Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31400
標題: 建立番茄斑萎病毒屬西瓜銀斑病毒逆向遺傳系統暨新德里番茄捲葉病毒東方甜瓜分離株機械接種特性、病原性及其對番茄感染決定因子之研究
Development of a reverse genetics system for Watermelon silver mottle tospovirus and studies of mechanical transmissibility, pathogenicity and tomato-infection determinant of Tomato leaf curl New Delhi begomovirus-oriental melon isolate
作者: 張賀雄
Chang, Ho-Hsiung
關鍵字: reverse genetics
逆向遺傳系統
Tospovirus
Watermelon silver mottle virus
Begomovirus
Tomato leaf curl New Delhi virus
nuclear shuttle protein
番茄斑萎病毒屬
西瓜銀斑病毒
菜豆金黃花葉病毒
新德里蕃茄捲葉病毒
核穿梭蛋白
出版社: 植物病理學系所
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摘要: 病毒與寄主的交互作用是病毒學家最感興趣的研究領域之一;但在這領域之中,病毒學家往往需要面對前所未有的挑戰;本論文即探討兩個嶄新的研究課題:(1) 發展植物負極性RNA病毒 (negative-strand RNA virus) 之逆向遺傳系統 (reverse genetics),(2) 應用具感染力DNA構築進行環狀單股DNA病毒的功能性基因體 (functional genomics) 研究。 逆向遺傳系統一詞在分子病毒學上,係指透過cDNA構築表現病毒基因體而產生病毒的研究系統。自1994年第一個負極性RNA病毒從cDNA構築產生後,相似的逆向遺傳系統被建立在許多屬於Mononegavirales目的病毒上。1996年逆向遺傳系統有了另一個大突破,具有三條基因體的Bunyaviridae科病毒被建立起逆向遺傳系統;緊接著,更為複雜、具有八條基因體的流感病毒 (influenza virus),其逆向遺傳系統也在1999年被完成。此後不論是具有單條基因體 (non-segmented genome) 或多條基因體 (segmented genome) 的負極性RNA病毒,均可以類似的研究策略建立逆向遺傳系統;逆向遺傳系統從而成為一個普遍、可靠且有力的研究工具,用來深入研究病毒的生活史、病毒蛋白的功能性角色以及病毒與寄主間的交互作用。然而截至目前為止,仍未有植物負極性RNA病毒被建立起有效率地、可供分子層次或基因體功能研究的逆向遺傳系統。番茄斑萎病毒屬病毒 (tospoviruses) 屬於Bunyaviridae病毒科的一員,是一群同樣具有外套膜包裹三條單股負極性RNA基因體 (single-stranded negative-sense RNA genome) 的植物病毒;理論上我們可以依循動物負極性RNA病毒在建立逆向遺傳系統上的經驗,以真核或原核的RNA表現系統及能表現病毒必須蛋白的轉基因植物,來建立番茄斑萎病毒屬病毒的逆向遺傳系統。在本論文中,我們發展T7 RNA聚合酶 (T7 RNA polymerase) 及RNA聚合酶II (RNA polymerase II) 表現系統,用以探討建立西瓜銀斑病毒 (Watermelon silver mottle tospovirus, WSMoV) 微型複製體 (minireplicon) 系統或逆向遺傳系統的相關研究。在T7 RNA聚合酶表現系統中,WSMoV的基因體RNA或帶有綠色螢光蛋白 (green fluorescence gene protein, GFP) 基因的S RNA相似物以生體外轉錄的方式進行表現,生體外轉錄產物以機械接種的方式接種至能表現L蛋白的N基因轉基因植物或WSMoV感染的植物上;然而不論在接種葉或系統葉上,均無法偵測到接種的RNA轉錄體被複製或累積的情形,在WSMoV感染的植物上,同樣無法偵測到GFP蛋白的表現;雖然在本研究中RNA的生體外轉錄及L與N蛋白的表現均屬可行,但RNA的接種效率卻是T7 RNA聚合酶表現系統操作上的一項阻礙。在RNA聚合酶II的RNA表現系統中,帶有GFP基因的S RNA相似物構築被選殖於35S啟動子 (promoter) 的調控下;我們以農桿菌滲透注射法使各DNA構築在植物上短暫表現RNA,但我們卻無法偵測到全長度RNA的表現;北方轉漬法 (northern blot) 的分析結果顯示,RNA聚合酶II的轉錄作用 (translation) 可能由於病毒RNA的基因間區 (intergenic region, IGR) 序列存在而被迫中止;雖然我們完成了去除IGR序列的構築,也表現出完整長度RNA產物,但接種至能表現L蛋白的N基因轉基因植物或WSMoV感染的植物上時,仍無法偵測到GFP蛋白的表現;推測其原因可能是,病毒蛋白與病毒RNA相似物間的交互作用或辨識發生問題。對於欲以這兩個RNA表現系統建立植物負極性RNA病毒逆向遺傳系統來說,病毒蛋白與RNA間交互作用發生的效率,是一個最主要需要克服的障礙。 新德里番茄捲葉病毒 (Tomato leaf curl New Delhi virus, ToLCNDV) 是一具兩條基因體DNA的菜豆金黃花葉病毒屬 (the Genus Begomovirus) 病毒,在番茄與瓜類作物上造成嚴重的危害。我們從表現出葉片捲曲、皺縮的罹病東方甜瓜植株中,分離出一個ToLCNDV分離株,並將其命名為新德里番茄捲葉病毒東方甜瓜分離株 (ToLCNDV oriental melon isolate, ToLCNDV-OM)。透過機械接種方式,傳播來自農桿菌接種 (agroinfection) 菸草所表現之病毒子代至東方甜瓜植株上,並表現與田間罹病植株相似病徵的結果,我們確定了ToLCNDV-OM的病原性及機械接種特性;其中讓人感到有興趣還有,在接種ToLCNDV-OM的番茄上,我們並未觀察到系統葉上病徵表現,同時也未被偵測到有病毒存在;進一步詳細的序列比對結果顯示,ToLCNDV-OM的核穿梭蛋白 (nuclear shuttle protein, NSP) N端分別較來自番茄 (ToLCNDV-Svr, NCBI GenBank accession No.: U15017) 及胡瓜 (ToLCNDV-Cuc, NCBI GenBank accession No.: AB330080) 的分離株短少13及28個胺基酸;針對比對結果,我們設計了增加ToLCNDV-OM NSP蛋白N端各13個胺基酸的病毒突變株,以農桿菌接種方式接種至菸草上,再將系統葉上產生的病毒子代以機械接種方式接種至番茄植株;接種三週後,我們可以在病毒突變株所接種的番茄系統葉上觀察到嵌紋及捲曲的病徵,並且以PCR (polymerase chain reaction) 方式偵測到該病毒突變株;此一結果顯示,ToLCNDV-OM NSP蛋白N端所短少的胺基酸片段,可能與病毒系統性感染番茄有關。 雖然建立番茄斑萎病毒屬病毒逆向遺傳系統極為困難且並未完全成功,一但成功將提供一個可以有效率操作病毒基因體的研究工具;建立菜豆金黃花葉病毒屬病毒的具感染力DNA構築相對較容易,且此系統的建立在功能性基因體研究上造成了莫大幫助。
The virus-host interaction is one of the most interesting topics for virologists. Often time that challenges virologists to embark on projects that are unprecedented. In this dissertation, two unprecedented projects were explored: (1) to develop a reverse genetics system for a plant negative-strand RNA virus and (2) to apply infectious DNA clones of a circular single-strand DNA virus for its functional genomics study. The term, reverse genetics, is used in molecular virology to describe the generation of viruses owning its genome derived from cDNA clones. Since the first development of a negative-strand RNA virus entirely from cDNA clones in 1994, similar reverse genetics systems have been established for several members of the Mononegavirales Order. A breakthrough of the reverse genetics development occurred in 1996 when a segmented virus, with three genomic segments, of the Bunyaviridae Family was generated from cloned cDNAs. Following that, a more complex influenza virus, whose genome contains eight RNA segments, was generated from cloned cDNAs in 1999. Whether the negative-strand RNA viruses are segmented or non-segmented, similar strategies of reverse genetics system were used by virologists. Thus, the reverse genetics system has become a popular, reliable and powerful tool to dissect the virus life cycle, the role of viral proteins and the interaction between viruses and hosts. To date, there is still no efficient routine reverse genetics system for detailed molecular investigation and functional genomics elucidation of any member of the plant negative-strand RNA viruses including tospoviruses. Tospoviruses, in the Family Bunyaviridae, are a group of enveloped plant viruses containing a tripartite single-stranded negative-sense RNA genome. In theory, we can follow the model of the reverse genetics applied in animal negative-strand RNA virus to develop a reverse genetics system for a tospovirus in conjunction with the eu-/pro-karyote RNA expression system and the transgenic plants that express the essential viral proteins. In this dissertation, we explored the development of minireplicon or reverse genetics systems for the Watermelon silver mottle tospovirus (WSMoV) via the development of the T7 RNA polymerase and RNA polymerase II RNA expression systems. In T7 RNA polymerase RNA expression system, the genomic RNAs of WSMoV or green fluorescence gene (GFP)-containing S-like RNA were in vitro expressed from DNA clones. The RNA transcripts, genomic RNAs mixture or S-like RNA alone, were mechanically inoculated onto L protein-expressed N transgenic plants or WSMoV-infected plant. There was no replicated RNAs detected in inoculated leaves and systemic leaves and no GFP expressed in WSMoV-infected plants. Although the in vitro RNA transcription and L and N proteins expression were functional, the efficiency of RNAs inoculation was indeed a stumbling block in T7 RNA polymerase expression system. For RNA polymerase II RNA expression system, GFP-containing S-like RNA were cloned and driven by 35S promoter. When RNAs transiently expressed from cDNA clones with agro-infiltration in plant tissue, the full-length S-like RNA was not detectable. Results of northern blot showed that the intergenic region (IGR) of viral RNAs might disrupt the transcription of RNA polymerase II. Although the construct of IGR deletion was made and full-length RNA was detected, the expression of GFP is not detected. It may be due to that there is no interaction or recognition between viral proteins and virus-like RNAs. The efficiency of protein-RNA interaction is the major and the most important barrier to both strategies for developing a reverse genetics system for plant negative-strand RNA viruses. Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus that causes extensive damage in tomato and cucurbitaceous plants. An isolate of ToLCNDV was isolated from an oriental melon plant exhibiting leaf curl and puckering symptoms, and was designated as Tomato leaf curl New Delhi virus oriental melon isolate (ToLCNDV-OM). The pathogenicity and mechanical transmissibility of ToLCNDV-OM was confirmed by mechanical inoculation of the progeny virions from the agroinfected Nicotiana benthamiana onto oriental melon which developed symptoms similar to those observed in the fields. The most interesting and significant discovery was that there were no symptoms observed and no virus detected on ToLCNDV-OM-inoculated tomato plants. Detailed sequence comparison showed that the N terminus of nuclear shuttle protein (NSP) of ToLCNDV-OM is shorter than those of the tomato isolate (ToLCNDV-Svr, NCBI GenBank accession No.: U15017) and cucumber isolate (ToLCNDV-Cuc, NCBI GenBank accession No.: AB330080) by 13 and 28 amino acids, respectively. A mutant, with the addition of 13 amino acids sequences to the N-terminal, was made to the NSP gene of ToLCNDV-OM. The progeny virions of these two mutants were individually mechanically inoculated from the agroinfected N. benthamiana to tomato plants. Three weeks post-inoculation, the systemic symptoms of mosaic and leaf curling on tomato were observed and the mutated viruses were detected by polymerase chain reaction. These results indicate that the N-terminal sequences, loss on the NSP of ToLCNDV-OM, may be involved in its ability to systemically infect tomato plants. Although it was difficult and only partially successful so far, the eventual development of a reverse genetics system for tospoviruses will be the efficient technique to manipulate viral genomes. On the contrary, the infectious DNA clones of begomoviruses are easier to establish and will contribute enormously for functional genomics study.
URI: http://hdl.handle.net/11455/31400
其他識別: U0005-1307201016202700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1307201016202700
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