杜仲CRISPR/Cas9基因编辑系统的建立

An efficient CRISPR/Cas9 mutagenesis system for Eucommia ulmoides

CRISPR/Cas9系统是一类有效的目标基因编辑工具,能够精准编辑植物基因组以改善植物的性状。该技术能够同时靶向一个或多个序列,切割后通过非同源末端连接或同源重组引入新的遗传变异。然而,CRISPR/Cas9技术对于木本植物仍为挑战,主要局限于基因组倍性与再生转基因植物的能力。为了建立一套有效的杜仲(Eucommia ulmoides)基因编辑系统,利用农杆菌介导的遗传转化,以杜仲EuFLC(Flowering Locus C)基因为靶标,通过设计4个向导RNAs(sgRNAs)靶向EuFLC的2个保守区域转化杜仲,在卡那霉素选择培养基上筛选4周后,分离出抗性株系,通过Sanger测序对其进行基因分型。结果表明:EuFLC的编辑效率约为5.62%,sgRNA1与sgRNA2介导的突变类型均为单个碱基的替换,其中sgRNA1编辑效率约为1.12%;sgRNA2编辑效率最高,约为4.49%,首次成功在杜仲细胞中实现基因编辑。本研究为杜仲基因功能鉴定与遗传改良提供了新的途径。

CRISPR/Cas9 system is an effective tool for gene editing,which can accurately edit plants genome to improve the traits of the plants.This technology allows targeting one or more sequences simultaneously,and introduces new genetic variations by non-homologous end joining or homologous recombination.However,CRISPR/Cas9 is still a challenge for woody plants,limited to their genome ploidy and the ability to regenerate transgenic plants.In order to establish an efficient editing system of Eucommia ulmoides,we designed 4 small guide RNAs(sgRNAs)targeting two conserved gene regions of EuFLC and transformed into E.ulmoides by agrobacterium-mediated genetic transformation.After four weeks culture on kanamycin selective medium,the resistant lines were isolated and genotyped by Sanger sequencing.The results showed that the editing efficiency of EuFLC was about 5.62%.The type of mutation mediated by sgRNA1 and sgRNA2 were a single base mutation,and the editing efficiency of sgRNA1 was about 1.12%;sgRNA2 had the highest editing efficiency about 4.49%.These results demonstrated that gene editing is being achieved for the first time in E.ulmoides.This study provided a new way for gene function research and genetic improvement of E.ulmoides.