| Paeonia Pall.is a perennial herb of the genus Paeonia in the family Paeoniaceae with great medicinal,horticultural,commercial,and cultural values.The roots of P.lactiflora can be used in traditional Chinese medicine as Paeoniae radix alba.Paeoniae radix alba is one of China’s traditional export commodities and an important strategic medicinal material for the implementation of China’s national poverty alleviation policy and rural revitalization strategy.The traditional production regions of Paeoniae radix alba are mainly in Zhongjiang of Sichuan,Bozhou of Anhui,Pan’an of Zhejiang,and Heze of Shandong.The current research on P.lactiflora is mostly focused on horticultural traits and pharmacological effects,but there is a lack of research on the quality formation and molecular mechanism of P.lactiflora from different regions.In this study,we used P.lactiflora from four major production areas as raw material and applied the research strategy of quality ecology on Chinese medicines(QECM)to determine the quality characteristics of P.lactiflora from three biological dimensions:quality-genetics-ecology,and the molecular mechanisms of their formation.Based on the integrated multi-omics analysis,we identified key genes and key(micro)ecological factors related to the quality formation of P.lactiflora in the different regions and thus elucidated the scientific insight of the quality formation mechanism and the authenticity of P.lactiflora.The main results are as follows:1.Using MaxEnt and GMPGIS models,the prediction of the global suitable habitat of P.lactiflora based on multiple environmental variables.The results showed that the suitable habitat of P.lactiflora in the world was distributed radially in two central zones.The eastern center is in eastern Asia,including China,Japan,and North Korea,and the western center was located along the Mediterranean coast.The Chinese "P.lactiflora belt"was mainly distributed in eastern and central China including Inner Mongolia,Heilongjiang,Liaoning,and Sichuan,and spans a large latitude.Distribution density and hotspot analysis indicated that the hotspots of P.lactiflora distribution in China were concentrated in eastern Sichuan,the junction of Anhui and Shandong,and Zhejiang.Fitting the middle and high suitability zones of P.lactiflora to the latest distribution zoning of Chinese herbs,the Chinese "P.lactiflora Belt" was defined in the northern and southern herb zones.Environmental variables affecting the distribution of P.lactiflora included isothermality,coefficient of variation of precipitation,average annual potential evapotranspiration,and wettest monthly precipitation.Global P.lactiflora suitability zones were predicted on a time scale for eight past-present-future periods.The results from the last three periods of the Last Glacial Maximum(LGM),the Last Interglacial(LIG),and the middle Holocene(MH)all showed that only one of the fitness zones of P.lactiflora is located in the center of eastern Asia,which should confirm that China is the center of region,evolution,development and diversity and natural distribution of P.lactiflora.The suitable areas of P.lactiflora are higher than the current ones in all 100 years of the future global warming scenario,indicating a better adaptive capacity of P.lactiflora in response to global climate change.The center-of-mass shift in each of the four periods under the shared socioeconomic path scenarios shifted toward the northeast.2.Based on LC-ESI-MS/MS non-targeted metabolomics,the secondary metabolism differences in the four main producing regions of P.lactiflora were comprehensively analyzed.A total of 197 secondary metabolites were detected.From the perspective of metabolites,the four producing areas can be divided into two groups:Sichuan-Anhui and Zhejiang-Shandong.The overall relative abundance of terpenoids in Sichuan was significantly higher than that in the other three regions.According to the distribution characteristics of secondary metabolites,six clusters were divided,among which cluster 3 and cluster 4 represent the two groups with the highest relative abundance in Sichuan.8desbenzoylpaeoniflorin could be regarded as a chemical marker representing Sichuan.In addition,the chemical quantitative results showed that the quality uniformity increased with the deepening of the processing method.Transcriptome revealed that P.lactiflora produced in Sichuan showed unique transcriptional characteristics,and the distance between Zhejiang and Shandong is relatively close.Transcriptome data were divided into six clusters according to gene expression levels,where clusters 3 and 4 represented the set of genes with high expression in Sichuan.The transcriptomic and metabolomic data were analyzed by sPLS-DA and O2PLS models.Key genes such as PB.14108.1 and PB.12728.1 and key metabolites related to 6,7,8-Tetrahydroxy-5-methoxyflavone(Zmhp003514)and Azaleatin(Lmdp003808)were screened to significantly affect the two data sets.Based on the three-generation full-length transcriptome data,29 protein sequences of terpene synthase(TPS),33 protein sequences of P450s,22 protein sequences of acyltransferase(BAHD),and 109 protein sequences of glycosyltransferase(UGT)were identified.Phylogenetic tree,motif position,conserved domain,and expression pattern analysis were performed on the identified sequences.The genes in the pathways of paeoniflorin and its derivatives were summarized,and a total of 188 pathway genes were sorted out.The key modules significantly associated with 11 paeoniflorin and its derivatives and the core genes(hub genes)in the modules were screened by the weighted co-expression network(WGCNA)to construct the co-expression network of hub genes.The top 20 important hub genes were screened by CytoHubba,including PB.34195.1(HMGR-MVA),PB.31670.1(MVD-MVA),PB.38966.1(MK-MVA),PB.37088.1(HMGS-MVA),PB.13639.1(ACAT-MVA),PB.37101.1(UGT-c).The correlation network of 286 key genes with 11 paeoniflorin and its derivatives was constructed.3.19 environmental variables such as climate,soil,UV radiation,drought evapotranspiration,land use,and vegetation cover were analyzed comparatively for the four regions.Through multiple variable analysis,spearman correlation analysis,linear regression,redundancy analysis,variance decomposition,random forest,longitude,range of mean annual temperature variation(Bio7),soil cation exchange(T_CEC_soil),and soil sand content(T_sand)were screened as significantly correlated with the metabolome and longitude,mean temperature in the wettest season(Bio8)and seasonal UV radiation(UVB2)had significant effects on the transcriptome.The effect of environmental variables on the root system of P.lactiflora was excluded through a pot experiment to explore the effect of germplasm genetic factors of P.lactiflora itself on the accumulation of active ingredients of roots.There were no significant differences in any of the major active ingredients and no significant differences in the expression of key enzyme genes in the potted group.A comprehensive comparison of gene expression between the potted and provenance groups showed that environmental factors have a higher effect on the accumulation of active ingredients in P.lactiflora roots than germplasm factors,which laterally proved that environmental variables were the dominant variables in the formation of P.lactiflora quality in different regions.Environmental factors including isothermality(Bio3),seasonal ultraviolet radiation(UVB2),land use(Gm-lc-v3-25),and soil organic carbon content(T_OC)were the dominant environmental variables for the formation of the quality of P.lactiflora in different places.4.The physical and chemical properties and soil metabolism of the four producing areas of P.lactiflora were determined by GC-MS and other methods,and the differences in soil properties in the four producing areas were compared.The differences in soil physicochemical,nutrient elements,and microbial enzyme activities among the four regions were significant.In all soil samples,85 soil metabolites were detected,including carbohydrates,lipids,and organic acids.Among them,the largest number of lipids was detected.Amplicon sequencing was used to study the composition and diversity of bacteria and fungi,the mechanism of community construction,and inter-kingdom interaction patterns of bacteria-fungi of P.lactiflora in bulk soil and rhizosphere.Most of the bulk soil bacteria were oligotrophs with high diversity,while the rhizosphere was enriched with copiotrophs with low diversity.Stochastic processes,especially dispersal limitation,are dominating the assembly of rhizosphere bacterial and fungal communities of P.lactiflora.The rhizosphere bacterial-fungal inter-kingdom network is more complex and stable than that of bulk soils.There are more cooperative interaction modes.Soil moisture(SWC),mean annual precipitation(MAP),soil pH,and Alternaria genus significantly influenced the accumulation of active ingredients in P.lactiflora. |
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