白花草木樨毛状根高效基因组编辑体系的建立

刘文文; 陈龙浩; 杨瑞娟; 苑筱一; 邢思年; 臧云; 吴凡; 张吉宇; 秦晓春; 付春祥

doi:10.11983/CBB25037

植物学报 >

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DOI: https://doi.org/10.11983/CBB25037

技术方法

白花草木樨毛状根高效基因组编辑体系的建立

刘文文 ,
陈龙浩 ,
杨瑞娟 ,
苑筱一 ,
邢思年 ,
臧云 ,
吴凡 ,
张吉宇 ,
秦晓春 ,
付春祥

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¹济南大学生物科学与技术学院, 济南 250022; ²中国科学院青岛生物能源与过程研究所, 山东能源研究院, 青岛新能源山东省实验室, 青岛 266101; ³兰州大学草地农业科技学院, 草种创新与草地农业生态系统全国重点实验室, 兰州 730000

收稿日期: 2025-03-06

修回日期: 2025-03-21

网络出版日期: 2025-05-14

基金资助

国家自然科学基金(No.U23A20216, No.32271752)、青岛新能源山东实验室“抓攻关”项目(No.QNESLKPP202302)、泰山学者项目

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Development of High-efficiency Genome Editing System for Hair Roots in Melilotus albus

LIU Wen-Wen ,
CHEN Long-Gao ,
YANG Rui-Juan ,
YUN Xiao-Yi ,
GENG Sai-Nian ,
ZANG Yun ,
WU Fan ,
ZHANG Ji-Yu ,
QIN Xiao-Chun ,
FU Chun-Xiang

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¹Shool of Biological Science and Technology, University of Jinan, Jinan 250022, China; ²Qingdao New Energy Shandong Laboratory, Shandong Energy Institute, Chinese Academy of Sciences,Qingdao Institute of Bioenergy and Process, Chinese Academy of Sciences, Qingdao 266101, China; ³State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730000, China

Received date: 2025-03-06

Revised date: 2025-03-21

Online published: 2025-05-14

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摘要

白花草木樨(Melilotus albus)具有粗蛋白含量高、耐逆性强等特点, 是优良饲草、轮作和水土保持作物。然而, 白花草木樨稳定遗传转化体系以及基因编辑体系尚未建立, 限制了其基因功能分析及新种质的创制。本研究基于35S::MtLAP1表达盒可诱导再生体花青苷累积进而裸眼“显红色”的原理, 建立了白花草木樨下胚轴毛状根快速诱导和筛选体系, 毛状根诱导率达62%, 阳性率达30.8%。此外, 利用携带有35S::MtLAP1表达盒且由拟南芥AtUbiquitin-10强启动子启动Cas9和sgRNA模块协同表达的基因编辑载体, 能通过上述毛状根体系实现白花草木樨MaPDS (Phytoene Desaturase)的高效编辑, 编辑效率达42.5%。本研究为白花草木樨基因功能研究及高品质种质新资源培育奠定了基础。

关键词： 白花草木樨; MaPDS基因; 毛状根; 基因组编辑

本文引用格式

刘文文 , 陈龙浩 , 杨瑞娟 , 苑筱一 , 邢思年 , 臧云 , 吴凡 , 张吉宇 , 秦晓春 , 付春祥 . 白花草木樨毛状根高效基因组编辑体系的建立[J]. 植物学报, 0 : 1 -0 . DOI: 10.11983/CBB25037

Abstract

INTRODUCTION: Melilotus albus (M. albus) is an excellent forage crop, suitable for crop rotation and soil and water conservation. However, the stable genetic transformation system and gene editing system of M. albus have not been reported, which limits its application in gene function analysis and the creation of new germplasm resources.

RATIONALE: The plant can produce hairy roots after infection by Agrobacterium rhizogenes harboring Ri plasmids. MtLAP1 expression can activate the anthocyanin synthesis process and then produce purple/red anthocyanin accumulation in the transgenic hairy roots.

RESULTS: This study verified that the 35S::MtLAP1 expression cassette can induce the accumulation of anthocyanins in regenerants, resulting in red color visible to the naked eye. When using the hypocotyls of Melilotus albus as explants, the induction efficiency of hairy root was as high as 62%, and the positive rate was as high as 30.8%. In addition, the research results showed that the gene editing vector, which carried the 35S::MtLAP1 expression cassette and co-expressed the Cas9 and sgRNA modules driven by the constitutive strong promoter of Arabidopsis Ubiquitin-10, could achieve an editing efficiency of 42.5% targeted to Phytoene Desaturase gene in M. albus.

CONCLUSION: This study successfully established a rapid inducing and screening system for positive hairy roots and developed an efficient genome-editing tool for M. albus, laying a foundation for functional gene studies and the development of high-quality new germplasm resources in this species.

Hairy root induction, the-naked-eye screening system and MaPDS gene editing in Melilotus albus.

Key words： Melilotus albus; MaPDS; hair roots; genome editing

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