Marker-free Transgenic Tobacco Lines with Concurrent Resistance to Whitefly-Borne DNA Geminivirus and Thrips-Borne RNA Tospovirus

以蚜蟲傳播之馬鈴薯Y病毒屬(Potyvirus)，薊馬傳播番茄斑萎病毒屬(Tospovirus)及粉蝨傳播之雙生病毒科(Geminivirade)，在全世界重要的經濟作物上造成相當嚴重的危害；而使用傳統的抗病育種無法有效對抗病毒所引起的病害，在缺乏自然的抗病株系下，使用遺傳工程方式所產生的轉基因抗病植株，則可以有效的控制植物病毒的危害。本研究針對在經濟作物上危害最嚴重的三大病毒屬，分別利用後轉錄基因沉寂策略(post-transcriptional gene silencing, PTGS)或同時以PTGS與轉錄基因沉寂策略(transcriptional gene silencing, TGS)來對抗RNA及DNA病毒。本論文總共分為三個章節，分述如下。 本論文第一章 「前人研究及研究目的」，主要蒐集整理近年來與本研究相關之研究論文及參考文獻，並概述本論文之目的與內容。 本論文第二章 「無篩選標幟且同時對粉蝨傳播DNA雙生病毒及薊馬傳播RNA蕃茄斑萎病毒具有抗性之轉基因菸草」，我們利用轉錄基因沉寂的方式，瞄準病毒的複製起始點及其啟動子，抑制其複製及轉錄作用，建立一個防治Geminiviridae之新方法。利用干擾性微小RNA(siRNA)誘導DNA甲基化的策略(RdDM)，將霍香薊黃脈病毒(Ageratum yellow vein virus, AYVV)含有雙向啟動子之基因間區域(intergenic region, IGR)設計成髮夾型RNA結構並將其放置在阿拉伯芥內含子(intron)基因序列中，以產生轉錄沉寂作用(TGS)於轉基因菸草植物中產生抗AYVV之抗性。將此構築結合薊馬所傳播之甜瓜黃斑病毒(MYSV)的不轉錄核鞘蛋白片段，使其誘導轉錄後基因沉寂(PTGS)以產生對甜瓜黃斑病毒的抗性。可抗霍香薊黃脈病毒(AYVV)之轉基因植株，接種甜瓜黃斑病毒(MYSV)來分析其抗病能力，有1株轉基因植株具有完全免疫抗性；而在siRNA分析中，也可測得甜瓜黃斑病毒核鞘蛋白siRNA的表現。本結果顯示，我們可以利用此RdDM及PTGS的新策略同時產生對ssDNA及ssRNA植物病毒產生轉基因抗性，並且可將選擇性標誌基因剔除，可減少生物安全疑慮。 本論文第三章 「同時對粉蝨傳播DNA雙生病毒具有抗性及對薊馬傳播RNA蕃茄斑萎病毒具有廣泛抗性之轉基因菸草」，將AYVV IGR髮夾型RNA結構放置在阿拉伯芥內含子基因序列中，此構築再結合至WSMoV的L基因之高保留性區域誘發轉基因植物產生對蕃茄斑萎病毒屬之廣泛抗性。而在接種的結果測試中，其轉基因植株可以同時廣泛抗西瓜銀斑病毒(WSMoV)及番茄斑點萎凋病毒(Tomato spotted wilt tospovirus, TSWV)；而具有抗TSWV的轉基因植株，其也同樣對於WSMoV 與AYVV具有良好之抗病能力。我們可以利用此RdDM及PTGS的策略同時產生對抗ssDNA病毒與廣泛抗ssRNA蕃茄斑萎病毒屬與無血清相關之不同病毒種。 本論文附錄一「利用轉基因產生同時抗兩個蚜蟲傳播及一個薊馬傳播的構築多重抗病毒之轉基因西瓜株系」，針對台灣瓜類上危害最嚴重的矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus, ZYMV), 木瓜輪點病毒西瓜型(Papaya ringspot virus type W, PRSV W)及西瓜銀斑病毒(Watermelon silver mottle virus, WSMoV)，以遺傳工程方法構築具有ZYMV、PRSV W病毒鞘蛋白及WSMoV病毒核鞘蛋白之轉基因西瓜，誘發轉基因植物產生後轉錄基因沉寂以達到多重抗病之轉基因西瓜株系。總共獲得13個轉基因西瓜株系，其中3個株系可以對ZYMV及PRSV W具有完全的抗性，其在北方點墨法中，偵測不到轉錄的RNA表現，但可以測得三個病毒片段構築的siRNA累積。因此可以證實其是藉由後轉錄基因沉寂所產生的抗性。Aphids-borne Potyvirus, thrips-borne Tospovirus and Whitefly-borne Begomovirus cause severe damages on crops worldwide. Incorporation of natural resistance gene into commercial cultivars is difficult by conventional breeding and requires many years to eliminate unfavorable characteristics, even if natural resistance source is available. Therefore, using genetic engineering to generate transgenic resistance provides an effective approach to control the diseases caused by plant viruses. In this study, we attempted to use post-transcriptional gene silencing (PTGS) or in combination of PTGS with transcriptional gene silencing (TGS) to generate transgenic resistance to Potyvirus and Tospovirus or concurrent resistance to a ssDNA Begomovirus and a Tospovirus on economic crops. This dissertation is divided into three chapters as described below. Chapter 1,'Literature review and research objectives'describes references are relevant to this study and the objectives of investigations. Chapter 2,'Generation of marker-free transgenic plants concurrently resistant to a DNA geminivirus and a RNA tospovirus' We tried to develop an RNA-directed DNA methylation (RdDM) transgenic system using a hairpin construct (int-hpIGR) of intergenic region (IGR) of Ageratum yellow vein virus (AYVV) residing in an intron of Arabidopsis to mediate TGS in tobacco plants. Furthermore, the int-hpIGR of AYVV IGR coupled with an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV) was constructed to induce PTGS against MYSV. The resistant lines showed better resistance to AYVV, when challenged with MYSV, one of the AYVV-resistant lines was immune to MYSV infection, in which the transgenic NP transcript was silenced and the corresponding siRNAs were detected. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses by TGS and PTGS with less biosafety concerns. Chapter 3,'Generation transgenic tobacco plants conferring resistance to a DNA geminivirus and broad-spectrum resistance to distinct RNA tospovirus species' Using the int-hpIGR of AYVV IGR coupled with an untranslatable antisense large (L) gene fragment containing the highly conserved RNA-dependent RNA polymerase (RdRp) region of WSMoV was created to induce PTGS against tospoviruses. When challenged with WSMoV and Tomato spotted wilt virus (TSWV), the TSWV-resistant lines were also highly resistant to WSMoV and AYVV. Hence, our novel approach provides a valuable way for concurrent control of ssDNA AYVV and broad-spectrum resistance to different distinct (-)ssRNA tospoviruses. Appendix I,'Generation of transgenic watermelon lines with resistance to two aphid-borne viruses and one thrips-borne tospovirus'thrips-borne Watermelon silver mottle virus (WSMoV), and aphid-borne Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type W (PRSV-W) are major limiting factors for watermelon production worldwide. An untranslatable construct containing truncated ZYMV coat protein (CP), PRSV-W CP and WSMoV nucleocapsid protein (NP) genes by genetic engineering to generation transgenic watermelon to control these viruses. A total of 13 transgenic watermelon lines were obtained. Among them, three transgenic lines conferred immunity to ZYMV and PRSV-W, of which the transgenic transcripts were not detected by northern blotting, but the accumulation of small interfering RNAs (siRNAs) were detected, indicating that the transgenic resistance was mediated by PTGS.目次 中文摘要 1 ABSTRACT 3 第一章 前人研究及研究目的 Chapter Ⅰ Literature review and research objectives 5 一、粉蝨傳播雙生病毒科之危害及其分類 6 二、豆類金黃嵌紋病毒屬之型態及基因體核酸之特性 7 三、雙生病毒基因沉寂靜默子 9 四、植物防禦DNA病毒之siRNA誘導DNA甲基化(siRNA directed DNA methylation)作用機制 13 1. 在植物細胞核內產生24nt siRNA 與DNA甲基化有關: 13 2. PTGS和TGS是有關聯的: 14 3. 去DNA甲基化為植物防禦雙生病毒的主要機制: 15 4. DNA病毒基因與甲基化可產生抗病防禦現象: 16 五、轉基因植物防禦DNA病毒之應用 16 六、番茄斑萎病毒屬之危害及其分類 18 七、蕃茄斑萎病毒屬之型態及基因體核酸之特性 19 八、植物防禦RNA病毒之後轉錄後基因沉寂 (Post-transcriptional gene silencing)作用機制 20 九、轉基因植物防禦RNA病毒之應用 21 十、研究目的與方法 22 十、參考文獻 26 第二章 同時對粉蝨傳播DNA雙生病毒及薊馬傳播RNA蕃茄斑萎病毒具有抗性之轉基因菸草 39 Chapter Ⅱ Generation of marker-free transgenic plants concurrently resistant to a DNA geminivirus and a RNA tospovirus 39 ABSTRACT 40 INTRODUCTION 41 MATERIALS AND METHODS 45 RESULTS 54 DISCUSSION 63 REFERENCES 69 TABLES AND FIGURES 74 第三章 同時對粉蝨傳播DNA雙生病毒具有抗性及對薊馬傳播RNA蕃茄斑萎病毒具有廣泛抗性之轉基因菸草 88 Chapter Ⅲ Generation of transgenic tobacco plants conferring resistance to a DNA geminivirus and broad-spectrum resistance to distinct RNA tospovirus species 88 ABSTRACT 89 INTRODUCTION 90 MATERIALS AND METHODS 93 RESULTS 97 DISCUSSION 101 REFERENCES 104 TABLES AND FIGURES 108 附錄一 利用轉基因產生同時抗兩個蚜蟲傳播及一個薊馬傳播的構築多重抗病毒之轉基因西瓜株系 117 Appendix I Generation of transgenic watermelon lines with resistance to two aphid-borne viruses and one thrips-borne tospovirus 117 ABSTRACT 118 INTRODUCTION 119 MATERIALS AND METHODS 122 RESULTS 127 DISCUSSION 132 REFERNCES 136 TABLES AND FIGURES 15