斑地锦EmPAL基因克隆、表达模式和酶活性分析

doi:10.3969/j.issn.1004-1524.20250609

浙江农业学报 ›› 2026, Vol. 38 ›› Issue (8): 1598-1609.DOI: 10.3969/j.issn.1004-1524.20250609

斑地锦EmPAL基因克隆、表达模式和酶活性分析

吴晴阳¹(), 桂明明², 宋美玲¹, 郭三保³, 张志高⁴, 黄胜和²^,^*()

¹ 抚州医药学院医学基础部, 江西抚州 344000
² 抚州医药学院江西省教育厅建昌帮中药研究重点实验室, 江西抚州 344000
³ 抚州医药学院药学系, 江西抚州 344000
⁴ 江西省烟草公司抚州市公司, 江西抚州 344000

收稿日期:2025-09-18 出版日期:2026-08-25 发布日期:2026-09-14
作者简介:吴晴阳,从事植物基因和生理学研究。E-mail:825166405@qq.com
通讯作者: *黄胜和,E-mail:hsh712@163.com
基金资助:
江西省教育厅科学技术研究项目(GJJ2205908);江西中医药高等专科学校科研项目(2024XJKY01)

Cloning, expression pattern, and enzyme activity analysis of the phenylalanine ammonia-lyase gene (EmPAL) in Euphorbia maculata

WU Qingyang¹(), GUI Mingming², SONG Meiling¹, GUO Sanbao³, ZHANG Zhigao⁴, HUANG Shenghe²^,^*()

¹ Department of Basic Medicine, Fuzhou Medical College, Fuzhou 344000, Jiangxi, China
² Key Laboratory of Jianchangbang Traditional Chinese Medicine Research of Jiangxi Education Institutes, Fuzhou Medical College, Fuzhou 344000,Jiangxi, China
³ Department of Pharmacy, Fuzhou Medical College, Fuzhou 344000,Jiangxi, China
⁴ Fuzhou Branch of Jiangxi Tobacco Company, Fuzhou 344000,Jiangxi, China

Received:2025-09-18 Published:2026-08-25 Online:2026-09-14

摘要/Abstract

摘要：

为解析药用植物斑地锦(Euphorbia maculata)药效成分黄酮类化合物生物合成途径结构基因苯丙氨酸解氨酶(phenylalanine ammonia-lyase,EmPAL)基因结构和蛋白质功能,为后续深入研究EmPAL提供参考,以斑地锦为材料,基于转录组数据结合cDNA 3'末端快速扩增(3'-RACE)和交错式热不对称PCR(Tail-PCR),克隆了EmPAL的开放阅读框(open reading frame, ORF)和启动子序列。采用生物信息学工具分析了EmPAL的理化性质、结构及系统发育关系。通过实时荧光定量PCR(RT-qPCR)检测EmPAL在不同组织中的表达模式。此外,还进行了原核表达并通过体外酶活性试验验证了其催化功能。结果表明,EmPAL基因ORF长度2 160 bp,编码719个氨基酸,其启动子含有光反应、胁迫应答元件和苯丙烷类元件等顺式作用元件。组织表达分析显示,EmPAL基因在斑地锦生殖生长期的根中表达量最高。EmPAL蛋白分子量为78.17 ku,等电点(pI)6.34,二级结构以α螺旋为主,三级结构为典型的“海马”状,四级结构为四聚体。原核表达与体外酶活性试验证实,EmPAL可催化苯丙氨酸生成反式肉桂酸,最适温度为60 ℃。本研究为深入解析EmPAL基因功能及其调控斑地锦中黄酮类化合物的生物合成提供了理论依据。

关键词: 斑地锦, EmPAL, 启动子, 克隆, 实时荧光定量PCR, 原核表达, 酶活性

Abstract:

This study aimed to elucidate the structure and protein function of the phenylalanine ammonia-lyase gene (EmPAL), a structural gene in the flavonoid biosynthesis pathway in the medicinal plant Euphorbia maculata, and to provide a basis for further functional investigation of EmPAL. Using E. maculata as the material, the open reading frame (ORF) and promoter sequence of EmPAL were cloned based on transcriptome data combined with 3'-RACE and Tail-PCR techniques. Bioinformatics tools were employed to analyze the physicochemical properties, structural characteristics, and phylogenetic relationships of EmPAL. The expression pattern of EmPAL in different tissues of E. maculata was examined using quantitative real-time PCR (RT-qPCR). Furthermore, prokaryotic expression was performed and its catalytic function was validated through in vitro enzyme activity assays. The results showed that the ORF of EmPAL was 2 160 bp in length, encoding 719 amino acids. The promoter region contained cis-acting elements related to light response, stress response, and phenylpropanoid metabolism regulation. Tissue-specific expression analysis revealed that EmPAL expression was highest in the roots during the reproductive growth period. The molecular weight of EmPAL protein is 78.17 ku, and its isoelectric point is 6.34. The secondary structure was predominantly composed of alpha helix, the tertiary structure exhibited a typical “sea horse” shape, and the quaternary structure was a tetramer. Prokaryotic expression and in vitro enzyme activity assays confirmed that EmPAL could catalyze the conversion of phenylalanine to trans-cinnamic acid, with an optimal reaction temperature of 60 ℃. This study provides a theoretical foundation for further investigation into the function of EmPAL and its role in regulating the biosynthesis of flavonoids in E. maculata.

Key words: Euphorbia maculata, EmPAL, promoter, cloning, RT-qPCR, prokaryotic expression, enzyme activity

中图分类号:

S567.219

吴晴阳, 桂明明, 宋美玲, 郭三保, 张志高, 黄胜和. 斑地锦EmPAL基因克隆、表达模式和酶活性分析[J]. 浙江农业学报, 2026, 38(8): 1598-1609.

WU Qingyang, GUI Mingming, SONG Meiling, GUO Sanbao, ZHANG Zhigao, HUANG Shenghe. Cloning, expression pattern, and enzyme activity analysis of the phenylalanine ammonia-lyase gene (EmPAL) in Euphorbia maculata[J]. Acta Agriculturae Zhejiangensis, 2026, 38(8): 1598-1609.

图/表 9

表1 引物序列

Table 1 Primer sequences

引物用途 Primer purpose	引物名称 Primer name	引物序列(5'→3') Primer sequence(5'→3')
cDNA合成cDNA synthesis	FZL	AAGCAGTGGTATCAACGCAGAGTTTTTTTTTTTTTTTTTTVN
cDNA 3'末端快速扩增	EmPAL-XF	ATCCGAGTTGAGCTGGCTG
3'-rapid amplification of cDNA ends,3'-RACE	EmPAL-RF	AGCAGTGGTATCAACGCAGAG
交错式热不对称PCR	EmPAL-SR1	AAGCTCTTTTTGCAAAGCACCACCTTGT
Thermal asymmetric interlaced polymerase chain reaction, Tail-PCR	EmPAL-SR2	ACGATGGACTCCAGTCCGGCCTTGCAAAGCACCACCTTG- TTTAGTTCTTCTATGA
	EmPAL-SR3	AGTTCTTCTATGAGAAGTGGCACCAAAACCAGTAG
	EmPAL-SR4	CCATCTCTGAGAAGAACCCAGATGG
	EmPAL-SR5	ACGATGGACTCCAGTCCGGCCTTGCAAAGCACCACCTTG- TTTAGTTCTTCTATGA
	EmPAL-SR6	AGTTCTTCTATGAGAAGTGGCACCAAAACCAGTAG
	ADF1	ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
	ADF2	ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
	ADF3	ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
	ADF6	ACGATGGACTCCAGAGCGGCCGCVNVNNCCTT
	ADF0	ACGATGGACTCCAGAG
ORF区克隆	EmPAL-OF	CAAACCAAGCTTTCCATTAATTCAGAT
ORF clone	EmPAL-OR	CAAAAGCTCACATATCTAATGCACAAAC
启动子克隆	EmPAL-PF	GTTGGATGAAGTCAAAACATAGGGAG
Promoter clone	EmPAL-PR	GCCGTTTTGGTGGATGGATTC
实时荧光定量PCR	EmPAL-qF	CGGGTTGCCTTCAAATCTAACG
RT-qPCR	EmPAL-qR	GTCAACCGACTCTTGTGTCTTTC
	EmGDI1-qF	TCTAGGTCCCGTTGACGAGT
	EmGDI1-qR	TCGAGAGCCTTCCCAGTGAT
异源表达	EmPAL-proF	CGAGCTCATGGAATCCATCCACCAAAACG
Heterologous expression	EmPAL-proR	CCCAAGCTTACAAATAGGAAGAGGAGCACCATTC

图1 EmPAL基因ORF及其启动子的克隆 M,DL 5 000;1,3'-RACE产物;2,第一次Tail-PCR第二轮产物;3,第一次Tail-PCR第三轮产物;4,第二次Tail-PCR第二轮产物;5,第二次Tail-PCR第三轮产物;6,EmPAL ORF序列PCR产物;7,EmPAL启动子序列PCR产物。

Fig.1 Cloning of EmPAL ORF and promoter M, DL 5 000; 1, PCR product of 3'-RACE; 2, Second round product of the first Tail PCR; 3, Third round product of the first Tail PCR; 4, Second round product of the second Tail PCR; 5, Third round product of the second Tail PCR; 6, PCR product of EmPAL ORF; 7, PCR product of EmPAL promoter.

图2 EmPAL蛋白的理化性质 A,EmPAL蛋白亲/疏水性预测;B,EmPAL蛋白的信号肽预测;C,EmPAL蛋白的跨膜域预测。

Fig.2 The physicochemical properties of EmPAL protein A, Hydrophobicity or hydrophilia prediction of EmPAL protein; B, Signal peptide prediction of EmPAL protein; C, Transmembrane domain prediction of EmPAL protein.

图3 EmPAL蛋白的结构 A,EmPAL蛋白的二级结构;B,EmPAL蛋白预测单体;C,EmPAL蛋白预测同型四聚体;D,EmPAL蛋白超家族。

Fig.3 Structure of EmPAL protein A, The secondary structure of EmPAL; B, Monomer of EmPAL protein prediction; C, Homotetramer of EmPAL protein prediction; D, EmPAL protein superfamily.

图4 EmPAL和其他不同植物PAL蛋白的系统发育树

Fig.4 Phylogenetic tree of EmPAL and other PAL proteins from different plants

图5 EmPAL蛋白与其他大戟科植物中同源蛋白的多序列比对绿色横线,N端域;红色横线,C端嵌入的屏蔽域;黑色方框,活性位点;黑色星号,同源序列。

Fig.5 Multiple sequence alignment of EmPAL protein and its homologues from other Euphorbiaceae plants Green line, N-terminal; Red line, C-terminal embedded shielding domain; Black box, Active site; Black asterisks, Conserved sequence.

图6 EmPAL启动子的顺式作用元件

Fig.6 The cis-acting elements in the promoter of EmPAL

图7 EmPAL的表达模式 A,EmPAL在不同时期转录组的FPKM;B,EmPAL在不同时期的相对表达量。柱上无相同小写字母表示差异显著(p<0.05),图8同。

Fig.7 Expression pattern of EmPAL in Euphorbia maculata A, FPKM of EmPAL transcriptome at different periods; B, Relative expression of EmPAL at different developmental periods. The absence of identical lowercase letters on the column indicates significant (p<0.05) differences, as shown in Figure 8.

图8 EmPAL原核表达和不同温度下的相对酶活性 A,EmPAL原核表达载体构建,M1、M2为DNA Marker(DL10 k),1为pET32a(+)质粒泳道,2为pET32a(+)-EmPAL单克隆菌液PCR,a为pET32a(+)质粒特异性条带,b为EmPAL的特异性条带;B,EmPAL在大肠埃希菌 BL21(DE3) 中诱导表达SDS-PAGE,c为重组蛋白;C,EmPAL在不同温度下的相对酶活性。

Fig.8 Prokaryotic expression and relative enzyme activity at different temperatures of EmPAL A, Construction of EmPAL prokaryotic expression vector, M1 and M2 are DNA markers (DL10 k), Lane 1 is the pET32a(+) plasmid, Lane 2 is the PCR of pET32a(+)-EmPAL monoclonal bacterial culture, a is the specific band of pET32a(+), and b is the specific band of EmPAL; B, EmPAL induced expression of SDS-PAGE in Escherichia coli BL21(DE3), c is a recombinant protein; C, Relative activity of EmPAL at different temperatures.

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斑地锦EmPAL基因克隆、表达模式和酶活性分析

Cloning, expression pattern, and enzyme activity analysis of the phenylalanine ammonia-lyase gene (EmPAL) in Euphorbia maculata

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