利用CRISPR/Cas13技术抑制SPCSV-RNase3介导的病毒协生作用及改良甘薯病毒病抗性的研究

甘薯褪绿矮化病毒(SPCSV)和甘薯羽状斑驳病毒(SPFMV)协生共侵染引起的甘薯病毒病(SPVD)严重危害我国乃至世界甘薯产业。目前尚无合适的遗传改良方法用于提高SPVD抗性。CRISPR/Cas13技术能够直接靶向干涉RNA，在防治植物RNA病毒病中具有较大应用前景。本项目拟利用该技术靶向干涉SPCSV-RNase3 (RNA沉默抑制子，SPVD致病关键因子)，希望通过抑制其介导的病毒协生作用来改良SPVD抗性。本项目首先以本氏烟作为研究材料，利用瞬时CRISPR/Cas13实验技术体系筛选有效Cas13-gRNAs组合, 并研究RNase3瞬时靶向干涉抑制病毒协生作用的效果；之后，通过遗传转化方式获得Cas13-gRNAs转基因甘薯并全面分析其SPVD抗性，最终获得SPVD抗性改良的甘薯新材料。本项目研究成果可为SPVD抗性遗传改良提供全新的思路方法、坚实的理论依据和可靠的技术体系。

Sweet potato virus disease (SPVD), which is caused by the co-infection of two RNA viruses (sweet potato chlorotic stunt virus: SPCSV and sweet potato feathery mottle virus: SPFMV), has great impact on the sweet potato industry in China and even all over the world. However, there is no suitable genetic improvement method to enhance SPVD resistance. Cas13 is a newly-discovered type VI CRISPR (Clustered regularly interspaced short palindromic repeats)-associated RNA-guided ribonuclease that cleaves RNA with high efficiency and specificity. This provides an improved tool for cleaving viral RNAs and protecting plants from the infection of RNA viruses. Therefore, CRISPR/Cas13 technique has great application in the prevention and control of plant RNA viruses. In this project, we intend to use CRISPR/Cas13 system to enhance SPVD resistance and this is mainly achieved by targeting SPCSV-RNase3, which is a viral RNA silencing suppressor as well as core pathogenic factor causing viral synergism. Firstly, we plan to use Nicotiana benthamiana as transient experimental system to screen efficient Cas13 enzymes and guide RNAs (gRNAs) and to evaluate the effect of transient RNase3-cleaving on the viral synergism. Furthermore, we intend to generate Cas13-gRNAs transgenic sweet potato by using of Agrobacterium tumefaciens-mediated transformation method. Subsequently, the SPVD resistance of Cas13-gRNAs transgenic sweet potato will be investigated comprehensively. The output of this project will provide new idea, solid theoretical basis and reliable technical system for the genetic improvement of SPVD resistance.