阅读说明：本技术 一种拟南芥中抗性代谢物Arabidopsides的纯化方法 (Method for purifying arabidopsis thaliana resistant metabolite Arabidopsis thaliana ) 是由 俞陆军 张雪飞 戴阳朔 周颖 肖仕 于 2020-03-16 设计创作，主要内容包括：本发明公开了一种拟南芥中抗性代谢物Arabidopsides的纯化方法。本发明应用高效液相色谱与质谱联用技术对拟南芥抗性代谢物Arabidopsides的乙醇浸出液进行抗性代谢物Arabidopsides检测,再应用硅胶柱分离系统进行分离,即可得到所述抗性代谢物Arabidopsides。本发明应用硅胶柱分离系统、高效液相色谱与质谱联用技术结合的方法,最大限度的成功纯化得到了包括Arabidopside A、Arabidopside B和Arabidopside D在内的至少3种类型的Arabidopsides。该方法高效提取纯化得到了多种抗性代谢物Arabidopsides,且该方法简单易行、成本低,对植物在防御病虫害的改善与进步方面具有重要指导意义。(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.)

1. A method for purifying arabidopsis resistant metabolite Arabidopsis is characterized in that the Arabidopsis resistant metabolite Arabidopsis is detected by applying a high performance liquid chromatography and mass spectrometry combined technology to ethanol leachate of the Arabidopsis resistant metabolite Arabidopsis, and then separated by using a silica gel column separation system, so that the Arabidopsis resistant metabolite Arabidopsis is obtained;

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, characterized in that it 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.

3. The purification method according to claim 2, 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%.

4. The purification method according to claim 2, 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%.

5. The purification process according to claim 2, wherein the washing solution is ethyl acetate: methanol 5: 1 is ethyl acetate: methanol 3: 1.

6. The purification method according to claim 2, wherein the separation method of step S4 is thin layer chromatography.

7. The purification method according to claim 2, wherein in the solution containing methanol and dichloromethane in step S5, the volume ratio of methanol to dichloromethane is 1: 1.

8. the purification method according to claim 2, wherein the volume ratio of chloroform, methanol and ammonium acetate in the mixed solution of step S3 is 300: 665: 35.

9. the purification method according to claim 2, wherein the extraction method of the ethanol leachate of arabidopsis thaliana in 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.

10. The purification method according to claim 9, wherein the Arabidopsis thaliana in step (1) is a lesion-inducing Arabidopsis thaliana.

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.

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.