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研究生:盧懷力
研究生(外文):Huai-Li Lu
論文名稱:台灣柑橘類病毒之診斷以及分子病理特性
論文名稱(外文):Diagnosis and Molecular Pathological Character of Citrus Viroids in Taiwan
指導教授:沈湯龍
指導教授(外文):Tang-Long Shen
口試委員:洪挺軒詹富智林乃君
口試日期:2013-05-08
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:61
中文關鍵詞:柑橘鱗砧類病毒啤酒花矮化類病毒柑橘季節變化序列分析番茄品種番茄退綠矮化類病毒
外文關鍵詞:Citrus exocortis viroidHop stunt viroidRT-PCRcitrusseasonal dynamicssequence alignmenttomato cultivarTCDVd
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類病毒是具有感染力的RNA並且可以造成多種植物病害,大小約介於250至400個核酸,單股但可經共價鍵結成環形,形成二級結構。植物與類病毒之間的交互作用至今仍然所知甚少,即使類病毒構造簡單,但卻可以造成農業損失。柑橘是重要的果樹作物之一,但遭受類病毒的危害甚廣,主要的病原是柑橘鱗砧類病毒(Citrus Exocortis Viroid)以及啤酒花矮化類病毒(Hop Stunt Viroid)。類病毒可藉由抽取RNA之後再依類病毒的保守序列設計引子對,以RT-PCR偵測。偵測對象可為實驗室中接種後的指示植物,或者是田間採集的樣本。實驗室接種的結果,藉由定量聚合酶連鎖反應(qPCR)偵測,顯示番茄的新葉含有較高的CEVd表現,但又少於莖部。自從1975年至今並沒有其他關於類病毒的報導,因此進行台灣各地的類病毒偵測,包括斗六、花蓮、新竹、屏東各地,偵測結果發現台灣存在另外兩種柑橘類病毒(Citrus bark cracking viroid,Citrus dwarf viroid)。田間樣本可被接種到番茄指示植物作為保存以及篩選,並且針對田間樣本進行選殖以及定序比對。經由序列比對,結果顯示在斗六田間採集到的樣本的CEVd序列曾發表於中國以及日本,推測此病原存在可能與地域性有關。比對採集地點的季節溫度,季節性的偵測顯示CEVd的量外界溫度呈現相似的趨勢,並且以qPCR進行定量測試,結果顯示CEVd量與溫度的升降有類似的動態曲線。因此以指示植物(番茄)進行熱處理,結果顯示高溫對於CEVd的表現量有抑制作用。為了研究CEVd在田間的傳播的其他路徑,接種CEVd到各種不同品種的番茄,但除了作為指示作物的品種,並沒有觀察到明顯病徵。番茄退綠矮化類病毒(Tomato Chlorotic Dwarf Viroid)序列和寄主與CEVd相似,為區別兩者,也完成TCDVd的接種以及開發偵測方法。

Viroids are the infectious RNA that lead to various plant growth disorder. Ranging from 250 to 400 nt, they are single stranded and closed covalently, forming a secondary structure. In spite of the simplicity of genome composition, viroids is of importance for the economic loss caused by them, although the pathogenesis and interaction between plant and viroids is rarely understood. Citruses are one of the most important fruit in economy. In many place citruses suffer from viroid disease. Citrus Exocortis Viroid (CEVd) and Hop Stunt Viroid (HSVd) are the two major citrus viroids, transmitted by mechanical injury or grafting. Viroids can be detected after TRIzol extraction detected by RT-PCR with primer designed according to conserved sequence. According to quantification in CEVd-infected tomato, new leaves contain more CEVd quantity than it in old ones yet less CEVd quantity than it in stems. Detection is available in lab inoculating indicators as well as field citrus sample. Since the last indigenous report was in 1975, detection to citrus in places in Taiwan, including Douliou, Hualian, Hsinchu, Pingtung, was performed. The result revealed there are two more citrus viroids(Citrus dwarf viroid, Citrus bark cracking viroid) that exist in Taiwan. Tomatoes are inoculated as indicator for pathogen maintenance and disease sieve. Cloning and Sequencing CEVd is necessary. The CEVd master sequence in Douliou field has been published from China and Japan; this result was probably due to geographical distribution. Seasonal sample collection reveals probable CEVd population change in accordance to temperature. Qiantitative result shows seasonal dynamics of CEVd. Therefore, heat treatment to indicators reveals that CEVd titer reduced in high temperature. To study the possible host of CEVd, the pathogenicity of CEVd to cultivars of tomato was test. No significant phenotype change was observed. As well, the other viroid of Tomato, Tomato Chlorotic Dwarf Viroid (TCDVd) which shares high similarity of sequence and host, was studied for distinguishing theses two. Inoculation of TCDVd and detection by RT-PCR and qPCR were done.

中文摘要……………………………………………………………………..………......i
英文摘要……………………………………………………………………..……..…..ii
目錄………………………………………………………………………………...…...iv
圖目錄………………………………………………………………………………….vii
Introduction…………………………………………………………………..1
Viroids…………………………………………………………………………...…1
Citrus exocortis viroid…………………………………………………………...…2
Citrus viroids……………………………………………………………………….3
The pathogenicity of viroids……..…………………………………………………4
Diagnosis of CEVd……………... …………………………………………………5
Viroid and environmental factor…………………………………………………...6
Research aim and rational………………………………………………………….6
Material and methods………………………………………………………………….8
Plant material, growth condition and viroid source………………………….......8
in vitro transcription and viroid inoculation……………………………………..8
Sampling methods of plants in laboratory and field survey……………………..8
Total RNA extraction……………………………………………………………9
RT-PCR and qPCR detection……………………………………………………9
Cloning CEVd sequence………………………………………………………..11
Construction of infectious dimeric viroid sequence……………………….…...11
Heat treatment to tomato………………………………………………………..12
Result…………………………………………………………………………………..13
CEVd-infected symptoms of indicator plants…………………………………..13
Citrus viroid field survey……………………………………………………….14
Development of quantitative PCR methods for detecting citrus viroids......…...14
Seasonal field survey of citrus viroids………………………………………….15
Effect of heat treatment on CEVd quantity…………………………………......16
CEVd sequence analysis in field survey……………………………………......17
CEVd-inoculation to cultivars of tomatoes…………………………………….17
Diagnosis of Tomato dwarf chlorotic viroid……………………………………18
Discussion……………………………………………………………………………20
CEVd causing symptoms in indicator plant and its distribution……………….20
Field survey and detection to citrus viroids…………………………………….21
Seasonal survey and the correspondence between viroid and temperature….....22
Heat treatment to CEVd-infected tomato……………………………..……….23
Sequence analysis of CEVd clones……………………………….…………….23
CEVd diagnosis in cultivars of tomatoes and TCDVd diagnosis………………24
Summary……………………………………………………………………......25
Tables……..……………………………………………………………………….26
Figures …………………………………………………………………………………29
Reference…………………………………………………………………….……….46
Appendix………………………………………………………………………………56


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