The chromosome-level reference genome assembly for Panax notoginseng and insights into ginsenoside biosynthesis

Panax notoginseng,a perennial herb of the genus Panax in the family Araliaceae,has played an important role in clinical treatment in China for thousands of years because of its extensive pharmacological effects.Here,we report a high-quality reference genome of P.notoginseng,with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb,produced with third-generation PacBio sequencing technology.This is the first chromosome-level genome assembly for the genus Panax.Through genome evolution analysis,we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis.We performed a detailed transcriptional analysis of P.notoginseng and explored genelevel mechanisms that regulate the formation of characteristic tubercles.Next,we studied the biosynthesis and regulation of saponins at temporal and spatial levels.We combined multi-omics data to identify genes that encode key enzymes in the P.notoginseng terpenoid biosynthetic pathway.Finally,we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P.notoginseng.The genetic information obtained in this study provides a resource for further exploration of the growth characteristics,cultivation,breeding,and saponin biosynthesis of P.notoginseng.

Molecular cloning and functional identification of sterol C24-methyltransferase gene from Tripterygium wilfordii

Sterol C24-methyltransferase(SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from Tripterygium wilfordii(Tw SMT1). Tw SMT1(Gen Bank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 k Da protein. The polypeptide encoded by the SMT1 cDNA was expressed and purified as a recombinant protein from Escherichia coli(E. coli) and showed SMT activity. The expression of Tw SMT1 was highly up-regulated in T. wilfordii cell suspension cultures treated with methyl jasmonate(Me JA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs(leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterolbiosynthesis of T. wilfordii and will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of T. wilfordii.

Molecular cloning and functional identification of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Tripterygium wilfordii

The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase(HDR) is the last step key enzyme of the methylerythritol phosphate(MEP) pathway,synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate,which is important for regulation of isoprenoid biosynthesis.Here the full-length cDNA of HDR,designated TwHDR(GenBank Accession No.KJ933412.1),was isolated from Tripterygium wilfordii for the first time.TwHDR has an open reading frame(ORF) of 1386 bp encoding461 amino acids.TwHDR exhibits high homology with HDRs of other plants,with an N-terminal conserved domain and three conserved cysteine residues.TwHDR cDNA was cloned into an expression vector and transformed into an Escherichia coli hdr mutant.Since loss-of-function E.coli hdr mutant is lethal,the result showed that transformation of TwHDR cDNA rescued the E.coli hdr mutant.This complementation assay suggests that the TwHDR cDNA encodes a functional HDR enzyme.The expression of TwHDR was induced by methyl-jasmonate(MJ) in T.wilfordii suspension cells.The expression of TwHDR reached the highest level after 1 h of MJ treatment.These results indicate that we have identified a functional TwHDR enzyme,which may play a pivotal role in the biosynthesis of diterpenoid triptolide in T.wilfordii.

Mechanistic analysis for the origin of diverse diterpenes in Tripterygium wilfordii

Tripterygium wilfordii is a valuable medicinal plant rich in biologically active diterpenoids,but there are few studies on the origins of these diterpenoids in its secondary metabolism.Here,we identified three regions containing tandemly duplicated diterpene synthase genes on chromosomes(Chr) 17 and 21 of T. wilfordii and obtained 11 diterpene synthases with different functions.We farther revealed that these diterpene synthases underwent duplication and rearrangement at approximately 2.3-23.7 million years ago(MYA) by whole-genome triplication(WGT),transposon mediation,and tandem duplication,followed by functional divergence.We first demonstrated that four key amino acids in the sequences of TwCPS3,TwCPS5,and TwCPSS were altered during evolution,leading to their functional divergence and the formation of diterpene secondary metabolites.Then,we demonstrated that the functional divergence of three TwKSLs was driven by mutations in two key amino acids.Finally,we discovered the mechanisms of evolution and pseudogenization of miltiradiene synthases in T.wilfordii and elucidated that the new function in TwMS1/2 from the terpene synthase(TPS)-b subfamily was caused by progressive changes in multiple amino acids after the WGT event.Our results provide key evidence for the formation of diverse diterpenoids during the evolution of secondary metabolites in T.wilfordii.