm its pathogenicity. The isolated virus induced systemic mosaic and enation symptoms on the leaves of back-inoculated radish plants. Semi-nested RT-PCR tests using Comoviridae degenerate primers ( Maliogka et al., 2004) amplified a cDNA fragment of expected size which highly homologous to the nucleotide sequence of Radish mosaic virus (RaMV). The isolated virus thus designated as Radish mosaic virus Taiwan isolate (RaMV-TW). Host range tests revealed that RaMV-TW caused symptoms of mosaic, rugose, enation and ringspot on various cruciferous crops. Virus particles were partially purified through clarification, PEG concentration, and sedimented by ultracentrifugation in a 40% sucrose cushion. The partially purified virus preparations contained numerous spherical virus particles with a diameter of about 30 nm. SDS-PAGE analysis on the partially purified sample indicating that the virus contains two coat protein subunits, i.e. large coat protein (LCP) and small coat protein (SCP), with relative molecular mass of 41 kDa and 25 kDa, respectively. Full length sequences of RNA1 and RNA 2 of RaMV-TW have been determined and deposited in GenBank with accession numbers of HM032712 and HM032711, respectively. The RNA 1 contains 6041 nt and encodes a polyprotein which is further cleaved into 5 funtional proteins including protease cofactor (Co-factor), helicase (Hel) , viral genome-linked protein (VPg), Protease (Pro), and RNA-dependent RNA polymerase (RdRp) at the order from N-terminus. The RNA2 consists of 4012 nt and contains an open reading frame (ORF) with two ATG start codons. The polyprotein derived from RNA 2 can also be further cleaved into 4 functional proteins including cofactor required for the replication of RNA 2 (CR) , movement protein (MP), large coat protein (LCP), and small coat protein (SCP) at the order from N-terminus. Based on the comparison of nucleotide sequences, RaMV-TW is identified as a new isolate of RaMV and highly homologous to other strains of RaMV. Among these, RaMV-TW is more closely related to strains RaMV-CA and RaMV-J than to RaMV-1.