RESUMO
Sequential catalysis by ent-copalyl diphosphate(CPS) and ent-kaurene synthase(KS) is a critical step for plants to initiate the biosynthesis of gibberellin with geranylgeranyl pyrophosphate(GGPP) as the substrate. This study mined the transcriptome data of Stellera chamaejasme and cloned two key diterpene synthase genes, SchCPS and SchKS, involved in the gibberellin pathway. The two genes had the complete open reading frames of 2 595 bp and 1 701 bp, encoding two hydrophilic proteins composed of 864 and 566 amino acid residues and with the relative molecular mass of 97.9 kDa and 64.6 kDa and the theoretical isoelectric points of 5.61 and 6.12, respectively. Sequence comparison and phylogenetic tree showed that SchCPS contained LHS, PNV, and DxDD motifs conserved in the CPS family and was categorized in the TPS-c subfamily, while SchKS contained DDxxD, NSE/DTE and PIx motifs conserved in the KS family and was categorized in the TPS-e subfamily. Functional validation showed that SchCPS catalyzed the protonation and cyclization of GGPP to ent-CPP, while SchKS acted on ent-CPP dephosphorylation and re-cyclization to ent-kaurene. In this study, the full-length sequences of SchCPS and SchKS were cloned and functionally verified for the first time, which not only enriched the existing CPS and KS gene libraries but also laid a foundation for the cloning and biosynthesis pathway analysis of more genes involved in the synthesis of active components in S. chamaejasme.
Assuntos
Alquil e Aril Transferases , Filogenia , Proteínas de Plantas , Thymelaeaceae , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/química , Thymelaeaceae/genética , Thymelaeaceae/enzimologia , Thymelaeaceae/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Sequência de Aminoácidos , Diterpenos do Tipo Caurano/metabolismo , Diterpenos do Tipo Caurano/química , Alinhamento de Sequência , Clonagem MolecularRESUMO
Tigliane type macrocyclic diterpenoids with special structures and diverse bioactivities are mainly extracted from plants of Euphorbiaceae and Thymelaeaceae. According to the different functional groups, they can be classified into types of phorbol esters, C-4 deoxyphorbol esters, C-12 deoxyphorbol esters, C-16 or C-17 substituted phorbol esters and others. Most of them present promising antiviral activities and cytotoxic activities and are expected to be developed as candidates for anti-AIDS, anti-tuberculosis, and anti-tumor clinical trials, demonstrating great potential for the application in healthcare. This paper reviews 115 novel tigliane-type diterpenoids discovered since 2013 and summarize their chemical structures and bioactivities, aiming to lay a foundation for further development and utilization of these compounds and provide new ideas for the development of clinical drugs.