臺灣博碩士論文加值系統

蝴蝶蘭是台灣花卉栽培業最重要的作物，無論內銷或外銷之種苗、切花、盆花皆有很大的市場，病毒病害是蘭花栽培業的主要障礙，其中又以喜姆比蘭嵌紋病毒（Cymbidium mosaic virus；CyMV）是公認影響蘭花最嚴重之病毒，防疫檢疫上有賴快速準確的病毒偵測診斷方法。本研究發展一不需要將組織均質化或萃取RNA，直接將樣本進行one-step RT-PCR反應，在三小時內完成偵測蘭花病毒的方法。在two step RT-PCR中以0.2 M lysine處理葉片汁液，抑制汁液中的去氧核醣核酸水解酵素（DNase），進行two step RT-PCR，汁液在稀釋105倍後仍能測到病毒存在。樣本加入template preparation solution（TPS），可使one step RT-PCR測得10-7 µg / µl之CyMV RNA，TPS可抑制植物組織中的酚類化合物氧化，減少多酚類化合物的產生，使PCR不受抑制。以此one step RT-PCR由台灣三個蘭園取218個蝴蝶蘭植株樣本，包括11個品種，其中104株檢測出感染CyMV病毒，檢出率最高的蝴蝶蘭品種為滿天紅及婚宴，分別為100%及93%；其次為小史帝夫和夕陽紅，分別為50%及10%；最低的為莎拉黃金及台糖火鳥，分別為6%及0%。另外對檢出病毒樣本CyMV coat protein gene片段以HhaI進行限制片段長度多型性分析，可將CyMV病毒株區分為A、B、C三群，滿天紅發現有同時被兩群（A/C或B/C）病毒株感染，夕陽紅和婚宴上檢出為A群，莎拉黃金為C群，小史帝夫則是A和B群各半。將35株台灣CyMV病毒株和40株已發表之CyMV病毒株的coat protein gene片段序列以NJ method建構系統樹，分析其類緣關係，系統樹有7個分支，台灣三個蘭園的CyMV病毒株在其中5個分支內都有出現，應是進出口貿易頻繁所帶進外來物種，造成本省CyMV strain-type的多樣性。牛記和台大的滿天紅的病毒株在第1 分支，與泰國和新加坡病毒株序列相近，一心的滿天紅病毒株則在第3分支，此分支尚包括台灣夕陽紅、婚宴和小史帝夫等品種，為廣佈三個蘭園與四個品種的重要病毒分支，且東南亞等其他國家尚未發現，可能屬於台灣本土之CyMV病毒株系。

Phalaenopsis is one of the most important ornamental crop in Taiwan. Cymbidium mosaic virus (CyMV) is the economically most important virus that affects the health and quality of orchid plants. The key point of health inspection and quarantine is a fast and highly sensitive method for the diagnosis of virus. The objective of this study was to establish a rapid, simple and sensitive one-step RT-PCR method that detects CyMV without nucleic acid isolation. Lysine inhibits the activity of DNase in the sap for detection of CyMV by two steps RT-PCR. Leaf sap of infested orchid was diluted and CyMV of 105 X sap dilution could be detected. To simplify the protocol, template preparation solution（TPS）was added to sap sample for detection of CyMV by one step RT-PCR. One-step RT-PCR could be used to detect 10-7 µg / µl CyMV RNA. TPS reduced the oxidation of phenolic compound to polyphenolic compound, which may interfere with PCR or RT-PCR. The application of the one step RT-PCR was use for the detection of 218 phalaenopsis plants including 6 cultivars from three orchid gardens in Taiwan, and there were 104 plants infected by CyMV. The detection rate of Phalaenopsis cultivars RedSky, Wedding, LittleBoy, Alisan, Salagold and Taisuco Firebird were 100%, 96%, 50%, 10%, 10%, and 0%, respectively. RFLP analysis of PCR products（CyMV coat protein gene）using HhaI grouped three subgroups. And some plants were doubly infected by subgroup A/C or B/C. Then, using the coat protein gene sequence of 35 Taiwan CyMV isolates and the published CyMV isolates I constructed the phylogenetic tree by neighbor-joining method. The phylogenetic tree had 7 clades. CyMV isolates from three orchid gardens in Taiwan spread in 5 clades. It is probably because the virus was imported to Taiwan together with orchids increasing the diversity of CyMV strain-type. The isolates from Niou-Ji and Tai-Da RedSky were in the No.1 clade, and close to Thailand and Singapore isolates. The isolates from I-Hsin RedSky were in the No.3 clade, also Alisan, Wedding and LittleBoy. So it is an important virus clade which included 4 cultivars from three orchid gardens and without any CyMV isolate from Southeast Asia. This clade could be the indigenous CyMV strain in Taiwan.

目 錄
目錄 i
表目錄 iv
圖目錄 v
中文摘要 I
英文摘要 III
前言 1
研究目的 10
材料與方法 11
一、供試材料來源及處理 11
（一）供試病毒與罹病蝴蝶蘭植株 11
（二）病毒接種 11
（三）病毒全量核酸萃取 11
（四）萃取罹病植株之全量RNA 12
（五）田間採樣 13
二、不同反轉錄-聚合酵素連鎖反應偵測方法之比較 14
（一）核酸引子的製作 14
（二）Two-step RT-PCR 14
1. 以純化之RNA進行two-step RT-PCR 14
2. 以蘭葉汁液進行two-step RT-PCR 15
（三）One-step RT-PCR 15
1. 以純化之RNA進行one-step RT-PCR 15
2. 以蘭葉汁液進行one-step RT-PCR 16
（四）PCR產物膠體電泳分析 17
三、田間CyMV病毒株之類緣關係分析 17
（一）限制片段長度多型性分析 17
1. 選取內切酵素 17
2. 內切酵素作用 18
3. RFLP產物膠體電泳分析 18
（二）CyMV cp gene序列的獲得 18
1. PCR產物直接解序 18
2. Cloning 18
a. 勝任細胞的製備 19
b. 接合作用 19
c. 轉形作用 20
d. 檢查插入質體的片段 20
（三）建構系統樹 20
四、核酸探針雜合偵測法 21
（一）RT-PCR產物的純化 21
（二）Digoxigenin（DIG）核酸探針之標識 21
（三）雜合反應 22
結果 24
一、接種測試 24
二、反轉錄聚合酵素連鎖反應 24
（一）Two-step RT-PCR偵測法 24
以帶毒蘭葉汁液直接進行two step RT-PCR 24
（二）One-step RT-PCR偵測法 26
三、核酸探針雜合偵測法 27
四、蘭園樣本檢測 28
（一）牛記蘭園採樣樣本檢測 28
（二）牛記、一心、台大蘭園採樣樣本檢測 28
五、蘭園病毒株系之類緣關係分析 28
（一）選取內切酵素對CyMV cp gene進行RFLP分析 28
（二）牛記蘭園樣本RT-PCR-RFLP分群結果 29
（三）牛記、一心、台大蘭園採樣樣本RT-PCR-RFLP分群結果 29
（四）建構類緣關係樹 30
討論 33
參考文獻 43





表目錄

表一、以核酸探針雜合偵測法與one step RT-PCR偵測感染CyMV之蘭葉汁液及純化之CyMV RNA靈敏度之比較 49
表二、牛記、一心、台大蘭園採樣樣本CyMV檢測結果 50
表三、已發表之CyMV cp gene區域進行HhaΙ限制酵素分析 51
表四、牛記、一心和台大蘭園CyMV病毒株系RFLP分群結果 53
















圖目錄

圖一、接種CyMV之蝴蝶蘭 54
圖二、以病葉汁液進行two step RT-PCR 55
圖三、葉片汁液以不同濃度lysine處理後，進行RT-PCR之比較 56
圖四、葉片汁液以不同體積lysine處理後，進行RT-PCR之比較 57
圖五、以lysine處理的two step RT-PCR之靈敏度測試 58
圖六、以病葉汁液進行one step RT-PCR 59
圖七、以lysine處理葉片汁液後進行one step RT-PCR 60
圖八、葉片汁液及lysine對one step RT-PCR的影響 61
圖九、帶毒的葉片汁液以TPS處理後進行one step RT-PCR 62
圖十、以TPS處理的one step RT-PCR之靈敏度測試 63
圖十一、使用455 bp核酸探針進行核酸雜合反應偵測病葉汁液中之CyMV 64
圖十二、牛記蘭園11種蝴蝶蘭品種58個葉片樣本檢測結果 65
圖十三、牛記蘭園樣本CyMV cp gene以HhaI作用後之圖譜 66
圖十四、採樣分離之CyMV cp gene序列解序後與已發表之序列以NJ method製作之親緣關係樹 67

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