A method for metabolomics research of selenium-enriched tobacco-rice rotation soil under different selenium levels based on LC-MS

Abstract

The application discloses a method for metabolomics research of selenium-rich tobacco-rice rotation soil under different selenium levels based on LC-MS, and comprises the following steps: step one, potting test design; step two, soil sample collection; step three, soil metabolite extraction; step four, instrument condition setting; step five, liquid chromatography-mass spectrometry detection; and step six, data processing and analysis. The application uses metabolomics to study the influence of different amounts of selenium on the content of 17 common amino acids and four organic acids in soil samples, thereby providing a theoretical reference for scientific fertilization of selenium in tobacco production, and being beneficial to the development of selenium-rich high-quality tobacco leaves, which has important significance in industry, agriculture and commerce.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of metabolomics research, and particularly relates to a method for metabolomics research of selenium-rich tobacco-rice rotation soil under different selenium levels based on LC-MS. BACKGROUND

[0002] Tobacco-rice rotation is a main planting system in tobacco-growing areas in the south of China, and one season of spring tobacco and one season of late rice are continuously planted in a year. This rotation mode is not only a water-flood rotation, but also a rotation of solanaceous crops and gramineous crops, which is beneficial to the inhibition of tobacco and rice diseases, and can improve soil physical and chemical properties and balance soil nutrients. At present, tobacco-rice rotation is an important water-flood rotation system in the south of China, and the total planting area in Anhui, Fujian, Hunan, Jiangxi, Guangdong and Guangxi provinces reaches 300,000 hm 2 , and tobacco stalks are returned to the field in tobacco-rice rotation areas, which has gradually become a common phenomenon.

[0003] Metabolomics refers to observing the change trend of metabolites within a certain time after external stimulation is applied, and analyzing the metabolism of organisms on the basis of the observation, and is a new discipline for qualitatively and quantitatively analyzing all metabolites of organisms in a certain growth period. Metabolomics broadens the research direction of life phenomena at the molecular level. Metabolomics takes each index of a sample as a variable, uses high-throughput measurement and data processing means, models the data and makes predictions, so as to study the change rule of metabolites of cells, tissues or living organisms after the external environment changes. Microbial metabolomics is an important direction of metabolomics research, and is widely used in microbial classification expression, mutant screening, metabolic pathway research and fermentation process monitoring, microbial decomposition of pollutants, metabolic performance of intestinal microorganisms and pathological relationship with the host, and the like.

[0004] Metabolomics, as a new research method, together with genomics, transcriptomics and proteomics, is an important part of systems biology for studying the composition, interrelation and dynamic change of each component structure of a biological system. The research target of metabolomics is mostly metabolites with a molecular weight less than 1000. At present, there are three common metabolomics analysis techniques, namely, nuclear magnetic resonance (NMR) technology, gas chromatography-mass spectrometry (GC-MS) combined technology and liquid chromatography-mass spectrometry (LC-MS) combined technology. According to the difference between the research object and the purpose, metabolomics is divided into four levels, namely, metabolite target analysis for detecting certain specific components, metabolite profile analysis for identifying specific structures and properties, metabolomics for qualitatively and quantitatively testing metabolites of a sample under certain conditions, and metabolite fingerprint analysis for rapidly identifying the whole sample.

[0005] Currently, metabolomics technology is mainly applied in the fields of medicine, food nutrition, microorganisms, plant functional genomics research, etc. Usually, only the metabolites of a single flora are used as research objects, and the analysis of microbial metabolites in the community is less required. In the study of soil environmental microorganisms, the situation of microorganisms in the soil environment is more complex, and various microbial communities and the life activities of various microorganisms form a complete metabolic system. Therefore, the breadth and complexity of this research far exceed that of a single flora. Although the study of soil microbial metabolism has certain difficulties, in recent years, with the emergence of new analysis methods and continuous improvement of analysis technology, such as chromatography, mass spectrometry, nuclear magnetic resonance, infrared spectroscopy, coulomb analysis, ultraviolet absorption, fluorescence scattering, emission detection, light scattering, etc. Analysis techniques can also be applied in the process of soil metabolite analysis.

[0006] The process of metabolomics analysis mainly includes sample extraction, sample pretreatment, data collection, data processing, and metabolite identification. In the early stage of the experiment, in order to ensure that the sample maintains a normal physiological state to obtain the original metabolite information, the freeze-drying technology is usually used to process the sample and store it at low temperature. Metabolites are usually extracted with organic solvents such as methanol, and then the types, states, and properties of metabolites in the sample are detected and collected using gas chromatography, liquid chromatography, capillary electrophoresis, etc. GC-MS and LC-MS have also been widely used in metabolomics research. GC-MS has strong separation ability and is mainly used to analyze easily vaporized substances. LC-MS has lower requirements for sample pretreatment and is usually used to analyze difficult-to-vaporize substances. Compared with GC and LC, GC-MS and LC-MS have higher efficiency and accuracy.

[0007] Nuclear magnetic resonance (NMR) technology has high integrity, can create experimental conditions similar to the normal physiological conditions of the sample, and maximize the original structure of the sample, but the sensitivity of this method is relatively low.

[0008] Liquid chromatography-mass spectrometry (LC-MS) has been widely used in metabolomics research due to its universal adaptability, high sensitivity, and simple pretreatment. Compared with nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS), LC-MS has been developed later, but its application has greatly improved the detection range and sensitivity. Therefore, in recent years, LC-MS has been favored by more and more people. LC-MS has the ability to detect hundreds of compounds including sugars, organic acids, amino acids, fatty acids, and a large number of plant secondary metabolites. Compared with NMR and GC-MS, LC-MS is easier to operate and can be widely used in substances that are not easy to volatilize, have poor thermal stability, are not easy to derivatize, and have relatively large molecular weight. At present, LC-MS has been widely used in the study of plant metabolic mechanisms, the mechanism of traditional Chinese medicine, drug action mechanism, microbial metabolites, and disease diagnosis. Through peak detection, peak alignment, and noise reduction, the data obtained by LC-MS can be simplified to intuitively and obviously reflect the differences between samples. The metabolite data measured by LC-MS are generally analyzed by principal component analysis (PCA). Using related software to classify data, the differences between data are reflected in the graph, so that the dispersion between samples and the aggregation reasons of different sample groups can be observed more conveniently.

[0009] Selenium is one of the essential trace elements for human body, which is closely related to the immune function and antioxidant capacity of human body. Since it was discovered by the Swedish chemist Berzelius in 1817, it has been considered as a toxin. C. A. Cameron (1880) and W. Knop (1884) successively studied the effect of selenium as a chemical element with similar properties to sulfur on plant growth. O. A Beath et al. (1934) found some high-selenium or high-selenium-tolerant plant species in the vegetation survey of Wyoming and named them as selenophyte. J. Pinsent (1954) found that selenium is an essential nutrient element for microorganisms such as Escherichia coli to maintain normal life activities. Rotruck (1973) first found that selenium is an essential component of red blood cell glutathione peroxidase. Since the 1980s, more and more attention has been paid to the relationship between selenium and environmental and food chain-related human diseases and health.

[0010] Selenium is an essential trace element for human body, and is known as "the spark of life", "the king of anti-cancer", "the element of longevity" and "the guardian of heart". Therefore, it has been widely concerned in the field of nutrition and agriculture. China is a large selenium-deficient country, about 72% of the country is deficient in selenium. Deficiency of selenium can lead to decline of human body's immunity and anti-aging ability, and even cause many diseases such as Keshan disease, Kaschin-Beck disease and cancer. Excessive selenium in the body can cause discomfort and even poisoning. In view of the deep influence of selenium on human health, appropriate supplementation of selenium has important effects on anti-oxidation, anti-cancer, improvement of immunity and the like. The most ideal way is to absorb selenium by plants and then supplement selenium through diet.

[0011] Appropriate amount of selenium can promote the growth of crops, improve the quality of crops, enhance the antioxidant capacity of crops and the resistance of crops to heavy metal toxicity. The problem of "smoking and health" has been a worldwide concern and research hotspot, and selenium can significantly reduce the content of tar and free radicals and carcinogens in smoke, and the selenium-rich cigarette can reduce the harm to the smoker's health. Therefore, increasing the selenium content of tobacco can increase the safety of tobacco. Selenium enters the smoker's blood with smoke, improves the blood selenium level, and thus enhances the human body's anti-cancer and anti-aging ability, and maintains the smoker's health. The selenium-rich cigarette is a low-toxicity and safe cigarette with high aroma and low tar, and is one of the most powerful cigarette varieties for market competition of the cigarette industry.

[0012] The paper "Effect of Exogenous Amino Acids on Amino Acid Content in Tobacco Leaves" published by Wu Xeping et al. pointed out that the addition of amino acids stimulated the growth of flue-cured tobacco, so that the flue-cured tobacco grew vigorously and the amount of synthesized amino acids increased. The paper "Study on Variation Regularity of Amino Acid Content in Flue-cured Tobacco" published by Liu Jingye et al. pointed out that the increase of the amount of amino acids was beneficial to improve the quality and aroma of flue-cured tobacco. The paper "Effect of Organic Acids on Physiological Metabolism and Nutrient Metabolism of Flue-cured Tobacco with Different Maturities" published by Zhang Yi et al. pointed out that organic acids could increase the content of total sugar and reducing sugar in tobacco leaves to improve the quality of tobacco leaves. The paper "Effect of Organic Acids on Growth and Physiological Metabolism of Flue-cured Tobacco" published by Du Jun et al. pointed out that malic acid could obviously promote the growth of tobacco plants and improve the activities of nitrate reductase and sucrose invertase in the early and middle stages of tobacco growth. The paper "Effect of Different Organic Acids on Aroma Quality of Flue-cured Tobacco Leaves with Different Maturities" published by Liu Shiliang et al. pointed out that malic acid could significantly increase the content of main aroma substances in tobacco leaves.

[0013] Therefore, how to reasonably control the content of selenium in soil to increase the content of amino acids and organic acids so as to produce selenium-rich and high-quality tobacco leaves has important significance in industry, agriculture and commerce. SUMMARY

[0014] The application provides a method for metabolomics research of selenium-rich tobacco-rice rotation soil at different selenium levels based on LC-MS, to solve the problem of how to reasonably control the selenium content of soil to increase the content of amino acids and organic acids based on the metabolomics research method, so as to produce selenium-rich high-quality tobacco leaves.

[0015] To solve the above technical problems, the application adopts the following technical solutions:

[0016] A method for metabolomics research of selenium-rich tobacco-rice rotation soil at different selenium levels based on LC-MS, comprising the following steps:

[0017] Step one: pot experiment design;

[0018] Step two: soil sample collection;

[0019] Step three: extraction of soil metabolites;

[0020] Step four: instrument condition setting;

[0021] Step five: liquid chromatography-mass spectrometry detection;

[0022] Step six: data processing and analysis.

[0023] Further, the pot experiment design method in step one comprises the following steps:

[0024] Weigh 15kg and put it into 18cm*40cm plastic pots respectively, and keep a 5cm water storage space between the barrel edge and the soil surface, add 5000mg / L selenium solution to each group of soil, so that the selenium content of the soil is: 0, 0.6, 1.2, 2.4, 4.8, 9.6mg / kg, 6 repetitions are set for each treatment, a total of 180 pots are placed in a random block arrangement, and each treatment is subjected to tobacco-rice rotation within one year, that is, one season of spring tobacco and one season of late rice are planted continuously in one year, and the soil samples are collected after the late rice is harvested.

[0025] Further, the soil sample collection method in step two comprises the following contents:

[0026] When collecting the soil sample, first remove the 3mm surface soil, use a soil drill to drill vertically downward to a depth of 20mm, avoid root residues, gravel impurities, and preliminarily crush and mix the soil sample into a sealed bag and store it in a-80℃ refrigerator;

[0027] The collected soil sample is divided into two parts and subjected to freeze-drying and natural air-drying treatment, and the dried sample is stored in a-80℃ refrigerator.

[0028] Further, the soil metabolite extraction method in step three comprises the following steps:

[0029] Weigh 1g of the crushed selenium-rich tobacco-rice rotation soil sample, add 5ml of 60% methanol water, shake thoroughly, and then ultrasonic extraction for 45min, shake every 15min during the period to ensure that the soil and methanol water are in full contact; 10000r·min -1 Centrifuge for 3min, pass through 0.22mu m organic filter membrane, and take 1.5ml of supernatant for detection.

[0030] Further, the instrument condition setting in step four includes mass spectrum condition setting and chromatographic condition setting.

[0031] Further, the mass spectrum condition setting is as follows:

[0032] Scan mode: MSEContinue positive ion, negative ion; ion source: ESI; mass scan range: 50-1200Da; atomizing gas: nitrogen; collision gas: argon; capillary voltage: 3kV; cone hole voltage: 40; source offset voltage: 80; ion source temperature: 100 DEG C; desolvation gas temperature: 450 DEG C; cone hole gas flow: 50L / h; desolvation gas flow rate: 650L / h; collision energy: low energy: off, high energy: 10-40V; calibration liquid: leucine enkephalin, positive ion: m / z 556.2771; negative ion: m / z 554.26154.

[0033] Further, the chromatographic condition setting is as follows:

[0034] Chromatographic column: Waters ACQUITY UPLC BEH C18; column temperature: 40 DEG C; column flow rate: 0.3ml / min; sample amount: 2mu L; mobile phase: A: 0.1% formic acid water, B: methanol.

[0035] Further, the specification of the chromatographic column is: 50mm*2.1mm, 1.7mu m.

[0036] Further, the liquid chromatograph-mass spectrometer in step five is Waters Xevo G2QTOF liquid chromatograph-mass spectrometer.

[0037] Further, the data processing and analysis in step six is that the data collected by LC-MS is analyzed by using Progenesis QI software.

[0038] The present application has the following beneficial effects:

[0039] (1) the amino acid comprehensive accumulation amount obtained is the largest when the soil selenium content is controlled at 4.87 mg / kg, and the amino acids play a stimulating role on the growth and development of flue-cured tobacco, so that the flue-cured tobacco grows vigorously, thereby the amino acid amount is increased, and the increase of the amino acid amount is beneficial to improving the quality and aroma of the flue-cured tobacco, so that when the soil selenium content is controlled at 4.87 mg / kg, the amino acid comprehensive accumulation amount obtained is the largest, which is most beneficial to improving the yield, quality and aroma of the flue-cured tobacco, thereby improving the economic benefits.

[0040] (2) the organic acid comprehensive accumulation amount obtained is the largest when the soil selenium content is controlled at 4.87 mg / kg, and these organic acids can improve the total sugar and reducing sugar content in the flue-cured tobacco to different extents, thereby improving the quality of the flue-cured tobacco and being beneficial to improving the added value of the flue-cured tobacco.

[0041] (3) the present application utilizes metabolomics to study the influence of different amounts of selenium on the content of 17 common amino acids and 4 organic acids in soil samples, provides a theoretical reference basis for scientific fertilization of selenium in tobacco production, is beneficial to the development of selenium-rich high-quality tobacco, and has important significance in industry, agriculture and commerce. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is a Loadings diagram.

[0043] Figure 2 is a PCA diagram of tobacco-rice rotation soil samples under different selenium levels, Figure 2 in which, 1 represents Se0, 2 represents Se2.4, 3 represents Se9.6, and QC represents a quality control sample group. DETAILED DESCRIPTION

[0044] I. Materials and methods

[0045] (I) Test materials

[0046] 1. Test soil

[0047] The test soil selected in the present application is a plough layer of tobacco-rice rotation soil with obvious horizons collected from a field in Dongshui Village, Chaodong Town, Fuchuan County, and the soil is crushed to remove impurities such as gravel.

[0048] 2. Test crops

[0049] The test rice variety is Yaxi 881, and the test flue-cured tobacco variety is Yunyan 87.

[0050] (II) Test design

[0051] 1. Pot experiment design

[0052] The pot experiment was used to study the difference of soil metabolism in tobacco-rice rotation under different selenium levels. The 15 kg soil was weighed and put into 18 cm x 40 cm plastic pots, and the 5 cm water storage space between the barrel edge and the soil surface was reserved. 5000 mg / L of selenium solution was added to each group of soil to make the soil selenium content: blank control (no selenium added), 0.6, 1.2, 2.4, 4.8, 9.6 mg / kg. Each treatment had 6 replicates, a total of 180 pots, and a randomized block arrangement was used.

[0053] Each treatment was planted in tobacco-rice rotation within one year, i.e. one season of spring tobacco and one season of late rice were planted continuously in a year, and the soil samples were collected after the late rice was harvested.

[0054] 2. Sample collection

[0055] When collecting soil samples, the 3 mm surface soil was removed, and the soil was drilled vertically downward to a depth of 20 mm using a soil drill to avoid root residues, gravel and other impurities. The soil sample was crushed and mixed, placed in a sealed bag, and stored in a -80°C freezer.

[0056] The collected soil samples were divided into two parts and subjected to freeze-drying and natural air-drying treatment, respectively. Liquid chromatography-mass spectrometry was used to analyze the composition, quantity, properties and change rules of metabolites. The dried samples were stored in a -80°C freezer.

[0057] 3. Extraction of soil metabolites

[0058] 1.000 g of crushed selenium-rich tobacco-rice rotation soil sample was weighed, 5 ml of 60% methanol water was added, and after thorough shaking, ultrasonic extraction was performed for 45 min, with shaking every 15 min during the extraction to ensure that the soil and methanol water were in full contact. 10000 r·min -1 Centrifugation for 3 min, 0.22 μm organic filter membrane, 1.5 ml supernatant for testing.

[0059] (Three) detection method

[0060] In this study, liquid chromatography-mass spectrometry was used to determine the content of 17 common amino acids and 4 organic acids in soil samples. The liquid chromatography-mass spectrometer used was Waters Xevo G2 QTOF liquid chromatography-mass spectrometer.

[0061] 1. Mass spectrometry conditions

[0062] Scan mode: MSE Continue positive ion, negative ion; Ion source: ESI; Mass scan range: 50-1200 Da; Nebulizer gas: nitrogen (N2, PEAK NM32LA nitrogen generator); Collision gas: argon (Ar, purity 99.999%); Capillary voltage: 3 kV; Sampling cone voltage: 40; Source offset voltage: 80; Source temperature: 100℃; Desolveation temperature: 450℃; Cone gas flow: 50 L / h; Desolvation gas flow: 650 L / h; Collision energy (CE): Low energy: off, high energy: 10-40 V; Lock mass: leucine enkephalin, positive ion: m / z 556.2771; negative ion: m / z 554.26154.

[0063] 2. Chromatographic conditions

[0064] Chromatographic column: Waters ACQUITY UPLC BEH C18 (50 mm x 2.1 mm, 1.7 μm); Column temperature: 40℃; Column flow rate: 0.3 ml / min; Injection volume: 2 μL; Mobile phase: A: 0.1% formic acid water, B: methanol. The parameters of the chromatographic separation gradient elution program are shown in Table 1.

[0065] Table 1. Chromatographic separation gradient elution program

[0066]

[0067] (IV) Data processing and analysis method

[0068] The data collected by LC-MS were analyzed by Progenesis QI software for principal component analysis, and the significantly changed small molecules in the sample were quantitatively identified. Progenesis QI can accurately identify the compounds with significant changes in the sample, and convert the data into graphics by virtue of its powerful visualization ability, so as to vividly display the data.

[0069] II. Results and analysis

[0070] (I) Effect of selenium level in tobacco-rice rotation soil on amino acids in soil

[0071] The peak areas of each amino acid obtained by LC-MS determination of tobacco-rice rotation soil samples under different selenium concentration gradients are shown in Table 2. Due to the difference between the actual measured soil selenium content and the planned one, 18 samples were selected according to the actual measured soil selenium content and the metabolites were detected. The results showed that most of the common amino acids were not detected in the selenium-rich tobacco-rice rotation soil, and only proline, threonine, glutamic acid, histidine, phenylalanine and tyrosine could actually respond. Among them, only the content of proline showed a relatively obvious trend with the change of selenium content in the soil samples. With the continuous increase of soil selenium content, the accumulation of proline in the soil samples also gradually increased, and reached a maximum value of 752 mg / kg when the soil selenium content reached 4.87 mg / kg. Then it gradually decreased with the increase of selenium content. While the contents of the other five amino acids were at the detectable level, but no obvious regularity was found between them and the soil selenium level. However, it was also found that when the soil selenium content reached 4.87 mg / kg, phenylalanine and tyrosine also reached the maximum value, 419 mg / kg and 696 mg / kg, respectively; threonine reached the second maximum value, 660 mg / kg; glutamic acid was at a medium value, 303 mg / kg. Therefore, in terms of the accumulation of the six amino acids, when the soil selenium content reached 4.87 mg / kg, the comprehensive accumulation of the six amino acids reached the maximum value. These amino acids played a stimulating role in the growth and development of flue-cured tobacco, making the flue-cured tobacco grow vigorously and thus increasing the synthesis of amino acids. The increase of amino acid content is beneficial to improve the quality and aroma of flue-cured tobacco. Therefore, when the soil selenium content is controlled at 4.87 mg / kg, the comprehensive accumulation of amino acids is the largest, which is most beneficial to improve the yield, quality and aroma of flue-cured tobacco, and thus improve the added value of flue-cured tobacco and the economic benefit.

[0072] Table 2: Detectable amino acids and their peak areas

[0073]

[0074]

[0075] (II) Effect of selenium level in tobacco-rice rotation soil on organic acids in soil

[0076] The three kinds of organic acids and the peak area related data measured at different selenium levels are shown in Table 3. As shown in Table 3, the organic acid in the soil sample is mainly glutaric acid, and the content is much higher than that of malic acid and butyric acid. The contents of glutaric acid, malic acid and butyric acid detected in the sample fluctuate, but do not show a clear change rule with the selenium content in the soil. However, it is found that when the selenium content in the soil reaches 4.87 mg / kg, the glutaric acid reaches the maximum value of 70118 mg / kg, the malic acid reaches the second maximum value of 2403 mg / kg, and the butyric acid reaches the upper middle value of 1869 mg / kg. Therefore, in terms of the accumulation of the three kinds of organic acids, when the selenium content in the soil reaches 4.87 mg / kg, the comprehensive accumulation of the three kinds of organic acids reaches the maximum value. These organic acids can improve the content of total sugar and reducing sugar in flue-cured tobacco to a certain extent, thereby improving the quality of flue-cured tobacco and increasing the added value of flue-cured tobacco. Therefore, when the selenium content in the soil is controlled at 4.87 mg / kg, the comprehensive accumulation of organic acids is the largest, which is most beneficial to improving the quality and increasing the economic benefit.

[0077] Table 3 Three kinds of organic acids and peak area

[0078]

[0079] (III) Principal component analysis results

[0080] The data collected by LC-MS is imported into Progenesis QI software for principal component analysis. Principal component analysis (PCA) is an effective mathematical statistics method, which is a method of using orthogonal transformation to convert a group of variables that may have correlation into a group of linearly uncorrelated variables, and the converted variables are called principal components. When studying the relationship between multiple variables, the more variables, the more difficult it is to extract effective information. In most cases, some variables have certain correlation, so the information reflected by the two variables can be considered as overlapping. The principal component analysis method is to check all variables and combine the original variables to form several new variables that are not related to each other under the condition of retaining the original information, so as to achieve the effect of reducing the dimension of data.

[0081] Since the identification results of amino acids and organic acids do not show obvious change trend, the Progenesis QI software is used for further PCA analysis of the detection results. Among them Figure 1 is a Loadings diagram, Figure 2The PCA plot of the soil samples of tobacco-rice rotation under different selenium levels. Each soil sample is shown as a corresponding point on the PCA plot, and the soil samples are divided into three groups according to different selenium concentrations, namely Se0, Se2.4 and Se9.6. According to the PCA plot, the differences in metabolites of the selenium-rich tobacco-rice rotation soil under different selenium concentration gradients can be compared intuitively. As can be seen from the PCA plot, there is a clear separation between the samples of different selenium levels. Although there is a certain deviation between the samples of the same selenium level, the samples of the same selenium level are concentrated in a certain area. Although the aggregation areas of the samples of different selenium levels are adjacent and relatively close, there is no obvious intersection. It can be seen that there is a significant difference between different groups. Therefore, it can be determined that the difference in selenium content has a significant impact on the types and contents of soil metabolites.

[0082] The relevant information of the substances that changed significantly with the change of the selenium concentration of the soil was screened out by the Progenesis QI analysis detection result, as shown in Table 4.

[0083] Table 4 Analysis results of sample metabolites

[0084]

[0085]

[0086]

[0087] Although the exemplary embodiments of the present application have been described and illustrated, it will be appreciated by those skilled in the art that various changes and substitutions can be made without departing from the spirit of the present application. In addition, many modifications can be made to adapt a particular situation to the teachings of the present application without departing from the central concept described herein. Therefore, the present application is not limited to the specific embodiments disclosed herein, but the present application can also include all embodiments within the scope of the present application and their equivalents.

Claims

1. A method for metabolomics research of selenium-enriched tobacco-rice rotation soil under different selenium levels based on LC-MS, characterized in that, The method comprises the following steps: Step 1: pot experiment design; Step 2: soil sample collection; Step 3: soil metabolite extraction; Step 4: instrument condition setting; Step 5: liquid chromatography-mass spectrometry detection; Step 6: data processing and analysis; The pot experiment design method in step 1 comprises the following steps: 15 kg of soil is weighed and respectively loaded into 18 cm*40 cm plastic pots, and a 5 cm water storage space between the barrel edge and the soil surface is reserved, 5000 mg / L of selenium solution is added to each group of soil, so that the selenium content of the soil is 0, 0.6, 1.2, 2.4, 4.8, 9.6 mg / kg, 6 repetitions are set for each treatment, a total of 180 pots are arranged in a random block arrangement, and each treatment is subjected to tobacco-rice rotation in one year, that is, one season of spring tobacco and one season of late rice are continuously planted in one year, and the soil sample is collected after the late rice is harvested; The soil sample collection method in step 2 comprises the following contents: When the soil sample is collected, the surface soil of 3 mm is removed, the soil at a depth of 20 mm is vertically drilled downward by using a soil drill, the root residues, gravel impurities are avoided, and the soil sample is preliminarily crushed and mixed uniformly and placed in a sealed bag and stored in a-80℃ refrigerator; The collected soil sample is divided into two parts and subjected to freeze-drying and natural air-drying treatment, and the dried sample is stored in a-80℃ refrigerator at low temperature; The soil metabolite extraction method in step 3 comprises the following steps: Take 1 g of crushed selenium-rich tobacco-rice rotation soil sample, add 5 ml of 60% methanol water, shake well, and ultrasonic extract for 45 min, shake every 15 min during the period to ensure that the soil and methanol water are in full contact; 10000r·min -1 Centrifuge for 3 min, pass through 0.22 μm organic filter membrane, and take 1.5 ml of supernatant for testing; The instrument condition setting in step 4 comprises mass spectrometry condition setting and chromatography condition setting; The mass spectrometry condition setting is as follows: Scan mode: MSEContinue positive ion, negative ion; ion source: ESI; mass scan range: 50-1200 Da; atomizing gas: nitrogen; collision gas: argon; capillary voltage: 3 kV; cone hole voltage: 40; source offset voltage: 80; ion source temperature: 100℃; desolvation gas temperature: 450℃; cone hole gas flow: 50 L / h; desolvation gas flow rate: 650 L / h; collision energy: low energy: off, high energy: 10-40 V; calibration liquid: leucine enkephalin, positive ion: m / z 556.2771; negative ion: m / z 554.26154; The chromatography condition setting is as follows: Chromatography column: Waters ACQUITY UPLC BEH C18; column temperature: 40℃; column flow rate: 0.3 ml / min; sample injection amount: 2 μL; mobile phase: A: 0.1% formic acid water, B: methanol.

2. The method for the metabolomics study of the selenium-enriched tobacco-rice rotation soil under different selenium levels based on LC-MS according to claim 1, characterized in that, The specification of the chromatography column is 50 mm*2.1 mm, 1.7 μm. 3.The method based on LC-MS for metabolomics study of selenium-enriched tobacco-rice rotation soil under different selenium levels according to claim 1, wherein, The liquid chromatography-mass spectrometry in step 5 is a Waters Xevo G2 QTOF liquid chromatography-mass spectrometry.

4. The method for the metabolomics study of the selenium-enriched tobacco-rice rotation soil at different selenium levels based on LC-MS according to claim 1, characterized in that, In step 6, the data processing and analysis are that the data collected by LC-MS is subjected to principal component analysis by using Progenesis QI software.

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