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標題: 台灣瓜類薊馬傳播病毒發生情形及利用L基因之高保留性區域誘發轉基因植物產生對番茄斑萎病毒屬之廣泛抗性
Occurrence of thrips-borne viruses infecting cucurbits in Taiwan and generation of broad-spectrum resistance in transgenic plants conferred by the conserved region of L genes of tospoviruses
作者: 彭瑞菊
Peng, Jui-Chu
關鍵字: Tospoviruses;番茄斑萎病毒屬;Watermelon silver mottle virus;Melon yellow spot virus;conserved region;西瓜銀斑病毒;甜瓜黃斑病毒;高保留性區域
出版社: 植物病理學系所
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A
以薊馬傳播之番茄斑萎病毒屬病毒(tospoviruses)為Bunyaviridae科中唯一可感染植物之一屬,在世界各地造成許多經濟作物嚴重的危害,為一全球重要性之植物病毒。西瓜銀斑病毒(Watermelon silver mottle virus,WSMoV)及甜瓜黃斑病毒(Melon yellow spot virus,MYSV)為番茄斑萎病毒屬 (Tospovirus) 之一員,是台灣瓜類作物栽培的主要限制因子之一。甜瓜及西瓜是台灣重要的經濟作物,本研究針對薊馬傳播的病毒在台灣瓜類作物上發生情形進行調查,並且進一步利用gtju,之高保留性區域誘發轉基因植物產生對番茄斑萎病毒屬之廣泛抗性。本論文總共分為四個章節,分述如下。
本論文第二章「薊馬傳播病毒在台灣甜瓜及西瓜發生之情形」,為了研究瓜類病毒的發生情形,從2007年7月至2009年12月收集台灣主要種植區中部和南部地區的疑似罹病毒樣品。先以抗N蛋白單株抗體進行酵素聯結免疫反應(ELISA) 測試,結果不確定者再以N基因專一性引子進行逆轉錄聚合酶鏈反應(RT-PCR)技術進一步鑑別WSMoV和MYSV。其中10,480甜瓜樣本有631(6%)和1906(18.2%)分別檢測出感染WSMoV和MYSV,只有17個為複合感染WSMoV和MYSV,檢測結果顯示台灣瓜田中主要之番茄斑萎病毒為MYSV。而西瓜1811個樣品中,有22.4%和9.2%分別檢測出感染WSMoV和MYSV,只有4個為複合感染WSMoV和MYSV。研究結果顯示台灣瓜類上WSMoV和MYSV複合感染情形極少,WSMoV在西瓜上感染嚴重,而MYSV 在甜瓜上感染普遍,但是MYSV的發生有越來越嚴重的趨勢,因此需積極尋求有效的防治方法。
本論文第三章「利用L基因之高保留性區域誘發轉基因植物產生對番茄斑萎病毒屬之廣泛抗性」,為利用WSMoV的L基因之高保留性區域誘發轉基因植物產生對番茄斑萎病毒屬之廣泛抗性,利用WSMoV L RNA對應產生的大蛋白 (L protein)複製酶的高保留性區域,建構了五種不同的構築包含以可轉譯性(WLm)與非轉譯性(WLmt、Wlmts、WlAs)、以及雙股RNA(WLmds)之構築形式建構於Ti載體中,利用農桿菌感染的方式將所構築之轉基因片段轉殖於菸草(Nicotiana benthamiana)或番茄 (Solanum esculentum) 細胞中育成轉基因作物,於溫室條件下接種WSMoV及其他tospoviruses,如番茄斑萎病毒(Tomato spotted wilt virus,TSWV)、花生黃化扇班病毒(Peanut chlorotic fan-spot virus,PCFV)、花生輪斑病毒(Groundnut ringspot virus,GRSV)及鳳仙花壞疽斑點病毒(Impatiens necrotic spot virus,INSV)等,轉殖菸草接種WSMoV抗性表現為46.7-70%,抗WSMoV的菸草再接種上述其他四種病毒的抗性表現為35.7-100%。由於轉殖植物體內偵測不到轉基因之mRNA,因此推測該廣泛抗性機制為後轉錄作用的基因沉寂現象(post-transcriptional gene silencing,PTGS),轉殖番茄有亦可以抗WSMoV及其他番茄斑萎病毒。由此結果可知利用WSMoV L RNA上的高保留性區域所構築的轉殖菸草及番茄可廣泛的抗番茄斑萎病毒屬的不同病毒。
本論文第四章「攜帶西瓜銀斑病毒L基因高保留性區域之無標幟轉基因於番茄及甜瓜誘發對番茄斑萎病毒屬之廣泛抗性」,將西瓜銀斑病毒L RNA上具有高保留性的區域以共轉殖策略利用農桿菌轉殖法產生無標幟基因 (marker-free) 轉基因植物。所構築的兩段共轉殖T-DNA片段分別位於單一農桿菌菌株中的同一轉殖載體上。經此轉殖系統所產生之L RNA上具有高保留性的區域構築反義股(antisence)及雙股(double-strand)的無標幟基因轉殖植物包括番茄及甜瓜,初步接種分析顯示亦可誘發對番茄斑萎病毒屬之廣泛抗性,可經由自交分離產生無篩選標幟基因(nptII) 的子代。

Thrips-borne tospoviruses, belonging to the only plant-infecting genus Tospovirus in the family Bunyaviridae that cause severe damages in economic crops worldwide, are globally important. Watermelon silver mottle virus (WSMoV) and Melon yellow spot virus (MYSV), the members of the genus Tospovirus, are of the major threats for the cultivation of cucurbits in Taiwan. Melon (Cucumis melo L.) and watermelon (Citrullus lanatus Thunb.) are economically important crops in Taiwan. In this study, the occurrence of thrips-borne viruses infecting melon in Taiwan was investigated, and the broad-spectrum resistance in transgenic plants conferred by the conserved region of L genes of tospoviruses was generated. This dissertation is divided into four chapters as described below.
Chapter 1, “ Literature review and research objectives” describes references relevant to this study and the objectives for the investigations.
Chapter 2, “Occurrence of thrips-borne viruses infecting melon and watermelon in Taiwan” describing the incidence of virus infection in melon fields, a field survey was conducted from July 2007 to December 2009. The symptomatic tissues were collected from the principal cultivated areas in central and southern Taiwan. The anti-N protein-monoclonal antibodies (MAbs) and the N gene-specific primer pairs can be used to identify WSMoV and MYSV. The uncertain ELISA results for tospoviruses were further verified by reverse transcription-polymerase chain reaction (RT-PCR) using N gene-specific primer pairs for WSMoV and MYSV. Among 10,480 melon samples tested, 631 (6%) and 1,906 (18.2%) were found infected with WSMoV and MYSV, respectively, and only 17 mixed infections by both WSMoV and MYSV. Our results indicated that MYSV is the major tospovirus to invade melon in Taiwan. On the other hand, among 1,811 watermelon samples assayed, 22.4% and 9.2% samples were singly infected with WSMoV and MYSV, respectively, and 4 samples were infected with both viruses. Our results indicated that mixed infection with the two thrips-borne viruses is rare. Moreover, host preference for both viruses is different; WSMoV prevails on watermelon whereas MYSV is more widespread on melon. We conclude that MYSV has become a serious threat for watermelon and melon production in Taiwan and the possible control measures are discussed.
Chapter 3, “Generation of broad-spectrum resistance in transgenic plants conferred by the conserved region of L genes of tospoviruses ”. In this investigation, the conserved region containing the RNA-dependent RNA polymerase (RdRp) motifs within the L gene of WSMoV, an important cucurbit-infecting tospovirus in Taiwan, was used as a transgene for transformation of Nicotiana benthamiana and Solanum esculentum mediated by Agrobacterium tumefaciens to generate broad-spectrum resistance to tospoviruses. Five different modified transgene constructs of the L gene conserved region, including WLm in a sense translatable orientation, WLmt and WLmts in non-translatable, WLmAs in antisense and WLmds in double-stranded inverted repeat, were used to evaluate broad-spectrum resistance in transgenic plants. A total of 46.7-70.0% transgenic N. benthamiana lines derived from these five transgenes showed resistance to WSMoV, and 35.7-100% of the WSMoV-resistant lines exhibited broad-spectrum resistance against four other serologically unrelated tospoviruses, including Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV) and Peanut chlorotic fan-spot virus (PCFV). Northern analyses indicated that the broad-spectrum resistance is mediated by post transcriptional gene silencing (PTGS). To validate the L conserved region resistance in vegetable crops, we have transferred all the transgenes constructs in transgenic tomato plants and the results indicated that this L conserved region generate high levels of resistance against WSMoV and other distinct tospoviruses. This is the first report that using a single nucleotide fragment corresponding to the L gene conserved region as a novel transgenic approach to trigger broad-spectrum resistance for controlling different tospoviruses at the genus level.
Chapter 4, “Broad-spectrum resistance to tospoviruses conferred by the marker-free transgene constructs containing the L gene conserved region of Watermelon silver mottle virus in tomato and melon”. Using the co-transformation method, the conserved region containing the RdRp motifs within L gene of WSMoV was constructed in a two T-DNAs binary vector and used to generate marker-free transgenic plants. Two constructs- MF-WLmAs and MF-3WLmds were evaluated for the broad-spectrum resistance. Our results preliminary indicated that the L gene conserved region is successfully used to generate marker-free transgenic tomato and melon lines conferring broad-spectrum resistance against different tospoviruses. The segregation and elimination of the selection marker nptII will be selected from the progenies after selfing.
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