生物技术通报 ›› 2026, Vol. 42 ›› Issue (2): 293-305.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0417

• 研究报告 • 上一篇    下一篇

当归4-香豆酸∶辅酶A连接酶基因家族的鉴定与功能表征

李勒松1,2,3(), 张金金1,2,3, 贺佳蝶1,2,3, 梁艳丽1,2, 杨生超1,3, 栗孟飞4, 赵艳1,2,3()   

  1. 1.云南农业大学西南中药材种质创新与利用国家地方联合工程研究中心 云南省药用植物生物学重点实验室,昆明 650201
    2.云南农业大学农学与生物技术学院,昆明 650201
    3.云南特色植物提取实验室,昆明 650106
    4.甘肃农业大学农学院,兰州 730070
  • 收稿日期:2025-04-20 出版日期:2026-02-26 发布日期:2026-03-17
  • 通讯作者: 赵艳,女,博士,教授,研究方向 :药用植物学;E-mail: zhaoyankm@126.com
  • 作者简介:李勒松,女,硕士研究生,研究方向 :药用植物学;E-mail: lls15126334094@163.com
  • 基金资助:
    国家自然科学基金项目(82460743);云南省科技计划项目(202304B1090009);云南特色植物提取实验室自主研究项目基金(2022YKZY001);云南省兴滇英才支持计划“青年人才”项目(XDYC-QNRC-2022-0219)

Identification and Functional Characterization of the 4-Coumarate∶coenzyme A Ligase Gene Family in Angelica sinensis (Oliv.) Diels

LI Le-song1,2,3(), ZHANG Jin-jin1,2,3, HE Jia-die1,2,3, LIANG Yan-li1,2, YANG Sheng-chao1,3, LI Meng-fei4, ZHAO Yan1,2,3()   

  1. 1.National-Local Joint Engineering Research Center on Germplasm Innovation & Utilization of Chinese Medicinal Materials in Southwest China, Key Laboratory of Medicinal Plant Biology, Yunnan Agricultural University, Kunming 650201
    2.College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201
    3.Yunnan Characteristic Plant Extraction Laboratory, Kunming 650106
    4.Agronomy College, Gansu Agricultural University, Lanzhou 730070
  • Received:2025-04-20 Published:2026-02-26 Online:2026-03-17

摘要:

目的 探究4-香豆酸∶辅酶A连接酶(4-coumarate:coenzyme A ligase, 4CL)在当归根部木质素生物合成中的作用,为通过遗传改良或栽培手段定向调控当归药材品质奠定基础。 方法 通过生物信息学方法鉴定当归4CL基因家族,开展蛋白理化性质、二级结构预测、基因结构、启动子顺式作用元件以及系统进化分析,通过大肠杆菌异源表达和体外酶活验证基因功能,利用实时荧光定量PCR分析基因表达特征。 结果 在当归基因组中共鉴定到12个As4CLs,编码415‒572个氨基酸,相对分子质量在45‒62 kD,等电点范围为5.47‒9.06,二级结构以无规则卷曲和α-螺旋为主。系统进化分析将As4CL6与As4CL7归在Group I,其余As4CLs均属于Group Ⅲ。氨基酸序列比对结果显示As4CLs含有2个功能保守结构域Box I和Box Ⅱ。启动子分析结果显示,As4CLs基因含有丰富的光响应元件、激素响应元件及非生物胁迫响应元件;体外酶活实验证明As4CL6与As4CL7均能够催化4-香豆酸、咖啡酸和阿魏酸生成相应的辅酶A硫酯,且两个酶均对阿魏酸的亲和力最强;RT-qPCR结果显示As4CL6As4CL7在当归抽薹根部的表达量高于未抽薹根部。 结论 成功克隆了As4CL6As4CL7基因,通过体外酶活实验证明2个重组蛋白对3种底物均具有催化活性。

关键词: 4-香豆酸:辅酶A连接酶, 当归, 抽薹, RT-qPCR

Abstract:

Objective Exploring the role of 4-coumarate: coenzyme A ligase (4CL) in lignin biosynthesis in the roots of Angelica sinensis (Oliv.) Diels, laying the foundation for targeted regulation of A. sinensis medicinal quality through genetic improvement or cultivation methods. Method The 4CL gene family of A. sinensis was identified using bioinformatics methods. The analysis on protein physicochemical properties, secondary structure prediction, gene structure, promoter cis-acting elements, and systematic evolution were conducted. The gene function was verified through heterologous expression in Escherichia coli and enzymatic assay in vitro, and its expression characteristics were analyzed by real-time quantitative PCR. Result A total of 12 As4CLs were identified in the genome of A. sinensis, encoding 415‒572 amino acids, relative molecular weights ranging from 45 to 62 kD, and isoelectric points ranging from 5.47 to 9.06. The secondary structure is mainly composed of irregular coils and α-helices. System evolution analysis categorizes As4CL6 and As4CL7 as Group I, while the remaining As4CLs belong to Group Ⅲ. The amino acid sequence alignment results showed that As4CLs contain two functionally conserved domains, Box I and Box Ⅱ. Promoter analysis showed that the As4CLs contain abundant light responsive elements, hormone responsive elements, and abiotic stress responsive elements. In vitro enzymatic assay showed that both As4CL6 and As4CL7 catalyzed 4-coumaric acid, caffeic acid, and ferulic acid to form corresponding coenzyme A thioesters, and both enzymes have the strongest affinity for ferulic acid. The RT-qPCR results showed that the expressions of As4CL6 and As4CL7 were higher in the roots of A. sinensis with bolting than in the roots unbolting. Conclusion The genes of As4CL6 and As4CL7 are successfully cloned, and enzymatic assay in vitro confirm that the two recombinant proteins demonstrate catalytic activity towards three substrates.

Key words: 4-coumarate: coenzyme A ligase, Angelica sinensis, bolting, RT-qPCR