Biosynthesis And Its Regulatory Mechanism Of Differential Metabolites In "Same Origin And Different Effect" Parts Of Ephedra Sinica

Ephedra sinica is a promising medicinal plant.Generally its herbaceous stem above ground and its root under ground are used as medicine respectively,and their effects are completely opposite,showing a typical "homologous and different effect" phenomenon.The studies showed that key active ingredients which affected the homologous medicine "mahuang" and "mahuanggen" mainly included ephedrine alkaloids,ephedradines,feruloylhistamine,ephedrannins,mahuannins and so on.To date,the studies for the formation mechanism of these significantly differential metabolites(SDM)between the stems and roots have focused on two directions.One was functional characterization of very few biosynthetic enzymes,and the other was rough construction of biosynthetic pathways.However,SDM biosynthetic pathways are still not complete,and the actual bioprocess and its regulatory mechanism are still unclear.Therefore,in this work,the proposed biosynthetic pathways were firstly summarized and perfected.The expression pattern of synthetic genes was studied via combining transcriptomics and metabolomics.Then the actual biosynthesis of SDM was revealed in E.sinica.Furthermore,the regulatory effects of TFs and miRNAs on SDM biosynthesis were elucidated.The results are as follows:1.An accurate identification method of E.sinica was established using DNA barcoding technology of combination rbcL and matK.The stable interspecific genetic variation sites were obtained based on the analysis of relatively large-scale sample data.The method would be used for distinguishing E.sinica and related species of the same genus.2.At first,based on the literature research and chemical logic,the possible biosynthetic intermediates and enzymes were summarized and speculated to further perfect the biosynthetic pathways of SDM.The stems and roots of E.sinica were divided into multiple segments respectively.A total of 57 SDM and biosynthetic intermediates were identified by non-targeted metabolomics of the segments.It was confirmed that ephedrine alkaloids,catechin and monoterpenoids were significantly enriched in the stems,while ephedradines,mahuannins and ephedrannins were significantly enriched in the roots.The storage preference of various SDM was clarified.59 biosynthetic gene families were identified and their expression were compared in each segment of E.sinica.The major contribution pathways to SDM formation were confirmed by transcriptome(mRNA)analysis.3.The main sites of enzymatic catalytic reactions in SDM biosynthesis of E.sinica,were analyzed by integrated metabolomics and transcriptomic(mRNA).It was found that monoterpenoids such as α-terpineol acetate were mainly synthesized in the stem,while ephedradines,mahuannins and ephedrannins were mainly synthesized in the root.Besides,metabolite transport between the stem and the root is likely to exist in the biosynthesis of other SDM such as ephedrine for participation in the target product synthesis.Ultimately,the actual SDM biosynthesis process of E.sinica was completely described,and hence the internal reason of SDM storage preference was explained.4.Based on transcriptome data(mRNA+miRNA),two regulatory factors(TFs and miRNAs)were identified,and the interactions between transcription factors and transcription factors,transcription factors and biosynthetic genes,miRNAs and transcription factors,and miRNAs and biosynthetic genes were predicted in E.sinica.Furthermore,the roles of the regulatory factors were analyzed according to the expression correlation and molecular function.The key regulatory network of miRNA-TF-gene was constructed.Finally,the regulatory mechanism of SDM biosynthesis was elucidated comprehensively.In the study,a method based on combined DNA barcoding was established to identify E.sinica,which could be used for avoiding counterfeit species circulating in the market.In order to interpret SDM formation mechanism in homologous parts of E.sinica,the real biological process of SDM synthesis was completely described.Finally SDM storage preference and its internal reasons were clarified,which provided a basis for guiding the practical production and a reference for the fine utilization of various tissue parts of E.sinica.Besides,the effects of two regulatory factors(TFs and miRNAs)on SDM biosynthesis were also explored.These results can deepen the understanding of microscopic biological phenomena in E.sinica,and lay a theoretical foundation for the green biosynthesis of these key active ingredients.