2009-12-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/655686摘要：本計畫之目標在利用生物技術及高解析度之共軛焦顯微影像技術，探討植物RNA病毒TMV於感染寄主植物過程中，阿拉伯芥寄主蛋白VIRAP與病毒複製累積之關係及參與病毒複製機制，病毒複製機制之瞭解，將有助於開發植物RNA病毒病害防治之嶄新標的。利用系統性的分析，本計劃之研究人員已篩選到一個與病毒複製累積相關的寄 主蛋白VIRAP，為進一步探討其如何影響病毒複製，本研究計畫以多螢光標定技術，探討VIRAP是否參與在VRCs的生合成，並利用高解析度之共軛焦顯微影像技術探討VIRAP和病毒複製蛋白，在細胞中分佈的相關性，以及是否具有交互作用，以進一步釐清VIRAP影響病毒複製的相關機制。 <br> Abstract: Plant host factors essential for viral replication could be targets for disease control and antiviral therapy. In the initial state of replication, viruses utilize their encoded limited protein(s) to change cellular compartmentalization and interact with host factors to assembly replication complex. To select host factors for antiviral strategy, Arabidopsis thaliana cDNA microarray was applied to monitor the protoplast transcriptome change in initial state of replication during transfection by Tobacco mosaic virus (TMV). One consistently up-regulated gene, named as VIRAP, from replica experiments was identified and further investigated. Double-strand RNAs specific to VIRAP were introduced in Arabidopsis protoplasts to induce RNAi, and TMV accumulation were not observed by northern blot hybridization in those protoplasts. Transgenic A. thaliana plants carrying VIRAP hairpin RNAs were generated. TMV was inoculated to the transgenic plants, and virus accumulation was partially inhibited in those transgenic plants. The virus accumulation levels are positively correlated with the knockdown degree of VIRAP. Following, virus replication complex (VRC) formations in wild type, VIRAP-silenced protoplasts and VIRAP-overexpressed cells will be further confirmed. Green and yellow fluorescent proteins will be fused to VIRAP and TMV P126, respectively. Those fusion proteins will be used to transfect A. thaliana protoplasts and cellular localizations will be assayed by confocal microscope examination. The mechanism about how VIRAP involved in TMV replication will be further understood through the results of confocal microscope examination.植物病毒病毒複製VRCs菸草嵌紋病毒P126阿拉伯芥寄主因子VIRAP農業生物技術共軛焦顯微鏡plant virusvirus replicationVRCsTMVP126Arabidopsishost factorVIRAPAgricultural Biotechnologyconfocal microscope