生菜CRISPR/Cas9基因编辑体系的建立

Establishment of CRISPR/Cas9-meidiated genome editing system in lettuce(Lactuca sativa L.)

CRISPR/Cas9基因编辑技术已经成功应用于多种植物基因组的编辑,但是在生菜中鲜有报道。本研究拟在生菜(Lactuca sati va L.)中建立CRISPR/Cas9基因编辑体系。首先根据生菜FANCM基因序列设计靶位点,构建Lsfancm-CRISPR/Cas9载体。然后以生菜子叶作为外植体,通过农杆菌侵染、共培养、愈伤组织诱导、芽再生及生根培养等过程,对生菜进行农杆菌介导的遗传转化,共获得24株T_0代转基因阳性植株。进一步对靶位点序列进行PCR并测序分析,发现4株转基因生菜的靶位点发生基因编辑,产生fancm杂合突变体,并且在T_1代植株中鉴定到纯合突变体。以上结果表明,本研究已成功将CRISPR/Cas9基因编辑体系有效地运用于生菜中,该体系的建立可为将来生菜的基因功能研究及分子育种提供重要的技术参考。

CRISPR/Cas9 system has been widely applied to genome editing in many plant species,but was rarely reported in lettuce.The present study was aimed to establish the CRISPR/Cas9-meidated genome editing system in lettuce（Lactuca saliva L.）.The lettuce FANCM gene was used for testing the system.According to the FANCM gene sequence,we designed the target site and successfully constructed the Lsfancm-CRISPR/Cas9 vector.Then we introduced the constructed Lsfancm-CRISPR/Cas9 plasmid into lettuce through Agrobacterium tumefaciens mediating genetic transformation approach with seedling cotyledons as explants and inoculating agrobacterium strain,co-cultivating,calli-inducing,shooting,and rooting as transformation steps.In total,24 transgenic positive plants were obtained in T0 generation by PCR verification.Further PCR detection and sequence analysis on target site demonstrated that 4 plants were genetically edited resulting in heterozygous fancm mutants.Furthermore,the homozygous mutants in T1 generation were obtained.The effective heritable genome editing system CRISPR/Cas9 established in this study will provide important resources not only for functional gene studies but also for genetic manipulation of lettuce breeding.