[Cloning and functional study of diterpene synthase genes in Rhododendron molle]

Rhododendri Mollis Flos is a Chinese herbal medicine derived from Rhododendron molle(Ericaceae), with diterpenoids being its primary bioactive constituents. Diterpene synthases are key enzymes in the biosynthetic pathway of diterpenoids. Transcriptome analysis of R. molle identified two ent-copalyl diphosphate synthase(CPS) genes(RmTPS33 and RmTPS64) and one ent-kaurene synthase(KS) gene(RmTPS65). Full-length cloning, bioinformatics analysis, and functional characterization of these three genes yielded the following results.(1) The open reading frames of RmTPS33, RmTPS64, and RmTPS65 were 2 280, 2 476, and 2 370 bp, encoding 759, 819, and 789 amino acid residues, respectively.(2)Multiple sequence alignment and phylogenetic analysis demonstrated that both RmTPS33 and RmTPS64 contained conserved CPS family motifs(LHS, PNV, and DxDD) and belonged to the TPS-c subfamily. RmTPS65 possessed conserved α-domain motifs(DDxxD and NSE/DTE) of class Ⅰ diterpene synthases and was classified into the TPS-e/f subfamily.(3) Functional assays confirmed that both RmTPS33 and RmTPS64 catalyzed the protonation-induced cyclization of GGPP to form ent-CPP, with RmTPS33 exhibiting higher catalytic activity than RmTPS64. RmTPS65 catalyzed the diphosphate elimination of ent-CPP to produce a single product, 16α-hydroxy-ent-kaurane. Furthermore, protein engineering of RmTPS33 and RmTPS64 suggested that their activity divergence stemmed from variant amino acids within functional domains. This study successfully cloned three diterpene synthases gene(RmTPS33, RmTPS64, and RmTPS65), expanding the current diterpene synthase gene repository and establishing a foundation for investigating biosynthetic pathway genes of active compounds in R. molle.