生物技术通报 ›› 2026, Vol. 42 ›› Issue (2): 293-305.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0417
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(
)
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:
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(
)
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As4CL6和As4CL7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As4CL6与As4CL7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。
李勒松, 张金金, 贺佳蝶, 梁艳丽, 杨生超, 栗孟飞, 赵艳. 当归4-香豆酸∶辅酶A连接酶基因家族的鉴定与功能表征[J]. 生物技术通报, 2026, 42(2): 293-305.
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.
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 |
表1 引物序列
Table 1 Primer sequences
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 |
表2 As4CLs蛋白理化性质
Table 2 Physicochemical properties of As4CLs protein
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 |
表3 As4CLs蛋白质二级结构分析
Table 3 Secondary structure analysis of As4CLs protein
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 |
图3 当归As4CLs基因家族的系统发育分析当归As4CLs家族成员用三角形标注。At:拟南芥;Os:水稻;Gm:大豆;Pt:毛白杨;Cs:茶树;Pp:白花前胡;Pc:欧芹;Dc:胡萝卜。蛋白名称及ID号见附表1
Fig. 3 Phylogenetic analysis of the As4CLs gene family in A. sinensisMembers of the As4CLs family in A. sinensis are labeled with triangles. At: Arabidopsis thaliana; Os: Oryza sativa; Gm: Glycine max; Pt: Populus tomentosa; Cs: Camellia sinensis; Pp: Peucedanum praeruptorum; Pc: Petroselinum crispum; Dc: Daucus carota. Protein names and ID numbers are listed in Supplementary table 1
图4 当归As4CLs基因结构和保守结构域A:基因结构;B:保守结构域;C:内含子与外显子结构
Fig. 4 The structure and conserved domains of the As4CLs gene in A. sinensisA: Gene structure. B: Conserved domain. C: Intron and exon structures
图7 电泳检测分析A:琼脂糖凝胶电泳检测扩增产物;B:As4CLs蛋白的SDS-PAGE分析;M:Marker;1:As4CL6;2:As4CL7;3:pET-28a-As4CL6重组蛋白;4:pET-28a-As4CL7重组蛋白
Fig. 7 Electrophoretic detection analysisA: Detection of amplified products by agarose gel electrophoresis; B: SDS-PAGE analysis of As4CLs protein. M: Marker; 1: As4CL6; 2: As4CL7; 3: purified pET-28a-As4CL6 protein; 4: purified pET-28a-As4CL7 protein
图8 As4CL6与As4CL7的功能表征A‒C:As4CL6、As4CL7体外酶促反应产物的HPLC分析;D‒F:产物标准品的LC-MS分析;G‒L:As4CL6、As4CL7体外酶促反应产物的LC-MS分析。CM、CF、FA分别代表4-香豆酸、咖啡酸和阿魏酸;CM-CoA、CF-CoA、FA-CoA分别代表4-香豆酰辅酶A、咖啡酰辅酶A和阿魏酰辅酶A
Fig. 8 Functional profiling of As4CL6 and As4CL7A‒C: HPLC analysis of in vitro enzymatic reaction products of As4CL6 and As4CL7; D-F: LC-MS analysis of product standards; G‒L: LC-MS analysis of in vitro enzymatic reaction products of As4CL6 and As4CL7. CM, CF, and FA refers to 4-coumaric acid, caffeic acid, and ferulic acid; CM-CoA, CF-CoA, and FA-CoA represent 4-coumaroyl CoA, caffeoyl CoA, and feruloyl CoA, respectively
图9 As4CL6与As4CL7的酶动力学分析A:4CL6对不同底物的非线性回归曲线;B:4CL7对不同底物的非线性回归曲线。CM、CF、FA分别代表4-香豆酸、咖啡酸和阿魏酸
Fig. 9 Enzyme kinetic analysis of As4CL6 and As4CL7A: Nonlinear regression curve of 4CL6 on different substrates. B: Nonlinear regression curve of 4CL7 on different substrates. CM, CF, and FA stand for 4-coumaric acid, caffeic acid, and ferulic acid
酶 Enzyme | 底物 Substrate | Km (μmol/L) | Vmax (μmol·L-1·min-1) | Vmax/Km (10-3/min) |
|---|---|---|---|---|
| As4CL6 | 4-香豆酸 | 60.73 | 0.088 83 | 1.462 7 |
| 咖啡酸 | 96.28 | 0.034 96 | 0.363 1 | |
| 阿魏酸 | 46.77 | 0.070 77 | 1.513 1 | |
| As4CL7 | 4-香豆酸 | 47.55 | 0.260 20 | 5.472 1 |
| 咖啡酸 | 166.10 | 0.244 70 | 1.473 2 | |
| 阿魏酸 | 39.61 | 0.282 20 | 7.124 5 |
表4 As4CL6与As4CL7的动力学参数
Table 4 Kinetic parameters of As4CL6 and As4CL7
酶 Enzyme | 底物 Substrate | Km (μmol/L) | Vmax (μmol·L-1·min-1) | Vmax/Km (10-3/min) |
|---|---|---|---|---|
| As4CL6 | 4-香豆酸 | 60.73 | 0.088 83 | 1.462 7 |
| 咖啡酸 | 96.28 | 0.034 96 | 0.363 1 | |
| 阿魏酸 | 46.77 | 0.070 77 | 1.513 1 | |
| As4CL7 | 4-香豆酸 | 47.55 | 0.260 20 | 5.472 1 |
| 咖啡酸 | 166.10 | 0.244 70 | 1.473 2 | |
| 阿魏酸 | 39.61 | 0.282 20 | 7.124 5 |
图10 As4CL6和As4CL7表达量分析UBP:未抽薹植株根部;BP:抽薹植株根部;** P<0.01
Fig. 10 Analysis of As4CL6 and As4CL7 expressionsUBP: Roots of unbolting plants; BP: roots of bolting plants;** P<0.01
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