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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張雅君(Ya-Chun Chang) | |
| dc.contributor.author | Yu-Wen Huang | en |
| dc.contributor.author | 黃裕雯 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:35:22Z | - |
| dc.date.available | 2019-08-21 | |
| dc.date.copyright | 2014-08-21 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-12 | |
| dc.identifier.citation | Ajjikuttira, P., Loh C. S., and Wong S. M., 2005. Reciprocal function of movement proteins and complementation of long-distance movement of Cymbidium mosaic virus RNA by Odontoglossum ringspot virus coat protein. Journal of General Virology 86: 1543-1553.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56569 | - |
| dc.description.abstract | 當植物病毒複合感染於同一植株時，所導致的寄主病徵加重、其中一個或雙方病毒累積量上升之現象稱為協力作用(synergistic interaction)。蕙蘭嵌紋病毒(Cymbidium mosaic virus, CymMV) 及齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)為兩種感染蘭科植物之重要病毒，其複合感染之情形在人工栽培之蘭園十分常見，並導致植株病徵加重的情況。前人研究顯示，當CymMV及ORSV複合感染蘭花的原生質體時，病毒累積量會較單獨感染高，然而其協力作用的分子機制仍尚未了解。本研究以圓葉菸草(Nicotiana benthamiana)作為寄主，當CymMV及ORSV複合感染於菸草原生質體時，仍保有協力作用的現象，且CymMV在複合感染時的累積量增加幅度較ORSV大；推論此現象係由病毒之基因靜默抑制子(silencing suppressor)參與調控。CymMV的triple gene block 1 (TGB1) 蛋白或ORSV的126 kDa (p126)蛋白為具有基因靜默抑制子潛力之蛋白，在16c轉基因菸草上同時表現綠色螢光蛋白與這兩種病毒蛋白時，發現p126具有較強之基因靜默抑制活性。當接種CymMV於短暫表現具基因靜默能力的p126之菸草植株時，發現p126對CymMV有提升病毒累積量的效果；但基因靜默活性低的TGB1亦能提升ORSV病毒累積量。由此推論p126與TGB1可能藉由不同的機制來達到提升病毒累積量的效果。進一步探討p126可能具有抑制子功效的功能區塊(domain)，發現此些功能區塊均無法獨立的抑制基因靜默，也無法幫助CymMV協力作用的進行。因此我們認為ORSV的p126蛋白藉由抑制轉錄後基因靜默，使得CymMV在單細胞層次的協力作用中得到較大的利益。 | zh_TW |
| dc.description.abstract | Synergistic interaction refers to a facilitating effect that the symptom and replication of one or both virus partners increase on mixedly infected host plant. Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are two economically important viruses infecting orchids. Mixed infection by these two viruses is a common phenomenon and leads to severe symptoms. Besides, when CymMV and ORSV were inoculated to orchid protoplasts simultaneously, the accumulation of virus increased as compared to the singly infected protoplasts. However, the molecular mechanism of the synergistic interaction is still unknown. In this study, CymMV and ORSV were proved to have synergistic interaction on Nicotiana benthamiana protoplasts which were mixedly inoculated. Additionally, the accumulation of CymMV increased much more than that of ORSV. We proposed that this interaction may be mediated by silencing suppressor of the viruses. Triple gene block 1 protein (TGB1) of CymMV and p126 protein of ORSV which are potential PTGS suppressors were assayed on 16c line GFP transgenic N. benthamiana plants. Our results indicated that p126 possessed stronger suppressor activity than TGB1. When CymMV was inoculated onto N. benthamiana plants transiently expressed with p126, the accumulation of CymMV increased. In contrast, TGB1, which does not possess the ability of silencing suppression, could also enhance the accumulation of ORSV. Accordingly, p126 and TGB1 could mediate the synergistic interaction between CymMV and ORSV but through different mechanisms. Furthermore, we dissected the potential silencing suppression function of each p126 domain. The results indicated that none of these domains could independently suppress PTGS and facilitate the synergistic interaction with CymMV. Hence, we concluded that the ability of silencing suppression was required for ORSV p126 to facilitate CymMV accumulation during mixed infection in N. benthamiana protoplasts. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:35:22Z (GMT). No. of bitstreams: 1 ntu-103-R01633001-1.pdf: 3194098 bytes, checksum: b78c75a1cd950544ec591af0c01fd3d0 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 誌謝 i
Abstract ii 中文摘要 iv Contents v 1. Introduction 1 2. Materials and Methods 8 2.1 Virus sources 8 2.2 Plant materials 8 2.3 Total RNA extraction 9 2.4 Multiplex RT-PCR for virus detection 10 2.5 plasmid construction 11 2.5.1 Construction of p35S promoter-driven full-length cDNA clone of CymMV 11 2.5.2 Construction of the T7 promoter-driven full-length cDNA clone of CymMV 13 2.5.3 Plasmid construction for protein transient expression in planta 14 2.5.4 Plasmid construction for virus agro-infection 15 2.6 Midipreparation 15 2.7 Infectivity assay 16 2.8 Western blot 17 2.9 In vitro transcription of T7 promoter-driven CymMV clone 18 2.10 Protoplasts isolation and inoculation 19 2.11 Northern blot 20 2.12 Agro-infiltration 21 2.13 GFP fluorescence image 23 3. Results 24 3.1 Construction of CymMV full-length infectious clones for different experiments 24 3.2 Synergistic effect of CymMV and ORSV in N. benthamiana protoplasts 26 3.3 Silencing suppressor ability of ORSV p126 protein and CymMV TGB1 protein 26 3.4 Synergism of p126 protein and CymMV as well as TGB1 protein and ORSV 28 3.5 Silencing suppressor ability of individual domains of ORSV p126 30 3.6 Synergism of individual domains of ORSV p126 and CymMV 31 4. Discussion 32 5. References 41 6. Tables 50 Table 1. Primers used in this study 50 7. Figures 52 Figure 1. Schematic representation of CymMV infectious cDNA clones in different vectors. 52 Figure 2. The map of pBIN61-myc-ko. 53 Figure 3. Infectivity assay of p35S-CymMV cDNA clones. 54 Figure 4. Northern blot analyses of viral RNA accumulation in N. benthamiana protoplasts singly and doubly infected by ORSV and CymMV transcripts. 55 Figure 5. RNA silencing suppression abilities of potential viral suppressors, ORSV p126 and CymMV TGB1, in N. benthamiana line 16c. 56 Figure 6. ORSV p126 protein enhanced the accumulation of CymMV RNAs and CP on N. benthamiana plants. 57 Figure 7. CymMV TGB1 protein enhanced the accumulation of ORSV RNAs and CP on N. benthamiana plants. 58 Figure 8. Schematic diagram of ORSV genome, ORFs and p126 different domains used for transient expression in planta. 59 Figure 9. RNA silencing suppression abilities of different domains of ORSV p126 in N. benthamiana line 16c. 60 Figure 10. CymMV accumulation in N. benthamiana plants with transient expression of different domains of ORSV p126. 62 8. Appendix 63 Figure A1. Alignment of the amino acid sequences of potexvirus TGB1s. 63 Figure A2. Alignment of the amino acid sequences of TMV and ORSV p126 methyltransferase (Met) domain. 64 Figure A3. Alignment of the amino acid sequences of TMV and ORSV p126 non-conserved I (NI) domain. 65 Figure A4. Alignment of the amino acid sequences of TMV and ORSV p126 non-conserved II (NII) domain. 66 Figure A5. Alignment of the amino acid sequences of TMV and ORSV p126 helicase (Hel) domain. 67 | |
| dc.language.iso | en | |
| dc.subject | 基因靜默抑制子 | zh_TW |
| dc.subject | 協力作用 | zh_TW |
| dc.subject | 齒舌蘭輪斑病毒 | zh_TW |
| dc.subject | 蕙蘭嵌紋病毒 | zh_TW |
| dc.subject | Cymbidium mosaic virus | en |
| dc.subject | silencing suppressor | en |
| dc.subject | synergistic interaction | en |
| dc.subject | Odontoglossum ringspot virus | en |
| dc.title | 齒舌蘭輪斑病毒及蕙蘭嵌紋病毒協力感染之研究 | zh_TW |
| dc.title | Synergism in infectivity between Odontoglossum ringspot virus and Cymbidium mosaic virus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林詩舜(Shih-Shun Lin),沈湯龍(Tang-Long Shen) | |
| dc.subject.keyword | 協力作用,齒舌蘭輪斑病毒,蕙蘭嵌紋病毒,基因靜默抑制子, | zh_TW |
| dc.subject.keyword | synergistic interaction,Odontoglossum ringspot virus,Cymbidium mosaic virus,silencing suppressor, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-13 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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