Study On The Mechanism Of Quality Integrated Difference Of Epimedium Sagittatum By Quality Integrating Metabolome And Transcriptome

ObjectiveBased on the previous results of our research group,three populations of Epimedium sagittatum(Sieb.et Zucc.)Maxim.with different chemical type were collected at the same vegetative stage for metabolome and transcriptome integration analysis,the synthesis pathway of active components of flavonols was mapped,candidate genes were screened,and the formation mechanism of quality differences was explored.Methods(1)Metabolomics analysis of E.sagittatum.Using widely targeted metabolomics,three groups with large differences in quality and chemical type of E.sagittatum were cultivated in homogeneous gardens for more than 3 years,i.e.the population with the vacuum chemical type(AHHS)in Anhui Huangshan,the ordinary population with common chemical type(JXWN)in Jiangxi Wuning and the population with the icariin major epimedin B high chemical type(HBLT)in Hubei Luotian,were sampled at the same trophic stage for whole characterization and relative quantification of leaf metabolites.Bivariate analysis of anthocyanin and total flavonoid glycosides was performed.Principal component analysis,cluster analysis,orthogonal partial leastsquares discrimination analysis and univariate statistical analysis were integrated to analyze the whole metabolites and differential metabolites of the three populations.KEGG functional annotation and enrichment analysis of differential metabolites were performed through the KEGG database.(2)Transcriptome analysis of E.sagittatum.The transcripts of three populations of E.sagittatum were sequenced using high-throughput transcriptome sequencing,and the transcriptome reference sequences were obtained by splicing the transcripts using Trinity software,and the transcripts were analysed by Corset hierarchical clustering using Corset software to obtain unigene for subsequent analysis.Genes were matched and functionally annotated in each sample using Diamond Blastx software based on 6major public databases(KEGG,NR,Swiss-Prot,GO,KOG,Trembl)and Hmmer software based on the Pfam database.CDS prediction of the assembled transcripts by Trans Decoder software,the expression-based quantification was then performed by RSEM software.Differentially expressed genes were obtained using DESeq2 for differential expression analysis between sample groups.Gene expression trends between populations were analysed based on K-means clustering analysis,and differentially expressed genes were annotated and functionally enriched using GO and KEGG databases.Transcription factors were predicted and annotated using i TAK software.(3)Mechanistic analysis of qualitative differences in metabolomic and transcriptomic integration.The biosynthetic pathway of flavonols was constructed based on differentially expressed genes,metabolites and literature research,cluster analysis of differentially expressed genes in this pathway were performed.KEGG pathway analysis,KEGG enrichment analysis and bivariate analysis were performed on differentially expressed genes and metabolites.Results with r > 0.8 were selected and visualized using the Cytoscape＿v3.10.0 to visually analyze gene-metabolite and metabolite-transcription factor correlation networks.Results(1)Metabolomics analysis of E.sagittatum.Based on widely targeted metabolomics,1020 metabolites were identified in three populations of E.sagittatum.Among them,there were 573 differential metabolites and 228 flavonoids differential metabolites,which were the largest proportion of the differential metabolites(39.8 %).Among the three anthocyanins identified,only the content of cyanidin-3,5-Odiglucoside(Cyanin)and cyanidin-3-O-(2’’-O-glucosyl)glucoside was significantly correlated with the content of epimedin C(P < 0.01),there was no significant correlation between epimedin A,epimedin B,icariin and anthocyanins.Using the AHHS population as a control group,most flavonoids,phenolic acids,terpenoids and other classes of the JXWN and HBLT populations were up-regulated.The AHHS population is an ideal control population for the study of mechanistic analysis of qualitative difference.KEGG functional annotation and enrichment analysis of the differential metabolites showed that all three differential subgroups of differential metabolites were significantly enriched in the flavonoid biosynthetic pathway,and the pathways significantly enriched in both AHHS vs HBLT and AHHS vs JXWN were the flavonoids and flavonol biosynthetic pathway and the flavonoids biosynthetic pathway.(2)Transcriptome analysis of E.sagittatum.Based on transcriptome sequencing technology,transcriptome analysis of nine samples from three populations at the same trophic stage obtained a total of 61.32 Gb of high-quality reads,the total number of bases of high-quality reads per sample exceeded 6.00 Gb.The assembly obtained179,679 expressed genes with an average sequence length of 869 bp.Unigene was compared and functionally annotated with seven major databases(KEGG,NR,SwissProt,Trembl,KOG,GO and Pfam databases)and a total of 91,388 genes(50.86 %)were annotated in least one database.Species annotations in the NR database indicated that the genetic relationship is close between E.sagittatum and Aquilegia coerulea E.James.Differential expression analysis of genes in three populations of E.sagittatum discovered the total number of differentially expressed genes for AHHS vs HBLT,AHHS vs JXWN and JXWN vs HBLT were found to be 18,569,9,657 and 18784,respectively.Among them,867,344 and 842 differentially expressed genes were annotated as transcription factors for AHHS vs HBLT,AHHS vs JXWN and JXWN vs HBLT,respectively.The three comparison subgroups had a total of 1201 differential genes,with AHHS vs JXWN having the fewest unique differential genes(1643)and AHHS vs HBLT having the most unique differential genes(3938).The KEGG database was used to enrich the transcriptomic data ofthree populations.A total of 140 KEGG metabolic pathways were annotated,and the pathway in which the differential genes were enriched in all three comparative subgroups was the circadian rhythm-plant pathway.(3)Mechanistic analysis of qualitative differences in metabolomic and transcriptomic integration.The metabolomic and transcriptomic data were integrated to identify eight enzymes(7 PAL,16 4CL,6 CHS,12 CHI,2 F3 H,13 FLS,2 Ao MT and26 GT)and 10 metabolites involved in the biosynthetic pathway of total flavonol glycosides with 84 differentially expressed genes to construct a total flavonol glycosides biosynthetic pathway.Among them,glycosyltransferase was the most differentially expressed gene in the synthetic pathway among the transcriptome data.Bivariate analysis of 10 metabolites,7 bioactive substances and differential genes in the biosynthetic pathway map showed that 56 genes were strongly correlated with epimedin A,epimedin B,epimedin C,icariin,2’’-O-rhamnosyl-icariside II,Icariside II,Sagittatoside B,Cinnamic acid,p-Coumaryl acids,Naringenin chalcone,Naringenin,Aromadendrin(Dihydrokaempferol),Kaempferol(3,5,7,4’-Tetrahydroxyflavone)and icarrin(r > 0.80).Bivariate analysis of 170 MYB,MYB-related,b HLH family transcription factors,bioactive components and metabolites in all phases of E.sagittatum showed that the 105 MYB,MYB-related and b HLH family transcription factors were strongly correlated with epimedin A,epimedin B,epimedin C,icariin,2’’-O-rhamnosyl-icariside II,icariside II,sagittatoside B,cinnamic acid,p-Coumaryl acids,Naringenin chalcone,Naringenin,Aromadendrin(Dihydrokaempferol),8-Prenylkaempferol and icarrin(r > 0.80).Cluster-4340.101633,the sequence of E.sagittatum transcription factor annotated in the NR database,was positively correlated(r > 0.8)with the accumulation of epimedin A,epimedin B and icaritin.Conclusion(1)Insights of quality evaluation studies.Widely targeted metabolomics provides a more comprehensive quality evaluation in different populations of E.sagittatum.The differences among the three populations are not only in total flavonol glycosides,but also in whole metabolites,which could be stably inherited.This difference further confirms that a more in-depth molecular study of the molecular mechanisms of the quality-dominant population HBLT and the Jiangxi local high-quality population JXWN is worth.The overall chemical composition of the three populations has a high degree of variability.Based on the holistic view of Chinese medicine,more in-depth pharmacological and pharmacokinetic studies and clinical trials on different varieties and origins of Epimedium are needed to improve the quality evaluation system of Epimedium and guide rational drug use.In addition,there was no significant correlation between the anthocyanin and epimedin A,epimedin B,icariin,and the HBLT population,which is chemically dominated by icariin and has strongly peaked by epimedin B,can be used to select varieties with high anthocyanin and suitable total flavonol glycosides content to improve the ornamental value and economic value,and to solve the problem of low economic efficiency of Epimedium cultivation.(2)Insights of mechanistic analysis of qualitative differences.The existence of significant differences in gene expression despite the uniform of environmental factors confirms that quality differences between populations of E.sagittatum are stably inherited.A large amount of genetic information is still unresolved in HBLT,which has great potential to be explored.In particular,for the HBLT population,a large number of differentially expressed genes in HBLT were screened in this study,the combination of metabolomics and enzymatic characterization data will hopefully lead to the discovery of functional genes and the elucidation of the biosynthesis of total flavonol glycosides and icarrin,providing a genetic basis for the heterologous production of flavonols active ingredients in Epimedium.The pathway that was enriched in all three comparative subgroups was the plant circadian pathway,suggesting that the accumulation of active ingredients in Epimedium may be closely related to circadian rhythms,but further confirmation is needed.It is suggested that selecting seed through circadian rhythm characteristics may provide a basis for breeding quality varieties of E.sagittatum.(3)Insight of biosynthetic pathway.Based on the constructed biosynthetic pathway of total flavonol glycosides,it is hypothesized that the O-methyltransferase of E.sagittatum is an S-adenosyl-L-methionine enzyme and that the low content of total flavonol glycosides in AHHS population may be limited by the S-adenosyl-Lmethionine.Glycosyltransferase is the most differentially expressed genes in the synthetic pathway,and the diversity of glycosyltransferase in Epimedium is an important reason for the high variety of flavonol glycosides components.In addition,it was noted that the annotated transcription factor E.sagittatum(Cluster-4340.101633)in NR database is positively correlated(r > 0.8)with the accumulation of epimedin A,epimedin B and icarrin,which may be candidate positive regulators of the active ingredients biosynthesis of E.sagittatum.