- 學位論文
齒舌蘭輪斑病毒缺失性RNA與鞘蛋白次基因體啟動子之分析與應用
Characterization and Application of Odontoglossum ringspot virus Defective RNA and Capsid Protein Subgenomic Promoter
摘要
蘭花為台灣重要之經濟花卉，其中又以蝴蝶蘭種苗為出口的最大宗。目前已知紀錄之蘭花病毒至少有28種，其中菸草嵌紋病毒屬(Tobamovirus)中的齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)為對蘭花最具威脅的病毒之一。缺失性RNA (defective RNA, dRNA)是RNA病毒經由基因體缺失重組後所形成的次病毒分子，此類型RNA必需有輔助病毒(helper virus)的協助，才能在植物體內進行複製、移行或包被；有些dRNA的高增殖力甚至能干擾輔助病毒的感染，使植物病徵減弱，因為這些特性，使得dRNA常成為病毒學家研究的對象或工具。本研究嘗試以缺失性RNA的形式製備ORSV之病毒載體，並利用鞘蛋白次基因體啟動子(subgenomic promoter)來表現綠螢光蛋白(enhanced green fluorescent protein, EGFP)。利用實驗室先前所構築的ORSV感染性選殖株，建立人為的ORSV缺失性RNA，接種於菸草原生質體中並篩選出感染性較佳的選殖株。結果顯示在人為構築的5種缺失性RNA中，以保留5’端1444個核苷酸及3’端1173個核苷酸之突變株lc的RNA累積量較多。另外藉由構築一系列ORSV刪除突變株，分別感染菸草原生質體以分析ORSV鞘蛋白基因的次基因體啟動子區域。實驗結果顯示，以鞘蛋白基因轉錄起始點為基準，-78到+32的序列位置為可能的啟動子區域，而-38到+12之序列為核心區域。另外，此區域序列在二級結構預測中會形成stem-loop的結構，而此二級結構對鞘蛋白次基因體啟動子之功能是必要的。綜合以上結果，將複製能力良好、並保留鞘蛋白次基因體啟動子的dRNA選殖株作為載體，攜帶EGFP基因，並測試其在植物單細胞層次的表現情形，經實驗確定此載體能成功表現EGFP。另外也嘗試接種於菸草植株，以分析此病毒載體之基本特性，並評估其應用潛力。
並列摘要
Orchids are important floral plants in Taiwan, and the Phalaenopsis is the most cultivated and exporting one. There are more than 28 viruses that have been reported to infect orchids. One of the most important viruses affecting orchid industry is the Odontoglossum ringspot virus (ORSV), belonging to the genus Tobamovirus. Defective RNAs (dRNAs) are RNA molecules that arise through the deletion and rearrangement of internal sequences from the genome of RNA viruses. Therefore, dRNAs require helper virus for their replication, movement or encapsidation. Some dRNAs with higher replication capability can even attenuate the symptoms caused by helper virus. Because of these unique traits, dRNAs become useful research tools for virologists. In this study, we try to develop viral vector base on ORSV artificial dRNA clone, and use capsid protein (CP) subgenomic RNA promoter to express green fluorescent protein. At first, we used previously constructed ORSV cDNA clone to create artificial dRNAs. The replication capability of the dRNA clones were then assayed in Nicotiana benthamiana protoplasts. One of dRNA clone lc which contains the ORSV genomic sequence of 5’ 1444 nts and 3’ 1173 nts showed high RNA accumulation in protoplast assays. In addition, we mapped the ORSV CP subgenomic RNA promoter using a series of deletion mutations. According to the results, CP subgenomic RNA promoter is located between -78 to +12 and the core active promoter is between -38 to +12, relative to the +1 transcription start site. In addition, computer predicted folding of these region revealed a unique stem-loop structure. This secondary structure is required for the normal function of CP subgenomic RNA promoter. Finally, we tried to use the ORSV artificial dRNA clone containing the CP subgenomic RNA promoter as a viral vector and the expression of EGFP in protoplasts was confirmed. We will evaluate basic characteristic and application possibility of this viral vector.