The Dissection Of Ginsenoside Metabolic Differences Controlled By Tissue,Development And Species Through Metabolomics And Transcriptomic Approaches

Ginseng is a traditional medicinal herb with a long history of cultivation,which is commonly associated with the dried roots of several species in the genus Panax of the family Araliaceae.Major commercial ginseng products are derived from P.ginseng and P.quinquefolius.Based on the effective protection,development and utilization of medicinal resources,comprehensive study of ginsenoside accumulation in other tissues and development stage have become a top priority.Ginseng and its popular metabolites,ginsenosides,are attracting more and more attention because of proposed potential health benefits including anti-stress,health promotion,maintenance and enhancement of the central and immune systems,anti-oxidation,prevention of certain chronic diseases and aging deterrence properties.Therefore,there were significant differences in the efficacy of P.ginseng and P.quinquefolius.The phenomenon of P.ginseng and P.quinquefolius trade confusion occurred,which P.quinquefolius was used for the sale of P.ginseng in the major ginseng trading market in China.It is seriously damaged the safety of ginseng’s consumption.Therefore,it was found that a method for rapid identification of two species to maintain a safe and stable ginseng trading market has become the major task.With the metabolic and transcriptome rapid development,they have been widely used to reveal the active components metabolomics regulation network and explore the main synthetic genes,respectively.Therefore,analysis the genus Panax metabolic regulation network and search different expression of genes in specific-tissues by two omics,have a significance meaning to ginsenoside synthesis regulation network and molecular mechanism.It also provides potential compounds for ginseng species evaluate and theoretical support.In this study,a sensitive ultra-performance liquid chromatography-electrospray ionization-mass spectrometry(UPLC-MS)method with a shorter chromatographic running time was developed and validated for simultaneous quantification of 10 ginsenosides.Comparing ginsenoside contents in various tissues during different developmental stages revealed tissues-specific accumulation of most ginsenosides.Most of the ginsenosides were found to accumulate with the developmental stage,and mainly accumulated in the main root,followed by lateral roots,stems,petioles and leaves.In the present study,the Rb2/Rbl ratio in P.quinquefolius was 0.05 and 1.1 in P.ginseng leaves.However,the ratio of Rb2/Rbl followed an opposite trend in stems with a ratio of 1.2 in P.ginseng and 0.1 in P.quinquefolius.Another significant factor was the level of the ginsenoside Rg3 in P.ginseng,which was nearly 80 times higher than in the petiole of P.quinquefolius.These results suggested that the Rb2/Rbl ratio in roots,leaves,and stems and the Rg3 content are good candidate metabolic markers for identifying the ginseng species within a diverse collection of ginseng accessions.In addition,the PCA showed that aboveground parts differed significantly between species and can be considered as species-specific markers rather than roots,so the valuable roots do not need to be sacrificed.This comprehensive survey can be used to identify ginseng species and developmental stages,as understanding the specific distribution of ginsenosides for the medicinal herb development,utilization and food safety to provide reliable scientific evidence.The tissue-specific profiling of primary and the secondary metabolites in two different species of ginseng has been performed using gas chromatography-mass spectrometry(GC-MS).The combination of multivariate statistics and mathematical modeling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways.The results showed that a total of 149 primary metabolites were identified from main roots,lateral roots,stems,petioles and leaves in P.ginseng and P.quinquefolius.Considering the classified metabolites,the most represented compound classes were sugars(14),acids(39),alcohols(14)and amino acids(29).Metabolites that were partially annotated were classified as unknown(24).The partial least squares-discriminate analysis(PLS-DA)revealed obvious metabolic discrimination among tissue-specific districts relative to cultivars.A total of 18 differential metabolites were identified.To verify whether our metabolic analysis provided enough information allowing the discrimination of each tissue when both species are compared,we decided to use a same supervised method.These compounds were identified in the main root,lateral root,stem,leave and petiole were 11,18,8,16 and 16,respectively.To investigate the contribution of tissue-specific C and N metabolism to the biosynthesis of ginsenosides,we examined the metabolic changes of different tissues.Our study demonstrated that the ginsenosides content was dependent on main roots and lateral roots energy metabolism and independent of leaves and petiole photosynthesis during ginsenosides accumulation.When the P.ginseng and P.quinquefolius were compared,the results indicated that higher rates of C assimilation to C accumulation are closely associated with ginsenosides accumulation in P.ginseng main roots and P.quinquefolius lateral roots,respectively.Taken together,our studies suggest that tissue-specific metabolites profiling dynamically varied during the course of ginsenosides accumulation,which may provide a new insight into the mechanisms of the ginsenosides accumulation at the metabolite level.The root,lateral,stem,leaf and petiole of the five-year P.ginseng are studied in this thesis.With high-throughput sequencing technology,the trancriptome dataset is built and the differentially expressed genes between five tissues are screened.The study in this paper will supply theory basis to find out functional genes,regulate ginsenosides accumulation genes,clarify ginseng efficacy material and breed good varieties.A total of 130.90 Gb Clean data,each sample 7.50 Gb Clean data,assembled 61.574 Unigene,28.956 Unigene annotations were obtained.By comparing the transcriptome data of the main root,lateral root,leaf,petiole and stem,the most significant group of genes with difference was screened,and 4233 genes were found between main root and leaf.Among them,2568 genes accumulated in the main root,1665 genes were highly expressed in the leaves.The GO function,COG analysis and KEGG metabolic pathway were analysis between the main roots and leaves were research.1.6605 in the GO function is divided into biological processes,cell composition and molecular function.1.595 COG notes were formed in difference genes of the main root and leaf,among which the DEGs were the most accumulation,and these genes were maintained to support the normal physiological function and participate in the basic regulation of plant.Followed by KEGG metabolic pathway analysis,the 4233 differences in the final expression of the gene to 111 metabolic pathways,mainly involved in the biosynthesis of carbon metabolism,amino acid biosynthesis and other biosynthesis between main root and leaf.By screening the differential genes in the main root and leaf,we screened the highly expressed genes with FPKM>1000,12,21 highly expression genes in main root and leaf,respectively.GBR5 was highly expression in main root,which was 504 times than leaf.It was identified as the major gene involved in ginsenoside biosynthesis.PgPR significantly expression with Re accumulation was consistency in main root,which means PgPR as the main synthetic Re ginsenoside regulation gene.In addition,phloem protein 2-2 play an important role in the plant defense mechanism as an internal part of the cell wall of higher plants,it the expression level in the leaf is 189 times higher than main root.Cytochrome P450 is thought to be a major regulation gene for the accumulation of PPT ginsenoside in leaf.