近年來，紅龍果已成為台灣市場重要之熱帶果樹，而田間調查結果顯示果園裡的植株幾乎全數帶有為害紅龍果之Potexvirus屬絲狀病毒，其中包含仙人掌X病毒(Cactus virus X, CVX)與紅龍果X病毒(Pitaya virus X, PiVX)。CVX普遍存在於多種仙人掌科植物，於2001年首度被記載可感染紅龍果，並造成輕微斑駁(mild mottling)病徵；而PiVX則由本實驗室於2008年發現、鑑定並命名，此二病毒於紅龍果上複合感染的現象非常普遍。為了瞭解CVX與PiVX在寄主植物體內是否有相互影響之情形，我們使用CVX與PiVX感染性選殖株為實驗材料，期望從複合感染時病毒複製之情形，以及在植株上之分布狀態，這兩層面探討兩病毒之關係。在CVX與PiVX對彼此複製之影響的研究上，我們從單細胞層次及組織層次進行探討，比較單獨及複合接種處理下兩病毒之RNA累積情形；使用之植物材料包含圓葉菸草(Nicotiana benthamiana)、白藜(Chenopodium quinoa)和紅龍果(Hylocereus undatus)之原生質體(protoplasts)，以及白藜與紅龍果植株。採用之分析策略為北方雜合分析法及反轉錄即時聚合酶鏈鎖反應，透過專ㄧ性RNA探針以及能區分CVX與PiVX之專一性引子對，偵測病毒之RNA累積。我們發現CVX與PiVX兩病毒複合感染於菸草原生質體、以及紅龍果原生質體與植株時，表現出協力作用；在感染白藜原生質體及植株時，則呈現拮抗作用。另一方面，我們構築了能表現螢光蛋白之病毒選殖株CVX-mCherry、CVX-EGFP及PiVX-EGFP，進行兩種病毒於植物體內分布之觀察。將CVX-mCherry與PiVX-EGFP複合接種至白藜或紅龍果植株上，經共軛焦螢光顯微鏡觀察，發現兩病毒於兩種寄主上皆表現出空間區隔之互斥現象。將上述結果與CVX-mCherry / CVX-EGFP以及CVX-mCherry / CymMV-EGFP複合感染白藜之結果比較，顯示此互斥現象在白藜組織上與病毒序列相似度呈正相關；相較之下，病毒在紅龍果植株表現之空間區隔現象是否與序列相似度有關，還有待確認。若將表現不同螢光基因之病毒以轉錄體型式接種至紅龍果原生質體，則可發現不少細胞皆同時表現兩種螢光訊號。綜合上述結果，我們認為當CVX與PiVX共同感染紅龍果時，應可進入同一細胞，並共創對彼此複製有利之環境，儘管於細胞間移動階段兩病毒並不容易移動至同一細胞內，如此的協力作用仍可能是田間紅龍果普遍受此二病毒複合感染的原因之一。此協力作用具寄主專一性，詳細機制還有待更多的研究探討釐清。

In recent years, pitaya has become an important tropical fruit in Taiwan markets. Field investigations showed that almost all pitaya plants in orchards are infected by viruses including two members of the genus Potexvirus, Cactus virus X (CVX) and Pitaya virus X (PiVX). CVX was first reported to infect pitaya, causing mild mottling, by Dr. R. F. Liou’s team in 2001. And PiVX was found, identified and named by our lab in 2008. Since mixed infections of CVX and PiVX on pitayas are very common, the aim of this study is to investigate whether there are interactions between these two viruses in host plants. The study was divided into two parts including viral replication and virus distribution in planta. To analyze viral RNA accumulation, the infectious clones of CVX and PiVX were singly or doubly inoculated to either protoplasts of tobacco (Nicotiana benthamiana), quinoa (Chenopodium quinoa) and pitaya (Hylocereus undatus) or seedlings of quinoa and pitaya. Through northern hybridization using specific RNA probes and RT-qPCR using specific primers of CVX and PiVX, we detected each viral RNA accumulation and found that these two viruses performed synergistic interaction when mixed infection on tobacco and pitaya but showed antagonistic interaction on quinoa. In the virus distribution study, viral infectious clones expressing different exogenous fluorescent proteins (CVX-mCherry, CVX-EGFP and PiVX-EGFP) were prepared and mechanically inoculated on leaves of quinoa and cotyledons of pitaya seedlings. According to the results of laser confocal fluorescence microscopy, CVX-mCherry and CVX-EGFP revealed stronger spatial separation than CVX-mCherry and PiVX-EGFP on quinoa inoculated leaves. Whereas, the signals of mix-inoculated CVX-mCherry and CymMV-EGFP showed completely overlapping on quinoa. The degree of mutual exclusion between two mix-inoculated viruses on quinoa is positively related to their sequence homology. However, the spatial separation observed on pitaya did not reveal relation with viral sequences and needs further study. When mix-inoculated with viral transcripts of CVX-mCherry / CVX-EGFP or CVX-mCherry / PiVX-EGFP, many pitaya protoplasts were found to express two fluorescence signals which means two viruses can infect the same cell during inoculation. According to these data, we propose that CVX and PiVX can enter the same cells simultaneously during mixed infection. After that, these two viruses create a friendly environment which is beneficial to each other. This may partially explain the common situation of mixed infection in field. The synergistic effect between CVX and PiVX is host specific, and the mechanism needs further experiments to clarify.

中文摘要………………………………………………………………………………...i
英文摘要……………………………………………………………………………...iii
壹、 前言
一、 Potexvirus屬病毒之介紹…………………………………………………......1
二、 紅龍果病毒之介紹………………………………………………………….5
三、 植物病毒之交互作用……………………………………………………….8
四、 研究動機…………………………………………………………………….11
貳、 材料與方法
一、 植物材料及栽種方式………………………………………………………13
二、 病毒cDNA株來源及質體DNA之中量製備……………………………....13
三、 生體外轉錄體之製備……………………………………………………….14
四、 原生質體之製備與接種
(一) 圓葉菸草原生質體之製備…………………………………………….15
(二) 白藜原生質體之製備………………………………………………….16
(三) 紅龍果原生質體之製備……………………………………………….16
(四) 計算原生質體數量…………………………………………………….17
(五) 原生質體之接種……………………………………………………….17
五、 白藜及紅龍果植株之機械接種…………………………………………….18
六、 植物全RNA之純化…………………………………………………………18
七、 探針設計與製備
(一) 探針設計及專一性測試……………………………………………….19
(二) RNA探針之製備………………………………………………………20
八、 北方雜合分析法…………………………………………………………….21
九、 反轉錄即時聚合酶鏈鎖反應……………………………………………….22
十、 帶有不同螢光基因之CVX與PiVX病毒株之構築………………………..23
十一、 螢光病毒載體於植物體之接種及顯微觀察………………………….24
參、 結果
一、 CVX與PiVX於單細胞之交互作用
(一) 於圓葉菸草原生質體………………………………………………….26
(二) 於白藜原生質體……………………………………………………….27
(三) 於紅龍果原生質……………………………………………………….27
二、 CVX與PiVX於植物體之交互作用
(一) 於白藜植株…………………………………………………………….28
(二) 於紅龍果植株………………………………………………………….28
三、 CVX-EGFP/mCherry、PiVX-EGFP之分布
(一) 螢光病毒載體測試…………………………………………………….29
(二) 於白藜植株(接種葉)…………………………………………………...29
(三) 於紅龍果植株………………………………………………………….31
(四) 於紅龍果原生質體…………………………………………………….31
肆、 討論
一、 複合感染有利CVX與PiVX於紅龍果及菸草細胞之RNA累積…………33
二、 CVX與PiVX之協力作用具寄主專一性…………………………………..35
三、 帶有不同螢光基因之CVX與PiVX於白藜接種葉上表現互斥作用……..37
四、 帶有不同螢光基因之CVX與PiVX於紅龍果組織及原生質體之表現…..38
伍、 參考文獻………………………………………………………………………….40
附圖…………………………………………………………………………………….48
附表…………………………………………………………………………………….68
附錄…………………………………………………………………………………….69

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