CN111420428B - Method for purifying arabidopsis thaliana resistant metabolite Arabidopsis thaliana - Google Patents
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Abstract
The invention discloses a method for purifying an arabidopsis thaliana anti-resistance metabolite Arabidopsis thaliana. The invention applies the high performance liquid chromatography and mass spectrometry combined technology to detect the arabidopsis resistant metabolite arabidopsis, namely arabidopsis resistant metabolites, in an ethanol leaching solution, and then applies a silica gel column separation system to separate, so as to obtain the arabidopsis resistant metabolite arabidopsis. The invention applies a method of combining a silica gel column separation system, a high performance liquid chromatography and a mass spectrum technology to successfully purify to the utmost extent to obtain at least 3 types of Arabidopsis including Arabidopsis A, Arabidopsis B and Arabidopsis D. The method has the advantages of high efficiency extraction and purification to obtain various resistant metabolites, simplicity, easy operation, low cost, and important guiding significance for improvement and progress of plant in defense against plant diseases and insect pests.
Description
Technical Field
The invention belongs to the technical field of plant metabolites. More particularly, relates to a method for purifying an arabidopsis thaliana anti-resistance metabolite Arabidopsis thaliana.
Background
Glycolipid-modifying complexes ars (arabidopsis) are a novel class of signal molecules that respond to plant stress. 12-oxo-phytodienoic acid (OPDA) and Demethylation-12-oxo-phytodienoic acid (dn-OPDA) are important precursors for synthesizing Jasmonates (JAs), and both play important roles as important signal molecules in plant growth and development and in responding to stress response. In Arabidopsis thaliana, a number of Arabidopsis thaliana containing Monogalactosylglycerol (MGDG) and digalactosylglycerolipid (DGDG) esters of OPDA and dn-OPDA have been identified. The resistant metabolites, Arabidopsis, which have been found so far are classified into the following 7 types according to the differences in the amount and combination of OPDA-or dn-OPDA-containing compounds: OPDA-dnOPDA MGDG (AR-A), OPDA-OPDAMGDG (AR-B), OPDA-dnOPDA DGDGDG (AR-C), OPDA-OPDA DGDGDGDG (AR-D); OPDA-OPDA-OPDA MGDG (AR-E), dnOPDA-OPDA-OPDA MGDG (AR-G). Research shows that the accumulation of the resistant metabolite Arabidopsis in Arabidopsis can be induced by hypoxiA stress, mechanical injury and anaphylactic reaction, and meanwhile, the exogenous application of AR-A can promote the senescence of leaves. In addition, AR-E contains at least 9 homologs that play important regulatory roles in plant senescence and in response to external stress.
Therefore, the resistant metabolite Arabidopsis can be used as a signal molecule for responding to plant stress, provides a new research object for a Jasmonic Acid (JA) signal path, and provides a theoretical basis for the function research of multiple hormone network regulation; meanwhile, based on the theoretical research that the resistant metabolite Arabidopsis can trigger the defense gene to play a role in protecting the plant body, the preparation of the resistant metabolite Arabidopsis provides a practical basis for the improvement and progress of the plant in the aspect of defending the plant diseases and insect pests.
In the methods for extracting and purifying plant metabolites mentioned in the prior art, most of the plant metabolites are separated and purified by extraction and chromatographic separation, so that the content of substances to be detected in a sample is increased, and the plant metabolites are detected by using a radioimmunoassay, an ultraviolet method or a fluorescence method. However, most of the methods have complicated procedures, much time consumption, large material consumption and high cost. The prior patent CN107870218A discloses a detection method of resistant metabolites Arabidopsis in rice, which applies a high performance liquid chromatography and mass spectrometry combined technology to detect the resistant metabolites Arabidopsis in rice, finally detects the AR-B and AR-F types of the resistant metabolites Arabidopsis, and performs qualitative and quantitative determination on the types. However, the patent only describes the detection method of the resistant metabolite Arabidopsis, and the substance in the plant can not be directly extracted and purified for experiment or production. Therefore, the further research on the extraction and purification method of the resistant metabolite Arabidopsis has important guiding significance in the aspect of defense against diseases and insect pests.
Disclosure of Invention
The invention aims to overcome the defects that the existing plant metabolites are difficult to purify and high in cost, and provides a method for purifying an arabidopsis thaliana resistant metabolite Arabidopsis thaliana. The invention is based on the detectable basis, uses a silica gel column separation system for separation, optimizes the component proportion of a washing liquid for washing the silica gel column, extracts and purifies at least 3 types of resistant metabolites Arabidopsis A, Arabidopsis B and Arabidopsis D from arabidopsis thaliana to the maximum extent, and provides a basis and a method for defending diseases and insect pests by using the resistant metabolites Arabidopsis D.
The invention aims to provide a method for purifying an arabidopsis thaliana anti-resistance metabolite Arabidopsis thaliana.
The above purpose of the invention is realized by the following technical scheme:
the invention provides a purification method of an arabidopsis resistant metabolite Arabidopsis, which comprises the steps of detecting the Arabidopsis of the resistant metabolite Arabidopsis by using an ethanol leaching solution of the Arabidopsis of the resistant metabolite Arabidopsis by using a high performance liquid chromatography and mass spectrometry combined technology, and separating by using a silica gel column separation system to obtain the Arabidopsis of the resistant metabolite;
wherein the resistant metabolite Arabidopsis is any one or more of Arabidopsis A, Arabidopsis B or Arabidopsis D.
Preferably, the method for purifying the arabidopsis thaliana resistant metabolite Arabidopsis thaliana comprises the following steps:
s1, evaporating ethanol in an arabidopsis thaliana ethanol leaching solution containing a resistant metabolite Arabidopsis thaliana to dryness to obtain an ethanol leaching solution;
s2, dissolving the ethanol extract obtained in the step S1 with methanol, adding the dissolved ethanol extract into a silica gel column, and washing the silica gel column with a washing liquid to obtain a dissolved solution;
s3, taking part of the dissolved solution obtained in the step S2, drying and dissolving to obtain a mixed solution, and detecting the resistant metabolite Arabidopsis by using a high performance liquid chromatography-mass spectrometry combined technology;
s4, according to the detection result of the step S3, taking dry substances dissolved in the flushing liquid, dissolving the dry substances in methanol, taking OPDA as a standard substance, separating and collecting substances in different areas;
s5, dissolving the substance obtained in the step S4 by using a solution containing methanol and dichloromethane, filtering, drying by blowing, and repeating the step S3;
s6, according to the detection result, performing preparative liquid phase separation, collecting components, repeating the step S3, and determining the position and the property of the Arabidopsis, so as to obtain the resistant metabolite Arabidopsis.
Preferably, the mobile phase a used in the combined hplc and mass spectrometry technique of step S3 is a mixed solution of methanol, acetonitrile, water and a volatile acid, wherein the volume ratio of methanol to acetonitrile is 1: 1, the volume fraction of the volatile acid is 0.1%.
Preferably, the mobile phase B is a mixed solution of a volatile acid and water, the volume fraction of the volatile acid being 0.1%.
More preferably, the volatile acid is formic acid or acetic acid.
Preferably, the flushing liquid is ethyl acetate: methanol 5: 1 is ethyl acetate: methanol 3: 1.
More preferably, the washing solution is ethyl acetate: methanol 3: 1.
Preferably, the method of separation in step S4 is thin layer chromatography.
Preferably, in the solution containing methanol and dichloromethane in step S4, the volume ratio of methanol to dichloromethane is 1: 1.
preferably, the volume ratio of chloroform to methanol to ammonium acetate in the mixed solution in step S3 is 300: 665: 35.
preferably, the method for extracting the arabidopsis thaliana ethanol leachate containing the resistant metabolite arabidopsis thaliana in the step S1 comprises the following steps:
(1) taking an arabidopsis plant, cleaning, and drying at 35-40 ℃;
(2) adding absolute ethyl alcohol into the dried sample in the step (1), and standing for 22-26 h in a closed environment to obtain an ethanol leaching solution;
(3) and (3) filtering the ethanol leachate obtained in the step (2), collecting filtrate, repeating the step (2), and collecting the filtrate, namely the arabidopsis thaliana ethanol leachate containing the resistant metabolite Arabidopsis thaliana.
Preferably, the arabidopsis thaliana in the step (1) is an arabidopsis thaliana subjected to a damage induction treatment.
Preferably, in step (1), drying is carried out at 37 ℃.
Preferably, in the step (2), standing is carried out for 24 hours in a closed environment.
The invention has the following beneficial effects:
the invention provides a method for purifying arabidopsis thaliana resistant metabolites, which is characterized in that a method combining a silica gel column separation system, high performance liquid chromatography and a mass spectrum technology is applied to successfully extract and purify at least 3 types of resistant metabolites, including arabidopsis A, arabidopsis B and arabidopsis D, to the maximum extent. The method is simple and easy to implement, has low cost, has wide popularization and application prospects, and has important guiding significance on improvement and progress of plant defense against plant diseases and insect pests.
Drawings
FIG. 1 is a graph showing the results of large-scale purification of Arabidopsis thaliana resistant metabolite Arabidopsis thaliana by different volume ratios of the washing solution; wherein EA represents ethyl acetate, EA: MeOH ═ 5: 1 represents the volume ratio (v/v) of ethyl acetate to methanol in the rinsing solution of the silica gel column of 5: 1, EA: MeOH ═ 3: 1 means that the volume ratio (v/v) of ethyl acetate to methanol in the washing liquid of the silica gel column is 3: 1, MeOH represents methanol.
FIG. 2 is a graph showing the results of measurement of the content of the resistant metabolite Arabidopsis in rice plants after brown planthopper feeding; wherein DJ represents a rice variety; oslox2 represents a loss-of-function mutation of gene lox2 in the context of rice variety DJ; oscoil a/b represents loss-of-function mutations of the genes coil a and coil b in the context of rice variety DJ.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 purification of Arabidopsis thaliana resistant metabolite Arabidopsis thaliana
In this example, the following materials and reagents were selected for preparation:
materials and reagents
Materials: arabidopsis thaliana;
mobile phase: a: methanol: acetonitrile 1: 1, adding formic acid with the volume fraction of 0.1 percent;
b: ultrapure water + 0.1% formic acid;
high performance liquid chromatography and mass spectrometry combined system: shimadzu 20A UFLC high performance liquid chromatography; AB SCIEX
5600+A mass spectrometry system;
a chromatographic column: PhenomenexKinetex C18150 x 2.1mm 2.6. mu.L.
Preparation of arabidopsis ethanol leachate containing resistant metabolite Arabidopsis thaliana
The extraction method of the arabidopsis thaliana ethanol leachate containing the resistant metabolite Arabidopsis thaliana comprises the following steps:
(1) taking 400g of arabidopsis thaliana plants aged all around, cleaning, and drying in a 37 ℃ drying oven;
(2) placing the dried sample in a triangular flask, adding absolute ethyl alcohol to immerse the sample, sealing the flask opening, and standing for 24h to obtain an ethanol leaching solution;
(3) filtering the ethanol leaching solution by using two layers of filter cloth, collecting the filtrate in a clean triangular flask, continuously adding absolute ethyl alcohol into the plant sample to immerse the sample, sealing the bottle mouth, and standing for 24 hours to obtain the ethanol leaching solution;
(4) repeating the step (3), and mixing the ethanol leachate obtained by filtering twice to obtain the arabidopsis ethanol leachate containing the resistant metabolite Arabidopsis;
wherein, the arabidopsis thaliana in the step (1) is subjected to damage induction treatment.
Purification of Arabidopsis thaliana resistant metabolite Arabidopsis thaliana
The method for purifying the arabidopsis thaliana resistant metabolite Arabidopsis thaliana comprises the following steps:
s1, evaporating ethanol in an arabidopsis ethanol leaching solution containing a resistant metabolite Arabidopsis by using a rotary evaporator in a water bath at 50 ℃ to dryness to obtain an ethanol leaching solution;
s2, dissolving the ethanol extract obtained in the step S1 with methanol, adding the dissolved ethanol extract into a silica gel column, flushing the silica gel column with 4 flushing fluids containing ethyl acetate and methanol in different volume ratios respectively, collecting the dissolved solutions obtained by dissolving the different flushing fluids, and drying a part of the dissolved solution by using nitrogen for later use;
wherein, the 4 kinds of washing liquids containing ethyl acetate and methanol with different volume ratios are respectively: methanol, ethyl acetate, and ethyl acetate in a volume ratio of: methanol 5: 1, and the volume ratio of ethyl acetate: methanol 3: 1;
s3, taking the other part of the dissolving solution obtained in the step S2, drying the dissolving solution by using nitrogen, and dissolving the dissolving solution by using methanol to obtain chloroform, methanol and ammonium acetate with the volume ratio of 300: 665: 35, placing the mixed solution with the total volume of 1.8mL in a brown bottle, and detecting the resistant metabolite Arabidopsis by using a high performance liquid chromatography-mass spectrometry combined technology;
s4, according to the detection result of the step S3, taking ethyl acetate as a volume ratio: methanol 3: 1, dissolving the dry substance dissolved in the mixed solution with methanol, taking OPDA as a standard substance, separating in a TLC plate by thin layer chromatography, developing with a bromocresol green developer, dividing the TLC plate into areas according to the positions of dark green, light yellow, no color development and the OPDA as the standard substance, and collecting substances in different areas;
s5, using methanol and dichloromethane in a volume ratio of 1: 1, dissolving the substances scraped from the TLC plate, filtering, drying by using nitrogen, and continuing to perform sample preparation detection by using the method in the step S3;
s6, obtaining the distribution situation of different components of the Arabidopsis in a TLC plate according to the detection result, separating the sample by a prepared liquid phase, collecting the components according to two factors of the ultraviolet absorption peak width and the retention time of the Arabidopsis, repeating the method of the step S3 to carry out sample preparation detection, determining the position and the property of the Arabidopsis, and correspondingly collecting to obtain the resistant metabolite Arabidopsis.
When the liquid phase is eluted by acetonitrile, the elution process is carried out for 30min, the liquid phase is divided into 6 time periods according to retention time, samples are collected every 5min, the method of the step S3 is repeated for sample preparation detection, and detection shows that the Arabidopsis shows large-area absorption peaks in 15-20 min. Accordingly, in the preparation of liquid phase separation, a peak area threshold value is set, and a large number of samples showing absorption peaks at this time period are collected and dried with nitrogen.
In step S3, the conditions for detecting the resistant metabolite arabidopsis by using the hplc and ms technique are as follows:
a chromatographic column: PhenomenexKinetex C18150 x 2.1mm 2.6. mu.L.
Liquid phase conditions:
mobile phase: a: methanol: acetonitrile 1: 1, adding 0.1% formic acid;
b: ultrapure water + 0.1% formic acid
Flow rate: 300 mu L/min; column temperature: 40 ℃; sample introduction amount: 10 μ L.
The liquid phase gradient conditions were as follows:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 10 | 20 | 80 |
| 25 | 0 | 100 |
| 30 | 0 | 100 |
| 30 | 50 | 50 |
| 40 | Terminate |
The parameters of the mass spectrum were set as: atomizing: 55 psi; auxiliary gas: 55 psi; air curtain air: 35 psi; ion source temperature: 30 ℃; electrospray voltage: 4500v, preparation of ion pairs (Arabidopsis/OPDA/dnOPDA) as: arabidopsis A: 773.5/291.1/263.1; arabidopsis B: 801.5/291.1/263.1; arabidopsis C: 935.5/291.1/263.1; arabidopsis D: 963.5/291.1/263.1; arabidopsis E: 1047.5/291.1/263.1; arabidopsis F: 759.5/291.1/263.1; arabidopsis G: 1075.5/291.1/263.1.
Through verification, in the purification method of the arabidopsis thaliana resistant metabolite Arabidopsis thaliana, a method combining a silica gel column separation system, high performance liquid chromatography and a mass spectrum combined technology is applied, and different types of resistant metabolite Arabidopsis thaliana can be successfully separated and purified.
The results of large scale purification of Arabidopsis thaliana by different volume ratios of the washing solution are shown in FIG. 1, and it can be seen that ethyl acetate: methanol 5: 1 can dissolve a large amount of arabidopsis D and a small amount of arabidopsis C; ethyl acetate: methanol 3: the rinse solution of 1 dissolved a large amount of Arabidopsis A, Arabidopsis B and a small amount of Arabidopsis G.
Therefore, the method can effectively extract and purify arabidopsis thaliana to obtain at least 3 types of resistant metabolites, namely arabidopsis A, arabidopsis B and arabidopsis D.
Example 2 determination of the content of the resistant metabolite Arabidopsis in Rice plants after Nilaparvata lugens feeding
Materials and reagents
Materials: rice DJ, oslox2, oscoil a/b;
mobile phase: a: methanol: acetonitrile 1: 1, adding formic acid with the volume fraction of 0.1 percent;
b: ultrapure water + 0.1% formic acid;
high performance liquid chromatography and mass spectrometry combined system: shimadzu 20A UFLC high performance liquid chromatography; AB SCIEX
5600+A mass spectrometry system;
a chromatographic column: PhenomenexKinetex C18150 x 2.1mm 2.6. mu.L.
Second, collecting plant material fed by brown planthopper and measuring content of Arabidopsis in the plant material
The method for collecting the plant materials fed by the brown planthopper and measuring the content of the Arabidopsis comprises the following steps:
(1) simultaneously culturing rice DJ, oslox2 and oscil a/b to four-leaf stage, and selecting 10 plants with consistent growth vigor; treatment group: taking 5 DJ, oslox2 and oscoil a/b from the plant, inoculating 10 brown planthopper nymphs of 4 instars to stems of each plant, and taking the plant stems as samples after 12 hours; control group: taking 5 DJ, oslox2 and oscil a/b respectively without inoculating brown planthopper, and taking plant stems as samples after 12 hours;
(2) weighing a fresh rice plant sample, adding 2.5 times volume of precooled methanol (containing 0.025 percent BHT) and uniformly mixing; adding 1.25 times volume of chloroform, and shaking on a shaker at 4 deg.C for 30 minutes;
(3) filtering, retaining the solution, adding 1.25 times of chloroform and 380mM KCL solution, and mixing uniformly; collecting an organic phase, adding chloroform for extraction once, and collecting the organic phase again; blowing the solution dry with nitrogen;
(4) dissolving with chloroform under shaking, filtering with nylon membrane with aperture of 0.22 μm, collecting the filtered solution, and collecting part of the solution to obtain final volume ratio of chloroform, methanol and ammonium acetate of 300: 665: 35, the total volume is 1.2mL, the mixture is placed in a brown bottle, and the resistant metabolite Arabidopsis is detected by using the high performance liquid chromatography and mass spectrometry combined technology.
2. Results of the experiment
The result of measuring the content of the resistant metabolite Arabidopsis in the rice body after the brown planthopper eats is shown in figure 2, and it can be seen that the content of the resistant metabolite Arabidopsis in the rice body is obviously improved after the brown planthopper eats the rice; shows that Arabidopsis B and Arabidopsis D in resistant metabolites Arabidopsis play an important role in the brown planthopper defense of rice. Meanwhile, in the JA synthetic mutant oslox2 and the JA signal deletion mutant osoil a/B with the rice variety DJ as the background, the contents of Arabidopsis B and Arabidopsis D are obviously lower than those of the wild type, which shows that the synthesis of the resistant metabolite Arabidopsis depends on the JA signal pathway.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A method for purifying arabidopsis thaliana resistant metabolite Arabidopsis thaliana is characterized by comprising the following steps:
s1, evaporating ethanol in an arabidopsis thaliana ethanol leaching solution containing a resistant metabolite Arabidopsis thaliana to dryness to obtain an ethanol leaching solution;
s2, dissolving the ethanol extract obtained in the step S1 with methanol, adding the dissolved ethanol extract into a silica gel column, and washing the silica gel column with a washing liquid to obtain a dissolved solution;
s3, taking part of the dissolved solution obtained in the step S2, drying and dissolving to obtain a mixed solution, and detecting the resistant metabolite Arabidopsis by using a high performance liquid chromatography-mass spectrometry combined technology;
s4, according to the detection result of the step S3, taking dry substances dissolved in the flushing liquid, dissolving the dry substances in methanol, taking OPDA as a standard substance, separating and collecting substances in different areas;
s5, dissolving the substance obtained in the step S4 by using a solution containing methanol and dichloromethane, filtering, drying by blowing, and repeating the step S3;
s6, according to the detection result, performing preparative liquid phase separation, collecting components, repeating the step S3, and determining the position and the property of the Arabidopsis, so as to obtain the resistant metabolite Arabidopsis;
wherein the resistant metabolite Arabidopsis is any one or more of Arabidopsis A, Arabidopsis B or Arabidopsis D.
2. The purification method according to claim 1, wherein the mobile phase A used in the combined HPLC-MS technology of step S3 is a mixed solution of methanol, acetonitrile, water and a volatile acid, and the volume ratio of methanol to acetonitrile is 1: 1, the volume fraction of the volatile acid is 0.1%.
3. The purification method according to claim 1, wherein the mobile phase B used in the combined HPLC-MS technique of step S3 is a mixed solution of a volatile acid and water, and the volume fraction of the volatile acid is 0.1%.
4. The purification process according to claim 1, wherein the washing solution is ethyl acetate: methanol 5: 1 is ethyl acetate: methanol 3: 1.
5. The purification method according to claim 1, wherein the separation method in step S4 is thin layer chromatography.
6. The purification method according to claim 1, wherein in the solution containing methanol and dichloromethane in step S5, the volume ratio of methanol to dichloromethane is 1: 1.
7. the purification method according to claim 1, wherein the volume ratio of chloroform, methanol and ammonium acetate in the mixed solution of step S3 is 300: 665: 35.
8. the purification method according to claim 1, wherein the extraction method of the arabidopsis ethanol leachate in the step S1 comprises the following steps:
(1) taking an arabidopsis plant, cleaning, and drying at 35-40 ℃;
(2) adding absolute ethyl alcohol into the dried sample in the step (1), and standing for 22-26 h in a closed environment to obtain an ethanol leaching solution;
(3) and (3) filtering the ethanol leachate obtained in the step (2), collecting filtrate, repeating the step (2), and collecting the filtrate, namely the arabidopsis thaliana ethanol leachate containing the resistant metabolite Arabidopsis thaliana.
9. The purification method according to claim 8, wherein the Arabidopsis thaliana in step (1) is a lesion-inducing Arabidopsis thaliana.
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