Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31433
標題: 矮南瓜黃化嵌紋病毒之協同與鞘蛋白基因上突變之弱系病毒被蚜蟲傳毒能力及具交互保護能力之評估與在台灣危害胡瓜之甜瓜黃斑病毒之鑑定
Aphid Transmissibility and Cross-Protection Eeffectiveness of Attenuated Mutants of Zucchini yellow mosaic virus with Double Mutations at HC-Pro and CP Genes, and Characterization of Melon yellow spot virus Infecting Cucumber ( Cucumis sativus L.) in Taiwan
作者: 趙佳鴻
Chao, Chia-Hung
關鍵字: cross-protection
交互保護
aphid transmissibility
double mutations
ZYMV
cucurbit plants
Tospovirus
MYSV
cucumber
Taiwan
蚜蟲傳播能力
雙重突變
矮南瓜黃化嵌紋病毒
葫蘆科作物
番茄斑尾病毒屬
甜瓜黃斑病毒
胡瓜
台灣
出版社: 植物病理學系所
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摘要: 矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus;ZYMV) 屬於馬鈴薯Y群病毒 (the genus Potyvirus),可感染超過 20 種之葫蘆科作物 (cucurbits),主要包括有甜瓜 (Cucumis melo L.)、胡瓜 (C. sativus L.)、西瓜 [Citrullus lanatus (Thunb) Matsum & Nakai] 及南瓜 (Cucurbita spp.),被認為是瓜類作物生長期間最大的危害因子,造成全世界葫蘆科作物嚴重的經濟損失。該病毒可藉由蚜蟲以非永續性之方式傳播,病毒鞘蛋白 (coat protein, CP) 及病毒協同性蛋白 (helper component-protease, HC-Pro) 參與該病毒之蚜蟲傳播。位在鞘蛋白N 端的三個胺基酸 (DAG motif) 被報導與病毒的專一性 (virus-specific function) 或寄主-媒介昆蟲-病毒 (host-vector-virus) 的作用有關,而DAG為一高保留序列,經過許多實驗証實鞘蛋白的 DAG係HC-Pro結合之位置與蚜蟲傳播病毒有關。為了能有效防治此病毒,本研究室已成功在ZYMV病毒之協同蛋白基因上進行定點突變,研發出病徵減弱之ZYMV輕症病毒ZAC,經實驗證明該輕症病毒能有效提供矮南瓜對強症病毒之抗性,具有應用於預防該病毒危害葫蘆科作物的潛力。本研究則進一步測定此輕症病毒被蚜蟲傳播能力;此外,為了提升該輕症病毒未來應用於田間的可能性與生物安全性,以相同的策略將另一個參與蚜蟲傳播病毒之鞘蛋白基因的DAG motif上進行定點突變,以研發喪失蚜蟲傳播能力之雙重基因定點突變輕症病毒,此輕症病毒可提供予農友作為預防重症病毒系統危害瓜類作物之生物防治工具,此輕症病毒具有潛力作為一種經濟、有效、且對環境友善的病毒病害生物防治策略。 本論文總共分成四個章節,第一章為前人研究,主要蒐集整理近幾年與本研究相關之參考文獻並概述本論文之目的與內容,第二章至第四章為主要架構。應用輕症矮南瓜黃化嵌紋病毒行交互保護來預防強症矮南瓜黃化嵌紋病毒在瓜類作物上造成的經濟損失。在第二章中,為了探討此輕症病毒在田間是否被蚜蟲傳播,進而對其他作物造成影響,因此進行蚜蟲傳播之實驗。首先飼養蚜蟲及利用蚜蟲傳播病毒技術來測定輕症病毒之蚜蟲傳播特性。結果顯示該輕症病毒的複製能力與重症病毒並無差異,且能提供作物良好預防重症病毒的危害,而且也無法被蚜蟲傳播,而且此輕症病毒在三種重要瓜類作物 (胡瓜、東方甜瓜、西瓜) 上具有相當優異之保護效果。在第三章中,為了在田間運用此輕症病毒系統能更穩定地達到預防效果,及降低生物安全性之風險,並且將輕症病毒運用在溫室內重要瓜類植物,探討其預防重症病毒之可行性。進一步定點突變能影響蚜蟲傳播病毒能力位於病毒鞘蛋白的基因位於N端的DAG 部位,試驗結果顯示所研發之五個雙重突變之輕症病毒會造成葉部斑駁繼而病徵消失的現象與原本之單一突變病毒類似及不被蚜蟲傳播之特性依舊存在。實驗結果顯示此五個雙重突變之輕症系統具良好之預防效果,極具潛力實際施用於田間葫蘆科作物上防治ZYMV且不危害環境。 在本文第四章中,甜瓜黃斑病毒 (Melon yellow spot virus, MYSV) 屬蕃茄斑點萎凋病毒屬 (Tospovirus) 病毒,可經由機械接種傳播,薊馬 (thrips) 為其主要傳播媒介。番茄斑點萎凋病毒不僅為全球重要性植物病毒,造成全世界作物及花卉等農產事業的損失,目前更嚴重影響到國內農業發展。本文第四章主要分析2007年於台灣中部地區發現的感病胡瓜植株,病株之葉部病徵為嵌紋、黃斑,進行分離純化後命名為TW-C1,經電子顯微鏡觀察、血清學及反轉錄核酸增幅試驗結果證實為番茄斑點萎凋病毒屬 (Tospovirus) 中甜瓜黃斑病毒 (Melon yellow spot virus, MYSV) 的新分離株 (MYSV TW-C1),MYSV TW-C1為台灣首次發現MYSV可感染胡瓜之報告,且MYSV TW-C1 已被證實可被南黃薊馬 (Thrips palmi Karny)傳播。
Zucchini yellow mosaic virus (ZYMV) is an aphid-borne potyvirus in the family Potyviridae. The virus is considered a major pathogen of cucurbits and causes serious yield losses in most region of the world where these crops are cultivated. In order to effectively control this virus, we recently developed a cross protection strategy to protect squash plants. The engineered Zucchini yellow mosaic virus ZYMV AC (ZAC) mutant that contains two mutations in its HC-Pro genes (R180→I, and E396→N) has been proven providing complete cross protection in squash plants. In this study, the DAG triplet of ZAC was modified; the aphid transmissibility and cross-protection effectiveness of the regulated mutants were examined. Lack of transmissibility is an ideal characteristic for mild viruses as a protective strain for cross protection, as it minimizes the risk of natural spread. The first chapter describes the objectives, main contents and previous studies related to this thesis. Chapter II of this study, lost of aphid transmissibility was found in attenuated ZYMV AC (ZAC) mutant, which was modified from ZYMV GAC mutant by removing the reporter green fluorescent protein (GFP) gene and the cloning sites, even though during the time when the level of virus accumulation were as high as that of the severe strain ZYMV-TN3. To test the feasibility for application of ZAC, the cross-protection effectiveness in major cucurbit crops were evaluated. Our results showed that the attenuated mutant ZAC provides complete cross protection against the severe strain ZYMV on watermelon, oriental melon and cucumber. We conclude that ZAC can be used for control of ZYMV against severe infection of ZYMV by cross protection in economically important cucurbit crops in Taiwan. In chapter III, in order to facilitate a safer system for application of cross protection and to minimize the possible risks, we constructed several mutations at a three amino acid motif Asp-Ala-Gly (DAG), which is highly conserved and essential for aphid-transmissibility of potyvirus. Five mutants including ZYMV-CP△DAG (DAG triplet deleted), ZYMV-CPKAG (Asp9→Lys), ZYMV-CPDTG (Ala10→Thr), ZYMV-CPDAS (Gly11→Ser) and ZYMV-CPDAL (Gly11→Leu) were generated through the modifications at the DAG motif of the coat protein (CP) gene of the wild type ZYMV TN3 by site-directed mutagenesis. The aphid transmissibility on squash was then tested under greenhouse conditions. The results of aphid transmission tests revealed that the five TN3 DAG mutants retain severe mosaic symptom on leaves, but all lose the aphid transmissibility. We further transferred the five DAG mutantions from ZYMV-TN3 (severe strain) to ZAC (attenuated strain) to create five mutants. When the attenuated ZAC DAG mutants were used to protect plants of watermelon, oriental melon and cucumber against the mechanical challenge of a severe recombinant virus ZYMV-Derp V at 5 or 10 days after the protective inoculation, complete cross protection was noticed. The five mutants induced transient leaf mottling followed by a recovery phenotype similar to that of ZAC. Our results indicated that the five double-attenuated mutants containing mutations both in HC-Pro and CP genes have a great potential to be used as a protective virus for control of ZYMV in cucurbits in a more environmental friendly way. Thrip-borne Tospovirus is a worldwide spread and causes great lost in agriculture, also an important limiting factor for cucurbit production in Taiwan. In chapter Ⅳ, "Characterization of Melon yellow spot virus infecting cucumber (Cucumis sativus L.) in Taiwan", we discovered abnormal cucumber plants showing masic, yellow spot symptom on leaf in field of the central region of Taiwan in 2007. Results obtained by analyses of electron microscopy, serology, reverse transcription-polymerase chain reaction and transmission by Thrips palmi indicated that the causal agent is Melon yellow spot virus (MYSV), a member of Watermelon silver mottle virus serogroup of the genus Tospovirus. This is the first report of cucumber naturally infected by MYSV in Taiwan.
URI: http://hdl.handle.net/11455/31433
其他識別: U0005-2308201010110800
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