洪挺軒臺灣大學：植物病理與微生物學研究所廖奕晴Liao, Yi-ChenYi-ChenLiao2007-11-272018-06-292007-11-272018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/58070近年來，觀察台灣流行於田間木瓜輪點病所引起之病徵與過去報告已經有所變異，病徵傾向嚴重化而容易產生畸型葉，且夏季病徵依然嚴重，顯示田間病毒系統可能產生改變。因此為了再次確認目前台灣田間PRSV的系統，由屏東採集了各種不同病徵型之木瓜病葉，並在白藜上進行三代單斑分離，再回接至感病種台農二號木瓜，依據在接種台農二號木瓜上所引起之不同病徵而可得到三個不同病徵型之病毒分離株，作為目前PRSV系統之代表。嚴重嵌紋系統(PRSV-SM train)可引起葉片嚴重嵌紋之病徵，此一系統為過去台灣主要之流行系統；畸形系統(PRSV-DF strain)主要可引起葉片嚴重扭曲變形之病徵，此系統為目前台灣南部主要木瓜產區所流行之系統。壞疽嵌紋系統(PRSV-SMN strain)引起葉片嵌紋，伴隨有壞疽的病斑出現並且容易快速萎凋，其病害嚴重度似乎更甚於其他系統，為一極具破壞力之系統。為釐清此三種PRSV系統之生物性與分子性差異，本論文除細加探討三系統於木瓜寄主上病徵之差別外，也配合電子顯微鏡的觀察，了解各系統在木瓜組織所引起之細胞組織學上的變異；並將三種不同病毒系統進行近全長核酸解序且比較其基因上的差異度（Identity）。電顯之細胞病理學觀察圓柱型形內含體（cylindeical inclusion）結果發現，PRSV-DF在細胞質有大量風車狀(pinwheel)與捲軸狀(scrolls) 內涵體，以及少量長直狀層板（laminated aggregates）的內涵體；PRSV-SMN除可見到大量風車狀 (pinwheel)、捲軸狀(scrolls) 內涵體外，尚可見短彎曲層板狀的內涵體，另還發現PRSV-SMN可在細胞質累積龐大之黑色帶狀病毒集合體；PRSV-SM系統可見到風車狀及大量捲軸狀的內涵體外，亦可見到大量的短直層板狀的內涵體。在序列分析上，除兩端點之極少部分序列外，此三個病毒系統已解序完成約9.8K。經由各基因之比對結果可發現，PRSV-SMN在大多數基因序列上與PRSV-SM、PRSV-DF差異性較大，在CP、NIb、NIa-Pro、P3、Hc-Pro、P1等基因上皆有明顯之變異；而目前台灣優勢的PRSV-DF與過去優勢PRSV-SM其基因則較相似。為了研發一般性或具系統特異性的RT-PCR快速偵測法，以應用於快速系統鑑別與田間感染生態研究，特以基因體序列分析結果作為基礎，先選出三個系統皆能通用的引子對”PRSV 857”應用於PRSV普測。另外選出三條特異性引子對 PRSV-SM517、PRSV-DF703與PRSV-SMN467，可分別對SM、DF與SMN系統做專一性RT-PCR增幅。經數百個樣品的測試已經證實其可正確而穩定的鑑別出不同PRSV系統。為了解不同病毒系統在不同的木瓜品種體內增殖情形、病徵表現型，以及各系統之交互作用關係，本論文挑選了目前台灣優勢之PRSV-DF與具潛在毀滅性之PRSV-SMN個別單獨或混合接種於「台農二號」、「台大十號」與「台大十一號」木瓜品系，發現在接種後兩星期病徵極不明顯之潛伏期即可以RT-PCR偵測到病毒複製的訊號，經6星期追蹤病毒複製及病徵演變情形，可證明台大十一號木瓜對於PRSV-DF系統具有非常高之耐受度，台大十號對PRSV-DF則為中度耐受度，目前最廣受台灣農民栽培之耐病性台農二號則最為罹病，RT-PCR的偵測結果與病徵之表現呈現一致性。另外亦證實同時接種PRSV-DF與PRSV-SMN至木瓜上，利用專一性引子對的偵測可發現PRSV-SMN複製優勢會抑制PRSV-DF之複製發展，而使得PRSV-DF之RT-PCR訊號會隨著感染時間慢慢減弱，似乎說明PRSV不同系統之間存在著相互競爭之關係。為能更經濟而有效地偵測PRSV，本論文亦從事單元抗體的研發，且已篩選到對矮南瓜純化的病毒具有高度反應之三個抗體細胞株，其在罹病與健康的矮南瓜純化液之間具有顯著差異性，但對罹病木瓜樣本的敏感度尚嫌不足；進行西方印漬法證實這些細胞株可以與病毒鞘蛋白36 KDa的位置具有強烈訊號，期再改進木瓜萃取緩衝液，以便實際應用在木瓜之偵測。Papaya ringspot is the most important disease of papaya in the world. It is caused by Papaya ringspot virus (PRSV, Potyvirus, Potyviridae). Compared to traditional symptoms in papayas incited by PRSV, present symptoms observed in the fields of Taiwan seem to be significantly different. It indicates that strain variation of PRSV probably occurs in recent years. In order to investigate current PRSV strains in Taiwan, disease samples with different symptom types were collected from orchards in Pingtung. Through 3-time successive isolation of single local lesions formed in Chenopodium quinoa, three distinct strains were obtained according to their induced symptom types in papayas. The SM strain, causing leaf severe mottling, was a common strain in Taiwan in the past; the DF strain, causing severe mottling with leaf-deformation, has recently become a new dominant strain; and the SMN strain, causing necrotic severe mottling and quick decline, is considered to be the most potentially destructive strain. For comparative genomic analysis, almost full lengths of genomic sequences of 3 PRSV strains have been determined. Molecular data demonstrate that genomic sequences are somewhat different among three strains. The one-step RT-PCR assay with strain-specific primers was developed for the detection and identification of various PRSV strains. These assays were also applied to monitor PRSV replicating in different papaya cultivars such as TN2, NTU10 and NTU11. The results in the inoculation test showed that PRSV could be accurately detected by RT-PCR even during the incubation period. Both symptom observation and RT-PCR detection revealed that the NTU11 cultivar showed high tolerance to PRSV (DF strain); NTU10 showed medium tolerance; and TN2 is a susceptible cultivar. In addition, the interaction tests between PRSV DF and SMN strains were also conducted in this thesis. The results indicated that the hosts infected with both DF and SMN produced more severe symptoms than those infected with individual DF or SMN. However, the RT-PCR assays with strain-specific primers demonstrated that SMN could interfere the replication of DF when they simultaneously infected papaya hosts. On the other hand, cytopathological studies in the PRSV-infected papayas (Tainoung NO.2) were further conducted by electron microscopy. DF induces pinwheel and scroll inclusions as well as few long laminated aggregates of virions in the diseased cells; SMN induces the pinwheels, scrolls, short curved laminated aggregates as well as huge black banded inclusions near nucleus；SM induces pinwheels, scrolls and short laminated aggregates. For more economic and efficient PRSV detection, the monoclonal antibody（McAb）against PRSV was also developed in the thesis. By using purified virus preparation of DF strain from Zucchini, Three stable McAb cell lines were obtained through virus purification, mouse-immunization and hybridoma technology. Positive results were obtained in the ELISA test with McAbs for detection of purified PRSV particles, and western blot also showed that the developed McAbs could react with coat protein of PRSV. However, the ELISA test in infected papaya tissues could not obtain satisfactory results. The extraction buffer of PRSV will be further improved for the adequate application of the McAbs in PRSV detection in host plants.中文摘要.............................................................................................................1 英文摘要……………………………………………………………………….3 目錄…………………………………………………………………………….5 壹、前言………………………………………………………………………..8 貳、前人研究………………………………………………………………….11 叁、材料與方法……………………………………………………………….17 一、試驗植物之來源……………………………………………………..17 二、田間病毒材料之收集及木瓜輪點病毒系統之分離與鑑別………..17 三、病毒之純化…………………………………………………………..17 1. 病毒純化之步驟………………………………………………...18 2. 電泳分離法……………………………………………………...18 四、穿透式電子顯微鏡觀察法…………………………………………19 1. 葉切片浸展法（Leaf dipping）…………………………………19 2. 超薄切片( Ultra-thin section)…………………………………...19 五、反轉錄聚合酶連鎖反應(RT-PCR)快速偵測木瓜輪點病毒（PRSV）技術之研發、改良與標準化……………………………………...21 1. RNA 抽取法…………………………………………………….21 2. RT-PCR 之增幅………………………………………………....22 3. PCR產物膠體分析………………………………………………23 六、基因全序列的選殖及具系統專一性引子對之設計………………24 七、木瓜輪點病毒系統感染生態試驗…………………………………24 八、單元抗體之製備……………………………………………………25 1. 動物免疫………………………………………………………...25 2. 培養機之製備…………………………………………………...25 3. 細胞融合……………………………………………………….. 26 4. 分泌抗體細胞生長穴之篩選………………………………….. 27 5. 單株融合瘤細胞系的產生與篩選…………………………….. 28 6. 融合瘤細胞之冷凍保存與解凍……………………………….. 28 7. 單元抗體之生產……………………………………………….. 29 8. 西方印漬法…………………………………………………….. 29 肆、結果……………………………………………………………………...31 一、分離株的收集與純系化……………………………………………31 二、病毒系統之生物性鑑別……………………………………………31 1. 病毒系統在藜葉上之病徵表現…………………………….......31 2. 病毒系統在矮南瓜上之病徵表現…………………………......32 3. 病毒系統在木瓜上之病徵表現…………………..………….....32 三、SMN、SM與DF三個不同系統之細胞病理學觀察………………...32 四、SMN、SM及DF三系統之近全長定序及序列分析………………...33 五、RT-PCR快速偵測技術之研發及改良……………………………..33 1. 核酸萃取方法之比較…………………………………………...33 2. Two step RT-PCR與one step RT-PCR增幅反應之比較……….34 六、研發具系統特異性的木瓜輪點病毒RT-PCR偵測法............... .... 34 1. 引子對之設計…………………………………………………...34 2. 系統特異性引子對之PCR黏合溫度試驗… ………………….35 3. 多元性RT-PCR（multiple RT-PCR）之偵測…… …………….36 七、不同木瓜輪點病毒系統感染的生態試驗..................... ...................36 1. 畸型嵌紋系統（DF）對於不同木瓜品系之致病性… ………..37 2. 壞疽嵌紋系統（SMN）對於不同木瓜品系之致病性…………37 3. DF/SMN複合感染對於不同木瓜品系之致病性……………38八、木瓜輪點病毒之單元抗體製備.......................................................39 1. 木瓜輪點病毒之純化…………………………………………...39 2. 單元抗體之製備………………………………………………...39 3. 西方印漬法（Western blot）…………………………………... 39 伍、討論………………………………………………………………………41 陸、參考文獻…………………………………………………………………48 柒、表…………………………………………………………………………55 捌、圖…………………………………………………………………………64 玖、附錄………………………………………………………………………90en-US系統分子鑑別木瓜輪點病毒Papaya ringspot potyvirusstrainmolecular differentiationother