Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35844
標題: 狐尾草嵌紋病毒載體之開發與應用
Development and Application of Foxtail Mosaic Virus-based Vector System
作者: Chang, Chao-Yuan
張肇源
關鍵字: Foxtail mosaic virus
狐尾草嵌紋病毒
viral vector
病毒載體
出版社: 生物科技學研究所
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摘要: 植物病毒載體的概念是根據植物病毒在植物體內可自動複製及系統性移動的特性所衍生而成。目前,植物病毒載體的主要應用有表現外源蛋白或胜肽及作為研究植物基因體功能的工具。其表現策略有兩種:一、利用重複外鞘蛋白次基因體啟動子 (CP sgRNA Promoter Duplication) 方式,將外源基因插入病毒基因體表現外源蛋白,或利用病毒誘導基因靜默 (Virus Induced Gene Silencing, VIGS) 方式,表現欲誘導基因靜默之基因片段進行植物基因體功能分析。二、利用胜肽展示系統 (Epitope Presentation System) 方式,將疫苗胜肽或蛋白融合於病毒外鞘蛋白,胜肽融合外鞘蛋白能在細胞內組裝成嵌合病毒顆粒 (Chimeric Virus Particles, CVPs),發展成高力價低成本的疫苗生產系統。本研究之主要目的是發展狐尾草嵌紋病毒 (Foxtail mosaic virus, FoMV) 載體,提高FoMV病毒載體的應用性。FoMV屬於馬鈴薯病毒群,其寄主範圍包括56種單子葉植物及35種雙子葉植物,其基因組成與馬鈴薯病毒X (Potato virus X, PVX) 相似。但FoMV會轉譯出5A蛋白,目前只知可以在生體內表現且與系統性的感染無關。為發展FoMV載體,本研究首先確定了FoMV 5A蛋白存在與否,皆不影響該病毒在植物的複製與不同寄主植物間的感染。其次,為建構胜肽展示系統,在FoMV外鞘蛋白的N端與C端分別融合C-myc及Foot-and-mouth disease virus (FMDV) VP1表面抗原序列,結果顯示在FoMV N端外鞘蛋白分別表現C-myc 及FMDV VP1表面胜肽抗原可獲得嵌合病毒顆粒F-Cmyc和FVP1,其中FVP1有裂解現象發生;而融合胜肽抗原於FoMV C端外鞘蛋白則無法獲得嵌合病毒顆粒。進一步,藉由胺基酸突變與表達較短胜肽的方式可有效的提高FVP1嵌合病毒穩定性。此外,藉由最佳化的病毒純化步驟可在短時間內獲得大量完整的嵌合病毒顆粒,其產量約為每克葉片可獲得1 mg之嵌合病毒顆粒,具有高度潛力應用於抗體與疫苗生產系統。另外,利用重複外鞘蛋白次基因體啟動子方式,發展FoMV為病毒誘導基因靜默 (FoMV-VIGS) 載體,作為分析植物基因體功能的工具。因此,本研究選用三個基因:表現類胡蘿蔔素合成必需的酵素phytoene desaturase (PDS)、葉綠素生合成關鍵酵素magnesium chelatase (SU)與負責負調控吉貝素訊號傳遞的蛋白slender rice 1 (SLR1)的基因作為基因靜默對象。結果顯示利用攜帶大麥PDS基因片段的FoMV-VIGS載體,可有效在大麥、高粱、蘇丹草、水稻、狐尾草、二穗短柄草等單子葉植物中造成葉片白化及黃化現象,而在雙子葉植物的菸草可利用帶有其SU基因片段而引發葉片白化現象發生。同時,證實當FoMV-VIGS載體攜帶大麥SLR1基因片段接種單子葉植物時,可在大麥與二穗短柄草產生葉片徒長現象。進一步藉由real-time RT-PCR證明該基因被靜默的效率達到40 %至80 %。此外,我們亦進一步發展為可同時針對兩個基因進行基因靜默的載體系統,由以上結果顯示:不論是單子葉與雙子葉植物的內生性基因 (PDS、GAPDH與SU) 或是轉殖基因 (mGFP) 均可有效抑制該基因mRNA的表現量,且均可造成其表現型發生。期望可進一步用於探討植物中相關基因調控或與代謝路徑相關的研究,以提高此VIGS載體之應用性。
The concept of plant viral vector is based on the characteristics of plant virus replication and systemic movement in plants. Currently, the major applications of plant viral vectors are used to express foreign proteins or peptides, and as a research tool for plant functional genomics. Two strategies are often used to achieve the above purposes. First, gene insertion vectors consisting of complete functional virus genomes with the addition of duplicated CP sgRNA promoter for the target protein or using virus-induced gene silencing (VIGS) approach used to analyze the gene function in plants. Second, epitope presentation system, a target epitope can be genetically fused to the CP protein gene which can form chimeric virus particles (CVPs), is used as potent vaccine cadidates. The effectiveness of CVPs as immunogen remains to be evaluated. The goal of this study is to develop a vector system based on Foxtail mosaic virus (FoMV) vector and to broaden the applications of FoMV vector system. FoMV, a member of the potexvirus genus, infects 56 species of the Poaceae and 35 dicot species. The difference between the gene organizations of FoMV and Potato virus X (PVX) is an ORF 5A present in FoMV, which is synthesized in plant and is not necessary for virus replication or systemic movement. We first confirmed that the presence or absence of 5A protein, will not affect virus infection and accumulation in plants. Secondly, we explore the potentiality of using C-myc and foot-and-mouth disease virus (FMDV) VP1 epitopes to fuse at the N- or C-terminus of FoMV coat protein. The results indicate that only N-terminal fusion of these two peptides can assemble into CPVs and replicate in plants. Further, we use amino acids mutations or peptides trancation strategies to improve the stability of chimeric FoMV virions (FVP1). A series of FVP1 (FVP1Q153N, FVP1Q153A and FVP1128-152) were successfully expressed in plants at the level of 1 mg/g of fresh leaf tissue. Finally, we have established FoMV as a VIGS vector for functional genomics studies in plant. We have chosen the magnesium chelatase (SU) gene from N. benthamiana, phytoene desaturase (PDS) gene from barley (Hordeum vulgare L. cv. Larker), which are essential for chlorophyll biosynthetic pathway and carotenoids production against photobleaching, and also the slender rice 1 (SLR1) from barley, which is a negative effecter on GA signaling, as the targets. Inoculation of recombinant FoMV carrying a fragment of the target gene of Nb-SU and HvPDS into N. benthamiana or barley resulted in a photo-bleached phenotype. The results showed that FoMV-VIGS vector carried HvPDS fragments or Nb-SU can effectively induce PDS or SU gene silencing in barley, sorghum, sudangrass, rice, foxtail, Brachypodium distachyon, and N. benthamiana plants to cause photo-bleached phenotype. We also demonstrated that FoMV-VIGS vectors triggered SLR1 gene silencing to cause barley and Brachypodium distachyon excessive growth phenomenon. The real-time RT-PCR results showed that mRNA level of target genes were reduced by 40-80%. Moreover, we developed FoMV-VIGS vector for multiple genes silencing in plants. The results demonstrated that FoMV-VIGS vectors can trigger two genes silencing at the same time and phenotype changes. In conclusion, FoMV-VIGS system can be used in the future to analyze the gene function in a broad host range.
URI: http://hdl.handle.net/11455/35844
其他識別: U0005-2907201315395100
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2907201315395100
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