斑地锦EmPAL基因克隆、表达模式和酶活性分析OA

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 per-formed 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 ele-ments related to light response,stress response,and phenylpropanoid metabolism regulation.Tissue-specific expres-sion analysis revealed that EmPAL expression was highest in the roots during the reproductive growth period.The mo-lecular weight of EmPAL protein is 78.17 ku,and its isoelectric point is 6.34.The secondary structure was predomi-nantly 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 cat-alyze 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.