碩士 / 國立臺灣大學 / 植物病理學研究所 / 90 / During investigating the effect of Zantedeschia mosaic virus (ZaMV) on the production of calla lilies, one of the uninoculated, tissue-cultured calla lilies was found exhibiting mild mosaic symptom. In indirect ELISA test, the result was positive for potyvirus group specific monoclonal antibody, but negative for Cucumber mosaic virus (CMV), Dasheen mosaic virus (DsMV), and ZaMV polyclonal antibodies. The flexuous, filamentous particles about 650 nm in length were observed by transmission electron microscope. The results of ELISA and electron microscopy indicated that the unknown virus infecting calla lily was a potyvirus. In the host range test, the virus only infected Philodendron selloum and Zantedeschia spp. causing systemic mosaic, respectively. To further characterize the virus and obtain its sequence information, several degenerate primers were designed according to the conserved amino acid sequences of the reported potyviruses and specific primers were designed after this viral sequences determined. Five fragments from different regions of viral genome were amplified using RT-PCR method. At least two clones from each fragment were obtained via TA cloning. When sequenced and piled up in GCG program, 8108 nucleotides of the virus were determined, including the C terminal region of HC-Pro, P3, 6K1, CI, 6K2, NIa, NIb, CP and 3’ untranslated region (3’UTR). The amino acid sequences of putative proteins were deduced by the GCG program and compared with twenty published potyvirus sequences. The amino acid sequence identities of the P3 protein among this virus and other potyviruses were 20 to 46 %, and the amino acid sequence identities of the VPg were 38-61%. Whereas, more conserved proteins of potyvirus, such as CI, NIa-Pro and NIb, had highest amino acid sequence identities up to 70% when compared with individual potyviruses. In addition, the highest amino acid sequence identities of CP between the unknown potyvirus and other potyviruses was 54%, and the nucleotide identities of 3’UTR were between 12 and 29%. Therefore, in terms of species demarcating criteria in the genus Potyvirus and the symptom on the calla lilies, this virus is considered as a new potyvirus and thus named Zantedeschia mild mosaic virus (ZaMMV).
On the other hand, in order to obtain the antiserum of ZaMMV, the primer pair, ZUNCPF2 and ZUNCPR1, based on the sequence of ZaMMV CP gene were designed. A fragment of 830 bp was amplified and cloned into pET-29a (+) expression vector. After transformed into Escherichia coli BL21 (DE3) and induced by IPTG, the CP of ZaMMV was expressed and purified to immunize rabbits. The specificity of the prepared ZaMMV antiserum was tested by western-blot assay. In the last part of this research, except for serological detection, three specific DNA probes were used to detect ZaMMV and the probe311, gave the best result of the specificity. Calla lilies collected from the market, field and Taiwan Seed Improvement and Propagation Station were detected using ELISA, dot-blot hybridization, the result indicated that ZaMMV existed in some of the these samples. When the same calla lily samples were detected with IC-RT-PCR, the result was reconfirmed. This finding suggested that ZaMMV might have already widely distributed in the field.
| Identifer | oai:union.ndltd.org:TW/090NTU00364012 |
| Date | January 2002 |
| Creators | Chin-Hsing Huang, 黃金興 |
| Contributors | Ya-Chun Chang, 張雅君 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 67 |
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