CN112034061A - Method for screening agriophyllum squarrosum ecotype with high medicinal active ingredient based on metabolome difference - Google Patents
Method for screening agriophyllum squarrosum ecotype with high medicinal active ingredient based on metabolome difference Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/8693—Models, e.g. prediction of retention times, method development and validation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention relates to the technical field of agriophyllum squarrosum variety screening, in particular to a method for screening an ecological type of a high medicinal active ingredient on the basis of a metabolome difference, which collects leaves, ears and stems of an ETL ecological type and a CC ecological type of two high-altitude wild agriophyllum squarrosum samples and researches the metabolome rule of the ETL ecological type and the CC ecological type, wherein the total number of the samples is 18; based on the extensive targeting metabolome technology platform, 506 metabolites were detected, of which 244 were known metabolites. The invention researches the difference of the metabolism groups among the leaves, ears and stems of the salicornia mongolica planted in different areas respectively by means of combining a UPLC-MS/MS detection platform, a self-built database and multivariate statistical analysis; the content of the overground part medicinal active ingredients, particularly flavonoids, of the ETL ecotype growing at high altitude is found to be obviously higher than that of the CC ecotype. According to the invention, the agriophyllum squarrosum ecotype with high medicinal activity is screened out through the difference analysis of the metabolites among different ecotypes, and superior clusters and varieties are provided for the de novo domestication of crops.
Description
Technical Field
The invention relates to the technical field of agriophyllum squarrosum variety screening, in particular to a method for screening an ecological type of a high medicinal active ingredient agriophyllum squarrosum based on metabolome difference.
Background
Salicornia bigelovii (L.) Moq, a wild good feed plant, is favored by both camel (Camulus bactrianus) goats (Capra aegagrus hircus) and sheep (Ovis aries) young shoots. Modern researches show that the husked rice seeds are rich in protein (21.59-25.5%), fat (7.7-11.8%), carbohydrate (32.5-51.2%) and cellulose (4.9-14.9%), and also contain physiological active substances such as chlorogenic acid, superoxide dismutase (SOD), isoflavone, saponin, alkaloid and the like which are beneficial to the human body, and trace elements such as calcium, magnesium, iron, zinc, selenium and the like. The substances have the effects of resisting oxidation, diminishing inflammation and the like, and have positive effects on adjusting the nutrition metabolism of animals, balancing the microbial community structure of the digestive tract, improving the immunity, increasing the product quality and the like. For example, flavonoids in the agriophyllum squarrosum can be used for resisting cancers, oxidation, bacteria and viruses, reducing blood pressure, blood fat and blood sugar and the like, can be used for preventing and treating a plurality of diseases, in particular isorhamnetin, has various cardiovascular and cerebrovascular protective effects of protecting endothelium, resisting atherosclerosis, myocardial ischemia, protecting cardiac muscle cells, inhibiting cardiac fibrosis, reducing blood pressure, resisting thrombus, platelet aggregation, resisting oxidation and the like, and has various effects of inhibiting differentiation of fat cells, resisting anoxia, resisting tumors, reducing blood sugar, resisting inflammation, resisting viruses and the like. Although relatively rich wild agriophyllum squarrosum resources exist in China, particularly in northwest and northeast regions, based on the analysis result of agriophyllum squarrosum population, significant genetic differentiation exists among wild populations of agriophyllum squarrosum in China. In the process of adapting to desert environment for a long time, the compounds form unique physiological, biochemical, morphological structure and cell tissue characteristics. For example, the contents of apigenin, naringenin and ferulic acid in the samphire in different altitude areas are different, so that more flavanols are synthesized downstream to resist strong UVB radiation and adapt to extreme high altitude habitats.
Although the nutritive value and the medical value of the agriophyllum squarrosum are high, the wild agriophyllum squarrosum is not easy to collect. Wild husked rice seeds are small, the fructification quantity and quality of single-plant husked rice are high in elasticity, at least, 2 grains can be fructified, at most, 80000 grains can be fructified, and fruits cannot be easily spread to a large distance. In winter and summer, when the wind power is higher, the sand rice stems are broken and are held into balls, the sand rice stems run everywhere by wind, and the seeds germinate and grow when encountering rainwater in the next year. In addition, the distribution of agriophyllum squarrosum at arid, high-temperature, barren edges of agriophyllum squarrosum, although 1300 years of history of eating and drinking, the wild agriophyllum squarrosum was not successfully acclimated due to the uncertainty and time consumption of the conventional acclimation method. With the research in recent years, the domestication technology of the agriophyllum squarrosum is greatly improved, for example, the gene flow among populations can be accelerated through a garden homogeneity experiment, and the adaptability of a target phenotype to a habitat is improved. Therefore, dominant group and variety screening is urgently needed to be carried out on the wild suaeda salsa germplasm resources so as to obtain high-yield and high-quality cultivated varieties.
Disclosure of Invention
Aiming at the problems, the invention provides a method for screening the agriophyllum squarrosum ecotype with high medicinal active ingredients based on the difference of metabolome, the agriophyllum squarrosum ecotype with high medicinal active ingredients is screened out through the difference analysis of metabolites among different ecotypes, and superior clusters and varieties are provided for the de novo domestication of crops.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for screening the ecological type of the high medicinal active ingredient of the agriophyllum squarrosum based on the difference of the metabolome comprises the following steps:
step 3, detecting and analyzing the metabolites in the step 2, and detecting by using a liquid chromatography tandem mass spectrometry platform;
and 4, performing qualitative and quantitative analysis.
Preferably, the step (2) specifically includes the following steps:
a. taking out the biological material sample which is frozen and preserved at ultralow temperature, and carrying out vacuum freeze drying on the sample;
b. grinding the dried sample for 1.5 minutes under the condition of 30Hz by using a grinding instrument;
c. weighing 100mg of powder;
d. dissolving the powder in 1.0mL of 70% methanol solution containing 0.1mg/L Lidocard;
e. the sample was placed in a refrigerator at 4 ℃ overnight with three vortexes to allow more complete extraction;
f. after extraction, centrifuging at 10000g for 10 minutes;
g. the centrifuged supernatant sample was aspirated, filtered through a 0.22 μm pore size microfiltration membrane, and stored in a vial.
Preferably, in step (3), a chromatographic column with a specification of 1.8 μm and 2.1mm x 100mm is adopted, ultrapure water (added with 0.04% acetic acid) is used as an aqueous phase, acetonitrile (added with 0.04% acetic acid) is used as an organic phase, and a gradient elution procedure is used.
Preferably, the step (4) specifically includes the following steps:
a. data set acquisition, peak identification and normalization were performed using Analyst 1.6.1 and multisquant 3.0.2 software;
b. comparing the detection result with a chemical standard database according to the retention time, the molecular weight, the mass spectrum fragment and the spectrum of the metabolite;
c. carrying out data preprocessing on the data by adopting pareto scaling, and carrying out principal component analysis based on SIMCA-P software to form a score map;
d. performing discriminant analysis by using a partial least square method to remove irrelevant differential variable information, and screening differential variables through an overload graph, S-Plot and variable weight values of model variables;
e. the reliability of the result is verified by adopting a cross verification and arrangement experiment model;
f. and acquiring KEGG ID of the differential metabolite by using KEGG metabolic pathway enrichment analysis on the obtained differential metabolite through MBRole, analyzing the enriched metabolic pathway, drawing an enrichment pathway histogram and labeling the important metabolite.
The invention collects the leaves, ears and stems of ETL ecotype (located in Bitala, Council county, Qinghai province) and CC ecotype (located in Changgai, Gansu province, Changhou county, China) of two high-altitude wild samplings, and researches the metabolome rule of the samples, wherein the total number of the samples is 18. Based on the extensive targeting metabolome technology platform, 506 metabolites were detected, of which 244 were known metabolites.
Meanwhile, the invention adopts a means of combining a UPLC-MS/MS detection platform, a self-built database and multivariate statistical analysis to respectively research the difference of the metabolic groups among the leaves, ears and stems of the agriophyllum squarrosum planted in different areas; the content of the overground part medicinal active ingredients, particularly flavonoids, of the ETL ecotype growing at high altitude is found to be obviously higher than that of the CC ecotype.
Compared with the prior art, the method for screening the agriophyllum squarrosum ecotype with the high medicinal active ingredient based on the metabolome difference has the following beneficial effects:
1. according to the invention, the agriophyllum squarrosum ecotype with high medicinal active ingredients is screened out through the difference analysis of the metabolites among different ecotypes;
2. the invention not only provides the molecular metabolism foundation formed by the agriophyllum squarrosum geology, but also provides superior groups and varieties for the de novo domestication of crops.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a schematic diagram comparing ETL and CC type Salicornia bigelovii metabolites of the present invention.
Detailed Description
The technical solution of the present invention is described below with reference to the accompanying drawings and examples.
Referring to fig. 1, a method for screening the ecotype of the high medicinal active ingredient of the agriophyllum squarrosum based on the metabolome difference comprises the following steps:
TABLE 1 Sago samples and grouping information
a. taking out the biological material sample which is frozen and preserved at ultralow temperature, and carrying out vacuum freeze drying on the sample;
b. the dried sample was ground for 1.5 minutes at 30Hz using a grinder (MM 400, Retsch);
c. weighing 100mg of powder;
d. the powder was dissolved in 1.0mL of 70% methanol solution containing 0.1mg/L of Lidoca (internal standard); all chemicals were either analytically pure or chromatographically pure. Of which methanol, acetonitrile and ethanol were purchased from Merck, germany. The double deionized ultrapure water was prepared using a Millipore product (Millipore, Bedford, Mass.) ultrapure water purification system. Chemical standards were purchased from BioBioPha and Sigma-Aldrich, USA, diluted with 70% methanol to various gradient concentrations before use, and stored in a-20 ℃ refrigerator for mass spectrometry.
e. The sample was placed in a refrigerator at 4 ℃ overnight with three vortexes to allow more complete extraction;
f. after extraction, centrifuging at 10000g for 10 minutes;
g. the centrifuged supernatant sample was aspirated, filtered through a 0.22 μm pore size microfiltration membrane, and stored in a vial.
And 3, detecting and analyzing the metabolites in the step 2, and detecting by using a liquid chromatography-tandem mass spectrometry platform (LC-MS/MS). The data acquisition instrument system mainly comprises an ultra-high performance liquid chromatography Shim-pack UFLC SHIMADZU CBM20A and tandem mass Applied Biosystems 4500 QTRAP. A Waters ACQUITY UPLC HSS T3C 18 column with a size of 1.8 μm, 2.1mm x 100mm was used. A gradient elution procedure was used with ultrapure water (0.04% acetic acid added) as the aqueous phase and acetonitrile (0.04% acetic acid added) as the organic phase.
Wherein, water at 0 min: the acetonitrile is 95: 5V/V; 5:95V/V at 11.0 min; 12.0min is 5:95V/V, 12.1min is 95: 5V/V; 15.0min is 95: 5V/V; the flow rate is 0.4 ml/min; the column temperature was 40 ℃ and the amount of sample was 5. mu.l.
During the analysis of the instrument, a quality control sample (QC) is inserted into each 10 samples to be analyzed so as to monitor the repeatability of the analysis process. Quality control samples (QC) were prepared by mixing sample extracts and used to analyze the reproducibility of samples under the same treatment. In an API 4500 QTRAP LC/MS system, the main parameters of the linear ion trap and the triple quadrupole rod include: electrospray ion source (ESI) temperature was 550 ℃, mass voltage was 5500V, curtain gas (CUR) was 25psi, and collision-induced ionization (CAD) parameter was set high. In the triple quadrupole (QQQ), each ion pair is scan detected based on an optimized Declustering Potential (DP) and Collision Energy (CE).
Step 4, qualitative and quantitative analysis; the method specifically comprises the following steps:
a. data set acquisition, peak identification and normalization were performed using Analyst 1.6.1 and multisquant 3.0.2 software;
b. comparing the detection result with a chemical standard database (comprising a self-built database (MWDB) and a public database) according to the retention time, the molecular weight, the mass spectrum fragment and the spectrum of the metabolite;
c. performing data preprocessing on data by adopting Pareto scaling (Pareto scaling), and performing Principal Component Analysis (PCA) on the basis of SIMCA-P software to form a score plot (scores plot);
d. removing irrelevant differential Variable information by using partial least squares discriminant analysis (PLS-DA), and screening differential variables through a Loading Plot (Loading Plot), S-Plot and Variable import in project (VIP) of model variables;
the VIP is used for measuring the influence strength and the interpretation capability of accumulation difference of each metabolite on classification and judgment of each group of samples, and the VIP is more than or equal to 1 and is a common differential metabolite screening standard. In addition, the present example also combines the fold difference (fold change) between groups to further screen the differential metabolites, the criteria are fold change ≥ 2 and fold change ≤ 0.5; plotting Morpheus heatmaps showing the differences in the identified metabolites in the two ecotypes;
e. the reliability of the results is verified by adopting Cross-validation (Cross-validation) and Permutation experiment (Permutation test) models; the obtained differential metabolite data are converted by Log2 to enhance the data uniformity and normalized, and the accumulation mode of the metabolite among different samples is subjected to clustering analysis (HCA) through R software (www.r-project. org /);
f. the KEGG metabolic pathway enrichment analysis is used for obtaining the KEGG ID of the differential metabolite by MBRole (metabolism Biological Role, http:// csbg, cnb, csic. es/MBRole /), analyzing the enriched metabolic pathway, drawing an enriched pathway histogram and labeling important concerned Metabolites.
Referring to fig. 2, in this example, 506 metabolites were co-detected, including 139 differential metabolic components in two ecotypes; PCA analysis shows that the metabolic spectra of three tissues (leaves, stems and ears) of two ecotypes (a, b and c) are obviously grouped, the metabolic products of each ecotype stem and ear are obviously differentiated, and the specific results are as follows:
1) ETL compared to the leaf metabolome of group CC: the metabolite contents of amino acids (4 types), polyphenols (2 types), nucleotides (6 types), flavones (2 types), vitamins (2 types), coumarins (2 types) and the like are obviously increased in the ETL group; while the contents of amino acids (2), flavones (4), vitamins (3) and lipids (3) are significantly reduced.
2) ETL versus CC spike metabolome: the metabolite contents of amino acid (3 types), polyphenol (1 type), nucleotide (4 types), flavone (2 types), coumarin (2 types) and the like are obviously increased in the ETL group; while the contents of amino acid (1), flavone (5) and lipid (4) are obviously reduced.
3) ETL compared to the CC group stem metabolome: the metabolite contents of amino acid (3 types), nucleotide (6 types), flavone (2 types), vitamin (2 types), coumarin (2 types) and the like are obviously increased in the ETL group; 5 flavonoid content in ETL group was significantly reduced.
In conclusion, the invention screens the ecological forms of the agriophyllum squarrosum with high medicinal active ingredients by the difference analysis of the metabolites among different ecoforms, and provides superior groups and varieties for the de novo domestication of crops.
The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Claims (4)
1. A method for screening the ecological type of the high medicinal active ingredient of the suaeda salsa based on the metabolome difference is characterized in that: the method comprises the following steps:
step 1, collecting and processing samples, namely respectively collecting an ETL ecological sample and a CC ecological sample; samples of the two varieties take 80% of mature seeds on the spike of the agriophyllum squarrosum as a collection period; respectively collecting fresh leaves, stems and ears on three plants collected in each ecotype, freezing in liquid nitrogen, and taking the three plants back to a laboratory to be stored in an ultra-low temperature refrigerator at minus 80 ℃ to be used as biological material samples;
step 2, carrying out metabolite extraction on the biological material sample in the step (1);
step 3, detecting and analyzing the metabolites in the step 2, and detecting by using a liquid chromatography tandem mass spectrometry platform;
and 4, performing qualitative and quantitative analysis.
2. The method for screening the ecotype of the high medicinal active ingredient of the samphire based on the metabolome difference as claimed in claim 1, wherein: the step (2) specifically comprises the following steps:
a. taking out the biological material sample which is frozen and preserved at ultralow temperature, and carrying out vacuum freeze drying on the sample;
b. grinding the dried sample for 1.5 minutes under the condition of 30Hz by using a grinding instrument;
c. weighing 100mg of powder;
d. dissolving the powder in 1.0mL of 70% methanol solution containing 0.1mg/L Lidocard;
e. the sample was placed in a refrigerator at 4 ℃ overnight with three vortexes to allow more complete extraction;
f. after extraction, centrifuging at 10000g for 10 minutes;
g. the centrifuged supernatant sample was aspirated, filtered through a 0.22 μm pore size microfiltration membrane, and stored in a vial.
3. The method for screening the ecotype of the high medicinal active ingredient of the samphire based on the metabolome difference as claimed in claim 1, wherein: in the step (3), a chromatographic column with the specification of 1.8 μm and 2.1mm x 100mm is adopted, ultrapure water (added with 0.04% acetic acid) is used as an aqueous phase, acetonitrile (added with 0.04% acetic acid) is used as an organic phase, and a gradient elution program is used.
4. The method for screening the ecotype of the high medicinal active ingredient of the samphire based on the metabolome difference as claimed in claim 1, wherein: the step (4) specifically comprises the following steps:
a. data set acquisition, peak identification and normalization were performed using Analyst 1.6.1 and multisquant 3.0.2 software;
b. comparing the detection result with a chemical standard database according to the retention time, the molecular weight, the mass spectrum fragment and the spectrum of the metabolite;
c. carrying out data preprocessing on the data by adopting pareto scaling, and carrying out principal component analysis based on SIMCA-P software to form a score map;
d. performing discriminant analysis by using a partial least square method to remove irrelevant differential variable information, and screening differential variables through an overload graph, S-Plot and variable weight values of model variables;
e. the reliability of the result is verified by adopting a cross verification and arrangement experiment model;
f. and acquiring KEGG ID of the differential metabolite by using KEGG metabolic pathway enrichment analysis on the obtained differential metabolite through MBRole, analyzing the enriched metabolic pathway, drawing an enrichment pathway histogram and labeling the important metabolite.
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