《植物生理学报》 2017, 53(4): 736-746

生菜CRISPR/Cas9基因编辑体系的建立

禹明森¹, 李翔¹, 高马也², 杨蕾², 倪迪安^2,*, 王应祥^1,*

¹复旦大学生命科学学院, 植物科学研究所, 上海200433; ²上海应用技术大学生态技术与工程学院, 上海201418

通信作者：倪迪安；E-mail: yx_wang@fudan.edu.cn; dani@sibs.ac.cn

摘　要：

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 sativa 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T₀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T₁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。

关键词：生菜; 农杆菌介导遗传转化; 基因编辑; CRISPR/Cas9

收稿：2017-03-13   修定：2017-04-12

资助：国家自然科学基金(31370347和31570314)、上海市绿化和市容管理局年度科技项目(G179910)和荷兰Rijk Zwaan种业公司。

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

YU Ming-Sen¹, LI Xiang¹, GAO Ma-Ye², YANG Lei², NI Di-An^2,*, WANG Ying-Xiang^1,*

¹Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China; ²School of Ecological Technology and Engineering, Shanghai Institute of Technology, Shanghai 201418, China

Corresponding author: NI Di-An; E-mail: yx_wang@fudan.edu.cn; dani@sibs.ac.cn

Abstract:

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 sativa 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.

Key words: Lactuca sativa L.; Agrobacterium-mediated genetic transformation; gene editing; CRISPR/Cas9