摘要： 基于CRISPR/Cas9的基因组编辑技术是一种新兴的分子修饰工具，可实现基因组特定位点碱基的缺失、插入或替换，造成基因功能丧失，现已广泛应用于基因功能的研究。为深入探讨甘蓝型油菜中BnSVP的生物学功能，以中双11号基因组（甘蓝型油菜参考基因组，v4.1）中的4个BnSVP为靶标基因，并针对不同染色体上的BnSVP基因，应用CRISPR-P 2.0软件共设计了12条特异sgRNA种子序列，以实现4个BnSVP同源基因的全部或部分敲除；以pYLCRISPR-Cas9P35S-H为基本载体，以AtU3b或AtU3d为sgRNA转录启动子，采用Golden gate cloning技术分别组装构建了6个双靶点CRISPR/Cas9植物表达载体。测序结果表明，6个CRISPR/Cas9植物表达载体各sgRNA表达盒DNA序列正确，双靶点组装顺序无误。研究结果为进一步原生质体瞬时表达和农杆菌介导的油菜遗传转化奠定了基础，同时也为应用CRISPR/Cas9基因组编辑系统研究植物多拷贝同源基因的功能提供参考。

关键词: 甘蓝型油菜, SVP基因, CRISPR/Cas9, 基因组编辑载体

Abstract: Targeted genome editing via CRISPR/Cas9 is emerging as a powerful tool for molecular modification in recent years,by which the target gene will lose functions because of deletion,insertion or substitution of several nucleotides in specific sites. It has been widely used to researches on gene function. To deeply elucidate the biological function of Short vegetative phase (SVP)in rapeseed(Brassica napus L.)by entire or partial knock-outs,totally twelve sgRNA seed sequences targeting four homologous copies of SVP distributed in Zhongshuang No.11 genome(Brassica napus reference genome,v4.1)were designed by using software CRISPR-P 2.0,and then six dual sgRNAs CRISPR/Cas9 genome editing constructs were developed employing pYLCRISPR-Cas9P35S-H as backbone and AtU3b or AtU3d as promoter with Golden Gate Cloning technology. Sequencing results demonstrated that all the recombinant plasmids showed the correct nucleotides and expression cassette assembly,which lay a solid foundation for further protoplast transient expression assay and Agrobacterium -mediated transformation of rapeseed. Meanwhile,this paper provides a strategy to study functions of multiple homologous genes in plants applying CRISPR/Cas9 genome editing system.

Key words: Brassica napus, Short vegetative phase gene, CRISPR/Cas9, Genome editing construct

中图分类号: 

• Q78