Development of Bamboo mosaic virus and its satellite RNA for virus-induced gene silencing vector

病毒誘導基因靜默(virus-induced gene silencing, VIGS)是目前常用於分析基因功能的技術，因其具有操作方便、快速、效率高、高專一性及可針對特定基因來分析等優點。竹嵌紋病毒衛星核酸(Bamboo mosaic virus satellite RNA, satBaMV)為馬鈴薯病毒群(Potexvirus)中唯一發現的衛星核酸，並已成功的發展為植物表現載體。本研究之目的為建立竹嵌紋病毒衛星核酸載體成為誘導基因靜默系統，以提供做為鑑定與竹嵌紋病毒(Bamboo mosaic virus, BaMV)生活史相關的寄主因子或研究植物基因體功能的分析工具。首先，實驗中利用與葉綠素(chlorophyll)合成相關的sulfur基因(SU)做為基因靜默的對象，並構築至竹嵌紋病毒衛星核酸載體，以DNA質體為接種源並採用機械接種方式與竹嵌紋病毒共同感染菸草(Nicotiana benthamiana)，接種14天後，在菸草的系統葉葉片出現白化的葉脈，證實竹嵌紋病毒衛星核酸的確能誘導基因發生靜默作用。同時，此載體系統也能藉由農桿菌注射方式來達到基因靜默的目的，除了可以靜默菸草內生的基因(endogenous gene)，也可以靜默轉殖的基因(transgene)。與菸草脆裂病毒(Tobacco rattle virus, TRV)相較之下，竹嵌紋病毒衛星核酸誘導基因靜默載體系統最大的優勢是能避免重要基因，例如熱休克蛋白90及70 〔heat shock protein (Hsp), Hsp90 and Hsp70〕，在基因靜默時造成植物的死亡。而且，此載體系統也能在單子葉植物中做為溫帶草本(temperate grass)的模式植物二穗短柄草(Brachypodium distachyon)引發基因靜默現象，非常適合做為探討雙子葉及單子葉植物之比較基因體學的工具。為了解決某些基因靜默後無法產生明顯的外觀型態，造成無法立即辨識是否引起基因靜默反應的困擾，同時也開發竹嵌紋病毒誘導基因靜默載體。同樣地，竹嵌紋病毒誘導基因靜默載體能夠在菸草、二穗短柄草及大麥(Hordeum vulgare)引起基因靜默。當竹嵌紋病毒及其衛星核酸兩個誘導基因靜默載體分別以SU及綠螢光蛋白(green flurocent protein, GFP)基因做為基因靜默對象，共同接種於轉殖綠螢光蛋白的菸草，證實兩個基因可以達成同時被靜默的目標，此系統也可做為研究兩個基因是否作用於相同途徑的工具。為了進一步瞭解核醣核酸複製酵素6 (RNA dependent RNA polymerase 6, RDR6)對竹嵌紋病毒及其衛星核酸誘導基因靜默載體引起基因靜默效率的影響，將這兩個基因靜默載體分別接種於RDR6缺失的菸草，實驗結果顯示，感染竹嵌紋病毒及其衛星核酸的RDR6缺失菸草會產生嚴重嵌紋和生長矮小的病徵，由北方雜合分析(Northern hybridization analysis)確認病毒的核醣核酸會累積於最上位之系統葉。竹嵌紋病毒誘導基因靜默載體的靜默效率在RDR6缺失的菸草中則會明顯降低，但是衛星核酸誘導基因靜默載體的靜默效率卻顯著提升。實驗結果建議，RDR6基因在竹嵌紋病毒及其衛星核酸侵入生長點上扮演重要角色。就我們所知，本研究是第一個將衛星核酸開發成為病毒誘導基因靜默的載體系統，並可運用於雙子葉及單子葉植物的基因靜默。Virus-induced gene silencing (VIGS) is recently used to analyze gene function in plants since it contains the advantages of rapid, efficient, and specific. The satellite Bamboo mosaic virus RNA (satBaMV) is the only one found in the potexvirus group, which has become a successful plant expression vector. To develop satBaMV-induced gene silencing as a tool for identification of host factors associated with Bamboo mosaic virus (BaMV) life cycles and analysis of gene function in plants, in this study, we have assessed the significance of sulfur gene (SU) essential for chlorophyll synthesis against yellow-photobleaching full-length cDNA by using satBaMV vector. Nicotiana benthamiana plants were co-inoculated with clones of BaMV and chimeric satBaMV by mechanical inoculation using plasmid DNA as inocula. The phenotype of N. benthamiana plants inducing VIGS of endogenous SU gene at 14 days post inoculation, showed silencing areas in the vein vicinity. The satBaMV vector also triggered gene silencing by agro-infiltration method. Compare with Tobacco rattle virus (TRV), the advantage of satBaMV vector could prevent the silencing plant to die, and when essential genes for plant development, such as heat shock protein 90 (Hsp90) and Hsp70 were silenced. In addition, the satBaMV vector could also induce gene silencing in temperate grass model plant of monocot purple false brome (Brachypodium distachyon). It could be used for studies of comparative functional genomics in both dicotyledonous and monocot plants. To compensate some genes targeted for gene silencing without displaying a visible phenotype, we simultaneously developed BaMV-induced gene silencing vector. It could induce gene silencing in tobacco, purple false brome, and barley (Hordeum vulgare). When the green fluorescent protein (GFP) transgenic N. benthamiana were co-inoculated with BaMV- and satBaMV-VIGS vector carrying genes of SU and GFP, respectively, the results demonstrated that simultaneously silencing of both genes can be accomplished. This system provides a genetic tool to investigate two genes in a single test. Further, in order to confirm RNA dependent RNA polymerase 6 (RDR6) effect on efficiency of BaMV- and satBaMV-VIGS vector, the RDR6-deficient N. benthamiana were inoculated with BaMV-VIGS vector or co-inoculated BaMV and satBaMV-VIGS vector. The results showed that RDR6-deficient N. benthamiana plants exhibited severe mosaic and stunting symptoms. Northern hybridization analysis revealed that the viral RNA of BaMV and satBaMV could be accumulated in the top-most leaves of RDR6-deficient N. benthamiana plants. The efficiency of BaMV-induced gene silencing was remarkable reduced, while the efficiency of satBaMV-induced gene silencing was relatively increased. The data supported that RDR6 gene is essential for BaMV and satBaMV to invade apical growth point. To the best of our knowledge, this is the first development of satellite RNA based vector system for virus-induce gene silencing applicable in monocotyledons and dicotyledonous.目次 中文摘要......................................................................................................i 英文摘要.....................................................................................................iii 壹、前言......................................................................................................1 一、基因靜默...............................................................................................1 二、病毒誘導基因靜默................................................................................2 三、竹嵌紋病毒...........................................................................................6 四、竹嵌紋病毒衛星核酸.............................................................................9 五、植物病毒的模式植物菸草....................................................................10 六、新興之溫帶草本模式植物二穗短柄草..................................................11 七、實驗目的.............................................................................................12 貳、材料與方法..........................................................................................14 一、植物材料..............................................................................................14 二、質體構築..............................................................................................14 三、植物接種方法.......................................................................................20 四、植物總量RNA之萃取............................................................................22 五、半定量反轉錄酶-聚合酶連鎖反應.........................................................22 六、即時定量聚合酶連鎖反應.....................................................................23 七、北方雜合分析.......................................................................................24 八、影像攝影方法.......................................................................................27 参、結果......................................................................................................29 第一章：發展竹嵌紋病毒衛星核酸成為病毒誘導基因靜默載體………….…29 一、BaMV及satBaMV之寄主範圍測試........................................................29 二、發展satBaMV成為基因靜默載體...........................................................29 三、satBaMV-VIGS載體誘導菸草內生基因靜默作用...................................31 四、satBaMV-VIGS載體抑制GFP轉殖菸草之GFP基因表現………..………32 五、比較satBaMV-與TRV-VIGS載體誘導基因靜默之異同……………….....33 六、satBaMV-VIGS載體可做為研究植物生長發育之逆向遺傳學工具..........34 七、satBaMV-VIGS載體抑制二穗短柄草基因之表現...................................35 第二章：研發竹嵌紋病毒誘導基因靜默為竹嵌紋病毒衛星核酸誘導 基因靜默載體之預報系統.............................................................................36 一、研發BaMV成為基因靜默載體................................................................36 二、運用BaMV-及satBaMV-VIGS載體同時誘導兩個基因產生靜默……..…37 三、BaMV-VIGS載體誘導二穗短柄草內生基因之靜默作用.........................38 四、BaMV-VIGS載體誘導大麥內生基因之靜默作用....................................39 第三章：RNA dependent RNA polymerase 6影響竹嵌紋病毒及其衛 星核酸誘導基因靜默載體之基因靜默效率....................................................40 一、BaMV及satBaMV能感染RDR6缺失菸草之新生葉.................................40 二、RDR6缺失菸草降低BaMV誘導基因靜默之效率.....................................40 三、RDR6缺失菸草增加satBaMV誘導基因靜默之效率……………….……...41 四、satBaMV-VIGS載體誘導RDR6缺失菸草之PCNA基因靜默....................42 肆、討論........................................................................................................44 伍、參考文獻.................................................................................................51 陸、表圖........................................................................................................6