請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47767完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 葉信宏(Hsin-Hung Yen) | |
| dc.contributor.author | Hsiang-Chia Lu | en |
| dc.contributor.author | 陸祥家 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:17:23Z | - |
| dc.date.available | 2011-08-16 | |
| dc.date.copyright | 2010-08-16 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-11 | |
| dc.identifier.citation | 第一章
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47767 | - |
| dc.description.abstract | 東亞蘭嵌紋病毒(Cymbidium mosaic virus) 隸屬於Flexiviridae科下的Potexvirus屬，為最主要為害蘭花的病毒。由於蘭花栽培已經成為台灣最重要的農業產業之一，所以相對的CymMV也成為經濟上相當重要的病毒。雖然Potexvirus屬已被廣泛的研究，但是針對CymMV所做的研究還是集中在病毒分離株的基因定序以及偵測。此論文中我收集了各種不同的CymMV分離株，除了發展一套病毒偵測之方法外，亦利用這些分離株進行基礎與應用兩方面的研究。在本論文的第一部分我針對同一病毒不同之分離株於宿主內不同之移動特性進行探討，透過這個研究不僅了解了參與CymMV病毒分離株移動的因子，同時也對相關的分子機制做一系列探討。另外我進一步利用無病徵的CymMV分離株構築病毒誘導基因靜默載體以進行蘭花的功能性基因體研究，同時也發展了一套策略克服蘭花生長期長而不利於研究的問題。這些結果詳細描述於論文的第2和第3章，同時先前也發表成兩篇論文: Virology, 388:147-159. 和Plant Physiol. 143:558-569. | zh_TW |
| dc.description.abstract | Cymbidium mosaic virus (CymMV) is a prevalent orchid virus and belongs to the genus Potexvirus in the family Flexiviridae (Adams et al., 2005b). Because the cultivation of orchids has become an important industry, CymMV is now a very economically important virus. Although potexvirus has been extensively studied, the study of CymMV remains focused on the isolate sequencing and detection. In my thesis, I have collected several CymMV isolates. Besides to develop the methods for detection of these isolates, I also utilized these isolates for basic and applied studies. In the first part of my thesis I established a system for the study of isolate-dependent host movement study. The studies revealed not only identify the factors involved in CymMV isolate dependent movement but also the molecular mechanism involved in CymMV movement. In addition, we utilized the collected symptomless CymMV isolates to develop virus-induced gene silencing (VIGS) vectors for orchid functional genomics studies. Beside the vector, we also develop a strategy to overcome the obstacles lie in the study of plants with long life cycle such as Phalaenopsis orchids. The results were detailed described in chapter 2 and 3 and published in two separated papers: Virology, 388:147-159 and Plant Physiol. 143:558-569. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:17:23Z (GMT). No. of bitstreams: 1 ntu-99-D93633005-1.pdf: 4997392 bytes, checksum: 71f2418d2ae1b97bd8c786a4178eb3a6 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 目 錄
口試委員會審定書……………………………………………………….........i 誌謝…………………………………………………………………. ………. ii 中文摘要……………………………………………………………………. iii 英文摘要…………………………………………………………………….. iv CHAPTER 1 INTRODUCTION……………………………………………7 1.1 Cymbidium mosaic virus……………………………………………..8 1.2 Taxonomy and phylogenetic analysis of CymMV…………………..8 1.3 Genome organization of CymMV…………………………………...9 1.4 RNA elements regulating potexvirus replication………………… .10 1.5 TGBs and virus cell to cell movement……………………………...11 1.6 Potexvirus viral vector development and application………………12 1.7 REFERENCE……………………………………………………...15 CHAPTER 2 Cymbidium mosaic potexvirus isolate-dependent host movement systems reveal two movement control determinants and the coat protein is the dominant………………………………………..………19 2.1 ABSTRACT………………………………………………………...21 2.2 INTRODUCTION…………………………………………………..22 2.3 MATERIALS AND METHODS……………………………………26 2.3.1 RNA extraction and northern blot hybridization……………….26 2.3.2 Construction of CymMV infectious clones and derived expression vectors………………………………………………26 2.3.3 Construction of CymMV recombinant clones………………….26 2.3.4 Construction of TGBps recombinant clones……………………27 2.3.5 Site-directed mutagenesis………………………………………28 2.3.6 Preparation and transfection of N. benthamiana protoplast…….29 2.3.7 Plants and virus inoculation…………………………………….30 2.3.8 RT-PCR…………………………………………………………30 2.3.9 Construction of CymMV CP expression clones………………..31 2.3.10 Expression and purification of CymMV-CP…………………..31 2.3.11 Protein-RNA pull-down assay………………………………...32 2.3.12 Whole-mount RNA analysis…………………………………..33 2.3.13 Construction of CymMV TGBp and CP yeast two-hybrid clones…………………………………………………………...33 2.3.14 Yeast two-hybrid system………………………………………33 2.4 RESULTS…………………………………………………………...35 2.4.1 Construction of CymMV infectious clones and derived expression vectors………………………………………………35 2.4.2 Replication of CymMV isolates in N. benthamiana protoplasts………………………………………………………35 2.4.3 Subliminal infection of CymMV-M2 in N. benthamiana……....36 2.4.4 Genome shuffling between CymMV-M1 and -M2 for complementation analysis………………………………………36 2.4.5 Mapping the amino acids on CymMV CP that are important for systemic infection of N. benthamiana plants…………………...37 2.4.6 Binding affinity assay between CymMV M1 and M2 CPs and RNAs…………………………………………………………...38 2.4.7 Interaction analysis of TGBps by use of yeast two-hybrid assay…………………………………………………………….39 2.4.8 Mapping the amino acids on TGBp1 and TGBp3 of CymMV that are important for systemic infection of N. benthamiana……….39 2.5 DISCUSSION………………………………………………………41 2.6 ACKNOWLEDGMENTS…………………………………………..45 2.7 REFERENCES……………………………………………………...46 Figure 2.1………………………………………………………………...53 Figure 2.2………………………………………………………………...55 Figure 2.3………………………………………………………………...57 Figure 2.4………………………………………………………………...59 Figure 2.5………………………………………………………………...61 Figure 2.6………………………………………………………………...63 Figure 2.7………………………………………………………………...65 Figure 2.8………………………………………………………………...68 Figure 2.9………………………………………………………………...70 Table 2.1……………………………………………………………….....72 CHAPTER 3 Strategies for Functional Validation of Genes Involved in Reproductive Stages of Orchids……………………………………………73 3.1 ABSTRACT………………………………………………………...75 3.2 INTRODUCTION………………………………………………….76 3.3 MATERIALS AND METHODS……………………………………79 3.3.1 Plants……………………………………………………………79 3.3.2 Virus isolates……………………………………………………79 3.3.3 Construction of infectious clones………………………………79 3.3.4 Construction of CymMV expression vectors…………………..80 3.3.5 RNA extraction and northern blot hybridization……………….81 3.3.6 RT-PCR…………………………………………………………82 3.3.7 In vitro transcription……………………………………………82 3.3.8 Real-time quantitative RT-PCR………………………………...82 3.3.9 Detection of siRNA…………………………………………….83 3.4 RESULTS…………………………………………………………..84 3.4.1 Construction of CymMV cDNA infectious clones……………..84 3.4.2 Construction of VIGS vectors…………………………………..84 3.4.3 Validation of CymMV vector in inducing gene silencing……...85 3.4.4 Validation of CymMV vector in inducing floral gene silencing………………………………………………………...86 3.4.5 Simultaneously knockdown of MADS-box genes……………...88 3.5 DISCUSSION………………………………………………………91 3.6 REFERENCES……………………………………………………...94 Figure 3.1……………………………………………………………... 100 Figure 3.2……………………………………………………………….102 Figure 3.3……………………………………………………………….104 Figure 3.4……………………………………………………………….106 Figure 3.5……………………………………………………………….108 Figure 3.6……………………………………………………………….110 Figure 3.7……………………………………………………………….112 Figure 3.8……………………………………………………………….114 Figure 3.9……………………………………………………………….116 Table 3.1………………………………………………………………...118 Table 3.2………………………………………………………………...119 Table 3.3………………………………………………………………...120 CHAPTER 4 CONCLUSIONS…………………………………………...122 | |
| dc.language.iso | en | |
| dc.title | 東亞蘭嵌紋病毒分離株移動機制的研究與病毒誘導基因靜默系統的建立 | zh_TW |
| dc.title | Studies the Cymbidium mosaic virus movement mechanism and establishment of a virus-induced gene silencing (VIGS) system | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 蘇鴻基(Hong-Ji Su),洪挺軒(Ting-Hsuan Hung),鄭秋萍(chiu-ping cheng),葉國楨(Kuo-Chen Yeh),吳素幸(Shu-Hsing Wu) | |
| dc.subject.keyword | 東亞蘭嵌紋病毒,病毒載體,移動機制,蝴蝶蘭, | zh_TW |
| dc.subject.keyword | CymMV,viral vector,movement,Phalaenopsis orchid, | en |
| dc.relation.page | 124 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-11 | |
| 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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