CN108211425A - A kind of chromatographic column and separation method isolated and purified for auximone - Google Patents

Abstract

The invention discloses a chromatographic column and a separation method for the separation and purification of plant auxin. The resin in the chromatographic column is immobilized with IAA affinity proteins, such as IAA7 protein, TIR1 receptor protein and TIR1-IAA co-receptor any of the proteins. The present invention breaks through the difficulty of effectively separating and purifying IAA from trace plant samples by traditional methods, solves the problems of complex operation, unenvironmental protection of organic extraction and low separation and purification efficiency of traditional methods, greatly improves the separation and purification efficiency of IAA, and is effective in plant In the case of a fresh weight of 10-50 mg, sufficient IAA can be quickly separated and purified from the plant homogenate in one step for detection by liquid phase-mass spectrometry. The separation and purification method in the present invention greatly simplifies the separation and purification process of IAA, has simple operation and low cost.

Description

A chromatographic column and separation method for separation and purification of plant auxin

technical field

The invention relates to a chromatographic column and a separation method for separating and purifying auxin, in particular to a chromatographic column and a separation method based on auxin affinity protein.

Background technique

Auxin (β-3-indoleacetic acid, referred to as IAA) is the earliest discovered among the five traditional plant hormones, and it is also a plant growth substance with very important biological significance and application value. IAA is ubiquitous in plants, and plant cells generally use tryptophan to synthesize IAA. High concentrations of IAA inhibit plant growth, and low concentrations promote plant growth. It is safe and harmless to humans and animals, and has no physiological effects. At present, the analysis of the function of IAA and its regulatory pathway has become a research hotspot at home and abroad. The premise of these studies is to know the content of IAA in different parts of plants. However, due to the low content of IAA in plants and its instability when exposed to light and heat, the recovery efficiency of traditional plant hormone IAA separation and purification methods is low, and it is difficult to meet the requirements of quantitative analysis of IAA in trace and trace plant samples. Moreover, traditional methods mostly use organic extraction to separate and purify IAA, which is complicated and not environmentally friendly. Therefore, it is of great significance to provide a new method for efficiently separating and purifying IAA from trace and trace plant samples.

Contents of the invention

The purpose of the present invention is to provide a chromatographic column and a separation method for the separation and purification of plant auxin, utilizing the biological characteristics of strong IAA binding ability and high specificity of IAA affinity protein, and applying it to the separation of IAA In chromatographic purification, a new IAA separation and purification method is formed by using a biological recognition system.

The technical scheme that the present invention takes is:

A chromatographic column for the separation and purification of auxin, the resin in the chromatographic column is immobilized with auxin affinity protein, and the preparation of the chromatographic column includes the following steps:

1) Injecting the auxin affinity protein into the resin, so that the affinity protein is fully combined with the resin, and obtaining the resin immobilized with the affinity protein after cleaning;

2) Inject the resin immobilized with auxin affinity protein into an empty protein purification gravity column, and obtain a chromatographic column after standing still.

Further, the auxin affinity protein is selected from IAA7 protein, TIR1 receptor protein, and TIR1-IAA co-receptor protein.

Further, the TIR1-IAA co-receptor protein is a TIR1-IAA7 co-receptor protein.

Further, the auxin has a protein tag.

Further, the protein tag is a GST tag.

A method for separating and purifying auxin using a chromatographic column of the present invention, comprising the following steps:

1) Inject the initial plant extract into the chromatographic column, release the excess liquid from the bottom of the purification column after the auxin is fully combined with the auxin affinity protein, clean the column bed, digest with enzymes, and collect the solution in the column bed;

2) Concentrating and drying the collected solution in the column bed to obtain a mixture of auxin and auxin affinity protein, redissolving the mixture, centrifuging and taking the supernatant to obtain an auxin solution.

Further, the preparation steps of the primary plant extract are as follows: after quick-freezing in liquid nitrogen, the plant sample is fully pulverized by a grinding method, added with a buffer, mixed evenly, and centrifuged, and the supernatant is the primary plant extract.

Further, the solvent used for redissolving the mixture is aqueous acetonitrile.

Further, the mass fraction of the acetonitrile aqueous solution is 20%.

A method for quantitatively analyzing auxin in plants, first using the method of the present invention to separate and purify the auxin. Then a liquid phase-mass spectrometer is used to detect and analyze the isolated and purified auxin.

The beneficial effects of the present invention are:

It breaks through the difficulty of effectively separating and purifying IAA from trace plant samples by traditional methods, solves the problems of complicated operation steps, non-environmental protection of organic extraction and low recovery efficiency of traditional methods, and greatly improves the efficiency of IAA separation and purification. In the case of 10-50 mg, sufficient IAA can be quickly separated and purified from plant homogenate in one step for detection by liquid phase-mass spectrometry. At present, the protein recombination technology is relatively mature, and it is convenient to obtain a large amount of active IAA affinity protein. The affinity protein resin mixture prepared at one time can be used for multiple times, which simplifies the preparation process of IAA separation and purification column. The separation and purification method in the present invention greatly simplifies the separation and purification process of IAA, has simple operation and low cost.

Detailed ways

After a lot of tests and analysis, the present invention finally selects three IAA affinity proteins: IAA7 protein, TIR1 receptor protein and TIR1-IAA co-receptor protein. The present invention will be further described below in conjunction with the examples, but not limited thereto.

Materials, Reagents and Methods

PCR-related products were purchased from Beijing Quanshijin Biotechnology Co., Ltd., DNA endonuclease was purchased from Takara Company, other molecular biology-related enzymes and reagents were purchased from Life Technologies Company, and conventional inorganic reagents were purchased from Shanghai Bioengineering Co., Ltd.; The carrier was purchased from Novagen, the Escherichia coli strain was purchased from Life Technologies, the Drosophila S2 cells and their medium were purchased from Life Technologies; the gravity column was purchased from bio-rad, and other consumables were Corning products; all reagents were of analytical grade. The experimental water was double distilled water. The main equipment includes GE protein purification system, Shimadzu 8060LC-MS liquid mass spectrometer.

Embodiment 1 IAA separation and purification method based on IAA7 protein

1. Expression and purification of IAA7 protein

Total RNA was extracted from Arabidopsis thaliana and cDNA was synthesized by reverse transcription, IAA7-specific primers were designed, and the cDNA was used as template for PCR amplification; the expression vector pGEX-KG with GST tag was used as the basic backbone to construct the prokaryotic expression vector of GST-IAA7 ; Prokaryotic expression vectors were transformed into Escherichia coli expression strains BL21, Tuner and Rosetta. Escherichia coli cells were cultured at 20°C and 0.4mmol/L IPTG concentration conditions to induce the expression of IAA7 protein; the Escherichia coli cells were collected, resuspended in PBS (pH7.4) buffer, and the cells were ultrasonically disrupted, and the supernatant was collected after centrifugation at 13000g, and then Inject the supernatant into GST Sefirose ^™ Resin resin, incubate for 30 min to fully bind the IAA7 protein to the resin, wash with 4°C pre-cooled PBS (pH7.4) buffer for 5 times, and place the resin at 4°C for use.

2. Preparation of IAA separation and purification column

Prepare an empty gravity column for protein purification, close the lower outlet, inject Sefirose ^™ Resin resin bound to IAA7 protein at a rate of 4 mL/min at 4°C, and let it stand for more than 8 hours to obtain an IAA separation and purification column. The IAA separation and purification column must be kept in a chromatographic cabinet at 4°C. If it is not used for a long time, lysozyme should be added to prevent the growth of colonies in the column bed.

3. Separation and purification of IAA

Determination of binding efficiency and recovery efficiency: configure 10ng/mL IAA standard sample, inject 1mL IAA standard sample into the IAA separation and purification column, incubate for 30min, recover the liquid from the bottom of the separation and purification column and use LC-MS to determine the IAA content; add 3mL to the column bed Pre-cooled PBS (pH7.4) buffer solution, then add 1U thrombin, incubate at 22°C for 30min, collect the liquid in the column, concentrate and dry to obtain a mixture of IAA and affinity protein, reconstitute the mixture with 20μL 20% acetonitrile, and centrifuge at 12000g for 10min Afterwards, the supernatant was taken for quantitative analysis of IAA with a liquid phase-mass spectrometer, and the recovery efficiency was calculated to be 28%.

Separation and purification of IAA in plant samples: After quick-freezing in liquid nitrogen, grind 50 mg of fresh plant samples by grinding method, add 2 mL of PBS (pH7.4) buffer solution, centrifuge at 12000 g, the supernatant is the initial plant extract, and the initial plant extract Inject into the IAA separation and purification column, incubate at 4°C for 30 minutes, release the excess liquid from the bottom of the purification column after the IAA and IAA7 protein are fully combined, wash the column bed with 3 times the volume of PBS (pH7.4) buffer, and finally digest with 1U thrombin After 30 min, the solution in the column bed was collected and concentrated and dried to obtain a mixture of IAA and affinity protein. The mixture was redissolved with 20 μL of 20% acetonitrile, centrifuged at 12000 g for 10 min, and the supernatant was taken for quantitative analysis of IAA with a liquid phase-mass spectrometer. Calculated, the recovery efficiency is 29%.

Example 2 IAA separation and purification method based on TIR1 receptor protein

1. Expression and purification of TIR1 protein

Total RNA was extracted from Arabidopsis thaliana and cDNA was synthesized by reverse transcription, TIR1-specific primers were designed, and the cDNA was used as a template for PCR amplification; the prokaryotic expression vector of TIR1 was constructed using the expression vector pGEX-KG with a GST tag as the basic backbone; The expression vectors were transformed into Escherichia coli expression strains BL21, Tuner and Rosetta. Escherichia coli cells were cultured at 20°C and 0.4mmol/L IPTG concentration to induce the expression of TIR1 receptor protein; the Escherichia coli cells were collected, resuspended in PBS (pH 7.4) buffer, and the cells were ultrasonically disrupted, and the supernatant was collected after centrifugation at 13000g. Then inject the supernatant into GST Sefirose ^TM Resin resin, incubate for 30 minutes to fully bind the GST-TIR1 protein to the resin, wash with 4°C pre-cooled PBS (pH7.4) buffer for 5 times, and place the resin at 4°C for later use.

2. Preparation of IAA separation and purification column

Prepare an empty gravity column for protein purification, close the lower outlet, inject Sefirose ^™ Resin resin bound to TIR1 receptor protein at a rate of 4 mL/min in an environment of 4°C, and let it stand for more than 8 hours to obtain an IAA separation and purification column. The IAA separation and purification column must be kept in a chromatographic cabinet at 4°C. If it is not used for a long time, lysozyme should be added to prevent the growth of colonies in the column bed.

3. Separation and purification of IAA

Determination of binding efficiency and recovery efficiency: configure 10ng/mL IAA standard sample, inject 1mL IAA standard sample into the IAA separation and purification column, incubate for 30min, recover the liquid from the bottom of the separation and purification column and use LC-MS to determine the IAA content; add 3mL to the column bed Pre-cooled PBS (pH 7.4) buffer solution, then add 1U thrombin, incubate at 22°C for 30min, collect the liquid in the column, concentrate and dry to obtain a mixture of IAA and affinity protein, redissolve the mixture with 20μL 20% acetonitrile, centrifuge at 12000g for 10min The supernatant was taken for quantitative analysis of IAA with a liquid phase-mass spectrometer, and the recovery efficiency was calculated to be 90%.

Separation and purification of IAA in plant samples: After quick-freezing in liquid nitrogen, crush 20 mg of fresh plant samples by grinding method, add 2 mL of PBS (pH7.4) buffer solution, centrifuge at 12000 g, the supernatant is the initial plant extract, and the initial plant extract Inject into the IAA separation and purification column, incubate at 4°C for 30 minutes, release excess liquid from the bottom of the purification column after IAA is fully combined with TIR1 receptor protein, wash the column bed with 3 times the volume of PBS (pH7.4) buffer, and finally use 1U thrombin Digested for 30 minutes, collected the solution in the column bed, concentrated and dried to obtain a mixture of IAA and affinity protein, reconstituted the mixture with 20 μL of 20% acetonitrile, centrifuged at 12000g for 10 minutes, and took the supernatant for quantitative analysis of IAA by liquid chromatography-mass spectrometry , after calculation, the recovery efficiency is 90%.

Example 3 IAA separation and purification method based on TIR1-IAA7 co-receptor protein

1. Expression and purification of TIR1-IAA7 co-receptor protein

Extract total RNA from Arabidopsis thaliana and synthesize cDNA by reverse transcription, design specific primers for TIR1 and IAA7, use cDNA as template for PCR amplification, apply SOE-PCR technology to fuse TIR1 and IAA7 protein genes; use expression vector with GST tag pGEX-KG and pIEx-3 were used as the basic framework to construct the eukaryotic expression vector of TIR1-IAA7; the eukaryotic expression vector was transfected into Drosophila embryonic cells S2, and the S2 cells were cultured in Drosophila medium at 28°C and expressed TIR1- IAA7 co-receptor protein; collect the Drosophila S2 cell culture fluid, concentrate the protein using an ultrafiltration tube, inject it into GST Sefirose ^TM Resin resin, incubate for 30 minutes to fully bind the TIR1-IAA7 co-receptor protein to the resin, and use a 4°C pre-cooled The PBS (pH7.4) buffer was washed 5 times, and the resin combined with the TIR1-IAA7 co-receptor protein was used to prepare an IAA separation and purification column, which was placed at 4°C for use.

2. Preparation of IAA separation and purification column

Prepare an empty gravity column for protein purification, close the lower outlet, inject Sefirose ^™ Resin resin bound to the co-receptor protein at a rate of 4 mL/min at 4°C, and let stand for more than 8 hours to obtain an IAA separation and purification column. The IAA separation and purification column must be kept in a chromatographic cabinet at 4°C. If it is not used for a long time, lysozyme should be added to prevent the growth of colonies in the column bed.

3. Separation and purification of IAA

Determination of binding efficiency and recovery efficiency: configure 10ng/mL IAA standard sample, inject 1mL IAA standard sample into the IAA separation and purification column, incubate for 30min, recover the liquid from the bottom of the separation and purification column and use LC-MS to determine the IAA content; add 3mL to the column bed Pre-cooled PBS (pH 7.4) buffer solution, then add 1U thrombin, incubate at 22°C for 30min, collect the liquid in the column, concentrate and dry to obtain a mixture of IAA and affinity protein, redissolve the mixture with 20μL 20% acetonitrile, centrifuge at 12000g for 10min The supernatant was taken for quantitative analysis of IAA with a liquid phase-mass spectrometer, and the recovery efficiency was calculated to be 96%.

Separation and purification of IAA in plant samples: After quick-freezing in liquid nitrogen, crush 10 mg of fresh plant samples by grinding method, add 2 mL of PBS (pH 7.4) buffer solution, centrifuge at 12000 g, the supernatant is the initial plant extract, and inject the initial plant extract into IAA separation and purification column, incubate at 4°C for 30 minutes, release excess liquid from the bottom of the purification column after IAA is fully combined with the co-receptor protein, wash the column bed with 3 times the volume of PBS (pH 7.4) buffer, and finally digest with 1U thrombin for 30 minutes , collect the solution in the column bed, concentrate and dry to obtain a mixture of IAA and affinity protein, redissolve the mixture with 20μL 20% acetonitrile, centrifuge at 12000g for 10min, take the supernatant and use a liquid phase-mass spectrometer for quantitative analysis of IAA. , The recovery efficiency is 95%.

Claims (10)

1. a chromatographic column for separation and purification of auxin, characterized in that, auxin affinity protein is fixed in the resin in the chromatographic column, and the preparation of the chromatographic column comprises the following steps: 1) Injecting the auxin affinity protein into the resin, so that the affinity protein is fully combined with the resin, and obtaining the resin immobilized with the affinity protein after cleaning; 2) Inject the resin immobilized with auxin affinity protein into an empty protein purification gravity column, and obtain a chromatographic column after standing still. 2. The chromatographic column according to claim 1, wherein the auxin affinity protein is selected from the group consisting of IAA7 protein, TIR1 receptor protein, and TIR1-IAA co-receptor protein. 3. The chromatographic column according to claim 2, wherein the TIR1-IAA co-receptor protein is a TIR1-IAA7 co-receptor protein. 4. The chromatographic column according to claim 1, characterized in that the auxin affinity protein has a protein label. 5. The chromatographic column according to claim 4, wherein the protein tag is a GST tag. 6. A method for separating and purifying auxin, characterized in that, using the chromatographic column according to any one of claims 1 to 5, comprising the following steps: 1) Inject the initial plant extract into the chromatographic column, release the excess liquid from the bottom of the purification column after the auxin is fully combined with the auxin affinity protein, clean the column bed, digest with enzymes, and collect the solution in the column bed; 2) Concentrating and drying the collected solution in the column bed to obtain a mixture of auxin and auxin affinity protein, redissolving the mixture, centrifuging and taking the supernatant to obtain an auxin solution. 7. The method according to claim 6, characterized in that, the preparation step of the initial plant extract is: after liquid nitrogen quick-freezing, fully pulverize the plant sample with a grinding method, add buffer and mix, and centrifuge, and the supernatant is the initial plant extract. Extraction. 8. The method according to claim 6, characterized in that the solvent used for the reconstituted mixture is aqueous acetonitrile. 9. The method according to claim 8, characterized in that the mass fraction of the acetonitrile aqueous solution is 20%. 10. A method for quantitative analysis of auxin in plants, characterized in that, comprising the following steps: 1) using the method described in any one of claims 6 to 9 to separate and purify auxin; 2) Using a liquid phase-mass spectrometer to detect and analyze the isolated and purified auxin.