The study of ATP synthase beta subunit on Bamboo mosaic virus and its satellite RNA infection in Nicotiana benthamiana

竹嵌紋病毒(Bamboo mosaic virus, BaMV)，分類上屬於Alphaflexiviridae科，Potexvirus屬。為單一核酸、正股的RNA病毒，全長6,366個核苷酸。在前人研究中，曾利用RNA親和性管柱層析法，發現在病毒複製複合體中的葉綠體ATP合成酶β單元蛋白(Chloroplast ATP synthase CF1 beta subunit, cpAtpB)，會與BaMV衛星核酸(-) 3'端啟動子結合。本研究利用本氏菸草(Nicotiana benthamiana)作為病毒寄主，探討植物cpAtpB蛋白與BaMV累積量之關係。為了觀察cpAtpB蛋白於BaMV感染的菸草中是否有表現量的差異，我們先行構築一段帶有cpAtpB全長之pET29a-cpAtpB質體，並轉型進入大腸桿菌(BL21)後利用IPTG誘導大量蛋白表現。利用SDS-PAGE分離及切割目標蛋白後，將純化的蛋白作為抗原並打入兔子體內製備抗體。在接種BaMV-S之菸草利用獲得之抗體進行西方墨點法，與健康菸草比較後，發現cpAtpB蛋白表現量沒有顯著性的差異。接著我們利用菸草脆裂病毒(Tobacco rattle virus, TRV)基因靜默技術(virus-induced gene silencing, VIGS)探討cpAtpB在BaMV感染過程中之重要性。選擇cpAtpB基因之3'端非轉譯區當作進行基因靜默的片段，利用RT-PCR測試基因靜默效率，結果使用此區域，cpAtpB基因無法被靜默掉，可能的原因推估為在葉綠體中未含有gene silencing的系統，使得基因無法被減弱。因此將測試cpAtpB蛋白過量表現，透過帶有橘色螢光蛋白(Orange fluorescent protein, OFP)及菸草signal peptide的cpAtpB蛋白質短暫表現於葉綠體中，並接種BaMV與satBaMV，觀察其對於病毒累積量的影響。結果得知，cpAtpB對於BaMV RNA量上沒有顯著性差異，而對於satBaMV RNA有明顯的增加。因此cpAtpB對於satBaMV累積量上是有明顯的關係。未來可進一步討論(1)BaMV RNA與cpAtpB結合 (2)BaMV與satBaMV共感染對於cpAtpB累積量的影響 (3)測試cpAtpB大量表現後in vitro RdRp complex transcription效率。並且觀察cpAtpB蛋白是否會影響其他病毒的感染性。Bamboo mosaic virus (BaMV) belongs to genus Potexvirus of family Alphaflexiviridae and is a single-stranded positive-sense RNA virus. The genome of BaMV is 6,366 kb with a 5'cap and a 3'poly (A) tail. In our previous study, using RNA affinity chromatography had confirmed the chloroplast ATP synthase CF1 beta subunit (cpAtpB) binds on satBaMV (-) 159 fragment. In this study, we use Nicotiana benthamiana as a model plant to observe the relationship between cpAtpB and BaMV accumulation. In order to observe the difference of cpAtpB expression in N. benthamiana after BaMV infection, we constructed a pET-29a vector with full length of cpAtpB, transformed into E. coli (BL21) and then used IPTG to induce the protein expression. SDS-PAGE was used to separate and purify the target protein for antiserum preparation in rabbit. Western blot results show no significant difference of cpAtpB protein accumulation between healthy and BaMV infected N. benthamiana. To further characterize the role of cpAtpB in BaMV infection cycle, we used the Tobacco rattle virus (TRV)-based virus induced gene silencing (VIGS) technology to knock down the expression of cpAtpB in N. benthamiana to reveal the relationship between cpAtpB and BaMV infection. We choose cpAtpB gene 3'UTR as a fragment to silence cpAtpB by VIGS. Test by RT-PCR for silencing efficiency revealed that cpAtpB gene can't not be silenced in this study, which might be no gene silencing system in the chloroplast. Therefore, BaMV or satBaMV accumulations were tested after overexpression of cpAtpB chloroplast targeted or signal peptide fused cpAtpB protein in N. benthamiana. The result showed that there is no impact on BaMV accumulation but satBaMV accumulation was increased, suggesting that cpAtpB might be involved in the satBaMV accumulation. In the future, we will discuss (1) Whether BaMV RNA binding to cpAtpB (2) Effects of BaMV and satBaMV co-infection on cpAtpB accumulation (3) Testing the RdRp complex transcription efficiency in cpAtpB overexpression. And, the effects of cpAtpB on other viruses infection will be investigated.目 錄 中文摘要 i 英文摘要 ii 壹、前言 1 一、竹嵌紋病毒介紹 1 二、宿主蛋白質參與病毒感染循環系統 2 三、Chloroplast ATP synthase β subunit protein 2 貳、材料與方法 4 一、製備cpAtpB抗體 4 (一)構築cpAtpB質體以大腸桿菌大量表現蛋白質製作抗體 4 (二)抽取BaMV複製複合體 5 (三)萃取植物總量蛋白 5 (四)聚丙醯胺膠體電泳(SDS-Polyacrylamide Gel Electrophoresis, SDS-PAGE) 5 (五)西方漬染免疫偵測(Western blot analysis) 6 二、病毒誘導基因靜默法(virus induced gene silencing, VIGS)靜默cpAtpB基因表達 6 (一)構築基因靜默質體 6 (二)農桿菌(Agrobacterium tumefaciens) C58C1製備與接種 7 (1)農桿菌勝任細胞製備 7 (2)電穿孔轉型 7 (3)農桿菌接種 8 (三)RNA萃取與Semi-Quantitative RT-PCR 8 三、暫時性表達(transient expression) 8 (一)構築質體在植物表達cpAtpB 8 (二)農桿菌(Agrobacterium tumefaciens) GV3850接種 9 (三)感染帶有BaMV與satellite BaMV基因的農桿菌 10 (四)北方墨點法分析與探針製備 10 (1)植物總量RNA之萃取 10 (2)核酸探針製備 11 (3)RNA電泳分離與轉漬 11 (4)雜合反應 11 參、結果 12 一、cpAtpB抗體製備 12 二、受BaMV感染後cpAtpB蛋白變化量 12 三、利用TRV病毒進行靜默cpAtpB基因的表現 13 四、短暫表達cpAtpB蛋白質後對BaMV或satBaMV感染能力之影響 13 肆、討論 14 伍、References 16 陸、圖表 2