柳枝稷木质素基因F5H的高效编辑
收稿日期: 2022-10-13
录用日期: 2023-01-16
网络出版日期: 2023-01-16
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
Received date: 2022-10-13
Accepted date: 2023-01-16
Online published: 2023-01-16
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。
邱锐, 何峰, 李瑞, 王亚梅, 邢思年, 曹英萍, 刘叶飞, 周昕越, 赵彦, 付春祥 . 柳枝稷木质素基因F5H的高效编辑[J]. 植物学报, 2023 , 58(2) : 298 -307 . DOI: 10.11983/CBB22240
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
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