Difference Of Gene Expression During The Process Of Differential Biosynthesis Of Main Active Metabolites In Roots And Stems Of Ephedra Sinica

Ephedra sinica Stapf.is a traditional Chinese medicine that has been used for over five thousand years.The plant is known for its opposite pharmacological activities of root and stem.The stem has the effect of dispersing cold by sweating and raising blood pressure,while the root has the effect of fixing the surface to stop sweating and lowering blood pressure.This phenomenon of "same origin,disparate efficacy" is closely related to the differences in the active ingredients of the rhizomes of E.sinica,and the specific active ingredients in the rhizomes of E.sinica and the molecular basis of their biosynthesis are still unclear,which seriously limits the development and utilization of E.sinica resources.Therefore,in this study,we used non-targeted metabolomic and transcriptomic techniques to systematically investigate the overall differential metabolites and key enzyme genes involved in the biosynthesis of differential metabolites in the roots and stems of E.sinica,and then analyze the molecular mechanism of differential biosynthesis of active metabolites in roots and stems of E.sinica.The findings list as follows:1.In order to elucidate the overall metabolic differences in roots and stems of E.sinica,the present study was conducted to examine the metabolome of roots and stems from E.sinica in May and September based on UPLC-QTOF-MS platform.Forty-five differential metabolites were screened and identified using multivariate statistical analysis,including ten alkaloid metabolites: Ephedrine,Pseudoephedrine,Nor（pseudo）ephedrine,Methylephedrine,Methylpseudoephedrine,Ephedradine A,Ephedradine B/D,Tetramethylpyrazine,Feruloylhistamine,Methcathinone;25 flavonoid metabolites:（+）-Catechin,（-）-Epicatechin,（Epi）afzelechin,Ephedrannin A,Mahuannin A/B/C/D,etc;six organic acids: Quinaldic acid,Ferulic acid,Benzoic acid,alpha-Linolenic acid,Trans-cinnamic acid,Salicylic acid;one coumarin: 6-Methylcoumarin;three monoterpenes:（L）-alpha-Terpineol,alpha-Terpinyl acetate,D-linalool 3-glucoside.Pathway enrichment analysis of these differential metabolites revealed that they were mainly distributed in pathways of phenylalanine metabolism,flavonoid biosynthesis,flavonoid and flavonol biosynthesis,and phenylpropane biosynthesis.2.In this study,the Illumina Hiseq^TM sequencing platform was used to sequence the non-referenced transcriptomes of roots and stems of E.sinica in May and September.The sequencing results were screened for differential genes,functional annotation and enrichment analysis,and a total of 112 differential genes related to the synthesis of active ingredients in the root of E.sinica were identified.Among them,16,54 and 26 differential genes were involved in synthesis of alkaloids,flavonoids and monoterpenes in E.sinica;and 24 differential genes were involved in synthesis of coumarins and organic acids,respectively.Key enzyme genes in the differentiation of root and stem active ingredient formation were annotated in these differential genes,Including,Phenylalanine aminolyase（PAL）,4-coumarate-coenzyme A ligase（4CL）,Chalcone synthase（CHS）,Chalcone isomerase（CHI）,Anthocyanin reductase（ANR）,and Hydroxymethylglutaryl Co A reductase（HMGR）.The expression levels and regulatory roles of these key enzyme genes were different in stems and roots.3.The metabolic pathways for synthesis of active ingredients in roots and stems of E.sinica were reconstructed by combined analysis of metabolomic and transcriptomic data.A total of three synthetic regulatory networks were obtained including regulatory networks for alkaloid biosynthesis,flavonoid biosynthesis,and biosynthesis of substances such as monoterpenes and organic acids.This study also found that the trends are same in relative contents of active ingredients and the expression of involved key enzyme genes.This study investigated the molecular and metabolic basis of the opposite medicinal functions between roots and stems,provide the foundation for improving molecular mechanism of differentia biosynthesis process of the main active metabolites in roots and stems of E.sinica,which provide the scientific for the safe and rational clinical application of Ephedra medicine.