Application Of High Throughput Sequencing Technology In Chinese Herbal Medicine Research

Traditional Chinese medicine（TCM）is the treasure of our traditional culture and the crystallization of Chinese national wisdom.In recent years,the rapid development of molecular biology technology,unveiled the mysterious veil of the genetic code of TCM gradually.As a cutting-edge biological tool,high-throughput sequencing（HTS）technology has played an important role in the identification of TCM,the acquisition of genetic information,the selection of resistance genes and the exploration of metabolic pathways of active components.The application of HTS to the research of TCM is a combination of traditional culture and modern technology,which may greatly promote the sustainable development of the TCM.Based on the research of several TCMs,that is Dioscorea opposita,Polygonum tinctorium and Solanum nigrum,we used HTS technology as well as the chemical and physiological analysis to research the diversity mechanism of polysaccharide in Dioscorea opposita,the interaction mechanism of rhizosphere microorganisms and rhizomes of continuous cropping Dioscorea opposita,the accumulation mechanism indigo and indirubin in Polygonum tinctorium,and the resistance mechanism of Solanum nigrum towards cadmium（Cd）stress.The details are as follows:1.Dioscorea opposita（Chinese yam）is an important medicinal plant and functional food.Its main active ingredient is polysaccharide.At present,most of the researches about the Chimese yam polysaccharide（CYP）were facus on its extraction,detection and pharmacological action,the mechanism of its biosynthesis is rarely reported.In order to explore the biosynthetic pathway of CYP and identify the candidated genes,we analyzed the CYPs in the leaves,stems,bulbils and rhizomes of Chinese yam as well as the transcriptomes of these tissues.It was found that the micromorphology and content of polysaccharides in the different tissues were different,indicating that CYPs have the diversity in these tissues.Transcriptome analysis selected 1,756 genes involved in polysaccharide biosynthesis.Moreover,64 of them were differentially expressed in different tissues.The differentially expressed genes（DEGs）involved in the biosynthesis of monosaccharides,can result in the production of different monosaccharides.Therefore,a variety of polysaccharides can be biosynthesized with different monosaccharides.This work explained the biosynthesis and diversity mechanism of polysaccharides at the gene level,and provided a theoretical basis for further research on the biosynthesis of polysaccharides by bioengineering.In addition,transcriptome data obtained in this work greatly enriched the gene pool of Chinese yam and provided valuable information for the follow-up studies.2.Although Chinese yam has important edible and medicinal value,serious continuous cropping obstacles restrict its large-scale cultivation and utilization.Continuous cropping（growing of a single crop species on a field year after year）can lead to shifts of the rhizosphere microbial community by releasing specific compounds,resulting in suppressed plant growth and decreased yield for many plants,particularly upon replanting.Here,we comprehensively investigated the synergistic response of the rhizosphere microbiome and the rhizome of Dioscorea opposita to continuous cropping by 16S rRNA gene amplicon and de novo transcriptome sequencing.Our results revealed that continuous cropping can induce a reconstruction of the rhizosphere microbiome and affect the rhizome’s gene expression in Dioscorea opposita.In particular,we found the diversity of the microbial community in replanted rhizosphere soil was reduced and the relative abundance of beneficial bacteria was decreased,which led to a decline of community function,an imbalance of microecology,and a degeneration in the growth microenvironment.Meanwhile,in the replanted rhizomes of Dioscorea opposita,some functional genes were differentially expressed to regulate the physiological processes of plant-pathogen interactions,hormone signal transduction and related metabolic pathways to resist microbial and environmental stress.We believe that an understanding of how rhizomes interact with the rhizosphere’s microbiota in continuously cropped systems can provide new insights for modulating microorganisms to promote agricultural production.3.Polygonum tinctorium（Polygonum tinctorium Ait.,P.tinctorium）is an important cultivated plant that produces the natural blue dye indigo and the medicinal metabolite indirubin.To explore the mechanism of indigo and indirubin accumulation,the leaves of P.tinctorium at different harvest times,namely July,August,and October,were investigated by metabolite and de novo transcriptome analyses.The results revealed that the indigo and indirubin content varied at different harvest times.Moreover,their contents were significantly higher（p<0.05）in August than in either July or October.Transcriptome analysis showed that the gene expression patterns of P.tinctorium leaves in August was different.Almost all the identified differentially expressed genes（DEGs）that were responsible for the biosynthesis of indigo and indirubin,including four anthranilate synthase（AS）genes,two indole-3-glycerolphosphate synthase（IGPS）genes,three tryptophan synthase（TS）genes and seven cytochrome P450（CYP450）genes were expressed at significantly higher levels in August（p<0.05）,which correlated with the increase of the two compounds.Overall,the results of this study provide useful information for understanding the accumulation mechanism of indigo and indirubin in P.tinctorium,which may promote the development of agricultural breeding and industrial production of P.tinctorium.Moreover,the sequence data obtained by de novo transcriptome sequencing of P.tinctorium provides a platform for the future molecular biological studies.4.Solanum nigrum（S.nigrum）is an important medicinal plant with strong vitality.It can grow well in harsh environment.S.nigrum not only has a variety of medicinal value,but also has the ability to resist and accumulate heavy metals,such as Cd.In order to explore the resistance mechanism of S.nigrum to Cd stress,we used different concentrations of CdCl₂（0μM,10μM,25μM,50μM and 100μM）to treat S.nigrum seedlings and then carried out a series of studies.The results showed that low concentration of CdCl₂ had little effect on the growth of S.nigrum seedlings.Moreover,with the increase of the CdCl₂ concentration,the contents of accumulated Cd in the S.nigrum seedlings were gradually increased,and the accumulation in leaves was always dominant.In order to explore the accumulation mechanism of S.nigrum leaves towards Cd,we sequenced the transcriptomes of the leaves.It was found that a lot of genes were involved in the biosynthesis of cell wall,antioxidant enzymes,amino acid,glutathione,transporter and transport protein etc,which provided important information for the detoxification and resistance mechanism of S.nigrum towards Cd stress.In addition,weighted gene co-expression network analysis（WGCNA）identified several important hub genes,such as ABC transporters,heat shock proteins（HSP）and Zinc finger proteins,which provide valuable resources for the study of S.nigrum and are expected to be used in the study of food safety and the repair of heavy metal pollution.