臺灣博碩士論文加值系統

在本研究中，多重逆轉錄聚合酶鏈反應（mRT-PCR）技術同時被使用在檢測葡萄中的病毒。 在2015年及 2016 年的5月至6月期間，從台灣彰化縣不同的葡萄園收集了五十個具有黃化或馬賽克症狀的葡萄葉樣品。根據從 NCBI 基因庫獲得的序列，新設計的特殊引子對被用於檢測啤酒花矮化類病毒（HSVd） ､澳大利亞葡萄樹病毒（AGVd） ､ 葡萄黃斑類病毒-1（GYSVd-1） ､葡萄黃斑類病毒-2（GYSVd-2） 及柑橘裂皮病類病毒。在比較了序列識別方面，樣品啤酒花矮化類病毒（HSVd-DY） 與基因庫的啤酒花矮化類病毒（HSVd）之類同的範圍為 37.6％〜99.7％。而樣品葡萄黃斑類病毒-1（GYSVd-1-DY）則類同範圍可達 82.9％〜99.7％。從啤酒花矮化類病毒（HSVd）序列之演化樹推衍出樣品葡萄黃斑類病毒-1（GYSVd-1-DY）更接近伊朗分離物（KF916041），樣品葡萄黃斑類病毒-1（ GYSVd-1-DY）則與中國分離物（JF746188）更密切相
關。

In this study a multiplexreverse-transcription polymerase chain reaction (mRT
PCR) technique is used simultaneously to detect viroids in grapevine. Fifty grapevine leaf samples with yellowing or mosaic symptoms were collected from difference vineyards at Changhua County, Taiwan during May to June, 2015-2016. Specific primer pairs for the detection of Hop stunt viroid (HSVd), Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2 (GYSVd-2) and Citrus exocortis viroid (CEVd) were selected from previous reports or primers were newly designed according to the sequences obtained from NCBI Genbank. Comparison of the sequence identity of HSVd-DY with other HSVd from GenBank ranged from 37.6% to 99.7%. While the sequence identity between GYSVd-1-DY with others ranged from 82.9% to 99.7%. A phylogenetic tree derived from the HSVd sequence indicated that HSVd-DY was closer to an Iran isolate (KF916041), while GYSVd-1-DY was close related to a China isolate (JF746188).

ABSTRACT iii
中文摘要 iv
ACKNOWLEDGEMENTS v
CONTENTS vi
LIST OF FIGURES iv
LIST OF TABLES x
LIST OF ABBREVIATION xii
Chapter 1. INTRODUCTION 1
1.1 Background of Research 1
1.2 Purposes of Research 2
1.3 Goals to Reach 3
1.4 Framework of Research 3
Chapter 2. LITERATURE REVIEWS 5
2.1 Structure and Classification of Viroid 5
2.1.1 Structure 5
2.1.2 Classification 7
2.2 Generation of Populations from Individual Viroid Variants 7
2.3 Origin and Evolution of Viroids 8
2.4 The Viroid Species Infect Grapevine 10
2.4.1 Hop Stunt Viroid 11
2.4.2 Grapevine Yellow Speckle 1, 2 13
2.4.3 Australian Grapevine Viroid 13
2.4.4 Citrus Excortis Viroid 13
2.5 The Detection Techniques in Viroid Disease 13
2.6 Elimination of Viroids from Plants 20
Chapter 3. STUDY METHODS 21
3.1 Source of Plant Materials 21
3.2 Extraction of RNA 22
3.3 Designation of Viroid-Specific Primer 23
3.4 The Single RT-PCR Reaction 28
3.5 Multiplex RT-PCR (mRT-PCR) 29
3.6 DNA Elution and Cloning 30
3.7 Phylogenetic Tree Construction 31
Chapter 4. RESULTS AND DISCUSSION 33
4.1 Results 33
4.1.1. Yield and quality of RNA extract 33
4.1.2. Primers Design for Detection 33
4.1.3. Detection of Grapevine viroids by single RT-PCR 33
4.1.4 Development of multiplex RT-PCR 34
4.1.5 T&A cloning and HindIII 35
4.1.6 Phylogenetic tree analysis 39
4.1.7 Nucleotide sequence identity 42
4.2 Brief Discussion of Results 46
Chapter 5. CONCLUSIONS AND SUGGESTIONS 49
5.1 Conclusions 49
5.2 Suggestions 51
REFERENCES 52
APPENDIX A: The rod like secondary structure of Potato Spindle tuber viroid showing the five domains
chacrateristic of members of the family Pospiviroidae: the terminal left (TL), pathogenicity (P),central (C), variable (V), and terminal right 60
APPENDIX B: Map and Sequence reference points of T&A Cloning Vector 61

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