張雅君臺灣大學：植物病理與微生物學研究所林怡礽Lin, I-JengI-JengLin2007-11-272018-06-292007-11-272018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/58073植物病毒要能成功地在植物體內完成感染，需要在植物細胞內複製，且透過原生質絲作細胞間的移動，以及經由維管束進行長距離的移動。大部分的RNA病毒需要表現自己的鞘蛋白才能有效率的系統性移動，也有些例子必須先被包被成病毒顆粒後才可以移動，而蕃茄叢生矮化病毒(Tomato bushy stunt virus，簡稱TBSV)是在特定寄主中不需要鞘蛋白即可系統性感染的特殊的一類。本論文以蕃茄叢生矮化病毒及胡瓜壞疽病毒(Cucumber necrosis virus，簡稱CNV)，兩者在基因結構及序列上相似度極高的Tombusvirus屬病毒為材料，以改良式綠色螢光蛋白(enhanced green fluorescent protein，簡稱EGFP)基因作為報導基因，利用插入(a)或是置換(b)鞘蛋白基因的方式，構築出pTBSV-EGFPa、pCNV*-EGFPa、pTBSV-EGFPb及pCNV*-EGFPb突變株。以TBSV與CNV野生株及其突變株為DNA模板進行生體外轉錄反應(in vitro transcription)，將所得的生體外轉錄體接種至菸草原生質體、菸草植株及不同品種的蕃茄植株上，再利用北方雜合反應(Northern hybridization)和即時聚合Successful infection in plants by plant viruses requires that viruses can accomplish a series of infection steps including replication in plant cells, cell-to-cell movement through plasmodesmata, and long-distance movement via vascular tissue. Most RNA viruses need coat protein expression to facilitate efficiently systemic movement and some viruses have to form virions in order to move within plants. Tomato bushy stunt virus (TBSV) is in a special group of plant viruses that coat protein is dispensable for systemic movement in some specific hosts. In this study, TBSV and Cucumber necrosis virus (CNV) that have the same genome organization and high sequence homology were chosen to construct viral vectors. Enhanced green fluorescent protein (EGFP) was used as a reporter gene. Insertion and replacement of the coat protein gene were applied to construct four mutants: pTBSV-EGFPa, pCNV*-EGFPa, pTBSV-EGFPb, and pCNV*-EGFPb. The wild-type TBSV and CNV clones and their derived mutant clones were used as DNA templates to perform in vitro transcription. Protoplasts and plants of Nicotiana benthamiana and different varieties of tomato plants were inoculated by the transcripts, respectively. Northern hybridization and Real-Time PCR were used to compare genomic RNA accumulation of the wild-type and mutants of TBSV and CNV in vivo. The EGFP expression in protoplasts as well as the movement of four viral mutants in plant were observed by detection of fluorescence with an epifluorescence microscope. The results revealed that all mutants could replicate in N. benthamiana protoplasts. The level of genomic RNA accumulation of CNV*-EGFPb in protoplast was about 1/10 of TBSV and CNV. Whereas, TBSV-EGFPa, CNV*-EGFPa, and TBSV-EGFPb were only about 1/465 amount of RNA in TBSV or CNV-infected protoplasts. Analysis of genomic RNA accumulation of these mutants in N. benthamiana plants showed that TBSV-EGFPa, CNV*-EGFPb, and TBSV-EGFPb accumulated to similar level as CNV, but about 1/8 amount of TBSV in inoculated leaves at three days post inoculation (dpi). The amount of CNV*-EGFPa RNA accumulated in inoculated leaves was about 1/865 amount of TBSV at 3 dpi. The symptoms caused by mutants were delayed as compared to wild type viruses. In addition, TBSV-EGFPa was able to move systemically and green fluorescence was observed in systemic leaves. Four cultivars of tomato plants could be infected with TBSV and TBSV-EGFPa. Both TBSV and TBSV-EGFPa infected tomato plant cultivar ‘Shin-Guang’ systemically and green fluorescence was observed in TBSV-EGFPa-infected systemic leaves. According to the experimental results, TBSV seems more suitable than CNV to be engineered as a gene expression vector. Furthermore, the mutant construct derived from TBSV that had foreign gene inserted into the 5’ end of coat protein gene could systemically infect N. benthamiana and tomato plants, and the foreign gene was able to express during infection. Accordingly, a TBSV-based vector can be used to express the interested genes in plants in the future.一、中文摘要……………………………………………………………1 二、英文摘要……………………………………………………………4 三、前言…………………………………………………………………7 四、前人研究……………………………………………………………9 1. TBSV與CNV之相關研究……………………………………10 (1) Tomato bushy stunt virus (TBSV)……………………10 (2) Cucumber necrosis virus (CNV) ………………………11 2. 植物病毒載體之相關研究…………………………………11 3. 報導基因(reporter gene)在病毒移動上研究的應用…13 五、材料與方法………………………………………………………14 1. 病毒材料……………………………………………………15 2. 構築TBSV與CNV之突變株…………………………………15 (1) pTBSV-EGFPa突變株之構築………………………………15 (2) pCNV*-EGFPa突變株之構築………………………………16 (3) pTBSV-EGFPb突變株之構築………………………………16 (4) pCNV*-EGFPb突變株之構築………………………………16 3. 試驗植物與栽種方法………………………………………17 (1) 菸草之栽種…………………………………………………17 (2) 蕃茄之栽種…………………………………………………17 4. 生體外轉錄體(in vitro transcript)之製備…………17 (1) 勝任細胞(competent cell)之製備………………………17 (2) 轉形試驗(transformation)………………………………18 (3) 質體DNA之中量製備(midipreparation)…………………18 (4) 限制酵素直線化質體(plasmid linearization)………19 (5) 生體外轉錄反應(in vitro transcription)……………20 5. 專一性核酸探針(probe)之製備…………………………20 (1) TBSV與CNV 3’端專一性核酸探針………………………20 (2) EGFP專一性核酸探針………………………………………21 6. 原生質體之製備、接種與分析……………………………21 (1) 原生質體之製備……………………………………………21 (2) 原生質體之接種……………………………………………22 (3) 原生質體全RNA(total RNA)之萃取與電泳分析…………23 (4) 北方轉漬法(Northern blotting)………………………23 (5) 北方雜合反應(Northern hybridization)………………24 7. 植株之接種與分析…………………………………………25 (1) 植株之接種方式……………………………………………25 (2) 植物全RNA之萃取與電泳分析……………………………25 (3) 病毒顆粒之純化與電泳分析………………………………26 (4) UA (uranyl acetate)陰染法(negative staining)及電子顯微鏡觀察(Electron Microscopy)…………………………………26 (5) 植物全蛋白質之粗萃取……………………………………27 (6) 聚丙烯醯胺凝膠電泳(SDS-PAGE)分析……………………27 (7) 西方轉漬分析法(Western blot analysis)……………27 8. EGFP螢光之分析……………………………………………28 (1) 原生質體之螢光分析………………………………………28 (2) 植物葉片之螢光分析………………………………………28 9. 即時聚合681649 bytesapplication/pdfen-US蕃茄叢生矮化病毒胡瓜壞疽病毒基因表現載體gene expression vectorsCucumber necrosis virusTomato bushy stunt virusotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58073/1/ntu-94-R90633003-1.pdf