| With the increase of market demand,the wild resources of licorice(Glycyrrhiza uralensis Fisch)in China are rapidly decreasing.Nowadays,large-scale cultivated licorice are planted in northwestern regions including Ningxia,Inner Mongolia,Gansu,and Xinjiang,and the quality of cultivated licorice in the market varies.In addition to a large number of clinical demand,the bioactive components of licorice such as glycyrrhizin is also widely used in the food and cosmetics industries.Therefore,it is particularly important to improve the quality of cultivated licorice.Licorice is a salt-tolerant medicinal material.The environment of salt stress is closely related to the quality of licorice.Moderate salt stress can promote the synthesis and accumulation of effective components of licorice and improve the quality.However,how salt stress affects the synthesis and accumulation of these bioactive components and the quality formation mechanism has not yet been elucidated.Therefore,the quality evaluation of licorice and the study of its quality formation mechanism are to relieve the shortage of wild licorice resources and improve the quality of cultivated licorice.1.Analysis of multiple bioactive constituents of licorice based on UFLC-QTRAP-MS/MS technology This project established a stable and reliable UFLC-QTRAP-MS/MS method concerning multi-component determination,to comprehensively evaluate the licorice quality from different habitats and different ecological types.Most wild and cultivated licorice were seperated clearly by cluster analysis,indicating different ecological types excert a great influence on the level of bioactive components.Wild and cultivated licorice from the Ningxia Yanchi area were divided into five parts,upper of taproot,middle of taproot,lower of taproot,lateral roots and rhizomes.The content distribution of bioactive compounds in different parts of wild and cultivated licorice was further studied.Wild licorice rhizomes have a stout shape,and the total bioactive compounds of them are most different from cultivated licorice rhizomes.The fluctuation trends of the content of bioactive compounds in all parts of taproot were similar in the two ecological licorice roots,but the trends of bioactive compounds in rhizomes and lateral roots were significantly different.The results showed that even if divided into different parts,wild and cultivated licorice are still clustered together.The overall contents in wild licorice was higher than those in cultivated ones,both in primary and secondary metabolites.Specifically,glycyrrhizin,liquiritin,isoliquiritin,liquiritin apioside,isoliquiritin apioside,neoliquiritin,licoflavone A and 4',7-dihydroxyflavone are higher in wild licorice.However,the content of liquiritigenin,echinatin,formononetin and licochalcone B showed opposite results.2.Metabolomics and iTRAQ proteomic analysis of wild and cultivated licorice 63 chemical components of wild cultivated licorice were identified based on non-targeted metabolomics by UFLC-Triple TOF-MS/MS,including triterpenes,flavonoids and their corresponding glycosides.Principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA)and t-test analysis showed that glycyrrhizin,licorice-saponin J2/G2,glycasperin D and dehydroglyasperin D could be regarded as chemical markers to distinguish wild and cultivated licorice.According to iTRAQ proteomic comparison of wild and cultivated licorice through,differentially expressed proteins(DEPs)involved in the metabolism of carbohydrates and important amino acids were screened out.Many abiotic stress-related enzymes were highly expressed in wild licorice as well,including bglB,bg1X,and oxidase.Combined with chemical analysis,the contents of proline and arginine are higher in wild-type.There is no significant difference in the content of isoliquiritigenin.Most flavonoid aglycones were fewer in wild licorice than in cultivated ones,while flavonoid glycosides were found with higher contents in wild-type,and generally the expression of related biosynthetic enzymes was not exactly consistent with the contents of downstream metabolites.3.Study on dynamic changes of physiological index,key enzyme gene expression and content of bioactive constituents of licorice under salt stress In the study of licorice quality formation mechanism,salinity stress experiment was carried out as follows,licorice seedlings were subjected to NaCl treatment in the field at four concentrations of high(200 mM NaCl),medium(100 mM NaCl),low(50 mM NaCl),and control(0 mM NaCl).It was found that the growth situation of the control group and the high salt stress group was weaker than the other two groups in respect of the morphological changes of licorice plants,.During the stress period,the physiological and biochemical changes including superoxide dismutase,peroxidase,glutathione reductase and catalase(SOD,POD,GR and CAT),the expression levels of key enzyme genes of ?-amyrin synthase and chalcone synthase(bAS and CHS)and the contents of 16 bioactive components were detected,including triterpenes and flavonoids in different treatment groups.The results showed that the expression of bAS and CHS and the content of bioactive components significantly increased in the 200 mM NaCl treatment group under short-term stress,but they were fluctuated greatly during the whole period.In fact,low-concentration salt-stress is most beneficial to the accumulation of active ingredients without damaging the normal growth of licorice.4.Multi-omics study of licorice under salt stress After 50-days of NaCl treatment,the 50 mM NaCl and control licorice samples were selected for comparative analysis of transcriptome study,TMT analysis,and non-targeted metabolic research.According to proteomics results,key DEPs in the synthesis pathway of the two major types(triterpenes and flavonoids)of bioactive components were chosen out.To be specific,phenylalanine ammonia lyase(PAL),cinnamate 4-hydroxylase(C4H),4-coumarate,CoA linkage Enzymes(4CL),chalcone synthase(CHS),chalcone isomerase(CHI),and flavonol synthase(FLS)were all highly expressed in the salt-treatment group.Three key enzymes for glycyrrhizin biosynthesis,bAS,CYP88D6 and CYP72A154,were significantly up-regulated in the stressed group as well.Based on the results of transcriptomics,key differentially expressed genes(DEGs)in the synthesis pathways of two major classes of bioactive constituents,triterpene and flavonoids,were screened.Among them,there are 79 DEGs related to flavonoid biosynthetic pathways,and 28 DEGs related to triterpene synthetic pathways.Except for one differentially expressed gene,the others were significantly up-regulated in the salt-stressed group.Experiments of PRM(qRT-PCR)verification of DEPs(DEGs)were carried out as well.According to the results of the non-targeted metabolic profile,121 secondary metabolites were identified according to UFLC-Triple TOF-MS/MS.It mainly includes triterpenoids and five types of flavonoids(flavones(flavonol),isoflavones,isoflavan,flavanone(flavanonol)and chalcones).As expected,the contents of flavonoid glycosides and most triterpenoid saponins were found with higher levels in the salt-stress group.The result of flavonoid aglycon content was opposite.5.Correlation analysis of multi-omics under salt stress The flavonoid and triterpenoid biosynthesis regulation network was established by combing the upstream DEPs(DEGs)with the content of bioactive metabolites.Combining the contents of 14 bioactive constituents,it was found that the expression of key DEPs is consistent with the content of flavonoid glycosides in the downstream,and opposite to the content of flavonoid aglycon in the upstream.Based on the analysis of comparative transcriptome and non-targeted metabolome of licorice between salt-stressed and the control groups,it was found that the expression trend of the DEGs was positively correlated with the relative content of glycosides and triterpenes,and 80%of differentially expressed UDP-glycosyltransferase genes were also significantly up-regulated in the salt-stressed group.Therefore,it is speculated that glycosyltransferases are the key factors that cause glycoside components to accumulate more in the salt stress group yet aglycon less.In addition,studies showed that MYB and bHLH transcription factor families regulate flavonoid biosynthesis and participate in abiotic stress.The results displayed that many of them were also significantly different between two groups.Two genes of glycosyltransferases(Glyur000289s00018722 and Glyur002678s00045101)with the greatest functional potential were eventually obtained through combined analysis of transcriptome and proteome.Based on the above results,our study finally revealed the logical relationship between the accumulation of bioactive metabolites and biosynthetic enzymes(genes)in licorice.We believe that the molecular mechanism by which salt stress environment affects the accumulation of bioactive components in licorice is not only the differential expression of upstream synthetases(genes),but also the characterization and functional research of downstream glycosyltransferases is worthy of deep study. |
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