植物学报 ›› 2023, Vol. 58 ›› Issue (2): 298-307.DOI: 10.11983/CBB22240

• 技术方法 • 上一篇    下一篇

柳枝稷木质素基因F5H的高效编辑

邱锐1,2, 何峰2, 李瑞2, 王亚梅2, 邢思年2, 曹英萍2, 刘叶飞1,2, 周昕越1, 赵彦1,*(), 付春祥2,*()   

  1. 1内蒙古农业大学草原与资源环境学院/农业农村部饲草栽培、加工与高效利用重点实验室/草地资源教育部重点实验室, 呼和浩特 010018
    2中国科学院青岛生物能源与过程研究所, 中国科学院生物燃料重点实验室, 山东能源研究院, 山东省能源生物遗传资源重点实验室, 青岛新能源山东省实验室, 青岛 266101
  • 收稿日期:2022-10-13 接受日期:2023-01-16 出版日期:2023-03-01 发布日期:2023-03-15
  • 通讯作者: *E-mail: zhaoyannmg@163.com;fucx@qibebt.ac.cn
  • 基金资助:
    中国科学院战略性先导科技专项(A类)(XDA26030301);内蒙古自治区关键技术攻关计划(2020GG0176);“科技兴蒙”重点专项(2020-科技兴蒙-草种业技术创新中心-2);国家自然科学基金(32160326)

Highly Efficient Gene Editing of Lignin Gene F5H in Switchgrass

Rui Qiu1,2, Feng He2, Rui Li2, Yamei Wang2, Sinian Xing2, Yingping Cao2, Yefei Liu1,2, Xinyue Zhou1, Yan Zhao1,*(), Chunxiang Fu2,*()   

  1. 1Key Laboratory of Grassland Resources of Ministry of Education/Key Laboratory of Forage Cultivation, Processing and Efficient Utilization of Ministry of Agriculture and Rural Areas/College of Grassland, Resources and Environment of Inner Mongolia Agricultural University, Hohhot 010018, China
    2Qingdao New Energy Shandong Laboratory, Shandong Key Laboratory of Energy Biogenetic Resources, Shandong Energy Institute, Key Laboratory of Biofuels, Chinese Academy of Sciences, Qingdao Institute of Bioenergy and Process, Chinese Academy of Sciences, Qingdao 266101, China
  • Received:2022-10-13 Accepted:2023-01-16 Online:2023-03-01 Published:2023-03-15
  • Contact: *E-mail: zhaoyannmg@163.com;fucx@qibebt.ac.cn

摘要: 柳枝稷(Panicum virgatum)是重要的C4多年生木质纤维素类生态能饲草。为了快速创制细胞壁转化效率高的能饲草新资源, 以异源四倍体柳枝稷品种Alamo为材料, 克隆了其木质素合成途径的阿魏酸-5-羟基化酶基因PvF5H, 并根据其序列设计编辑靶点, 用于构建CRISPR/Cas9-PvF5H编辑载体, 最后通过农杆菌(Agrobacterium tumefaciens)介导的遗传转化方法, 获得了59株柳枝稷转基因阳性植株。测序分析表明, PvF5H在94.9%的转基因植株中被编辑, 纯合编辑效率为55.4%。该研究建立了高效的柳枝稷基因编辑系统, 实现了对细胞壁品质相关靶基因的有效编辑, 为今后能饲草新品种的培育奠定了基础。

关键词: 能饲草, 柳枝稷, 木质素, F5H, 基因编辑

Abstract: Switchgrass (Panicum virgatum) is an important C4 perennial lignocellulosic bioenergy and forage crop. In order to rapidly develop novel germplasm of switchgrass with high cell wall conversion rate, we cloned the ferulic acid 5-hydroxylase gene (PvF5H) from the heterotetraploid switchgrass cultivar Alamo. According to the PvF5H sequence, the editing target sgRNA was designed and used to construct CRISPR/Cas9-PvF5H editing vector. Finally, 59 positive transgenic switchgrass lines were generated by Agrobacterium tumefaciens mediated transformation. Sequencing analysis showed that the PvF5H was edited in most of the transgenic lines (94.9%), and the homozygous editing efficiency was 55.4%. Thus, we have successfully established a highly efficient gene editing system for switchgrass. It will facilitate manipulating target genes involved in cell wall quality and can be employed to breed novel switchgrass cultivars for production of biofuels and fodders in future.

Key words: bioenergy and forage grass, switchgrass, lignin, F5H, gene editing