| Purpose:This study intends to explore the change rule and correlation of inorganic elements in Chinese herbal medicine and soil during the growth of G.scabra by analyzing and measuring the expression of inorganic elements,active components and key enzyme genes in different growth stages of G.scabra,and combining the obtained ecological factors（temperature,rainfall,average annual sunshine hours,water potential,relative humidity,etc.）,Identify and analyze the variety of chemical components and the accumulation and change of the content of nine representative components in G.scabra,determine the best harvest time,measure and analyze the relative expression of key enzyme genes,integrate the data of various factors,analyze the response relationship of inorganic elements,chemical components and key enzyme genes to ecological factors during the growth of G.scabra medicinal organs,and clarify the impact of ecological factors on the expression of key enzyme genes,Then regulate the absorption of inorganic elements and the synthesis of active components of gentian,and speculate the correlation between them,which is of great significance for clarifying the adaptation of gentian to the environment at different growth stages,helping to clarify the harvesting time of gentian,shorten the harvesting time of cultivated gentian,and realize the controllable quality of gentian and the rapid development of medicinal resources.Material and method:1.Materials:The experimental samples of gentian in this study were collected from Group 8,Ying’emen Town,Qingyuan County,Liaoning Province.The seeds were directly seeded.The seeds were collected every 15 days or so from May 10,2021.The medicinal materials were sampled at five points,and the soil samples were collected from the rhizosphere soil by the"shaking root method".The four point method was used for sampling[11].The gentian was divided into two years old and three years old.There were 22 batches in total.The growth period was divided into six periods,including seedling stage（5.10,5.25）In the early growth stage（6.10,6.25）,peak growth stage（7.10,7.25）,late growth stage（8.10,8.25）,flowering stage（9.10,9.25）,and late flowering stage（10.10）,the sample number of 2-year old on May10 is 2S1,the sample number of 3-year old on May 10 is 3S1,where"2"is 2-year old,"S1"is May 10,2021,and the number is 2S11,3S11.After simple treatment and washing,the samples are naturally dried.The root of G.scabra is classified according to different growth periods and stored for future use.Some fresh leaves are powdered by a freezing grinder,and then stored in an ultra-low temperature refrigerator at-86℃for standby.The sample was identified as G.scabra Bge by Professor Yin Haibo of Liaoning University of Traditional Chinese Medicine.Sample photos are shown in Figures 1-31 at the end of this paper.2.Method:2.1 Determination methods for physical and chemical properties of medicinal materials and soil:The determination methods of various mineral element ions in gentian medicinal materials and rhizosphere soil are shown in Table 1,and the biological repeat of the tested samples is 3.2.2 Determination of effective ingredients in G.scabra:HPLC-QTOF-MSMS was used to qualitatively identify various compounds in the whole growth period of 2-year-old and 3-year-old G.scabra,and the content of nine representative components was determined,including Gentiopicroside（Iridoid）,Swertiamarin（Iridoid）,Swertiamarin（Iridoid）and Loganic acid（Iridoid）,Kaempferol（Flavones）,Gentianine（Alkaloids）,Ursolic acid（Triterpenes）,Gentiobiose（Saccharides）,and Syringaresinol 4’-O in lignans-β-D-glucopyranoside（Lignans）.2.3 Determination of G.scabra key enzyme gene expression:RT qPCR method was used to determine the relative expression of six key enzyme genes in G.scabra MEP synthesis pathway,including GPPS,GES,G10H,10-HGO,DXS,DXR.2.4 Acquisition of environmental factors:A small meteorological station is used to set up at the gentian harvest site for this experiment to obtain the ecological factor data of the gentian cultivation base（temperature,rainfall,annual average sunshine hours,water potential,relative humidity,etc.）.2.5 Correlation analysis:Spearman correlation analysis was conducted on the physical and chemical properties of gentian medicinal materials and soil,content of terpenoids,relative gene expression,and environmental factors to clarify the correlation between various factors.Results:1.There are 13 inorganic elements in the medicinal materials,and the content of G.scabra in the late growth stage accumulates significantly.Except P,Ca,Mg,the content of other 10elements in the 2-year-old medicinal materials is higher than that in the 3-year-old medicinal materials.The accumulation and change of physical and chemical elements in rhizosphere soil20 were mainly in seedling stage and vigorous growth stage.The contents of organic matter,humus,humic acid,humin,N,P,K,Cd,Pb in 3-year-old soil were higher than those in2-year-old soil.According to the mean value comparison,the order of the enrichment ability of G.scabra to inorganic elements is:Fe>Cd>Mn>Ca>Mg>Zn>Cu>N>Pb>K>P>Hg>Na.Among the trace elements of G.scabra in different years,the enrichment ability to Fe is strong;Among heavy metal elements,the enrichment ability of Cd is strong;The grade analysis of inorganic element content in G.scabra showed that P,K and Fe were the highest,Mg,Mn,Zn and Cd were the middle,and Ca,Na,Cu and Hg were the lowest.The soil with high content of P,K,Mg and Zn should be selected for G.scabra cultivation,and the content of Cd should be strictly controlled.2.In the physical and chemical properties of 3-year-old G.scabra,Pb Zn/Fe showed a significant positive correlation;Organic matter humus/humin,humus humin and Na Mn in soil have significant positive correlation,while N-Mg has significant negative correlation.2.60 compounds were identified qualitatively,including 25 iridoids（compounds or peaks 5,8,10,12,14,15,16,17,18,19,23,24,25,26,32,34,35,36,37,38,39,42,46,47,48）,11flavonoids（compounds or peaks 1,3,6,9,21,22,29,33,43,45,49）,1 alkaloid（compounds or peaks 31）There are 9 lignans（compounds or peaks 7,11,20,28,30,40,41,44,51）,6triterpenoids（compounds or peaks 27,54,55,56,57,60）,2 Saccharides（compounds or peaks2,4）and 6 other classes（compounds or peaks 13,50,52,53,58,59）.However,24 and 19compounds in 2-and 3-year old G.scabra were discontinuous in different growth periods.。3.Quantitative analysis of 9 characteristic components of G.scabra at different growth stages,The results showed that the order of its content was Gentiopicroside>Syringaresinol-4’-O-beta-D-glucopyranoside>Swertiamarin>Gentiobise>Swertioside>Ursolic acid>Loganic acid>Kaempferol>Gentianine.Gentiopicroside（Iridoid）,as the main active ingredient of G.scabra,has a trend of continuous decline from seedling stage to early growth stage,increasing from vigorous growth stage to late flowering stage,and reaching the highest content at flowering stage.It is recommended to harvest at flowering stage;The content of Sweroside（Iridoid）increased from seedling stage to vigorous growth stage,and decreased after vigorous growth stage;The content of Swertiamarin（Iridoid）increased before the end of growth,and decreased after the end of growth;The content of Loganic acid（Iridoid）gradually accumulated from the seedling stage to the end of growth,and gradually decreased from the end of growth to the end of flowering;The content of Gentiobiose（Saccharides）had no obvious change before the end of growth,and the content of Gentiobiose（Saccharides）increased sharply at flowering,which was much higher than that at other growth stages;The content of Syringaresi-nol-4’-O-beta-D-glucopyranoside（Lignans）has a large change,among which the content accumulation changes are more obvious in the seedling stage and the vigorous growth stage.The content of Ursolic acid（Triterpenes）increases significantly at the end of growth,reaching the highest level.The content of Kaempferol（Flavones）increases more at the early stage of flowering,and the content of Gentianine（Alkaloids）is mainly synthesized at the seedling stage.Compared with the contents of active ingredients in the2-year-old and 3-year-old G.scabra,the contents of Gentiopicroside,Swertiamarin（Iridoid）,Gentiobiose（Saccharides）and Syringaresinol-4’-O-beta-D-glucopyranoside（Lignans）in the 2-year-old G.scabra are generally lower,while the contents of Sweroside（Iridoid）and Loganic acid（Iridoid）,Ursolic acid（Triterpenes）,Kaempferol（Flavones）The content of Gentianine（Alkaloids）is on the high side.4.The results of the relative expression of six key enzyme genes in MEP pathway showed that the expression of DXS gene in 2 and 3 year old G.scabra was mainly up-regulated,but its relative expression difference was the largest.The difference of DXS gene expression in 2year old G.scabra reached 60 times at the early stage of flowering;The expression of10HGO gene is mainly up-regulated in 2-year-old G.scabra,and down-regulated in3-year-old G.scabra;The expression of DXR gene is mainly down-regulated in 2-year-old G.scabra,and up-regulated in 3-year-old G.scabra.The expression of H10G gene in 2-year-old and 3-year-old G.scabra is mainly down-regulated,and the expression of GPPS gene is mainly up-regulated.The relative expression of DXR,H10G and GPPS is significantly increased during the vigorous growth period,which is consistent with the change of the content of iridoid components,and the overall trend is"down-up-down";The relative expression of the GES gene is mainly down-regulated,but in the vigorous growth period,the expression of the GES gene in the 2-year-old G.scabra is down-regulated,and the expression in the 3-year-old G.scabra is up-regulated.The consistent content changes are Gentiobiose and Ursolic acid.By comparing the relative expression trend of the same gene at 2 and 3years old,it can be seen that the relative expression trend of 10HGO,DXR and other genes in2 and 3 years old G.scabra is opposite.For example,at the growth and flowering stages,the relative expression of DXR gene in 2 years old G.scabra is significantly decreased,while the relative gene expression of DXR in 3 years old G.scabra is significantly increased;After the end of growth,2Y 10-HGO gene expression was significantly up-regulated,while 3Y10-HGO gene expression was significantly down-regulated.5.Based on the correlation between the inorganic elements,active components,gene expression and ecological factors in the 2-and 3-year-old G.scabra,with the change of ecological factors,the three ecological factors of atmospheric pressure,sunshine hours and temperature have a significant impact on the expression of six genes.At the same time,the expression of 10-HGO gene in the six genes has a very significant positive correlation with the synthesis of active components.At the same time,the Na,Mn The contents of inorganic elements such as Hg,Cd and Pb were significantly negatively correlated with the active components,and the contents of inorganic elements P and Mg in 3-year-old G.scabra were significantly positively correlated with the active components.Conclusion:1.G.scabra has a strong ability to enrich Fe,Mn,Ca and Cd;There are many pairs of related elements such as Zn-Fe in G.and rhizosphere soil;Taking the physical and chemical properties as reference,the best time for scoring the inorganic element content of 2-and3-year-old G.scabra was determined to be the vigorous growth stage and the seedling stage,respectively.Cu and Ca are the key elements to distinguish G.scabra and rhizosphere soil in different years;According to the results of one-way ANOVA and OPLS-DA,Fe and K were the key elements of G.scabra and rhizosphere soil in different growth periods;The content of harmful elements in G.scabra during the whole growth period is lower than the limit value,and the medicine is safe.2.Qualitative identification showed that 60 compounds were identified,but 24 and 19compounds in 2-and 3-year old G.scabra were intermittent in different growth periods.It was speculated that there might be biotransformation between different compounds,or that the compound only played a role in the corresponding period and would not be synthesized later.3.The 9 representative compounds all exist in the whole growth period.The quantitative determination results show that the accumulation of 9 representative compounds is different in different growth periods,and the content of iridoid compounds is much higher than that of other 5 compounds in each growth period,which is its main component.According to the changes in the content of iridoid,the flowering period is the highest,which can be used as the best harvest time of G.scabra,which is basically consistent with the best harvest time specified in the Pharmacopoeia.In different growth stages,the contents of Gentiopicroside（Iridoid）,Swertiamarin（Iridoid）,Syringaresinol-4’-O-beta-D-glucopyranoside（Ligans）and Gentiobiose（Saccharides）are relatively high,and the change trend of the contents in the2-year-old and 3-year-old G.scabra is consistent.It is speculated that lignans,Saccharides and iridoids play a synergistic role to enhance the medicinal efficacy of G.scabra;During the whole growth period,the content of iridoid compounds accumulates the most in the vigorous growth and late growth period.The content of lignans accumulates mainly in the seedling stage and the vigorous growth period.The content of triterpenoids increases more in the late growth period.The content of Saccharides and flavonoids changes greatly in the late growth and flowering period,and the content of alkaloids changes significantly in the seedling stage.On the whole,the accumulation time of various compounds concentrated in seedling stage and late growth stage.4.In different growth stages,with 2S1and 3S1samples as the control,the relative expression of six key genes had significant differences（P<0.05）.In the period with large differences,the expression of DXS and GPPS was mainly up-regulated,the expression of GES and H10G was mainly down-regulated,and the expression of DXR was first up-regulated and then down-regulated,taking the growth peak as the dividing point,The expression of 10HGO was down regulated before the vigorous growth period in the 2-year-old G.scabra,then up regulated,and mainly down regulated in the 3-year-old G..The increase of gene expression is greater in the vigorous growth stage and early flowering stage.5.There is a close correlation between the content of inorganic elements,the content of active components,the expression of key enzyme genes and the external environmental factors in Gentiana gentiana at different growth stages.The results show that the content of active components is significantly related to the expression of 10-HGO gene,the content of Mn,Hg,Cd in inorganic elements,and the atmospheric pressure,sunshine hours,temperature and soil temperature in environmental factors.By increasing the expression level of 10-HGO gene,the contents of P and Mg in the inorganic elements of the 3-year-old gentian can be increased,and the contents of Na,Mn,Hg,Cd,Pb and other inorganic elements in the 2-year-old gentian can be reduced appropriately.Cultivation at high pressure,less sunlight and low temperature can promote the synthesis of the active components of gentian. |
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