| Panax japonicus var.major also known as “Zhuzi Shen”,is a traditional Chinese medicinal herb.Its main active ingredient is total saponins,but the key genes and expression patterns of the triterpenoid saponin biosynthetic pathway in P.japonicus var.major is still unclear.The identification of key genes involved in triterpenoid saponin biosynthesis in P.japonicus var.major is of great significance for improving its yield and quality.In this study,transcriptome and metabolome analysis techniques were used to reveal the biosynthetic pathways and regulatory mechanisms of triterpenoid saponins in P.japonicus var.major at both transcriptome and metabolome levels.The main results are as follows:(1)Transcriptome analysis was conducted on the roots,stems,leaves,and fruits of P.japonicus var.major,generating 228.9 Gb of data through the next generation sequencing.216,108 Unigenes were got via de novo assembly,of which 96,902 had functional annotations,including 211 Unigenes potentially involved in triterpenoid saponin biosynthesis.Furthermore,135,044 SSRs and 56,544 CDS sequences were identified.Enrichment analysis revealed that the majority of the differentially expressed genes were associated with growth and development,as well as secondary metabolite synthesis.(2)Based on the transcriptome results,507 PJ＿NBS genes were identified.GO and KEGG annotation results revealed that PJ＿NBS genes play a significant role in various defense reactions in P.japonicus var.major,with a small number of genes involved in the MAPK signaling pathway.In comparison to other Panax species,P.japonicus var.major possesses the highest number of NBS genes,and the greatest proportion of these genes are annotated as defense response genes.A total of 12 identified conserved domains and 5 unknown conserved domains were found in the NBS genes.Notably,there are significant differences in the sequences of the same conserved domains among various types of NBS genes.The phylogenetic tree reveals that most NBS genes have independent branches,and the RNL gene class originated earlier than those of Arabidopsis thaliana and Camelina sativa.Expression analysis demonstrated significant differences in the expression of NBS genes across tissues.The stem has been found to contain the greatest number of expressed genes.Furthermore,several NBS genes exhibited high similarity to known genes in public databases,such as ROQ1,AT1G74360,GSO1,and ERECT.These identified PJ＿NBS genes may play a pivotal role in plant disease resistance responses.(3)A total of 1,161 compounds were identified in P.japonicus var.major using LC-MS/MS untargeted metabolomics.PCA and PLS-DA analyses demonstrated high experimental stability,reliable data,and significant differences between various samples,making them suitable for subsequent analysis.A total of 792 significantly different metabolites were identified across different tissues of P.japonicus var.major,including ginsenoside F2,Ro,Rb1,Rd,Rg1,Rg3,Rf,and chikusetsusaponin Iva.These saponins are the major distinguishing compounds between the roots compared to stems,leaves,and fruits,with the majority being more abundant in the roots,except for Rg1 and Rf,which are present in lower amounts than in other tissues.Hierarchical clustering analysis revealed that metabolic activity primarily occurs in leaves and fruits,while stems function as transporters for metabolites.Enrichment analysis of different metabolites indicated that the synthesis and metabolism pathways of carbohydrates and amino acids were the most significant.(4)In P.japonicus var.major,17,19,and 23 candidate genes were identified as being involved in the MVA,MEP,and 2,3-oxidosqualene synthesis pathways,respectively.Gene expression analysis revealed that the MVA pathway genes were highly expressed in the roots,while MEP and 2,3-oxidosqualene synthesis pathway genes were predominantly expressed in the leaves.This suggests that triterpenoid saponins are primarily synthesized in the roots and leaves.A total of 311 CYP450 and147 UGT genes were identified,with 113 and 74 genes containing complete domains,respectively.Functional annotation analysis showed that CYP450 and UGT genes were mainly associated with the synthesis of secondary metabolites,and 30 UGT genes were annotated to the triterpenoid pathway.Some conserved motifs in A-type and non-A-type CYP450 genes displayed genetic variation or mutation,with A-type motifs being more conserved.In addition,the analysis of expressions revealed that the majority of them were highly expressed in both the roots and leaves,indicating that these two tissue types are the main locations for the synthesis of triterpenoid saponins.(5)Correlation analysis of saponin compounds and candidate genes involved in triterpenoid saponin biosynthesis demonstrated that various saponins were grouped into four distinct branches.Based on the correlation analysis,three UGT genes were ultimately determined to be highly correlated with five ginsenosides and were likely involved in their biosynthesis.PJ＿UGT013,PJ＿UGT092,and PJ＿UGT146 exhibited high similarity to UGT94Q2(51.7%),UGT74AE2(56.2%),and UGT73AH1(68.1%),respectively.The expression levels of three UGT genes were quantified using RT-q PCR,and the results revealed that all of the genes were expressed at high levels in both roots and leaves.In summary,this study utilized transcriptomics and metabolomics approaches to examine the biosynthesis pathway of triterpenoid saponins in P.japonicus var.major at both transcriptome and metabolome levels,identifying key genes and uncovering the molecular regulatory mechanism.Additionally,it offers theoretical and data support for further research on the biosynthesis and functional genes of triterpenoid saponins in P.japonicus var.major. |
