| OBJECTIVE:(1)The contents of Peucedanum Peucedanum essence I,Peucedanum Peucedanum essence II,praeruptorin A,praeruptorin B,and praeruptorin E before and after bolting were determined by HPLC.The changes of the contents of five coumarins in Peucedanum praeruptorum Dunn before and after bolting were discussed.(2)The comparative transcriptome analysis of Peucedanum praeruptorum Dunn in different growth stages and different ecological environments was carried out by using high-throughput sequencing technology.Combined with co expression network,the expression patterns of key genes of coumarin synthesis in Peucedanum praeruptorum Dunn before and after bolting were analyzed,and the internal mechanism of bolting and flowering affecting the synthesis and transport of coumarins in one-year and two-year Peucedanum Peucedanum was explored.(3)The differential gene expression level of coumarin synthesis pathway of Peucedanum Peucedanum before and after bolting was analyzed by fluorescence quantitative PCR.23 key enzyme genes were selected for gene expression determination,and the expression patterns of key enzyme genes of coumarin synthesis before and after bolting were compared,which provided a basis for exploring the biosynthesis pathway of coumarins in Peucedanum Peucedanum.METHODS:(1)The samples of Peucedanum Peucedanum before and after bolting were collected,and the contents of Peucedanum praeruptorum Dunn essence I,Peucedanum Peucedanum essence II,Peucedanum praeruptorin A,praeruptorin B,and praeruptorin E before and after bolting were determined by HPLC.(2)The experimental samples from different harvest periods,different bolting times and different slope directions were collected.The transcriptome sequencing combined with co-expression analysis was used to study the regulatory mechanism of the key genes related to the coumarin biosynthesis.(3)The samples of Peucedanum Peucedanum before and after bolting were collected,and the expression of23 differentially expressed genes were analyzed by fluorescence quantitative PCR to analyze the biosynthetic pathway of coumarins in Peucedanum praeruptorum Dunn.RESULTS:(1)The contents of praeruptorin A,praeruptorin B,and praeruptorin E before bolting were higher than that after bolting.The content of peucedanocoumarin I after bolting was higher than that before bolting,and the difference of peucedanocoumarin II between pre-and post-bolting was relatively small.The proportion of five chemical components before bolting is praeruptorum essence I:praeruptorum A: Peucedanum Peucedanum essence II: Peucedanum praeruptorum B: praeruptorum E = 1:25:4:9:11,and the proportion after bolting is 1:10:3:2:4.(2)The gene expression profiles of P.praeruptorum under pre-and post-bolting periods were compared and analyzed in different harvesting periods and different light intensities.A series of key genes involved in the metabolism and transportation of phenylpropanoid were screened out.Differential gene enrichment analysis and co-expression analysis further revealed the connection between bolting progress and secondary metabolic pathways.(3)The KEGG database was used to annotate and analyze the biological functions of differential genes in this study.The results indicated that among the differentially expressed genes before and after bolting,there were 137 genes involved in the biosynthesis of coumarin,among which 56 were up-regulated unigenes and 81 were down-regulated unigenes.CONCLUSION:(1)In contrast to bolting,the content of praeruptorin A,praeruptorin B,and praeruptorin E is higher than that after bolting.The content of peucedanocoumarin I after bolting was higher than that before bolting,and the difference of peucedanocoumarin II between pre-and post-bolting was relatively small.After bolting and flowering,the contents of praeruptorin A,praeruptorin B,and praeruptorin E in the roots of P.Peucedanum were significantly reduced,indicating that coumarin metabolism after bolting had changes,and the expression of key enzymes involved in the coumarin pathway decreased.(2)The ressults also suggested that most of the peroxidases involved in monomer biosynthesis are down-regulated.CHS gene that involved in the biosynthesis of flavonoids is up-regulated.4CL and HCT that involved in the synthesis of phenylpropanoid showed a down-regulated trend,while the ABC transporter genes involved in the transport of coumarins showed an up-regulated expression.The phosphatidylinositol signaling and apoptosis genes are the main differential genes,which may be involved in the process of programmed cell death(PCD)of root cells.(3)Based on the changes in gene expression levels and chemical composition before and after bolting,it is speculated that these key enzymes could play a role in the regulation of P.praeruptorum coumarin. |
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