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

doi:10.3969/j.issn.1004-1524.20250609

Acta Agriculturae Zhejiangensis ›› 2026, Vol. 38 ›› Issue (8): 1598-1609.DOI: 10.3969/j.issn.1004-1524.20250609

• Horticultural Science • Previous Articles Next Articles

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 Online:2026-08-25 Published:2026-09-14

Abstract

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

CLC Number:

S567.219

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.

Figures/Tables 9

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

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.

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.

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.

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

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.

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

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.

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

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