Molecular cloning, characterization and detection of dasheen mosaic virus and a new potyvirus infecting aroids

• Main Authors: Chen, Ying-Lien, 陳穎練
• Other Authors: Ya-Chun Chang
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/89695498354902875693

碩士 === 國立臺灣大學 === 植物病蟲害學系 === 86 === The genome of dasheen mosaic virus (DsMV), belonging to the genus Potyvirus , is a single-stranded, positive-sense RNA molecule of ca. 10 kb. DsMV is the most important virus in aroid plants. Inoculation results indicated that Philodendron selloum and Chenopodium amaranticolor can be used as a propagation host and a local lesion host of DsMV, respectively. DsMV virions from a taro isolate (DsMV-TW) and a calla lily isolate (DsMV-ZAN) were separately purified. The average length of DsMV-TW virions is 813 nm. The estimated molecular weights of the coat proteins of DsMV-TW and DsMV-ZAN, determined by Western blot analysis were 44 and 43 kDa, respectively. Partial genome fragments of DsMV-TW amplified by RT-PCR were cloned and sequenced. The region of 1,893 nucleotides contains a part of the NIb gene (630 nt), the complete coat protein (CP) gene (990 nt), the 3'' non-coding region (248 nt) and a poly (A) tail (25 nt). Comparison of amino acid sequence of the CP gene of DsMV-TW with that of published DsMV isolates revealed that they all contain deletion/insertion, tandem repeats and a proline-rich region at the N terminal region of their CP genes, an unusual feature of DsMV compared to other potyviruses. A new potyvirus, distinct from DsMV in sequence, was cloned from a virus infecting Dieffenbachia picta cv. Rudolph Roehrs and Zantedeschia sp. cv. Black Magic, and tentatively named Zantedeschia mosaic virus (ZaMV). This is the first sequence report for a potyvirus infecting aroid plants distinct from DsMV. The sequence of the 3''-terminal 1,619 nucleotides of ZaMV-ZAN was determined. This region contains a part of NIb gene (627 nt), the complete CP gene (849 nt), the 3'' non-coding region (124 nt) and a poly (A) tail (19 nt). Percent similarities of amino acid sequence between the CP gene of ZaMV-ZAN and that of other viruses in Potyvirus is 59-73%. From the sequence of ZaMV-ZAN, a putative cleavage site of the NIb and the CP (Q/S), the DAG triplet and conserved amino acid motifs of the CP gene were found. In addition, there were potyvirus-specific cytoplasmic inclusions found in epidermal cells of ZaMV-infected calla lily. According to the above results, ZaMV is a new virus species belonging to the genus Potyvirus. In order to develop a detection system for DsMV and ZaMV, ELISA, RT-PCR and dot-blot hybridization were chosen to detect these two viruses. The data of ELISA did not clearly indicate the appearance of two viruses. This might be due to the interference by the sticky materials of aroid plants. Although RT-PCR gave high sensitivity of detection, the reactions were also affec ted by the sticky materials of plant and viral mix infection. The meth od of dot-blot hybridization could clearly detect the mix infection of DsMV and ZaMV, its sensitivity was uq to 5 ng of total RNA, and its detection was not affected by the materials of aroid plants. Therefore, dot-blot hybridization with specific DNA probes is the best detection method for detecting DsMV and ZaMV in aroid plants.