Mining And Functional Verification Of Key Genes In The Synthesis Pathway Of Eleutherococcus Senticosus Triterpene Saponins

Triterpene saponins,as the main active substances of many medicinal plants,have pharmacological activities such as anti-cancer,anti-inflammatory,antioxidant,and prevent the occurrence of cardiovascular and cerebrovascular diseases.The medicinal plant of the family Araliaceae,Eleutherococcus senticosus,is a traditional medicinal plant with abundant triterpene saponins in China.However,there are few reports on the analysis of the metabolic pathways of the triterpene saponins of Eleutherococcus senticosus.Therefore,it is important to explore the key genes of triterpenoid saponin biosynthesis and analyze its metabolic pathway.In this experiment,whole-genome sequencing was performed and evolutionary events were analyzed.The key gene for triterpene saponin biosynthesis,OSC,was mined by genome sequence and its function was further verified by using tobacco transient transformation system.The species of different triterpene saponins and their specific accumulation were analyzed by metabolomics,and the key genes CYP and UGT involved in triterpene saponin biosynthesis in Eleutherococcus senticosus were mined by combined WGCNA and phylogenetic tree analysis,which laid the foundation for the study of triterpene saponin metabolic pathways in Eleutherococcus senticosus,with the main findings as follows:(1)The genome of Eleutherococcus senticosus at the chromosome level was obtained based on the second generation,third generation and Hi-C techniques with about 1.39 G and 24 chromosomes,and 51,243 genes were annotated,and 15 key OSC genes for triterpenoid saponin biosynthesis were mined.The evolutionary relationships were analyzed and two whole-genome replication events were found to have occurred.(2)The 12 target genes obtained from cloning were successfully constructed into tobacco expression vectors,and the gene products were expressed by tobacco transient transformation system,and four OSC synthases with different functions were finally identified by GC-MS and NMR,among which EsOSC2 functioned as cycloartenol synthase,EsOSC1,EsOSC5,EsOSC8,EsOSC15 functioned as β-amyrin synthase,EsOSC13 functions as lupeol synthase,and EsOSC4 and EsOSC12 function as tirucalla synthase.Among them,tirucalla synthase was found for the first time in Eleutherococcus senticosus,and the results provided a new direction for the subsequent metabolic pathway analysis and metabolomic analysis.(3)The qualitative and quantitative analyses of the triterpene saponins of Eleutherococcus senticosus were performed by TOF.68 triterpene saponins were detected and their tissue distribution specificity was investigated by quantitative analysis,which showed that most of the triterpene saponins accumulated in the leaves and fruits of Eleutherococcus senticosus.In combination with the transcriptome sequencing results,WGCNA was used to correlate the transcriptome and metabolome of Eleutherococcus senticosus to obtain key candidate CYP and UGT genes involved in the biosynthesis of Eleutherococcus senticosus triterpenoid saponins,and the candidate genes were further screened by phylogenetic tree analysis,and 29 candidate CYP genes and 5 UGT genes were finally analyzed.In this study,a chromosomal-level genome of 1.39 Gb in size was assembled,15 OSC genes were mined,and four OSC synthases were resolved,and tirucalla synthase was resolved for the first time in Eleutherococcus senticosus.A phylogenetic tree and WGCNA analysis predicted 29 candidate CYP genes and 5 UGT genes,laying the foundation for the analysis of triterpenoid saponin metabolic pathways in Eleutherococcus senticosus.