Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31050
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dc.contributor李國欽zh_TW
dc.contributor柯文雄zh_TW
dc.contributor陳慶忠zh_TW
dc.contributor葉錫東zh_TW
dc.contributor王惠亮zh_TW
dc.contributor張清安zh_TW
dc.contributor林長平zh_TW
dc.contributor陳煜焜zh_TW
dc.contributor.advisor詹富智zh_TW
dc.contributor.author鄭尤琇zh_TW
dc.contributor.authorZheng, You-Xiuen_US
dc.contributor.other中興大學zh_TW
dc.date2008zh_TW
dc.date.accessioned2014-06-06T07:40:59Z-
dc.date.available2014-06-06T07:40:59Z-
dc.identifierU0005-2308200710425500zh_TW
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W., Ko, N. J., Wisler, G. C., Elliott, M. S., and Wong, S. M. 1990. Viruses of orchids and their control. Plant Dis. 74:621-625. 第三章 1. Bairoch, A., and Bucher, P. 1994. PROSITE: recent developments. Nucleic Acids Res. 22:3583-3589. 2. Bandla, M. D., Campbell, L. R., Ullman, D. E., and Sherwood, J. L. 1998. Interaction of tomato spotted wilt tospovirus (TSWV) glycoproteins with a thrips midgut protein, a potential cellular receptor for TSWV. Phytopathology 88:98-104. 3. Baulcombe, D. C. 2004. RNA silencing in plants. Nature 431:356-363. 4. Bayne, E. H., Rakitina, D. V., Morozov, S. Y., and Baulcombe, D. 2005. Cell-to-cell movement of potato potexvirus X is dependent on suppression of RNA silencing. Plant J. 44:471-482. 5. Biederer, T., Volkwein, C., and Sommer, T. 1997. Role of Cue1p in ubiquitination and degradation at the ER surface. Science 278:1806-1809. 6. Brigneti, G., Voinnet, O., Li, W. X., Ji, L. H., Ding, S. W., and Baulcombe, D. C. 1998. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17:6739-6746. 7. Brodsky, L. I., Ivanov, V. V., Kalaidzidis, Y. L., Leontovich, A. M., Nikolaev, V. K., Feranchuk, S. I., and Drachev, V. A. 1995. GeneBee-NET:Internet-based server for analyzing biopolymers structure. Biochemistry 60:923-928. 8. Brodsky, L. I., Vasiliev, A. V., Kalaidzidis, Y. L., Osipov, Y. S., Tatuzov R. L., and Feranchuk, S. I. 1992. GeneBee: the program package for biopolymer structure analysis. Dimacs 8:127-139. 9. Bucher, E., Sijen, T., de Haan, P., Goldbach, R., and Prins, M. 2003. Negative-strand tospoviruses and tenuiviruses carry a gene for a suppressor of gene silencing at analogous genomic positions. J. Virol. 77:1329-1336. 10. Chen, C. C., Chen, T. C., Lin, Y. H., Yeh, S. D., and Hsu, H. T. 2005. A chlorotic spot disease on calla lilies (Zantedeschia spp.) is caused by a tospovirus serologically but distantly related to Watermelon silver mottle virus. Plant Dis. 89:440-445. 11. Chen, K., Xu, Z., Yan, L., and Wang, G. 2007. Characterization of a new strain of Capsicum chlorosis virus from peanut (Arachis hypogaea L.) in Chinazh_TW
dc.identifier.urihttp://hdl.handle.net/11455/31050-
dc.description.abstract蝴蝶蘭 (Phalaenopsis sp.) 是廣受世界各國民眾歡迎的觀賞植物,也是台灣最重要的外銷蘭花之一;迄今,至少有 29 種以上感染蘭花的病毒被記錄,其中東亞蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV)、齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV)、胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV)、蘭花斑點病毒 (Orchid fleck virus, OFV) 及石斛蘭葉脈壞疽病毒 (Dendrobium vein necrosis virus, DVNV) 曾被報導感染蝴蝶蘭,而其中的 CymMV、ORSV及CMV 是三個被報告發生台灣蝴蝶蘭的病毒。蝴蝶蘭呈現疑似病毒感染的黃化輪斑 (chlorotic ringspot) 病徵在台灣中、南部已被發現至少十年之久,過去一直不知其感染因子為何,但因此病徵常出現在台灣所外銷的蝴蝶蘭上,故一直被國際蘭界稱為『Taiwan virus』;我們自具有黃化輪斑及壞疽斑點 (necrotic spot) 的蝴蝶蘭上取得一包有套膜 (envelope) 的球形病毒分離株 (91-蘭-1),且此病毒可與西瓜銀斑病毒 (Watermelon silver mottle virus, WSMoV) 抗血清反應;該病毒之 L RNA 高度保留區 (conserved region of L RNA) 及核鞘蛋白 (nucleocapsid, N) 基因序列顯示 91-蘭-1 是辣椒黃化病毒 (Capsicum chlorosis virus, CaCV, Tospovirus),故將其定名為 CaCV-Ph;進一步以生物資訊軟體分析 CaCV-Ph 基因特性,得知病毒蛋白與病毒的寄主範圍、媒介昆蟲及地理分佈有密不可分之關係;此外,在進行 23 種蝴蝶蘭品系 (cultivar) 之CaCV-Ph 抗性測試及調查 CaCV-Ph 田間發生率之後,發現有三個蝴蝶蘭品系表現出對 CaCV-Ph 具有耐受性 (tolerance) 或抗性 (resistance)。另於葉片出現黃化斑點 (chorotic spot) 之蝴蝶蘭上取得一長絲狀病毒分離株 (7-2),此病毒可與廣泛性偵測馬鈴薯Y病毒屬 (Potyvirus) 病毒之單株抗體反應,在完成包含鞘蛋白 (coat protein, CP) 基因之RNA 3’ 區域的定序後,得知 7-2 是一新的 potyvirus 並進而將其命名為蝴蝶蘭黃化斑點病毒 (Phalaenopsis chlorotic spot virus, PhCSV)。此外,另一球型病毒分離株 (92-蘭-1) 分離自呈現黃化輪紋 (chlorotic ring) 病徵之蝴蝶蘭且可與康乃馨斑駁病毒 (Carnation mottle virus, CarMV) 抗血清反應,經聚合酶 (polymerase) 基因高度保留區及 CP 基因之定序,確認 92-蘭-1 屬於 CarMV 並將其更名為 CarMV-Ph。蝴蝶蘭病毒病害的發生直接威脅到台灣各大蘭園及花卉產業,為了防除多種病毒病害,我們發展一個新的轉基因抗病策略,經由可產生小片段干擾 RNA (small interference RNA, siRNA) 的合成核酸片段 (synthetic nucleoties) 發展具有多重病毒抗性的轉基因植物;依據此策略,我們已產生可同時抗二或三個番茄斑萎病毒屬 (Tospovirus) 病毒的轉殖合成核酸片段之 R0 轉基因菸草,而轉有合成N基因之 18-17 株系 (line) R1 子代對番茄斑萎病毒 (Tomato spotted wilt virus, TSWV) 及花生輪點病毒 (Groundnut ringspot virus, GRSV) 則表現出高度抗性;這些結果意謂著可產生 siRNA 的合成核酸片段之轉基因策略可提供對多種番茄斑萎病毒屬病毒的抗性,而此一策略未來可能可以進一步推展到產生具多重病毒抗性之蘭花甚至其他作物上。總括來說,我們已鑑定三種可分別引起黃化輪斑 (chlorotic ringspot)、黃化斑點 (chlorotic spot) 及黃化輪紋 (chlorotic ring) 等病徵之蝴蝶蘭病毒,並命名為 CaCV-Ph、PhCSV及CarMV-Ph;此外,本研究之結果更提出一套利用可產生 siRNA 的合成核酸片段發展具多重病毒抗性之轉基因植物的可能新策略。zh_TW
dc.description.abstractPhalaenopsis orchids are popular ornamental plants worldwide and they are currently the most important exporting orchids for Taiwan. At least 29 orchid-infecting viruses have been reported, and among these, Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), Cucumber mosaic virus (CMV), Orchid fleck virus (OFV) and Dendrobium vein necrosis virus (DVNV) were reported to be able to infect Phalaenopsis orchids. CymMV, ORSV, and CMV are the only three viruses that have been reported to infect Phalaenopsis orchids in Taiwan. Phalaenopsis orchid plants bearing virus-like symptoms of chlorotic ringspots have been observed in central and southern Taiwan for more than a decade. As the causal agent of this orchid disease was unclear, the syndrome was always described as “Taiwan virus” by orchid industry people since they were frequently observed on orchids from Taiwan. A virus culture (91-orchid-1) with quasi-spherical enveloped particles was isolated from Phalaenopsis orchid showing chlorotic spots with centric necrosis and positively reacted with an antiserum against Watermelon silver mottle virus (WSMoV). The sequences of the conserved region of L RNA and nucleocapsid (N) gene indicated that 91-orchid-1 was an isolate of Capsicum chlorosis virus (CaCV, Tospovirus) and was therefore designated as CaCV-Ph. We investigated the genomic information of CaCV-Ph via bioinforamtic analyses and found that the tospoviral proteins are related with the hosts, insect vectors and geographic distributions of topoviruses. Moreover, after challenging 23 Phalaenopsis cultivars with CaCV-Ph and surveying the incidence of CaCV-Ph in nurseries, three cultivars were found to be tolerant or resistant to CaCV-Ph. A second virus culture (7-2) with long filamentous particles was isolated from the Phalaenopsis orchid showing chlorotic spots and found to react with a commercialized potyvirus-specific monoclonal antibody. The sequence of 3' terminal region containing coat protein (CP) gene indicated that 7-2 was a new potyvirus and therefore named as Phalaenopsis chlorotic spot virus (PhCSV). In addition, another virus culture (92-orchid-1) with isometric particles was collected from the Phalaenopsis orchid showing chlorotic rings and reacted with the antiserum against Carnation mottle virus (CarMV, Carmovirus). The sequences of the conserved region of polymerase gene and CP gene indicated that 92-orchid-1 was a CarMV isolate and the virus isolate was thereby designated as CarMV-Ph. The occurrence of these viruses in Phaelenopsis orchids has become a problem for the economically important nurseries and floral industry in Taiwan. To control multiple viral diseases, a new strategy was developed for generating transgenic plants with broad-spectrum resistance to viruses via siRNA-generating synthetic nucleotides. The transgenic R0 tobacco plants with synthetic nucleotides resisted to two or three topsoviruses were generated. The R1 progeny of line 18-17 with synthetic N gene showed high resistance to Tomato spotted wilt virus (TSWV) and Groundnut ringspot virus (GRSV). These results indicated that the siRNA-generating synthetic nucleotide can provide resistance to multiple tospoviruses and this strategy might be able to apply for the development of orchids or ther crops with broad-spectrum viral resistances in the future. In this study, we have identified and characterized three new Phalaenopsis orchids-infecting viruses, CaCV-Ph, PhCSV and CarMV-Ph that induced symptoms of chlorotic ringspots, chlorotic spots and chlorotic rings, respectively, on Phalaenopsis orchids. In addition, we proposed a possible new strategy for generating transgenic plants with multi-virus resistance using siRNA-generating synthetic nucleotides.en_US
dc.description.tableofcontents摘要......................................................1 Abstract .................................................3 Chapter 1. Introduction and literature review.............5 第一章、前言及前人研究 Chapter 2. Identification and characterization of a tospovirus causing chlorotic ringspots on Phalaenopsis orchids .................................................29 第二章、引起蝴蝶蘭黃化輪點斑之番茄斑萎病毒屬病毒之鑑定及特性分析 Abstract ................................................30 Introduction ............................................31 Materials and methods ...................................31 Virus sources and maintenance ...........................31 Electron microscopy .....................................32 Host range and back-inoculation .........................32 Purification of nucleocapsid protein and production of polyclonal antibody to 91-orchid-1 (CaCV-Ph) ............33 Immunoblotting ..........................................33 ELISA ...................................................34 Molecular cloning and Sequence analysis .................35 Results .................................................36 Symptoms and virus isolation ............................36 Host range and back-inoculation .........................36 Electron microscopy .....................................37 Serological analysis ....................................37 Molecular cloning and sequence analysis .................38 Discussion ..............................................39 References ..............................................41 Tables and Figures ......................................45 Chapter 3. Complete nucleotide sequence of Capsicum chlorosis virus isolate Phalaenopsis (CaCV-Ph) and the prediction of the unexplored genetic information of tospoviruses ............................................53 第三章、辣椒黃化病毒蝴蝶蘭分離株之基因體解序及番茄斑萎病毒屬病毒未知基因功能之測定 Abstract ................................................54 Introduction ............................................55 Materials and methods ...................................56 Virus source ............................................56 Purification of viral dsRNAs ............................56 Strategies for cloning and sequencing the S, M and L RNAs of CaCV-Ph .........................................56 Analyses of viral RNA sequences .........................57 Results .................................................58 Sequencing and analysis of S RNA ........................58 Sequencing and analysis of M RNA ........................59 Sequencing and analysis of L RNA ........................59 Multiple sequence alignments and homology analysis of the CaCV-Ph genome ..........................................59 Topology of the glycoprotein precursors of tospoviruses..60 Discussion ..............................................61 References ..............................................66 Tables and Figures ......................................73 Chapter 4. Survey of the incidence of Capsicum chlorosis virus (CaCV-Ph) in the Phalaenopsis nurseries and resistance evaluation of current Phalaenopsis cultivars ...............................................83 第四章、辣椒黃化病毒於蘭園發生之調查與蝴蝶蘭對辣椒黃化病毒蝴蝶蘭分離株抗病性之分析 Abstract ................................................84 Introduction ............................................85 Materials and methods ...................................86 Virus sources and maintenance............................86 Inoculation of Phalaenopsis hybrids with CaCV-Ph ........86 Phalaenopsis orchids sampling for CaCV survey ...........86 ELISA ...................................................86 Results .................................................87 Symptom and susceptibility of Phalaenopsis orchids to CaCV-Ph .................................................87 CaCV survey of Phalaenopsis orchids .....................88 Discussion ..............................................88 References ..............................................90 Tables and Figures ......................................94 Chapter 5. Identification and characterization of a potyvirus causing chlorotic spots on Phalaenopsis orchids ................................................102 第五章、引起蝴蝶蘭黃斑之馬鈴薯Y病毒屬病毒之鑑定及特性分析Abstract ...............................................103 Introduction ...........................................104 Materials and methods ..................................104 Virus sources and maintenance ..........................104 Electron microscopy ....................................105 Host range and back-inoculation ........................105 Virus purification and production of polyclonal antibody to isolate 7-2 (PhCSV) .................105 ELISA ..................................................106 Immunoblotting .........................................106 Molecular cloning and Sequence analysis ................107 Results ................................................107 Symptoms and virus isolation ...........................107 Host range and back-inoculation ........................108 Electron microscopy ...................................108 Serological analysis ...................................109 Molecular cloning and sequence analysis ................109 Discussion .............................................110 References .............................................112 Tables and Figures .....................................116 Chapter 6. Phalaenopsis orchid showing chlorotic rings, a new diseasecaused by Carnation mottle virus ............123 第六章、康乃馨斑駁病毒引起之蝴蝶蘭黃化輪紋病害 Abstract ...............................................124 Introduction ...........................................125 Materials and methods ..................................125 Virus sources and maintenance ..........................125 Electron microscopy ....................................125 Host range..............................................126 Purification of viral particles and production of polyclonal antibody to 92-orchid-1 .....................126 Immunoblotting .........................................127 ELISA ..................................................127 Molecular cloning and sequence analysis ................128 Results ................................................129 Symptoms and virus isolation ...........................129 Host range .............................................129 Electron microscopy ....................................130 Serological analysis ...................................130 Molecular cloning and sequence analysis ................130 Discussion .............................................131 References .............................................134 Tables and Figures .....................................137 Chapter 7. Developing transgenic plants with broad-spectrum resistance to viruses by expressing designed siRNA-generating synthetic nucleotides ............................................144 第七章、藉人工設計可產生短片段干擾RNA之合成核苷酸片段以發展可廣泛性抗多種病毒之轉基因植物 Abstract ...............................................145 Introduction ...........................................146Materials and methods ..................................147 Virus source ...........................................147 siRNA prediction and designing synthetic nucleotides ...147 Transgene constructions and plant transformation .......148 PCR analysis of transgenic plants ......................149 Transgenic R1 plants screening .........................149 Inoculation of transgenic plants .......................149 ELISA of transgenic plants .............................149 Results ................................................150 Designed and amplified the synthetic siRNA-generating nucleotides ............................................150 Generation of transgenic plants expressing synthetic nucleotides ............................................151 A synthetic nucleotide targeting to TSWV, GRSV and INSV confers resistance to TSWV and GRSV ....................151 A synthetic nucleotide targeting to WSMoV and CaCV confers resistance to both viruses .............................152 Multiple resistances in R1 progeny plants ..............152 Discussion .............................................152 References .............................................154 Tables and Figures .....................................159 Chapter 8. Conclusions .................................169 第八章、結論zh_TW
dc.language.isoen_USzh_TW
dc.publisher植物病理學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2308200710425500en_US
dc.subject病毒zh_TW
dc.subjectPhalaenopsisen_US
dc.subject鑑定zh_TW
dc.subject廣泛性zh_TW
dc.subject轉基因zh_TW
dc.subject抗病zh_TW
dc.subject人工合成核苷酸zh_TW
dc.subjectVirusesen_US
dc.subjectIdentificationen_US
dc.subjectTransgenic Approachen_US
dc.subjectBroad-Spectrum Resistanceen_US
dc.subjectDesigned siRNA-Generating Synthetic Nucleotidesen_US
dc.title新蝴蝶蘭病毒之鑑定及其特性之研究暨以可產生短片段干擾RNA之人工合成核苷酸片段發展新式可廣泛性抗多種病毒之轉基因抗病策略zh_TW
dc.titleIdentification and Characterization of New Phalaenopsis orchid-infecting Viruses and Developing New Transgenic Approach for Broad-Spectrum Resistance to Viruses by Expressing Designed siRNA-Generating Synthetic Nucleotidesen_US
dc.typeThesis and Dissertationzh_TW
Appears in Collections:植物病理學系
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