CRISPR/Cpf1与CRISPR/Cas9介导的广谱抗棉花曲叶病毒差异比较

Cotton leaf curl virus (CLCuV) is kind of geminivirus which seriously threaten to cotton production. In previous study, we found that Cotton leaf curl Multan virus (CLCuMuV) were detected in many malvaceous plants, which is threatening cotton production in Xinjiang. Therefore, it is imperative to cultivate cotton varieties which have broad-spectrum resistant to CLCuV. In this project, CRISPR/Cas system will be adopted to targeting different conserved sequence of coding replication initiation protein (Rep) or non-coding region (IR) in the genomes of multiple CLCuV. The gRNA specific to Rep and IR sequences were delivered into cotton by Agrobacterium tumefaciens-mediated transformation, respectively. Virus replication and resulting resistance in transgenic plants will be analyzed based on comparison of targeting Rep and IR sequences for cleavage by CRISPR/Cpf1 and CRISPR/Cas9. It will be helpful for us to determine the roles of difference target sequences and CRISPR/Cas system in CRISPR/Cas mediated transgenic cotton resistance. The editing efficiency of Cpf1 and Cas9 targeted to for cleavage different targeting sequences will be investigated, respectively. It will conductive to us confirm the correlation between targeted sequences and editing system and obtain CRISPR/Cas system suitable for transgenic cotton resistance to CLCuV.

棉花曲叶病毒（CLCuV）是严重威胁棉花生产的一类双生病毒。本课题组前期研究发现，木尔坦棉花曲叶病毒（CLCuMuV）已在新疆多种锦葵科植物上侵染危害，正在对新疆棉花构成严重威胁，因此培育广谱抗棉花曲叶病毒棉花品种势在必行。为此，本项目拟通过基因编辑技术构建分别靶向棉花曲叶病毒复制酶相关蛋白基因（Rep）和基因间隔区（IR）保守序列的CRISPR/Cpf1和CRISPR/Cas9系统，利用农杆菌介导的遗传转化方法转化棉花。通过比较CRISPR/Cpf1和CRISPR/Cas9系统分别靶向Rep区和IR区对病毒复制的影响及获得的抗性差异，明确靶位点选择及不同编辑系统对抗性的影响；通过比较Cpf1和Cas9对不同靶位点的编辑作用及编辑效率，明确靶位点与编辑系统之间的相关性，筛选适于抗棉花曲叶病毒的基因编辑系统，对棉花广谱抗棉花曲叶病毒育种具有重要意义。

木尔坦棉花曲叶病毒（Cotton leaf curl Multan virus，CLCuMuV）是引起棉花曲叶病（Cotton leaf curl disease，CLCuD）的主要病原之一，该病害严重影响棉花的生产。已有研究表明CRISPR/Cas9系统靶向DNA病毒可有效抑制病毒的积累。本研究针对CLCuMuV基因组DNA-A和DNAβ的复制及致病关键区域分别设计靶位点，成功构建由CRISPR/Cas9 和CRISPR/FnCpf1系统分别介导的8个单靶点编辑载体；为提高编辑效率，在此基础上进一步构建4个由35s启动子驱动的双靶点编辑载体，并在本氏烟中瞬时表达载体进行病毒接种实验，结果显示4个瞬时表达基因编辑载体均能够减轻CLCuMuV症状；为了明确棉花中CRISPR/Cas9 和CRISPR/FnCpf1编辑系统靶向CLCuMuV的差异，继而构建了由GhU6.9驱动的双靶点载体，并建立棉花子叶的瞬时表达体系，利用qRT-PCR验证棉花双靶点载体在子叶中能够瞬时表达Cas9和FnCpf1基因，病毒接种实验表明4个编辑载体在棉花中瞬时表达对CLCuMuV的病毒积累量降低了82.72%~87.94%，表明CRISPR/Cas9系统与CRISPR/Cpf1系统均能在棉花中有效抑制病毒积累。.为探索棉花的高效遗传转化，使基因编辑技术在棉花抗病毒研究中得到应用。利用纳米材料介导的棉花花粉磁转染方法分别将CRISPR/Cas9和CRISPR/FnCpf1介导的4个双靶点载体导入棉花基因组，成功结棉铃169个，所获1642粒种子播种后检测筛选出27株转基因阳性棉花，将为下一步棉花抗病毒转基因研究奠定基础。