棉花中不同植物病毒介导的VIGE体系的研究

Study on VIGE system mediated by different plant viruses in cotton

植物病毒介导sgRNA的传递与表达相比传统转化完整的编辑载体进行基因编辑具有巨大优势,因为sgRNA的表达可以伴随着病毒的复制和移动而快速扩增、累积,从而产生高效的基因编辑效率。为在棉花中开发应用更多植物病毒介导的基因编辑系统(virus-induced genome editing,VIGE)。本研究以超表达Cas9(Cas9-OE)棉花作为VIGE受体,在棉花中分别建立了棉花叶皱缩病毒(cotton leaf crumple virus,CLCrV)和烟草脆裂病毒(tobacco rattle virus,TRV)介导的VIGE系统。首先CLCrV和TRV介导的VIGE均可以靶向敲除棉花GhBsrk1和GhMAPKKK2基因A亚组和D亚组基因组序列,验证了这2个系统的可行性和有效性。进一步量化这2个系统对GhBsrk1和GhMAPKKK2基因的编辑效率结果发现,CLCrV和TRV介导的VIGE均能够产生高效的基因编辑效率,并且2种系统基因编辑效率不存在显著差异。本研究还探究了使用棉花内源U6启动子和拟南芥U6启动子驱动sgRNA对VIGE基因编辑效率的影响。结果显示,这2个启动子介导的VIGE均能够产生高效的基因编辑效率,并且基于这2种启动子介导的VIGE系统基因编辑效率不存在显著差异。以上结果预示着可以开发更多的植物病毒载体应用于棉花基因编辑的研究,进而获得高效的sgRNA筛选体系。

Plant virus-mediated sgRNA delivery and expression has tremendous advantage over traditional transformation of intact editing vectors for gene editing,because sgRNA expression can be rapidly amplified and be accumulated along with virus replication and movement,resulting in efficient gene editing efficiency.In this study,to develop and apply more plant virus-mediated gene editing(VIGE)systems in cotton,the overexpressing Cas9(Cas9-OE)cotton was used as VIGE receptor and the cotton leaf crumple virus(CLCrV)and tobacco rattle virus(TRV)mediated VIGE systems were established in cotton.First of all,both CLCrV and TRV-mediated VIGE could target and knock out the Gh Bsrk1 and GhMAPKKK2 in subgroup A and subgroup D genomic sequences,which verified the feasibility and effectiveness of these two systems.Further quantificaiton of the editing efficiency of Gh Bsrk1 and GhMAPKKK2 genes by these two systems showed that both CLCrV and TRV-mediated VIGE could produce high-efficiency gene editing efficiency,and there was no significant difference in gene editing efficiency between the two systems.This study also explored the effect of sgRNAs driven by cotton endogenous U6 promoter and Arabidopsis U6 promoter on VIGE gene editing efficiency.The results showed that both promoter-mediated VIGE were able to produce high-efficiency gene editing efficiency,and there was no significant difference in gene editing efficiency based on these two promoter-mediated VIGE systems.The above results indicate that more plant virus vectors can be developed for the application of cotton gene editing research,thus obtaining an efficient sgRNA screening system.