[Functional identification and analysis of Ad4CL1 gene from Angelica dahurica var. formosana]

4-coumarate: coenzyme A ligase(4CL) is a key rate-limiting enzyme regulating the phenylpropanoid metabolic pathway in plants. It catalyzes the conversion of hydroxycinnamic acids into coenzyme A thioesters, providing precursors for the synthesis of secondary metabolites such as lignin, coumarins, and flavonoids. This study investigated the enzymatic characteristics and expression profile of Ad4CL1 in Angelica dahurica var. formosana, aiming to provide targets for elucidating the molecular mechanisms of coumarin biosynthesis and stress-resistance breeding. Using the cultivated variety "Chuanzhi No. 2" of A. dahurica(Apiaceae) as material, the Ad4CL1 gene(cDNA full length 1 632 bp) was cloned, encoding 534 amino acids. Bioinformatics analysis showed that the Ad4CL1 protein has a molecular weight of ～59.55 kDa, an isoelectric point(pI) of 5.54, and consists of 50.28% random coil, 30.02% α-helix, and 19.71% extended chain, without transmembrane domains or signal peptides. Phylogenetic analysis showed that Ad4CL1 is most closely related to A. sinensis 4CL. A prokaryotic expression system successfully induced soluble Ad4CL1 protein. Enzyme kinetic analysis confirmed that Ad4CL1 catalyzes the conversion of p-coumaric acid and caffeic acid into the corresponding coenzyme A thioesters. With these substrates, the optimal catalytic pH of Ad4CL1 protein was 7.0, and the optimal catalytic temperatures were 45 ℃ for p-coumaric acid and 40 ℃ for caffeic acid, with significantly higher affinity for p-coumaric acid than for caffeic acid. The results of qRT-PCR analysis revealed that Ad4CL1 expression in roots was significantly higher than that in leaves(2.94-fold, P<0.000 1), and its expression was dynamically regulated under abiotic stresses: flooding and shading significantly inhibited expression; salt stress promoted upregulation at later stages; and drought first suppressed expression and later induced upregulation. In conclusion, this study functionally identified the Ad4CL1 gene in A. dahurica var. formosana for the first time. Ad4CL1 exhibits substrate-specific catalytic activity, and its expression is dynamically regulated by tissue development and abiotic stress.