Transcriptional And Metabolic Analysis Of Aboveground And Underground Parts Of Gentiana Rhodantha

Gentiana rhodantha Franch.is a perennial herb of Gentiana genus.It is published in Chinese Pharmacopoeia(2020 edition).The plant is used for the treatment of tuberculosis and cholecystitis.G.rhodantha has the effects of clearing heat and promoting diuresis and detoxification.It is used to treat acute jaundice hepatitis,dysentery,wind-heat common cold,stomach pain and other diseases.At present,most researches of G.rhodantha have been focused on extraction technology and content determination.There are few researches on the biosynthesis mechanism of secondary metabolites in G.rhodantha.Even now the analysis of the synthetic pathway of secondary metabolites in G.rhodantha and the discovery of key enzyme genes has yet been done.Therefore,this study,using the aboveground part and the underground part of G.rhodantha as materials,which will systematically analyze the differences in the accumulation of main medicinal substances in the aboveground and underground parts of Gentiana tinctoria by using transcriptome and metabolome methods.This study will have important theoretical and practical significance for future studies of G.rhodantha.The main results are as follows:(1)Transcriptome analysis of the six samples of G.rhodantha was performed using high-throughput sequencing technology.A total of 39.65 Gb of sequencing data was obtained,and 112526 genes were obtained after assembly.By comparing the different transcriptomes in different stages,we found that a total of 15539 genes were differently expressed,of which 7730 were up-regulated and 7809 were down-regulated.Compared with the aboveground part stage and the underground part stage,a total of 80 genes were annotated the iridoid biosynthetic pathway,of which 27 genes were up-regulated and 53 genes were down-regulated.And 72 genes were annotated the flavonoid biosynthetic pathway,of which 25 genes were up-regulated and 47 genes were down-regulated(2)Using secondary metabolomics,a total of 632 compounds were identified in the aboveground part and the underground part of G.rhodantha.A total of 250 differentiated secondary metabolites were obtained by variable importance projection analysis and differential multiple numerical screening.Compared with the aboveground part stage and the underground part stage,there were 50 up-regulated and 200 down-regulated metabolites.The differentiated secondary metabolites were annotated,by comparing with KEGG,a total of 2 metabolites were noted in iridoid biosynthetic pathway and 15 metabolites were noted in flavonoid biosynthetic pathway.It was found that mangiferin and swertia glycosides were synthesized mainly in the aboveground part of G.rhodantha.(3)The genes and metabolites related to iridoterpene synthesis in the transcriptome and metabolome of G.rhodantha were thoroughly explored and the cluster analysis and correlation analysis were carried out on the differential genes and metabolites annotated in the synthetic pathway.In aboveground part,a total of 4iridoid biosynthetic pathway metabolites and 34 genes were main expression while 11 genes were main expression in underground part of G.rhodantha.On this basis,finally,5 genes involved in iridoid biosynthetic pathway by advancing in key enzymes of the iridoid pathway.(4)The biosynthetic pathway of flavonoids was annotated.A total of 72 genes and 15 differential metabolites were annotated in flavonoid biosynthetic pathway.Cluster analysis and correlation analysis were performed for the differentially annotated genes and metabolites in the synthetic pathway.There were 2 metabolites primary synthesis in underground part of G.rhodantha.Others were primary synthesis in aboveground part of G.rhodantha.Combining with flavonoid gene-metabolic network interaction map,8 important genes involved in flavonoid biosynthetic pathway were screened.(5)Fluorescence quantitative expression analysis of 13 key genes was performed,and it was found that 13 key genes were significantly expressed in the aboveground part.In the iridoid pathway,10 HGO,DXS and DXR genes showed a predominance expression in the flowers of G.rhodantha.,two HMGR genes exhibited high expression level in the stems of G.rhodantha.and HMGR gene in the leaves of G.rhodantha.In flavonoid pathway,two CHS genes were significantly expressed in stem,one FLS gene and two CHI genes in were significantly expressed in flowers,and the other three genes were significantly expressed in leaves.