臺灣博碩士論文加值系統

Tospoviruses 乃一群可以經由薊馬傳播之植物病毒，受害之作物病徵通常極為明顯，對生長與品質造成嚴重影響。根據文獻報導目前有三種可能危害蘭花之 tospoviruses 存在；包括台灣已經發生之番椒黃化病毒（Capsicum chlorosis virus, CaCV）、蕃茄斑萎病毒（Tomato spotted wilt virus, TSWV）及尚未但可能入侵台灣之鳳仙花壞疽斑點病毒（Impetiens necrotic spot virus, INSV）。此三種病毒的檢測與鑑別技術便攸關此類病毒病害之發生與傳播是否能被精準的掌握與控制。傳統 tospoviruses 的鑑定是以其 S RNA 互補股所對應之核鞘蛋白（nucleocapsid protein，NP）為檢測之標的與分類之依據。本研究基於相關文獻指出 tospovirus S RNA 之正意股所對應之非結構性蛋白（Non-structural protein, NSs）會累積在感病細胞內形成內含體（inclusion），故擬嘗試探討以 NSs 蛋白作為此等病毒檢測或鑑定標的之可行性。同時針對此等病毒間其 NSs 蛋白之血清類緣關係進行比對，供檢測應用之參考。研究策略乃將感染本省蝴蝶蘭最普遍之 CaCV 及發生於荷蘭蝴蝶蘭上之 INSV 及 TSWV 所對應之 NSs 蛋白以細菌表達外源蛋白之技術，將其大量誘導純化並製備成多元抗體，進行血清學研究。另外將另一種普遍發生於台灣瓜類作物且與 CaCV 同屬 NP 蛋白第四血清型之西瓜銀斑病毒（Watermelon silver mottle virus, WSMoV）之 NSs 蛋白加以純化供血清類緣關係比較。根據上述四種 tospoviruses 之已知 S RNA 基因體序列，設計可分別增幅各病毒 NSs 基因之四組專一性引子對，將四種病毒之 NSs 基因加以增幅，並分別選殖於 pGEM-T Easy（Promega﹐USA）或pCRII-TOPO （Quiagen, USA）載體上，經分別解序並分析後證實利用NSs 基因序列分析所獲得之 tospovirus 親緣關係與演化趨勢結論與文獻上針對 NP 基因所做之相同分析結論幾近相符。分析發現同種病毒之 NSs 基因在核苷酸及胺基酸序列相同度（identity）均落於 94

Tospoviruses is a group of plant viruses characterized in their distinct thrips-borne transmissibility. There have been more than 30 different tospoviruses reported globally and most of them induce very severe symptoms on their host plant that result evident loss in yield and quality. Among the three tospoviruses known to infect Phalaenopsis, Capsicum chlorosis virus (CaCV) was currently endemic in Taiwan. The other two including Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) have not yet been found in Taiwan to infect Phalaenopsis. Therefore the latter two tospoviruses are considered as quarantine pathogens for Taiwan’s Phalaenopsis industry. Therefore, the accuracy of the techniques applied to the detection and diagnosis is a major concern on the surveillance and control of the these virus diseases of orchids. Traditionally, the nuclear capsid protein (NP) was used as the major target of detection and taxonomic criteria for tospoviruses. While recently there was literature suggesting the non-structure protein (NSs) encoded in the S-RNA of tospoviruses could be used an alternative target. This study was therefore aiming at clarification of the feasibility of using NSs as a target for quick differentiation of those Phalaenopsis-infecting tospoviruses. In addition, serological relationship of NSs proteins among tospoviruses was also studied due to its importance in application of NSs for virus detection. Our study took the approach of using bacteria expressed protein system to obtain purified NSs protein encoded by the three Phalaenopsis infecting tospoviruses for their antibody production. We also included Watermelon silver mottle virus (WSMoV), the type species of the 4th NP serogroup in tospoviruses, to study the serological variation among NSs proteins of tospoviruses. The study started by the design of primer pairs specific respectively to these tospoviruses to amplify their NSs genes, which were then cloned in pGEM-T Easy (Promega, USA) or pCRII-TOPO (Quiagen, USA) plasmids and sequenced. Analyses of these NSs gene sequences found that those viruses in the same NP serogroup were also closely related among their NSs proteins. The nucleotide and amino acid sequence identities within the same NP serogroup were 70-79

目錄
書名頁
碩士論文授權書
論文口試委員會審定書(中文版)
論文口試委員會審定書(英文版)
中文摘要 -------------------------------------------- Ⅰ
英文摘要 -------------------------------------------- Ⅴ
致謝 ------------------------------------------------ IX
目錄 ------------------------------------------------ XI
表目錄 --------------------------------------------- XVI
圖目錄 -------------------------------------------- XVII
壹、 前言 --------------------------------------------- 1
貳、 前人研究 ----------------------------------------- 5
一、蘭花之簡介 ------------------------------------ 5
二、台灣蘭花產業之發展現況 ------------------------ 5
三、蘭花病毒病害之發生與對產業的影響 -------------- 7
四、可能影響蘭花之番茄斑委病毒屬病毒之特性 -------- 8
（一）tospoviruses 之特性 ------------------------ 9
（二）番椒黃化病毒
（ Capsicum chlorosis virus﹐CaCV ）-------- 17
（三）番茄斑點萎凋病毒
（ Tomato spotted wilt virus﹐TSWV ）-------- 18
（四）鳳仙花壞疽斑點病毒
( Impatiens necrotic spot virus, INSV ) ------ 20
（五）西瓜銀斑紋病毒
( Watermelon silver mottle virus, WSMoV ) --- 20
參、材料與方法 --------------------------------------- 22
一、病毒來源 ------------------------------------- 22
二、四種病毒 NSs 基因之增幅、選殖與序列分析 ------ 22
（一）設計四種病毒 NSs 基因引子對 ------------ 22
（二）反轉錄聚合酶鏈鎖反應（RT-PCR） ---------- 22
（三）電泳分析 -------------------------------- 23
（四）PCR 產物之純化 ------------------------- 23
（五）PCR 增幅片段之選殖 --------------------- 24
（六）選殖株之篩選 ---------------------------- 25
（七）質體之純化 ------------------------------ 26
（八）酵素剪切 -------------------------------- 26
（九）定序 ------------------------------------ 27
三、四種 tospoviruses NSs 基因表現載體構築---------- 27
（一）限制酶處理（restriction enzyme treatment）----- 27
（二）接合反應（ligation）------------------------ 27
（三）四種 tospoviruses NSs 基因表現載體之構築
與轉型 --------------------------------- 28
四、NSs基因選殖 --------------------------------- 30
（一）四種 tospoviruses NSs蛋白之選殖策略 ------- 30
（二）構築四種 tospoviruses NSs 基因之表現載體
之引子對設計 --------------------------- 30
五、細菌表現蛋白之誘導與純化 --------------------- 32
（一）表現蛋白之誘導 -------------------------- 32
（二）西方轉漬（Western blotting）分析 ------------ 33
（三）大量表現蛋白之純化 ---------------------- 34
（四）表現蛋白質之回收與純化 ------------------ 34
六、免疫抗血清製備 ------------------------------- 35
（一）免疫抗血清製備 -------------------------- 35
（二）SDS免疫擴散反應測試 -------------------- 35
（三）免疫球蛋白（immunoglobin, IgG）製備 ------- 36
七、酵素結合免疫吸附法（ELISA）-------------------- 36
肆、結果 ---------------------------------------------- 38
一、四種 tospoviruses NSs 基因之增幅與選殖 --------- 38
二、四種 tospoviruses NSs 基因之定序與親緣分析 ----- 38
三、利用細菌表現載體表達四種 tospoviruses 之 NSs基因
--------------------------------------------- 39
（一） 四種 tospoviruses NSs 表現載體之構築
--------------------------------------- 40
（二） 表現載體之轉型與篩選
--------------------------------------- 40
（三） 表現蛋白之誘導及電泳分析 --------------------------------------- 41
（四） 四種 tospoviruses NSs 不同構築組合之大量表現
與純化 -------------------------------- 42
（五） NSs 蛋白抗體之血清學特性比較 --------------------------------------- 43
1. SDS-免疫擴散法（SDS-immunodiffusuin test）
-------------------------------------- 43
2.西方轉漬（Western blotting）試驗 ---------- 43
3.ELISA反應試驗 ------------------------ 44
（六）應用 NSs 蛋白抗血清於 CaCV 病毒檢測之探討
--------------------------------------- 45
1. 不同稀釋倍數下之蝴蝶蘭病汁液與 CaCV NSs
血清在 ELISA 下之反應 ---------------- 45
2.應用 ELISA 檢測蝴蝶蘭病 -------------- 45
3.蝴蝶蘭黃化輪斑病徵之不同位置與 CaCV NSs
蛋白累積之關係 ----------------------- 46
伍、討論 ---------------------------------------------- 47
陸、參考文獻 ------------------------------------------ 54
表---------------------------------------------------- 70
圖---------------------------------------------------- 75








表目錄
表一、增幅四種 tospovirus NSs 基因所使用之引子對名稱、
序列、增幅片段之預期長度及黏合反應溫度 ---------- 70
表二、構築六種不同重組表現蛋白所使用之引子對名稱、
序列、增幅片段之預期長度及黏合反應溫度 ---------- 71
表三、篩選具嵌入序列之 pGEM-T Easy 及 pCR 2.1-TOPO
載體之成功轉型菌株所使用之引子對 --------------- 72
表四、四種 tospoviruses NSs 基因與 GenBank 上其他
Tospovirus 屬病毒 NSs 基因全長序列之核苷酸序
列相同度比對 ----------------------------------- 73
表五、四種 tospoviruses NSs 基因與 GenBank 上其他
Tospovirus 屬病毒 NSs 基因全長序列之胺基酸序
列相同度比對 ----------------------------------- 74







圖目錄
圖一、成功選型 pGEN- T Easy 載體之 CaCV NSs 基因菌株
篩選-------------------------------------------- 75
圖二、成功選型 pCR 2.1-TOPO 載體之 TSWV NSs 基因菌株
篩選 ------------------------------------------- 76
圖三、成功選型 pCR 2.1-TOPO 載體之 INSVV NSs 基因菌株
篩選 ------------------------------------------- 77
圖四、成功選型 pCR 2.1-TOPO 載體之 WSMoV NSs 基因菌
株篩選 ----------------------------------------- 78
圖五、Tospovirus NSs-核苷酸序列與 GenBank 上已登錄之演化
親源樹狀圖 ------------------------------------- 79
圖六、Tospovirus NSs-胺基酸序列與 GenBank 上已登錄之演化
親源樹狀圖 ------------------------------------- 80
圖七、CaCV 全長 NSs 基因序列構築於表現載體 pET-28b（＋）
上之上下游相關位置與序列 ----------------------- 81
圖八、CaCV NSs 基因之 N 端序列構築於表現載體 pET-28b（＋）
上之上下游相關位置與序列 ----------------------- 82
圖九、CaCV NSs N 端部分序列構築於表現載體 pET-28b（＋） 上
之上下游相關位置與序列，此轉譯架構含蘭花 CymMV 鞘
蛋白之 N 端 177 bp 及 CaCV NSs 之 N 端 519 bp --- 83
圖十、TSWV 全長 NSs 基因序列 構築於表現載體 pET-28b（＋）
上之上下游相關位置與序列 ----------------------- 85
圖十一、INSV 全長 NSs 基因序列構築於表現載體 pET-28b（＋）
上之上下游相關位置與序列 --------------------- 86
圖十二、WSMoV 全長 NSs 基因序列構築於表現載體 pET-28b（＋）上之上下游相關位置與序列 ----------------- 87
圖十三、成功轉型表現載體 CaCV NSFL/pET-28b（＋）
之菌株篩選 ----------------------------------- 88
圖十四、成功轉型表現載體 CaCNSN/pET-28b（＋）
之菌株篩選------------------------------------ 89
圖十五、成功轉型表現載體 CyCaNSN/pET-28b（＋）
之菌株篩選 ----------------------------------- 90
圖十六、成功轉型表現載體 TSWV NSFL/pET-28b（＋）
之菌株篩選 ----------------------------------- 91
圖十七、成功轉型表現載體 INSV NSFL/pET-28b（＋）
之菌株篩選 ----------------------------------- 92
圖十八、成功轉型表現載體 WSMoV NSFL/pET-28b（＋）
之菌株篩選 ----------------------------------- 93
圖十九、利用電泳（SDS-PAGE）及西方轉漬（Western blotting）分析表現蛋白載體 CaNSFL/pET-28b(＋) 經 IPTG 誘導後之表現蛋白---------------------------------- 94
圖二十、利用電泳（SDS-PAGE）及西方轉漬（Western blotting）分析表現蛋白載體 CaNSN/pET-28b(＋) 經 IPTG 誘導後之表現蛋白 --------------------------------- 96
圖二十一、利用電泳（SDS-PAGE）及西方轉漬（Western blotting）分析表現蛋白載體 CyCaNSN/pET-28b(＋) 經 IPTG 誘導後之表現蛋白 -------------------------- 98
圖二十二、利用電泳（SDS-PAGE）及西方轉漬（Western blotting）分析表現蛋白載體 TSNFL/pET-28b(＋) 經 IPTG 誘導後之表現蛋白 ---------------------------- 100
圖二十三、利用電泳（SDS-PAGE）及西方轉漬（Western blotting）
分析表現蛋白載體 INNSFL/pET-28b(＋) 經 IPTG 誘
導後之表現蛋白 ---------------------------- 102
圖二十四、利用電泳（SDS-PAGE）及西方轉漬（Western blotting）
分析表現蛋白載體 WSNSFL/pET-28b(＋) 經 IPTG
誘導後之表現蛋白 -------------------------- 104
圖二十五、CaCV 三種不同構築所表現之重組 NSs 蛋白於純化前
後之分子量與免疫特性確認 ------------------ 106
圖二十六、三種不同 tospoviruses 包括 TSWV、INSV 及 WSMoV
之全長 NSs 基因序列構築載體所表現之重組非結構
性蛋白於純化前後之分子量與免疫特性確認 ---- 108
圖二十七、CaCV 三種不同構築方式所表現之重組 NSs蛋白之抗
原性確認 ---------------------------------- 110
圖二十八、利用 CaCV NSs 蛋白部分 N 端序列所製備之抗體（anti-CaNSN）於西方轉漬分析與其他三種 tospoviruses（TSWV、INSV 及 WSMoV）純化 NSs 表現蛋白及感染也片反應之結果 ---------------- 112
圖二十九、利用 TSWV NSs 蛋白序列所製備之抗體（anti-TSNSFL）於西方轉漬分析與其他三種 tospoviruses（CaCV、INSV 及 WSMoV）純化 NSs 表現蛋白及感染也片反應之結果 --------------- 114
圖三十、利用 TSWV NSs 蛋白序列所製備之抗體（anti-INNSFL）
於西方轉漬分析與其他三種 tospoviruses（CaCV、INSV
及 WSMoV）純化 NSs 表現蛋白及感染也片反應之結果----------------------------------------------- 116
圖三十一、利用 SDS 免疫擴散法分析 CaCV、TSWV 及 INSV 重組NSs蛋白抗血清之血清反應特性 ---------- 118
圖三十二、利用 CaV 部分 NSs N 端序列所製備之抗體於酵素連結免疫吸附法（ELISA）與其同源及三種異源 tospoviruses 之純化 NSs 表現蛋白之反應結果 ------------------------------------------- 119
圖三十三、利用 TSWV NSs 序列所製備之抗體於酵素連結免疫吸
附法（ELISA）與其同源及三種異源 tospoviruses 之純
化NSs 表現蛋白之反應結果 ----------------- 120
圖三十四、利用 INSV NSs 序列所製備之抗體於酵素連結免疫吸
附法（ELISA）與其同源及三種異源 tospoviruses 之純化NSs 表現蛋白之反應結果 --------------- 121
圖三十五、應用 CaCV NSs 蛋白部分 N 端序列所製備之抗體於酵素連結免疫吸附法（ELISA）檢測不同稀釋倍數下之蝴蝶蘭黃化輪斑病徵組織研磨之反應結果 ------ 122
圖三十六、利用酵素連結免疫吸附法（ELISA 及 RT-PCR）測試蝴蝶蘭不同黃化輪斑部位中 NSs 及 NP 蛋白與基因之累積 ------------------------------------ 123
圖三十七、利用三種不同 tospoviruses 之 NSs 及 NP 蛋白抗體於酵素連結免疫吸附法下檢測八株呈現典型黃化輪斑病徵之蝴蝶蘭樣品之結果 -------------------- 125

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