以侵染性克隆技术为核心的反向遗传学分析是现代分子病毒学研究中最基本、最重要的技术手段，然而迄今为止，国际上尚未有植物负链RNA病毒侵染性cDNA克隆成功构建的报道，这严重制约了该类病毒的基础和应用研究。申请者实验室在前期工作中，以弹状病毒科苦苣菜黄网病毒（SYNV）为模式，建立了高效的SYNV微型复制子（minireplicon）反向遗传学体系，为全长侵染性克隆构建打下了坚实的基础。本项目拟在此基础上，进一步优化微型复制子工作效率，并依次构建微型基因组（minigenome）和全长cDNA克隆，逐步解决影响克隆侵染性的技术难点，如启动子利用、末端序列的忠实性和病毒组份多，模板长伴随的低效问题，最终获得SYNV侵染性cDNA克隆，并建立反向遗传学技术体系。本项目的实施可望突破破制约植物负链RNA病毒研究的主要技术障碍，推动其它重要作物负链病毒的反向遗传学研究，具有重要的理论意义和应用价值。

Infectious clone-based reverse genetics approach is the most fundamental and essential technique in modern molecular virology. Although reverse genetics has been applied to animal negative-strand RNA viruses for nearly 20 years, unfortunately, application of similar approach to plant counterparts has not been accomplished so far, which greatly limits our ability to genetically manipulate plant negative-strand viruses. Previously, our laboratory has established an efficient minireplicon-based reverse genetics approach to study a plant rhabdovirus, Sonchus yellow net virus (SYNV), which set up a solid platform for construction of full-length infectious clone. On the basis of this approach, the present study aimed to further optimize the SYNV minireplicon derivatives, and to sequentially construct more complex minigenome and full-length clone. We use bottom-up approaches and design multiple experimentation schemes to tackle down the most serious technical hurdles of reverse genetics analysis of plant negative-stranded viruses, including promoter design, authetic 5' and 3'-termini sequences and inefficiency inherent in multi-components and long template. Our approach should also be applicable to other members of plant negative-strand viruses which collectively cause an enormous number of economically important crop diseases and facilitate the fundamental and applied studies of those viruses.