Gene Mining Of Paeoniflorin Biosynthesis Related Enzymes Based On Joint Transcriptional And Metabolomic Analysis

Paeoniflorin is a characteristic chemical component of plants in Paeoniaceae,with various pharmacological activities such as analgesic,sedative,anti-inflammatory,immunomodulatory,antioxidant,and antibacterial effects.However,the biosynthesis pathway of paeoniflorin has not been fully elucidated,especially the post-modification stage that determines the structural and functional specificity of paeoniflorin.This lack of understanding has hindered the comprehensive development and utilization of paeoniflorin.Therefore,in this study,metabolomics and transcriptomics technologies were used to systematically analyze the changes in metabolites and gene expression levels in the roots,stems,leaves,petals,and seeds of Paeonia ostii and Paeonia lactiflora,in order to clarify the molecular mechanism of paeoniflorin biosynthesis.The main results obtained are as follows:(1)Using comprehensive targeted metabolomics,the characteristic metabolites of P.ostii and P.lactiflora in their roots,stems,leaves,petals,and seeds were studied.A total of 796 compounds were identified in these five organs,including 275 flavonoids,accounting for 34.55%;259 phenolic acids,accounting for 32.54%;93 terpenoids,accounting for 11.68%;88 alkaloids,accounting for 11.06%;17 tannins,accounting for2.14%;15 coumarins and 9 lignans,accounting for 3.02%,as well as 40 compounds that did not belong to these seven main categories,accounting for 5.03%.Among the five organs of P.ostii,seven unique compounds were identified in the roots,and two unique compounds were identified in the seeds.Among the five organs of P.lactiflora,one unique compound was found in the stems,and two unique compounds were found in the seeds.Among the 93 detected terpenoids,there were 53 triterpenoids,26 monoterpenoids,and 14 terpenes.Among the 26 identified monoterpenoids,there were a total of 11 compounds including paeoniflorin and its derivatives.Based on the structural analysis,paeoniflorgenin and 8-debenzoylpaeoniflorin were identified as precursors of paeoniflorin.(2)Transcriptome sequencing technology was used to study the relative expression level of genes in the roots,stems,leaves,petals and seeds of P.ostii and P.lactiflora.A total of 367 Gb data and 55369 expression genes were obtained.8516(KEGG: 15%),6883(Pathway: 12%),28953(Nr: 52%),28921(Uniprot: 52%),20795(GO: 38%),2804(KOG:5%)and 19987(Pfam: 36%)genes were annotated in 7 databases,respectively.KEGG annotation showed that,7302 genes were annotated into the metabolism category,accounting for the largest proportion;GO annotation shows that gene functions are mainly concentrated in carbohydrate metabolism processes,membrane components,and ATP binding;In KOG classification,the proportion of general functional prediction categories is the highest;The Nr annotation results indicate that Vitis vinifera has the highest homology with P.ostii and P.lactiflora;Principal component analysis showed that P.ostii and P.lactiflora root,stem,leaf,petal and seed had high repeatability within each tissue group,and there was a large difference between different tissue groups.The sequencing data results could be used for subsequent functional analysis and gene screening.(3)By using a combined analysis of metabolomics and transcriptomics,based on gene annotation results and functional gene conserved domain features,a total of 599 CYP450,268 2ODD,and 237 UGT genes were identified as the major functional genes involved in the post-modification stage of paeoniflorin.A phylogenetic tree was constructed for the237 UGT genes,and 19 UGT genes related to monoterpenoid biosynthesis were selected.Correlation analysis was performed between the functional genes and the precursors of paeoniflorin synthesis,paeoniflorgenin and 8-debenzoylpaeoniflorin,resulting in the identification of 21 candidate CYP450 genes,13 candidate 2ODD genes,and 14 candidate UGT genes.Successfully cloned 5 highly expressed UGT and performed crude enzyme extraction.In summary,the use of comprehensive targeted metabolomics identified 796 metabolites in five organs of P.ostii and P.lactiflora,enriching and improving the chemical composition research of these two plants.The terpenoid backbone biosynthesis pathway was identified as a key pathway significantly enriched with genes and metabolites.Based on the screening of conserved domains of genes and correlation analysis between genes and metabolites,21 candidate genes of CYP450,13 of 2ODD,and 14 of UGT were obtained,laying the foundation for subsequent gene function verification and analysis of the biosynthesis pathway of paeoniflorin.