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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 gene editing technology has been successfully applied to a variety of plant genome editing, but rarely reported in lettuce. This study aims to establish a CRISPR / Cas9 gene editing system in lettuce (Lactuca sati va L.). According to the sequence of lettuce FANCM gene, the target site was designed and Lsfancm-CRISPR / Cas9 vector was constructed. Then, using lettuce leaves as explants, Agrobacterium-mediated genetic transformation of lettuce was carried out through the process of Agrobacterium infection, co-culture, callus induction, bud regeneration and rooting culture, and 24 T0 transgenic positives Plant. Further PCR and sequencing analysis of the target site sequences revealed that gene editing was performed at the target sites of four transgenic lettuce plants to generate fancm heterozygous mutants and homozygous mutants were identified in the T 1 plants. The above results indicate that this study has successfully applied the CRISPR / Cas9 gene editing system to lettuce. The establishment of this system will provide important technical reference for gene function research and molecular breeding of lettuce in the future.