Abstract
Neopicrorhiza scrophulariiflora (Pennell) D.Y.Hong, an endangered perennial herb, is rich in triterpenes, iridoids, and phenolic compounds, which exhibit significant pharmacological effects. However, the molecular mechanisms of triterpenoid biosynthesis in N. scrophulariiflora remain unclear. Here, transcriptomic and metabolomic analyses were performed to investigate the triterpene content in different tissues and the expression patterns of key enzyme-encoding genes related to triterpenoid biosynthesis. We functionally characterized eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid biosynthesis, among which NsOSC2 is a bifunctional enzyme capable of catalyzing the conversion of 2,3-oxidosqualene to β-amyrin and α-amyrin. Additionally, an efficient regeneration system and a stable genetic transformation system were established for N. scrophulariiflora. These findings reveal key genes in triterpenoid biosynthesis, providing a theoretical foundation for the future production of key triterpenoids in N. scrophulariiflora through synthetic biology approaches.