水稻条纹病毒（RSV）等细胞质负链RNA病毒严重危害作物生产，然而，该类病毒致病机理研究受制于缺乏反向遗传学技术体系。负链RNA病毒的最小侵染单元为核糖核蛋白复合体，目前常用的RNA病毒cDNA克隆转录方案难以有效用于该类病毒的遗传工程操作。为此，课题组在前期构建了基于T7 聚合酶（T7 Pol）和RNA聚合酶III（Pol III）的体内转录方法。本项目拟据此设计两套技术方案：1）通过植物体内表达的T7 Pol转录病毒RNA，并利用不依赖帽子结构的翻译元件促进病毒核心蛋白的表达；2）优化Pol III转录病毒RNA的技术方案，并解决35S启动子转录核心蛋白mRNA伴随的剪接问题，提高其表达效率。在此基础上，建立基于T7 Pol和Pol III转录的RSV反向遗传学体系。本项目的实施有望突破制约该类病毒遗传操作的技术障碍，为构建全长侵染性cDNA 克隆打下基础，推动其生物学和病理学研究。

Rice stripe tenuivirus (RSV) and related cytoplasmic plant negative stranded RNA (NSR) viruses account for enormous crop diseases worldwide; however, our understanding of their pathogenesis has been greatly hindered by the unavailability of reverse genetics approach for this group of viruses. The minimal infection unit of NSR viruses are viral ribonucleoprotein complex (RNP) consisting of viral genome and replication-related core proteins including nucleocapsid protein and RNA-dependent RNA polymerase. The state of the art studies indicate that the commonly used T7 polymerase (T7 Pol) in vitro transcription and RNA polymerase II (Pol II) in vivo transcription systems are not widely suitable for genetic engineering of cytoplasmic NSR viruses. In order to tackle down this technical problem, we have developed T7 Pol in vivo transcription system and RNA polymerase III in vivo transcription systems in plants during our pilot studies. The current project aims to employ two approaches on the basis of the two systems, respectively, to build RSV reverse genetics systems. First, to construct the T7 Pol based-RSV reverse genetic system, we plan to use T7 Pol expressed in Nicotiana benthamiana plants to direct in vivo transcription of RSV minireplicon RNA. Cap-independent translation enhancer sequence derived from a varieties of positive stranded RNA viruses will be used to facilitate the expression of T7 transcripts encoding viral core proteins. Second, for the Pol III-based RSV reverse genetic system, Pol III promoter controlled transcription of RSV minireplicon RNA will be optimized. Meanwhile, 35S promoter controlled expression of viral core proteins will be improved by mitigation of RNA polymerase II transcription-coupled mRNA splicing. Our studies promise to resolve the most serious technical hurdle plaguing genetic manipulation of plant NSR viruses, and should provide a foundation for construction of full-length infectious cDNA clone that are essential for virus biology and pathology characterization.