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

doi:10.13560/j.cnki.biotech.bull.1985.2025-0417

Abstract

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

LI Le-song, ZHANG Jin-jin, HE Jia-die, LIANG Yan-li, YANG Sheng-chao, LI Meng-fei, ZHAO Yan. Identification and Functional Characterization of the 4-Coumarate∶coenzyme A Ligase Gene Family in Angelica sinensis （Oliv.） Diels[J]. Biotechnology Bulletin, 2026, 42(2): 293-305.

Figures/Tables 15

References 46

[1]	严辉, 段金廒, 尚尔鑫, 等. 当归不同部位入药功效取向差异的化学物质基础与药性关联性研究 [J]. 中草药, 2014, 45(21): 3208-3212.
	Yan H, Duan JA, Shang EX, et al. Study on chemical materials and drug nature association of efficacy orientation of different parts from Angelicae Sinensis Radix [J]. Chin Tradit Herb Drugs, 2014, 45(21): 3208-3212.
[2]	董涵. 低温调控当归生长及主效活性物质生物合成的机制研究 [D]. 兰州: 甘肃中医药大学, 2022.
	Dong H. Mechanism of cool temperature regulating growth and main metabolites biosynthesis in Angelica sinensis [D]. Lanzhou: Gansu University of Chinese Medicine, 2022.
[3]	吕成龙, 李会会, 史永洁, 等. 中药当归现代研究进展及其质量标志物的预测分析 [J]. 中国中药杂志, 2022, 47(19): 5140-5157.
	Lyu CL, Li HH, Shi YJ, et al. Research progress of Angelicae sinensis and predictive analysis on its quality markers [J]. China J Chin Mater Med, 2022, 47(19): 5140-5157.
[4]	赵静, 夏晓培. 当归的化学成分及药理作用研究现状 [J]. 临床合理用药杂志, 2020, 13(6): 172-174.
	Zhao J, Xia XP. Research status of chemical constituents and pharmacological effects of Angelica sinensis [J]. Chin J Clin Ration Drug Use, 2020, 13(6): 172-174.
[5]	国家药典委员会. 中华人民共和国药典-一部: 2020年版 [M]. 北京: 中国医药科技出版社, 2020.
	National Pharmacopoeia Committee. People’s republic of China (PRC) pharmacopoeia-part I: 2020 edition [M]. Beijing: China Medical Science Press, 2020.
[6]	梁盈, 袭晓娟, 刘巧丽, 等. 阿魏酸及其衍生物的生理活性及应用研究进展 [J]. 食品与生物技术学报, 2018, 37(5): 449-454.
	Liang Y, Xi XJ, Liu QL, et al. Research progress on the physiological activity and application of ferulic acid and its derivatives [J]. J Food Sci Biotechnol, 2018, 37(5): 449-454.
[7]	Kitashiba H, Yokoi S. Genes for bolting and flowering [M]. Compendium of Plant Genomes, The Radish Genome, 2017: 151-163.
[8]	Li ML, Cui XW, Jin L, et al. Bolting reduces ferulic acid and flavonoid biosynthesis and induces root lignification in Angelica sinensis [J]. Plant Physiol Biochem, 2022, 170: 171-179.
[9]	Liu L, Liu C, Hou XL, et al. FTIP1 is an essential regulator required for florigen transport [J]. PLoS Biol, 2012, 10(4): e1001313.
[10]	黎洁. 光周期阶段当归抽薹开花的调控机制研究 [D]. 兰州: 甘肃农业大学, 2021.
	Li J. Study on regulation mechanism of bolting and flowering of Angelica sinensis at photoperiodic stage [D]. Lanzhou: Gansu Agricultural University, 2021.
[11]	Sulis DB, Jiang X, Yang CM, et al. Multiplex CRISPR editing of wood for sustainable fiber production [J]. Science, 2023, 381(6654): 216-221.
[12]	Chanoca A, de Vries L, Boerjan W. Lignin engineering in forest trees [J]. Front Plant Sci, 2019, 10: 912.
[13]	Liu QQ, Luo L, Zheng LQ. Lignins: Biosynthesis and biological functions in plants [J]. Int J Mol Sci, 2018, 19(2): 335.
[14]	吴萍, 王晓宇, 郭俊霞, 等. 早期抽薹对白芷生长发育和品质的影响 [J]. 中国农业科技导报, 2023, 25(12): 58-66.
	Wu P, Wang XY, Guo JX, et al. Effects of early bolting on growth and quality of Angelica dahurica [J]. J Agric Sci Technol, 2023, 25(12): 58-66.
[15]	刘双利, 许永华, 王晓慧, 等. 防风抽薹开花的研究进展 [J]. 人参研究, 2016, 28(6): 52-56.
	Liu SL, Xu YH, Wang XH, et al. Research progress on the early bolting and flowering of Divaricate saposhnikovia Root [J]. Ginseng Res, 2016, 28(6): 52-56.
[16]	Lavhale SG, Kalunke RM, Giri AP. Structural, functional and evolutionary diversity of 4-coumarate-CoA ligase in plants [J]. Planta, 2018, 248(5): 1063-1078.
[17]	Sun SC, Xiong XP, Zhang XL, et al. Characterization of the Gh4CL gene family reveals a role of Gh4CL7 in drought tolerance [J]. BMC Plant Biol, 2020, 20(1): 125.
[18]	Liu H, Guo ZH, Gu FW, et al. 4-Coumarate-CoA ligase-like gene OsAAE3 negatively mediates the rice blast resistance, floret development and lignin biosynthesis [J]. Front Plant Sci, 2017, 7: 2041.
[19]	Trantas E, Panopoulos N, Ververidis F. Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae [J]. Metab Eng, 2009, 11(6): 355-366.
[20]	Wang YC, Yi H, Wang M, et al. Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase: stilbene synthase [J]. J Am Chem Soc, 2011, 133(51): 20684-20687.
[21]	Lin YH, Sun XX, Yuan QP, et al. Combinatorial biosynthesis of plant-specific coumarins in bacteria [J]. Metab Eng, 2013, 18: 69-77.
[22]	Li Y, Kim JI, Pysh L, et al. Four isoforms of Arabidopsis 4-coumarate: CoA ligase have overlapping yet distinct roles in phenylpropanoid metabolism [J]. Plant Physiol, 2015, 169(4): 2409-2421.
[23]	Sun HY, Li Y, Feng SQ, et al. Analysis of five rice 4-coumarate: coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice [J]. Biochem Biophys Res Commun, 2013, 430(3): 1151-1156.
[24]	Reinprecht Y, Yadegari Z, Perry GE, et al. In silico comparison of genomic regions containing genes coding for enzymes and transcription factors for the phenylpropanoid pathway in Phaseolus vulgaris L. and Glycine max L. Merr [J]. Front Plant Sci, 2013, 4: 317.
[25]	Rao GD, Pan X, Xu F, et al. Divergent and overlapping function of five 4-coumarate/coenzyme a ligases from Populus tomentosa [J]. Plant Mol Biol Report, 2015, 33(4): 841-854.
[26]	Li MZ, Guo LL, Wang YR, et al. Molecular and biochemical characterization of two 4-coumarate: CoA ligase genes in tea plant (Camellia sinensis) [J]. Plant Mol Biol, 2022, 109(4/5): 579-593.
[27]	侯冲, 晁楠, 戴明洁, 等. 桑树4CL基因家族的筛选和Mm4CL2的功能研究 [J]. 蚕业科学, 2022, 48(1): 18-24.
	Hou C, Chao N, Dai MJ, et al. Screening of 4CL family genes in mulberry and functional study of Mm4CL2 [J]. Acta Sericologica Sin, 2022, 48(1): 18-24.
[28]	Gong JM, Sun SC, Zhu QH, et al. Gh4CL20/20A involved in flavonoid biosynthesis is essential for male fertility in cotton (Gossypium hirsutum L.) [J]. Plant Physiol Biochem, 2024, 208: 108484.
[29]	Zhou X, Cao J, Liu XM, et al. Cloning and functional analysis of Gb4CL1 and Gb4CL2 from Ginkgo biloba [J]. Plant Genome, 2024, 17(2): e20440.
[30]	Douglas C, Hoffmann H, Schulz W, et al. Structure and elicitor or u.v.-light-stimulated expression of two 4-coumarate: CoA ligase genes in parsley [J]. EMBO J, 1987, 6(5): 1189-1195.
[31]	Liu TT, Yao RL, Zhao YC, et al. Cloning, functional characterization and site-directed mutagenesis of 4-coumarate: coenzyme a ligase (4CL) involved in coumarin biosynthesis in Peucedanum praeruptorum Dunn [J]. Front Plant Sci, 2017, 8: 4.
[32]	张苛苛, 谭宇萍, 徐欢欢, 等. 菘蓝4-香豆酸: 辅酶A连接酶全基因家族的鉴定及表达分析 [J]. 中国中药杂志, 2024, 49(2): 361-369.
	Zhang KK, Tan YP, Xu HH, et al. Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase [J]. China J Chin Mater Med, 2024, 49(2): 361-369.
[33]	朋冬琴, 罗蜜蜜, 郭欣慰, 等. 当归实时荧光定量PCR内参基因筛选 [J]. 中草药, 2024, 55(1): 269-278.
	Peng DQ, Luo MM, Guo XW, et al. Selection of reference genes for quantitative real-time PCR analysis in Angelica sinensis [J]. Chin Tradit Herb Drugs, 2024, 55(1): 269-278.
[34]	何磊, 严希, 袁圆, 等. 辣椒4CL基因家族成员的鉴定与生物信息学分析 [J]. 分子植物育种, 2022, 20(8): 2478-2484.
	He L, Yan X, Yuan Y, et al. Identification and bioinformatics analysis of the 4CL gene family members in pepper (Capsicum annuum) [J]. Mol Plant Breed, 2022, 20(8): 2478-2484.
[35]	郑飞雄, 陈俊宇, 江林琪, 等. 掌叶覆盆子4CL基因家族鉴定及表达分析 [J]. 农业生物技术学报, 2024, 32(2): 311-321.
	Zheng FX, Chen JY, Jiang LQ, et al. Identification and expression analysis of 4CL gene family in Rubus chingii [J]. J Agric Biotechnol, 2024, 32(2): 311-321.
[36]	Wang YY, Guo LH, Zhao YJ, et al. Systematic analysis and expression profiles of the 4-coumarate: CoA ligase (4CL) gene family in pomegranate (Punica granatum L.) [J]. Int J Mol Sci, 2022, 23(7): 3509.
[37]	Kienow L, Schneider K, Bartsch M, et al. Jasmonates meet fatty acids: functional analysis of a new acyl-coenzyme A synthetase family from Arabidopsis thaliana [J]. J Exp Bot, 2008, 59(2): 403-419.
[38]	De Azevedo Souza C, Barbazuk B, Ralph SG, et al. Genome-wide analysis of a land plant-specific acyl: coenzymeA synthetase (ACS) gene family in Arabidopsis, poplar, rice and Physcomitrella [J]. New Phytol, 2008, 179(4): 987-1003.
[39]	Ehlting J, Büttner D, Wang Q, et al. Three 4-coumarate: coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms [J]. Plant J, 1999, 19(1): 9-20.
[40]	Hamberger B, Hahlbrock K. The 4-coumarate: CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes [J]. Proc Natl Acad Sci USA, 2004, 101(7): 2209-2214.
[41]	Lavhale SG, Joshi RS, Kumar Y, et al. Functional insights into two Ocimum kilimandscharicum 4-coumarate-CoA ligases involved in phenylpropanoid biosynthesis [J]. Int J Biol Macromol, 2021, 181: 202-210.
[42]	Zhang GH, Yu ZM, Yao B, et al. SsMYB113, a Schima superba MYB transcription factor, regulates the accumulation of flavonoids and functions in drought stress tolerance by modulating ROS generation [J]. Plant Soil, 2022, 478(1): 427-444.
[43]	Yuan CX, Li LS, Zhou PH, et al. Decoding the root lignification mechanism of Angelica sinensis through genome-wide DNA methylation analysis [J]. J Exp Bot, 2025, 76(9): 2573-2589.
[44]	Lindermayr C, Möllers B, Fliegmann J, et al. Divergent members of a soybean (Glycine max L.) 4-coumarate: coenzyme A ligase gene family [J]. Eur J Biochem, 2002, 269(4): 1304-1315.
[45]	Zhao Y, Liu GZ, Yang F, et al. Multilayered regulation of secondary metabolism in medicinal plants [J]. Mol Hortic, 2023, 3(1): 11.
[46]	Gao QQ, Zhang JJ, Cao JT, et al. MetaDb: a database for metabolites and their regulation in plants with an emphasis on medicinal plants [J]. Mol Hortic, 2024, 4(1): 17.

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	备注 Note
As4CL6-F	cagcaaatgggtcgcggatccATGGGGGATTGTGTAGCACC	基因克隆
As4CL6-R	cagtggtggtggtggtggtgTTATTTGGGAAGATCACCGGATGC	Gene cloning
As4CL7-F	cagcaaatgggtcgcggatccATGGATACCAAAACAAACAAGAAACAAG
As4CL7-R	cagtggtggtggtggtggtgTTAGTTTTGAGGAAGATCGCCAGC
q-As4CL6-F	TTCGTCAGGTACCACAGGAC	实时荧光定量
q-As4CL6-R	CCCCTGCTCTAAGTCCACAA	RT-qPCR
q-As4CL7-F	ATAAAATCGGGTGCGTGTGG
q-As4CL7-R	TGCAGCCAACCTTCTTTGTC
EEF1G-F	GTCCCAGCAGCCAAAAAGTC	内参基因
EEF1G-R	TCTGCCTTGGGCAATTCCTT	Reference genes

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	备注 Note
As4CL6-F	cagcaaatgggtcgcggatccATGGGGGATTGTGTAGCACC	基因克隆
As4CL6-R	cagtggtggtggtggtggtgTTATTTGGGAAGATCACCGGATGC	Gene cloning
As4CL7-F	cagcaaatgggtcgcggatccATGGATACCAAAACAAACAAGAAACAAG
As4CL7-R	cagtggtggtggtggtggtgTTAGTTTTGAGGAAGATCGCCAGC
q-As4CL6-F	TTCGTCAGGTACCACAGGAC	实时荧光定量
q-As4CL6-R	CCCCTGCTCTAAGTCCACAA	RT-qPCR
q-As4CL7-F	ATAAAATCGGGTGCGTGTGG
q-As4CL7-R	TGCAGCCAACCTTCTTTGTC
EEF1G-F	GTCCCAGCAGCCAAAAAGTC	内参基因
EEF1G-R	TCTGCCTTGGGCAATTCCTT	Reference genes

基因名 Gene name	基因ID Gene ID	氨基酸数量 Number of amino acids （aa）	分子量 Molecular weight （Da）	等电点 Isoelectric point	不稳定系数 Instability index	亲水系数 Hydrophilicity coefficient	亚细胞定位Subcellular localization
As4CL1	AS01G01317	541	59 348.05	8.90	33.77	0.068	过氧化物酶体
As4CL2	AS02G00709	502	54 877.60	6.84	35.33	0.109	过氧化物酶体
As4CL3	AS04G00278	572	62 011.86	6.22	54.87	-0.061	过氧化物酶体
As4CL4	AS04G02110	470	50 783.66	7.60	40.56	0.077	过氧化物酶体
As4CL5	AS05G03160	415	45 242.42	6.16	33.56	0.134	过氧化物酶体
As4CL6	AS06G02531	548	60 207.84	5.47	34.15	0.057	过氧化物酶体
As4CL7	AS07G02833	549	60 205.70	5.87	33.28	-0.013	过氧化物酶体
As4CL8	AS07G03471	475	51 047.82	9.06	45.65	-0.004	过氧化物酶体
As4CL9	AS08G00981	568	62 771.36	6.71	37.89	0.065	过氧化物酶体
As4CL10	AS09G00363	543	59 783.05	8.76	39.09	-0.042	过氧化物酶体
As4CL11	AS09G01563	544	59 805.70	7.08	37.46	-0.100	过氧化物酶体
As4CL12	ASUnG00581	550	60 229.63	5.70	35.50	0.024	过氧化物酶体

基因名 Gene name	基因ID Gene ID	氨基酸数量 Number of amino acids （aa）	分子量 Molecular weight （Da）	等电点 Isoelectric point	不稳定系数 Instability index	亲水系数 Hydrophilicity coefficient	亚细胞定位Subcellular localization
As4CL1	AS01G01317	541	59 348.05	8.90	33.77	0.068	过氧化物酶体
As4CL2	AS02G00709	502	54 877.60	6.84	35.33	0.109	过氧化物酶体
As4CL3	AS04G00278	572	62 011.86	6.22	54.87	-0.061	过氧化物酶体
As4CL4	AS04G02110	470	50 783.66	7.60	40.56	0.077	过氧化物酶体
As4CL5	AS05G03160	415	45 242.42	6.16	33.56	0.134	过氧化物酶体
As4CL6	AS06G02531	548	60 207.84	5.47	34.15	0.057	过氧化物酶体
As4CL7	AS07G02833	549	60 205.70	5.87	33.28	-0.013	过氧化物酶体
As4CL8	AS07G03471	475	51 047.82	9.06	45.65	-0.004	过氧化物酶体
As4CL9	AS08G00981	568	62 771.36	6.71	37.89	0.065	过氧化物酶体
As4CL10	AS09G00363	543	59 783.05	8.76	39.09	-0.042	过氧化物酶体
As4CL11	AS09G01563	544	59 805.70	7.08	37.46	-0.100	过氧化物酶体
As4CL12	ASUnG00581	550	60 229.63	5.70	35.50	0.024	过氧化物酶体

基因名 Gene name	α-螺旋 Alpha helix （%）	β-折叠 Beta turn （%）	延伸链 Extended strand （%）	无规则卷曲 Random coil （%）
As4CL1	30.87	7.76	19.59	41.77
As4CL2	33.27	7.97	20.32	38.45
As4CL3	29.72	6.82	17.66	45.80
As4CL4	32.77	7.66	19.79	39.79
As4CL5	30.60	7.95	20.00	41.45
As4CL6	29.96	6.99	19.49	43.57
As4CL7	30.28	6.97	19.45	43.30
As4CL8	30.95	8.21	20.21	40.63
As4CL9	28.87	6.51	17.96	46.65
As4CL10	31.68	7.00	19.15	42.17
As4CL11	30.33	6.99	19.67	43.01
As4CL12	30.55	6.91	19.45	43.09