| Summary: | 碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 103 === 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.
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