蕃茄叢生矮化病毒與胡瓜壞疽病毒作為基因表現載體之研究

Abstract

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.