Cloning and functional study of AdOPT1, a gene for synthesizing isoimperatorin from Angelica dahurica var. formosana

Abstract

Angelica dahurica var. formosana (ADF) is a perennial medicinal plant in the Apiaceae family. Its dried roots are rich in furanocoumarins, which exhibit significant pharmacological activities and development potential. The main coumarins in ADF are imperatorin and isoimperatorin. The gene responsible for imperatorin biosynthesis has been reported, but the gene catalyzing isoimperatorin synthesis remains unknown. This study identified a candidate gene, AdOPT1 (1230 bp), from the transcriptome data of ADF through homology-based comparison with AkPT1 in Angelica keiskei, showing 93 % similarity. In vitro functional assays revealed that AdOPT1 catalyzes the oxygen-prenylation reaction between DMAPP and bergaptol to produce isoimperatorin. The kinetic parameters of AdOPT1 for bergaptol and DMAPP were found to be Km bergaptol = 112.9 ± 17.83 μM, Km DMAPP = 1.97 ± 0.08 μM, respectively. Molecular docking and alanine scanning analysis identified critical residues 45Lys, 124His, and 395Trp for AdOPT1 catalytic activity. Under various abiotic stresses such as flooding, salt, drought, and shading treatment, the expression level of AdOPT1 gene in ADF significantly increased, suggesting its involvement in ADF stress resistance. Additionally, transient overexpression of AdOPT1 significantly increased isoimperatorin content, with 3.26-fold higher than the control-group and 3.53-fold higher than the wild-type. It further confirmed that AdOPT1 is responsible for catalyzing the biosynthesis of isoimperatorin in ADF. This provides a molecular theoretical basis for making the biosynthesis pathway of furanocoumarin more comprehensive and helps to breed new high-quality ADF varieties in the future.