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CN109136166B - Method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis - Google Patents

Method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis Download PDF

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CN109136166B
CN109136166B CN201810759252.0A CN201810759252A CN109136166B CN 109136166 B CN109136166 B CN 109136166B CN 201810759252 A CN201810759252 A CN 201810759252A CN 109136166 B CN109136166 B CN 109136166B
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聂燕芳
李云锋
王振中
叶智坚
邹小桃
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Abstract

本发明属于蛋白质组学技术领域，具体首次公开了一种适于双向电泳的水稻叶片质膜磷酸化蛋白的提取方法，包括水稻叶片粗质膜的提取、水稻叶片质膜的纯化及水稻叶片质膜磷酸化蛋白质的富集等步骤。本发明所述提取方法操作简单、提取效率高、成本低，所获的水稻叶片质膜纯度可达93%以上，从质膜蛋白质中富集的质膜磷酸化蛋白质得率为3.33～3.89%。该方法适合于质膜磷酸化蛋白提取困难、干扰杂质多的植物材料；同时所得质膜磷酸化蛋白杂质和干扰物较少，可满足双向电泳的要求，所获双向电泳图谱背景清晰、蛋白质点分布均匀、纵向拖尾和水平横纹现象较少，且具有良好的重复性；该方法适用于单子叶植物质膜磷酸化蛋白质组的制备与分析。

Figure 201810759252

The invention belongs to the technical field of proteomics, and specifically discloses for the first time a method for extracting phosphorylated protein from rice leaf plasma membrane suitable for two-dimensional electrophoresis, including the extraction of rice leaf crude plasma membrane, the purification of rice leaf plasma membrane and the Enrichment of membrane phosphorylated proteins, etc. The extraction method of the invention has the advantages of simple operation, high extraction efficiency and low cost, the purity of the obtained rice leaf plasma membrane can reach more than 93%, and the yield of plasma membrane phosphorylated protein enriched from the plasma membrane protein is 3.33-3.89% . The method is suitable for plant materials with difficult plasma membrane phosphorylated protein extraction and many interfering impurities; meanwhile, the obtained plasma membrane phosphorylated protein has less impurities and interfering substances, which can meet the requirements of two-dimensional electrophoresis, and the obtained two-dimensional electrophoresis pattern has a clear background and protein spots. Uniform distribution, fewer vertical tails and horizontal streaks, and good repeatability; this method is suitable for the preparation and analysis of monocot plasma membrane phosphorylated proteomes.

Figure 201810759252

Description

Method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis
Technical Field
The invention relates to the technical field of proteomics, in particular to a method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis.
Background
Protein phosphorylation refers to phosphorylation of a protein by a protein kinase and subsequent phosphorylation by a phosphatase. Protein phosphorylation, one of the most important forms of post-translational modification of proteins, regulates almost all of the vital activities within a cell, and plays an extremely important role in regulating cell signaling processes in particular. The phosphoproteomics refers to the analysis method of proteomics, which is used for observing the modification state and the change of the phosphoprotein in cells on the whole so as to analyze the regulation and control effect of specific phosphorylation modification on the life process and the molecular mechanism of the phosphorylation modification; the content mainly comprises enrichment, detection, identification, quantification and the like of phosphorylated proteins. Among them, the 2-fractional gel electrophoresis (2-DE) technique is still the most mature and classical protein separation technique in the study of phosphoproteomics, and the research strategy is to purify and enrich the phosphoprotein first and then separate the phosphoprotein by electrophoresis and the like. The 2-DE technology has the advantages of high throughput, high resolution, good repeatability and the like, can clearly reflect the molecular weight and isoelectric point information of protein spots, and is one of the most commonly adopted methods in plant phosphorylation proteomics research.
Receptors and early reaction proteins which are related to the functions of recognizing various external stress signals and participating in signal transduction and the like exist on the plant cell plasma membrane, and the phosphorylation of the plasma membrane protein is proved to be widely involved in the reactions of signal recognition and the like to external stress (such as pathogenic bacteria and the like); therefore, the extraction and purification of rice cytoplasmic membrane phosphorylated protein are developed, which is beneficial to comprehensively understanding rice phosphorylated proteome, screening functional phosphorylated protein and the like. In the existing research on the plant plasma membrane phosphorylation proteome, the most adopted strategies are as follows: separating and purifying a plant plasma membrane, performing enzymolysis and phosphorylated peptide enrichment on plasma membrane protein, and performing protein phosphorylation modification analysis through chromatographic separation and mass spectrometry; its advantages are high solubility of peptide segment, high enriching specificity, and low cost.
At present, no reports related to rice plasma membrane phosphorylated proteome exist, and the main reasons are that the monocotyledon plasma membrane is difficult to purify, the abundance of plasma membrane phosphorylated protein is low, and the loss of protein is easily caused by the hydrophobicity of the plasma membrane protein. Therefore, it is necessary to develop a separation technology system for rice plasma membrane phosphorylated proteins suitable for dimensional electrophoresis analysis.
Disclosure of Invention
The invention aims to overcome the blank of the existing rice plasma membrane phosphorylated proteome separation and purification technology, and provides a method for extracting rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis for the first time, which specifically comprises the steps of separation and purification of rice leaf plasma membrane, separation and purification of plasma membrane protein and Al (OH)3Incubation, washing of non-phosphorylated protein, elution of phosphorylated protein, acetone precipitation and the like, thereby obtaining the rice leaf plasma membrane phosphorylated protein with high purity.
Another objective of the invention is to provide an extraction buffer for rough rice leaf membranes.
Another objective of the invention is to provide a buffer solution for enriching the rice leaf plasma membrane phosphorylated protein.
In order to achieve the purpose, the invention is realized by the following scheme:
due to the characteristics of the phosphorylated protein itself, there are still some technical difficulties in the analysis of the phosphorylated protein: first, the abundance of most phosphorylated proteins in cells is low; secondly, the variability of phosphorylation, such that different forms of phosphorylation of a protein exist under different conditions; thirdly, the existing analysis method lacks dynamic analysis of phosphorylation sites, so that partial sites are difficult to identify; the hydrophobicity of the plasma membrane protein itself easily causes the loss of the protein; finally, the presence of phosphatase enzymes leads to dephosphorylation during sample preparation. Therefore, aiming at the problems, the invention optimizes and improves the existing phosphorylated protein extraction method and the buffer solution and the extracting solution used in the preparation process, so that the purity of the finally enriched plasma membrane phosphorylated protein is greatly improved, and the finally enriched plasma membrane phosphorylated protein contains fewer impurities and interferents and is suitable for the subsequent two-dimensional electrophoresis analysis.
Therefore, the invention claims an extraction buffer for rough membranes of rice leaves, which comprises the following components: BTP-MES buffer solution with a final concentration of 50mmol/L, sucrose with a final concentration of 250-500 mmol/L, EDTA with a final concentration of 0.5-10 mmol/L, 10-20% v/v glycerol, 0.1-1% w/v BSA with a final concentration of 1-10 mmol/LPMSF of 0.1-2 mmol/L of DTT, 0.6-5% w/v PVP k-30, NaF of 10-50 mmol/L of final concentration, beta-mercaptoethanol of 5-15 mmol/L of final concentration, NaMoO of 1-2 mmol/L of final concentration4·2H2O, NaVO with final concentration of 0.05-2 mmol/L3Sodium pyrophosphate with the final concentration of 2.5-10 mmol/L and 0.01-0.02% w/v Brij 56; the pH value of the rice leaf rough membrane extraction buffer solution is 7.6-7.8.
According to the invention, the components are preferably compounded to obtain the extraction buffer solution for the rough plasma membrane of the rice leaf, so that the degradation of plasma membrane protein can be effectively inhibited, the solubility of the plasma membrane protein is increased, and the loss of the protein is reduced. Wherein, EDTA and PMSF are protease inhibitors to prevent protein degradation; DTT, PVP k-30 and beta-mercaptoethanol are reducing agents, and disulfide bonds of protein molecules can be broken to increase the solubility of the protein; NaF, NaMoO4·2H2O、NaVO3The sodium pyrophosphate is a phosphatase inhibitor, can prevent dephosphorylation of target protein, is easy to remove, and does not influence subsequent two-dimensional electrophoresis analysis; poly (oxyethylene) cetyl ether, Brij56, is an ionic detergent that increases the solubility of plasma membrane proteins.
Preferably, the extraction buffer comprises the following components: BTP-MES buffer with a final concentration of 50mmol/L, sucrose with a final concentration of 250mmol/L, EDTA with a final concentration of 2mmol/L, 10% v/v glycerol, 0.5% w/v BSA, PMSF with a final concentration of 10mmol/L, DTT with a final concentration of 2mmol/L, 0.6% w/v PVP k-30, NaF with a final concentration of 40mmol/L, beta-mercaptoethanol with a final concentration of 15mmol/L, NaMoO with a final concentration of 1mmol/L4·2H2O, NaVO with final concentration of 1mmol/L3Sodium pyrophosphate at a final concentration of 10mmol/L, 0.02% w/v Brij56, pH 7.8.
The invention also claims a buffer solution for enriching the rice leaf plasma membrane phosphorylated protein, which comprises the following components: MES with the final concentration of 20-50 mmol/L, imidazole with the final concentration of 10-40 mmol/L, urea with the final concentration of 7-8 mol/L, potassium aspartate with the final concentration of 0.1-0.2 mol/L, sodium glutamate with the final concentration of 0.1-0.2 mol/L, 0.2-4% w/vCHAPS and the pH value of 6.0-6.3.
Wherein the miaowAzoles are phosphatase inhibitors; CHAPS is an ionic detergent and can improve the solubility of hydrophobic proteins in plasma membranes; the potassium aspartate and the sodium glutamate can enhance Al (OH)3The specific adsorption effect on the phosphorylated protein can improve the separation efficiency of the phosphorylated protein and the non-phosphorylated protein.
Preferably, the buffer comprises the following components: MES with a final concentration of 30mmol/L, imidazole with a final concentration of 30mmol/L, urea with a final concentration of 8mol/L, potassium aspartate with a final concentration of 0.1-0.15 mol/L, sodium glutamate with a final concentration of 0.1-0.15 mol/L, 0.25% w/v CHAPS and a pH value of 6.1.
The invention also claims a method for extracting the rice leaf plasma membrane phosphorylated protein suitable for dimensional electrophoresis, which comprises the following steps:
s1, extracting a rough membrane of a rice leaf: grinding the rice leaves into powder, adding the extraction buffer solution according to the mass-to-volume ratio of 4:13, and grinding until homogenate is obtained; filtering, centrifuging at 11500 Xg for 20min, centrifuging the supernatant at 87000 Xg for 40min, and suspending the precipitate in phase buffer to obtain crude membrane solution;
s2, purifying a rice leaf plasma membrane: adding the crude membrane solution obtained in the step S1 into a double aqueous phase system, uniformly mixing, centrifuging at 1000 Xg for 5min, taking the upper phase, and repeating for three times; centrifuging, collecting the upper phase solution, diluting with 10 times volume of phase buffer solution, centrifuging at 120000 Xg for 1h, and collecting precipitate; suspending the precipitate in plasma membrane preservation solution, centrifuging at 120000 Xg for 1h, and repeating twice; dissolving the precipitate with any one of the above buffer solutions to obtain purified plasma membrane protein;
s3, enrichment of rice leaf plasma membrane phosphorylated proteins: incubating the plasma membrane protein obtained in S2 at 20 deg.C for 2h, centrifuging at 15000 Xg for 15min, and collecting the supernatant; adding the supernatant to Al (OH)3And (4) incubating for 2 h; centrifuging at 6000 Xg for 10min, and collecting precipitate; adding any one of the above buffer solutions into the precipitate, mixing, centrifuging at 6000 Xg for 5min, removing supernatant, and repeating for 4 times; adding an elution buffer solution into the precipitate, uniformly mixing, and incubating at room temperature for 2 h; centrifuging at 10000 Xg for 5min, collecting supernatant, and enriching plasma membrane phosphorylated protein by DOC-TCA-acetone precipitation method.
The invention adopts a 6.3% w/w PEG 3350/Dextran T-500 two-phase distribution system to purify the rice leaf plasma membrane, and obtains a high-purity plasma membrane, wherein the purity is more than 93%; with Al (OH)3As a substrate, rice leaf plasma membrane phosphorylated protein is enriched, and the enrichment yield is 3.31-3.88%; the rice leaf plasma membrane phosphorylated protein is subjected to two-dimensional electrophoresis analysis, and through Pro-Q Diamond dyeing and image scanning, the 2-DE map of the rice leaf plasma membrane phosphorylated protein has good repeatability, clear background and uniform protein point distribution, and can meet the research requirements of subsequent analysis of phosphorylated proteomes; Pro-Q Diamond staining and silver nitrate staining are adopted to analyze the same 2-DE gel, and the result shows that the enriched rice leaf plasma membrane phosphorylated protein has high specificity.
In the process of extracting the rough rice leaf membranes, NaF and NaMoO are added4·2H2O、NaVO3Phosphatase inhibitors such as sodium pyrophosphate and imidazole can effectively inhibit the degradation of phosphorylated protein in the protein extraction process, and an ideal 2-DE map is obtained; in addition, because a large amount of hydrophobic protein exists in the plasma membrane and is difficult to dissolve, the ionic detergents Brij56, CHAPS and NP-40 are added in sequence in the processes of extracting and purifying the plasma membrane, so that the solubility of the plasma membrane protein is improved, and protein spots in an obtained 2-DE map are clear and uniformly distributed.
Preferably, the steps S1 and S2 are both performed at 4 ℃.
Preferably, the DOC-TCA-acetone precipitation method in S3 comprises the following specific steps: adding 2% w/v sodium deoxycholate into the supernatant, mixing uniformly, adding 100% w/v TCA, mixing uniformly, and standing on ice overnight; centrifuging at 18000 Xg for 15min, and removing supernatant; suspending and precipitating with 25% w/v TCA, centrifuging at 18000 Xg at 4 deg.C for 15min, and collecting precipitate; suspending the precipitate in 0.01mol/LTris-HCl solution containing 80% acetone at pH 7.5, and standing at-20 deg.C for more than 30 min; centrifuging at 4 deg.C for 10min, and removing supernatant; suspending the precipitate in 90% acetone, centrifuging at 4 deg.C for 10min at 20000 × g, and removing supernatant; and (5) placing the precipitate on ice for air drying to obtain the rice leaf plasma membrane phosphorylated protein. When the prepared rice leaf plasma membrane phosphorylated protein is used specifically, a solubilization buffer solution (7mol/L urea, 2mol/L thiourea, 2% v/v NP-40, 65mmol/L DTT) containing 1% w/v DTT is dissolved for standby.
Preferably, the phase buffers in S1 and S2 comprise the following final concentrations of the respective components: 5mmol/LK2HPO4-KH2PO4Buffer solution, 250mmol/L sucrose, 3mmol/L KCl, 1mmol/L DTT; the pH of the phase buffer was 7.8.
Preferably, the aqueous two-phase system in S2 comprises the following components: 6.3% w/w Dextran T-500/PEG 3350, sucrose to a final concentration of 250mmol/L, KCl to a final concentration of 3mmol/L, KH to a final concentration of 5mmol/L2PO4(ii) a The pH value of the aqueous two-phase system is 7.8.
Preferably, the plasma membrane preservation solution in S2 comprises the following components at the final concentrations: 5mmol/L BTP-MES buffer solution, 250mmol/L sucrose, 2mmol/L KCl, 1mmol/L DTT; the pH value of the plasma membrane preservation solution is 7.8.
Preferably, the elution buffer in S3 comprises the following final concentrations of the components: 0.3mol/L potassium pyrophosphate and 8mol/L urea; the pH of the elution buffer was 9.0.
Compared with the prior art, the invention has the following beneficial effects:
the extraction method provided by the invention is simple to operate, high in extraction efficiency and low in cost, the purity of the obtained rice leaf plasma membrane can reach more than 93%, and the yield of plasma membrane phosphorylated protein enriched from the plasma membrane protein is 3.33-3.89%. The method is suitable for plant materials with difficult extraction of plasma membrane phosphorylated protein and much interference impurities; meanwhile, the obtained plasma membrane phosphorylated protein has fewer impurities and interferents, can meet the requirements of dimensional electrophoresis, and has clear background, uniform protein point distribution, less longitudinal trailing and horizontal cross striation phenomena and good repeatability; the method is suitable for preparing and analyzing the monocotyledon plasma membrane phosphorylated proteome.
Drawings
FIG. 1 shows the binding of different specific inhibitors to rice leaf plasma membrane H in example 2+-effect of ATPase activity.
FIG. 2 is a silver nitrate staining 2-DE map of rice leaf plasma membrane phosphorylated protein in example 2.
FIG. 3 is the analysis of the goodness of fit of the rice leaf phosphorylated protein 2-DE profile in example 2 after staining by different methods; wherein A is the result of staining the gel with Pro-Q Diamond; b is the result of silver nitrate staining of the gel; c, marking the points in A as green by using analysis software; d is marking the points in the B picture as red using the analysis software; e is the result of superimposing C and D using the analysis software.
FIG. 4 is a Venn plot of the 2-DE profile protein spot goodness of agreement of Pro-Q Diamond staining with silver staining in example 2; wherein, yellow is the number of protein points of Pro-Q Diamond stained 2-DE map; green is the number of protein points of silver-stained 2-DE map.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 extraction method of phosphorylated protein from plasma membrane of rice leaf suitable for dimensional electrophoresis
1. Rice planting and leaf collection
The rice is indica rice line CO 39. The rice is sowed in a plastic pot containing rice field soil and is irradiated for 14 hours at 24-26 ℃ (about 250 mu mol.m)-2·s-1) Is grown in the growth chamber. And (4) performing sampling when the 4 th leaf is completely unfolded according to the management of a conventional method. Taking the 3 rd and 4 th leaves respectively, quickly weighing the leaves, quickly freezing the leaves by liquid nitrogen, and storing the leaves in a refrigerator at the temperature of 70 ℃ below zero for later use.
2. Extraction of rough rice leaf membranes
80g of rice leaves were weighed and ground to powder with liquid nitrogen. 260mL of extraction buffer (containing 50mmol/LBTP-MES buffer, 250mmol/L sucrose, 2mmol/L EDTA, 10% v/v glycerol, 0.5% w/v BSA, 10mmol/L PMSF, 2mmol/L DTT, 0.6% w/V-v PVP k-30, 40mmol/L NaF, 15mmol/L beta-mercaptoethanol, 1mmol/L NaMoO4·2H2O，1mmol/L NaVO310mmol/L sodium pyrophosphate, 0.02% w/v Brij56, ph7.8), ground to a homogenate. Filtering with a filter screen with aperture of 260 μm, centrifuging the filtrate at 11500 Xg for 20min, and collecting supernatant; the supernatant was centrifuged at 87000 Xg for 40min and the pellet was suspended in phase buffer (5 mmol/LK)2HPO4-KH2PO4Buffer solution, 250mmol/L sucrose, 3mmol/L LKCl, 1mmol/L DTT, pH7.8), and the obtained sample is the crude membrane solution. All the above operations were carried out at 4 ℃. Wherein EDTA and PMSF are protease inhibitors; DTT, PVP k-30 and beta-mercaptoethanol are reducing agents, and disulfide bonds of protein molecules can be broken to increase the solubility of the protein; NaF, NaMoO4·2H2O、NaVO3Sodium pyrophosphate is a phosphatase inhibitor; poly (oxyethylene) cetyl ether (Brij 56) is an ionic detergent that increases the solubility of plasma membrane proteins.
3. Purification of plasma membranes
Adding the above crude membrane solution into aqueous two-phase system (containing 6.3% w/w Dextran T-500/PEG 3350, 250mmol/L sucrose, 3mmol/L KCl, 5mmol/L KH)2PO4pH7.8), centrifuging at 1000 Xg for 5min, collecting the upper phase, and repeating for three times. Diluting the upper phase solution obtained by the last centrifugation with 10 times volume of phase buffer solution, centrifuging at 120000 Xg for 1h, and taking the precipitate; the pellet was suspended in plasma membrane stock (5mmol/L BTP-MES buffer, 250mmol/L sucrose, 2mmol/L KCl, 1mmol/L DTT, pH7.8), centrifuged at 120000 Xg for 1h, repeated twice; the precipitate was dissolved in incubation buffer (30mmol/LMES, 30mmol/L imidazole, 8mol/L urea, 0.1mol/L potassium aspartate, 0.1mol/L sodium glutamate, 0.25% w/v CHAPS) and the resulting sample was the purified plasma membrane. All the above operations were carried out at 4 ℃. Wherein imidazole is a phosphatase inhibitor; CHAPS is an ionic detergent and can improve the solubility of hydrophobic proteins in plasma membranes; the potassium aspartate and the sodium glutamate can enhance Al (OH)3Specific adsorption on phosphorylated proteins.
4. Enrichment of rice leaf plasma membrane phosphorylated protein
Will 8mg plasma membrane protein was incubated at 20 ℃ for 2h on a shaker, centrifuged at 15000 Xg for 15min and the supernatant was taken. The supernatant was added to 2g of Al (OH)3In the middle, incubating for 2h on a shaking table; centrifuging at 6000 Xg for 10min, and collecting precipitate. 15mL of washing buffer (30mmol/LMES, 30mmol/L imidazole, 8mol/L urea, 0.15mol/L potassium aspartate, 0.15mol/L sodium glutamate, 0.25% w/v CHAPS, pH 6.1) was added to the precipitate, vortexed, centrifuged at 6000 Xg for 5min, the supernatant removed, and repeated 4 times. Adding 20mL of elution buffer (0.3mol/L potassium pyrophosphate, 8mol/L urea, pH 9) into the precipitate, mixing uniformly, and incubating for 2h at room temperature in a shaking table; centrifuging at 10000 Xg for 5min, and collecting supernatant. Adding 2% w/v sodium deoxycholate, and mixing by vortex; then 100% w/v TCA was added, mixed well and left on ice overnight. Centrifuge at 18000 Xg for 15min, remove supernatant. Suspending and precipitating with 25% w/v TCA, and centrifuging at 18000 Xg for 15min at 4 deg.C; and taking the precipitate. Suspending the precipitate in 0.01mol/L Tris-HCl solution (pH 7.5, containing 80% acetone) and standing at-20 deg.C for more than 30 min; centrifuging at 4 deg.C for 10min, and removing supernatant; the pellet was resuspended in 1mL 90% acetone, centrifuged at 20000 Xg at 4 ℃ for 10min, and the supernatant removed. The precipitate was air dried on ice. The protein concentration was determined by dissolving it in a solubilization buffer containing 1% w/v DTT (7mol/L urea, 2mol/L thiourea, 2% v/v NP-40, 65mmol/L DTT) until use.
Example 2 two-dimensional electrophoresis analysis and specificity analysis of phosphorylated protein of rice leaf plasma membrane
1. Analysis of rice leaf plasma membrane purification effect
The rice leaf plasma membrane is purified by adopting a 6.3% w/w PEG 3350/Dextran T-500 two-phase distribution system, and the result shows that the yield of the plasma membrane protein obtained by purification is about 2.71-3.21% after 100mg of crude membrane protein is subjected to two-phase distribution for 3 times.
With different inner membrane marker enzymes (H)+ATPase) crude and purified plasma membranes were treated to evaluate the purity of the plasma membranes. The method comprises the following specific steps: mu.g of plasma membrane protein was added to 0.5mL of BTP-MES buffer (containing 30mmol/L of BTP-MES pH 6.5, 5mmol/L of MgSO4，50mmol/L KCl，0.02％(w/v)Brij 58，5mmol/L Na2-ATP), the reaction is started, incubated at 30 ℃ for 30min and 2.5mL of terminator [ containing 0.5 mmol-L PVP k30，86mmol/L(NH2OH)2·H2SO4，5.3mmol/L EDTA-Na20.2% v/v concentrated H2SO4，1.3％w/v(NH4)2MoO4]The reaction was terminated, and then 0.25mL of a developing solution (50mmol/L Na) was added rapidly2CO36.47mol/L NaOH) for 30min, and the absorption value at A720nm was measured; plasma membrane protein added with boiling for 30min was used as blank control. The activity inhibition rate is determined by adding 0.5mmol/L Na into the reaction system3VO4、50mmol/L KNO3、1mmol/L NaN3And 1mmol/L Na2MoO4Then, the absorbance at A720nm was measured, and H inhibited by each inhibitor was calculated+-the proportion of ATPase activity in the total activity. Wherein, Na3VO4Is plasma membrane H+-specific inhibitors of ATPase; KNO3Vacuolar membrane, endoplasmic reticulum membrane and Golgi apparatus membrane H+-specific inhibitors of ATPase; NaN3Is mitochondria and chloroplasts H+Inhibitors specific for ATPase, Na2MoO4Is lysosomal intimal H+Specific inhibitors of ATPase.
The results show (as shown in FIG. 1), that plasma membrane H+Sensitivity of ATPase to vanadate increased from 43.5% to 93.9%, H of type V (tonoplast, endoplasmic reticulum membrane, Golgi membrane, etc.)+-ATPase vs. KNO3The sensitivity of the strain is reduced from 66.8% to 8.2%, and the type F (mitochondria and chloroplasts) H+-ATPase vs. NaN3The sensitivity of the enzyme decreased from 71.9% to 6.4%, and the non-specific phosphatase (lysosome) was sensitive to Na2MoO4The sensitivity of (A) is reduced from 42.5% to 5.2%; the results show that the 6.3 percent PEG 3350/Dextran T-500 aqueous two-phase system can be used for effectively purifying the rough plasma membrane of the rice leaf, reducing other inner membrane impurities and obtaining a high-purity plasma membrane, wherein the purity is more than 93 percent.
2. Yield of rice leaf plasma membrane phosphorylated protein
Purified 8mg plasma membrane protein was mixed with 2g Al (OH)3Incubation, washing of non-phosphorylated proteins, elution of phosphorylated proteins and the likeAnd (3) enriching to obtain 265-310 mu g of plasma membrane phosphorylated protein, namely, the enrichment yield of the plasma membrane phosphorylated protein is 3.31-3.88%.
3. Two-dimensional electrophoresis analysis of rice leaf plasma membrane phosphorylated protein
In order to further determine whether the purified rice leaf plasma membrane phosphorylated protein is suitable for two-dimensional electrophoresis (2-DE), 2-DE and Pro-Q Diamond staining and image scanning were performed to obtain a 2-DE map of the rice leaf plasma membrane phosphorylated protein (shown in FIG. 2). The two-dimensional electrophoresis comprises the following steps: taking a plasma membrane phosphorylated protein sample (150 mu g) and hydrating in a solubilization buffer solution for 16h, and carrying out first phase gradient isoelectric focusing electrophoresis, wherein the focusing parameters are as follows: boosting the pressure at 300V for 0.5h at a low speed; boosting the pressure at a low speed of 500V for 0.5 h; boosting the pressure slowly for 0.5h at 1000V; rapidly boosting the pressure for 1.5h at 5000V; 8000V fast boost focus 60000 Vh. The concentration of the second SDS-PAGE was 12% and the electrophoresis parameter was 16 mA/gel. The same gel was stained sequentially by Pro-Q Diamond staining and silver staining. The gel was scanned using a Typhoon Trio multifunctional laser scanner.
PDQuest 8.0 software is used for analyzing the protein, and the result shows that 296 plasma membrane phosphorylated protein spots are in total in the 2-DE map and have good repeatability; meanwhile, the 2-DE map has clear background, protein spots are uniformly distributed, and the phenomena of longitudinal tailing and horizontal transverse striation are not obvious, so that the research requirement of subsequent analysis of the phosphorylated proteome can be met.
4. Specificity analysis of rice leaf plasma membrane phosphorylated protein
To examine the specificity of the method described in example 1 for the enrichment of plasma membrane phosphorylated proteins in rice leaf plasma membranes, Pro-Q Diamond staining and silver nitrate staining were performed on the same 2-DE gel, and overlay analysis was performed on the staining patterns of the same 2-DE gel by different methods using PDQuest 8.0 image analysis software (as shown in FIG. 3). The results show that: Pro-QDiamond stained plasma membrane phosphorylated protein spots were 296 (FIG. 3A) and total protein spots were 291 (FIG. 3B).
Protein spots in the Pro-Q Diamond staining pattern (FIG. 3A) were labeled green (FIG. 3C), and protein spots in the silver nitrate staining pattern (FIG. 3B) were labeled red (FIG. 3D); the two labeled maps were then superimposed (FIG. 3E) to analyze the goodness of fit of the protein spots in the two maps. The results show that: the overlay shows that most protein spots are in different shades of yellow, indicating that the protein is phosphorylated to a relatively high degree; part of protein spots are green, which shows that the protein of the protein spots is low in relative abundance and high in phosphorylation degree; the red color of only and individual protein spots indicates that the protein spot is a non-phosphorylated protein, or that the protein spot has a high relative abundance of protein and a low degree of phosphorylation (FIG. 4).
By combining the results, the rice leaf plasma membrane phosphorylated protein enriched by the extraction method of the invention has high specificity.
Comparative example 1
A method for extracting phosphorylated protein from plasma membrane of rice leaf, which is different from that in example 1 except that the extraction buffer solution adopted in the step 2 for extracting rough plasma membrane of rice leaf (lack of NaF and NaMoO)4·2H2O、NaVO3Sodium pyrophosphate), the rest steps are the same.
The formula of the buffer solution for extracting the rough plasma membrane of the rice leaf adopted in the comparative example is as follows: BTP-MES buffer at a final concentration of 50mmol/L, sucrose at a final concentration of 250mmol/L, EDTA at a final concentration of 2mmol/L, glycerol at a final concentration of 10% v/v, BSA at a final concentration of 0.5% w/v, PMSF at a final concentration of 10mmol/L, DTT at a final concentration of 2mmol/L, 0.6% w/v PVPk-30, beta-mercaptoethanol at a final concentration of 15mmol/L, 0.02% w/v Brij56, pH 7.8.
And (4) analyzing results: analysis of the purification effect of the rice leaf plasma membrane shows that the purity of the plasma membrane reaches 93.52%, the yield of the purified plasma membrane protein is about 2.94-3.02%, and the method has no obvious difference compared with the example 2. However, the enrichment yield of phosphorylated protein of the rice leaf plasma membrane is only 2.18%, which is obviously lower than that of example 2. The result shows that the crude plasma membrane extraction buffer solution is lack of NaF and NaMoO4·2H2O、NaVO3And phosphatase inhibitors such as sodium pyrophosphate, plasma membrane phosphorylated proteins are degraded.
Comparative example 2
A method for extracting phosphorylated protein from a rice leaf plasma membrane comprises the same steps except that an extraction buffer used in the step 2 for extracting a rough rice leaf plasma membrane is different from that in the step 1 (NaF and sodium pyrophosphate are lacked).
The formula of the buffer solution for extracting the rough plasma membrane of the rice leaf adopted in the comparative example is as follows: BTP-MES buffer with a final concentration of 50mmol/L, sucrose with a final concentration of 250mmol/L, EDTA with a final concentration of 2mmol/L, 10% v/v glycerol, 0.5% w/vBSA, PMSF with a final concentration of 10mmol/L, DTT with a final concentration of 2mmol/L, 0.6% w/v PVP k-30, beta-mercaptoethanol with a final concentration of 15mmol/L, NaMoO with a final concentration of 1mmol/L4·2H2O, NaVO with final concentration of 1mmol/L30.02% w/v Brij56, pH 7.8.
And (4) analyzing results: analysis of the purification effect of the rice leaf plasma membrane shows that the purity of the plasma membrane reaches 93.47%, the yield of the purified plasma membrane protein is about 2.96-3.11%, and the method has no obvious difference compared with the example 2. However, the enrichment yield of phosphorylated protein of the rice leaf plasma membrane is only 2.83%, which is obviously lower than that of example 2. NaF and sodium pyrophosphate are acid phosphatase inhibitors and have poor effect on alkaline phosphatase; the result shows that when the crude plasma membrane extraction buffer solution is lack of sodium fluoride and sodium pyrophosphate, the plasma membrane basic phosphorylated protein is easy to degrade.
Comparative example 3
A method for extracting phosphorylated protein from plasma membrane of rice leaf, which is different from that in example 1 except that the extraction buffer solution adopted in the step 2 for extracting rough plasma membrane of rice leaf (lack of NaF and NaMoO)4·2H2O、NaVO3Sodium pyrophosphate, Brij 56), the rest of the procedure was the same.
The formula of the buffer solution for extracting the rough plasma membrane of the rice leaf adopted in the comparative example is as follows: BTP-MES buffer with a final concentration of 50mmol/L, sucrose with a final concentration of 250mmol/L, EDTA with a final concentration of 2mmol/L, 10% v/v glycerol, 0.5% w/vBSA, PMSF with a final concentration of 10mmol/L, DTT with a final concentration of 2mmol/L, 0.6% w/v PVP k-30, beta-mercaptoethanol with a final concentration of 15mmol/L and a pH value of 7.8.
And (4) analyzing results: analysis of the purification effect of the rice leaf plasma membrane shows that the purity of the plasma membrane reaches 93.66%, and compared with example 2, the purity of the plasma membrane has no obvious difference. However, the yield of plasma membrane protein obtained by purification is about 2.18 to E2.35%, significantly lower than example 2. Meanwhile, the enrichment yield of the rice leaf plasma membrane phosphorylated protein is only 1.56%, which is obviously lower than that of the example 2. The results show that when the crude plasma membrane extraction buffer lacks the ionic detergent Brij56, the solubility of plasma membrane proteins is reduced, resulting in reduced plasma membrane protein yield; lack of NaF, NaMoO4·2H2O、NaVO3And phosphatase inhibitors such as sodium pyrophosphate, the phosphorylated protein is easily degraded, and the enrichment yield of plasma membrane phosphorylated protein is reduced.
Comparative example 4
A method for extracting phosphorylated protein from plasma membrane of rice leaf comprises the same steps except that step 3, the incubation buffer used in the purification of the plasma membrane is different from that used in example 1 (lacking imidazole, sodium glutamate and potassium aspartate).
The incubation buffer solution for enriching the rice leaf plasma membrane phosphorylated protein adopted by the comparative example comprises the following components: MES at a final concentration of 30mmol/L, urea at a final concentration of 8mol/L, 0.25% w/v CHAPS, pH 6.1.
And (4) analyzing results: the enrichment yield of the rice leaf plasma membrane phosphorylated protein is 3.90%, and compared with example 2, the enrichment yield has no obvious difference. However, SDS-PAGE results show that the prepared phosphorylated protein contains more impurities (non-phosphorylated protein), and the subsequent two-dimensional electrophoresis analysis results are influenced. Since imidazole, sodium glutamate and potassium aspartate can reduce Al (OH)3Non-specific adsorption of histidine-rich proteins and acidic amino acids, thus resulting in Al (OH) in the absence of the three components3The degree of specific adsorption to phosphorylated proteins is reduced, and the purity of the obtained phosphorylated proteins is also reduced.
Comparative example 5
A method for extracting phosphorylated protein from plasma membrane of rice leaf comprises the same steps except that the incubation buffer used in the purification of the plasma membrane in step 3 is different from that in example 1 (the concentrations of sodium glutamate and potassium aspartate are respectively 0.05 mol/L).
The incubation buffer solution for enriching the rice leaf plasma membrane phosphorylated protein adopted by the comparative example comprises the following components: MES with a final concentration of 30mmol/L, imidazole with a final concentration of 30mmol/L, urea with a final concentration of 8mol/L, potassium aspartate with a final concentration of 0.05mol/L, sodium glutamate with a final concentration of 0.05mol/L, 0.25% w/v CHAPS, pH 6.1.
And (4) analyzing results: the enrichment yield of phosphorylated protein of the rice leaf plasma membrane is 2.89%, which is lower than that of the example 2. The results show that Al (OH) can be enhanced by potassium aspartate and sodium glutamate3Specific adsorption of phosphorylated proteins, therefore in the case of reduced potassium aspartate and sodium glutamate concentrations, Al (OH)3The specific adsorption degree of the phosphorylated protein is reduced, resulting in a reduction in the enrichment yield of the phosphorylated protein.
Comparative example 6
A method for extracting phosphorylated protein of rice leaf plasma membrane is the same as that in example 1 except that the washing buffer used in the step 4 for enriching the phosphorylated protein of rice leaf plasma membrane is different (the concentrations of sodium glutamate and potassium aspartate are respectively 0.20mol/L), and the other steps are the same.
The cleaning buffer solution for enriching the plasma membrane phosphorylated protein of the rice leaf adopted by the comparative example comprises the following components: MES with a final concentration of 30mmol/L, imidazole with a final concentration of 30mmol/L, urea with a final concentration of 8mol/L, potassium aspartate with a final concentration of 0.20mol/L, sodium glutamate with a final concentration of 0.20mol/L, 0.25% w/v CHAPS, pH 6.1.
And (4) analyzing results: the enrichment yield of phosphorylated protein of the rice leaf plasma membrane is 3.01 percent, which is lower than that of the example 2. The results show that Al (OH) can be enhanced by potassium aspartate and sodium glutamate3Specific adsorption of phosphorylated proteins, and therefore increased concentrations of potassium aspartate and sodium glutamate, may result in binding to Al (OH)3The phosphorylated protein is difficult to elute, resulting in a decrease in the yield of the phosphorylated protein.
Comparative example 7
A method for extracting phosphorylated protein of a rice leaf plasma membrane comprises the same steps except that an elution buffer solution adopted in the enrichment of the phosphorylated protein of the rice leaf plasma membrane in the step 4 is different from that in the example 1 (0.15mol/L potassium pyrophosphate);
the elution buffer for enriching the plasma membrane phosphorylated protein of the rice leaf adopted in the comparative example comprises the following components: 0.15mol/L potassium pyrophosphate, 8mol/L urea, pH 9.
And (4) analyzing results: the enrichment yield of phosphorylated protein of the rice leaf plasma membrane is 2.88 percent, which is lower than that of the example 2. Due to potassium pyrophosphate to Al (OH)3The substrate has strong affinity, and when the concentration of potassium pyrophosphate is reduced, the substrate may be bonded to Al (OH)3The phosphorylated protein is difficult to elute, resulting in a decrease in the yield of the phosphorylated protein.
It should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the scope of the present invention, and those skilled in the art can make other variations or modifications based on the above description and ideas, and all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (8)

1.一种水稻叶片粗质膜的提取缓冲液，其特征在于，包含以下组分：终浓度50mmol/L的BTP-MES缓冲液，终浓度250～500mmol/L的蔗糖，终浓度0.5～10mmol/L的EDTA，10～20％v/v甘油，0.1～1％w/v BSA，终浓度1～10mmol/L的PMSF，终浓度0.1～2mmol/L的DTT，0.6～5％w/v PVP k-30，终浓度10～50mmol/L的NaF，终浓度5～15mmol/L的β-巯基乙醇，终浓度1～2mmol/L的NaMoO4·2H2O，终浓度0.05～2mmol/L的NaVO3，终浓度2.5～10mmol/L的焦磷酸钠，0.01～0.02％w/v Brij 56；所述水稻叶片粗质膜提取缓冲液的pH值为7.6～7.8。1. the extraction buffer of a crude plasma membrane of rice leaf, is characterized in that, comprises the following components: the BTP-MES buffer of final concentration 50mmol/L, the sucrose of final concentration 250～500mmol/L, the final concentration 0.5～10mmol EDTA/L, 10-20% v/v glycerol, 0.1-1% w/v BSA, PMSF at a final concentration of 1-10 mmol/L, DTT at a final concentration of 0.1-2 mmol/L, 0.6-5% w/v PVP k-30, NaF with a final concentration of 10-50 mmol/L, β-mercaptoethanol with a final concentration of 5-15 mmol/L, NaMoO 4 ·2H 2 O with a final concentration of 1-2 mmol/L, and a final concentration of 0.05-2 mmol/L of NaVO 3 , sodium pyrophosphate with a final concentration of 2.5-10 mmol/L, and 0.01-0.02% w/v Brij 56; the pH of the rice leaf crude plasma membrane extraction buffer is 7.6-7.8. 2.根据权利要求1所述的提取缓冲液，其特征在于，所述提取缓冲液包括以下组分：终浓度50mmol/L的BTP-MES缓冲液，终浓度250mmol/L的蔗糖，终浓度2mmol/L的EDTA，10％v/v甘油，0.5％w/v BSA，终浓度10mmol/L的PMSF，终浓度2mmol/L的DTT，0.6％w/v PVPk-30，终浓度40mmol/L的NaF，终浓度15mmol/L的β-巯基乙醇，终浓度1mmol/L的NaMoO4·2H2O，终浓度1mmol/L的NaVO3，终浓度10mmol/L的焦磷酸钠，0.02％w/v Brij56，pH值为7.8。2. extraction buffer according to claim 1, is characterized in that, described extraction buffer comprises following components: the BTP-MES buffer of final concentration 50mmol/L, the sucrose of final concentration 250mmol/L, final concentration 2mmol /L EDTA, 10% v/v glycerol, 0.5% w/v BSA, 10 mmol/L final PMSF, 2 mmol/L final DTT, 0.6% w/v PVPk-30, 40 mmol/L final NaF, β-mercaptoethanol with a final concentration of 15 mmol/L, NaMoO 4 ·2H 2 O with a final concentration of 1 mmol/L, NaVO 3 with a final concentration of 1 mmol/L, sodium pyrophosphate with a final concentration of 10 mmol/L, 0.02% w/v Brij56, pH 7.8. 3.一种适于双向电泳的水稻叶片质膜磷酸化蛋白的提取方法，其特征在于，包括如下步骤：3. an extraction method of the rice leaf plasma membrane phosphorylated protein suitable for two-dimensional electrophoresis, is characterized in that, comprises the steps: S1.水稻叶片粗质膜的提取：将水稻叶片研磨成粉末，按质量体积比为4:13加入权利要求1或2任一所述的提取缓冲液，研磨至匀浆；过滤、于11500×g离心20min，然后取上清液于87000×g离心40min后，再将沉淀悬浮于相缓冲液中，即得粗质膜溶液；S1. Extraction of the coarse plasma membrane of rice leaves: grinding the rice leaves into powder, adding the extraction buffer according to any one of claims 1 or 2 in a mass-to-volume ratio of 4:13, and grinding to a homogenate; g centrifugation for 20 min, then the supernatant was centrifuged at 87,000 × g for 40 min, and then the precipitate was suspended in the phase buffer to obtain a crude plasma membrane solution; S2.水稻叶片质膜的纯化：将S1所得粗质膜溶液加入到双水相体系中混匀，于1000×g离心5min，取上相，重复三次；离心后取上相溶液，用10倍体积的相缓冲液稀释，于120000×g离心1h，取沉淀；将沉淀悬浮于质膜保存液中，于120000×g离心1h，重复两次；将沉淀用水稻叶片质膜磷酸化蛋白富集缓冲液溶解，即得纯化的质膜蛋白质；S2. Purification of rice leaf plasma membrane: Add the crude plasma membrane solution obtained from S1 into the aqueous two-phase system and mix well, centrifuge at 1000 × g for 5 min, take the upper phase, and repeat three times; after centrifugation, take the upper phase solution, use 10 times The volume of phase buffer was diluted, centrifuged at 120,000 × g for 1 h, and the precipitate was taken; the precipitate was suspended in the plasma membrane preservation solution, and centrifuged at 120,000 × g for 1 h, repeated twice; the precipitate was enriched with rice leaf plasma membrane phosphorylated protein The buffer solution is dissolved to obtain purified plasma membrane protein; S3.水稻叶片质膜磷酸化蛋白质的富集：将S2所得质膜蛋白质于20℃孵育2h，于15000×g离心15min，取上清；将上清加入到Al(OH)3中，孵育2h；于6000×g离心10min，取沉淀；向沉淀中加入水稻叶片质膜磷酸化蛋白富集缓冲液混匀，于6000×g离心5min，去上清，重复4次；在沉淀中加入洗脱缓冲液，混匀后于室温下孵育2h；于10000×g离心5min，取上清，采用DOC-TCA-丙酮沉淀法富集质膜磷酸化蛋白质；S3. Enrichment of plasma membrane phosphorylated proteins in rice leaves: Incubate the plasma membrane proteins obtained from S2 at 20 °C for 2 h, centrifuge at 15000 × g for 15 min, and take the supernatant; add the supernatant to Al(OH) 3 and incubate for 2 h ; Centrifuge at 6000 × g for 10 min, take the precipitate; add rice leaf plasma membrane phosphorylated protein enrichment buffer to the precipitate, mix well, centrifuge at 6000 × g for 5 min, remove the supernatant, repeat 4 times; add elution to the precipitate Buffer, after mixing, incubate at room temperature for 2 hours; centrifuge at 10,000 × g for 5 min, take the supernatant, and use DOC-TCA-acetone precipitation to enrich plasma membrane phosphorylated proteins; 所述水稻叶片质膜磷酸化蛋白富集缓冲液包含以下组分：终浓度20～50mmol/L的MES，终浓度10～40mmol/L的咪唑，终浓度7～8mol/L的尿素，终浓度0.1～0.15mol/L的天冬氨酸钾，终浓度0.1～0.15mol/L的谷氨酸钠，0.2～4％w/v CHAPS，pH值为6.0～6.3。The rice leaf plasma membrane phosphorylated protein enrichment buffer comprises the following components: MES with a final concentration of 20-50 mmol/L, imidazole with a final concentration of 10-40 mmol/L, urea with a final concentration of 7-8 mol/L, 0.1-0.15mol/L potassium aspartate, final concentration 0.1-0.15mol/L sodium glutamate, 0.2-4% w/v CHAPS, pH value 6.0-6.3. 4.根据权利要求3所述的提取方法，其特征在于，S3中所述DOC-TCA-丙酮沉淀法的具体步骤如下：向上清液中加入2％w/v脱氧胆酸钠混匀，再加入100％w/v TCA，混匀后于冰上放置过夜；于18000×g离心15min，去上清；用25％w/v TCA悬浮沉淀，于4℃下18000×g离心15min，取沉淀；将沉淀悬浮于pH为7.5、含80％丙酮的0.01mol/L Tris-HCl溶液中，于-20℃放置30min以上；于4℃下离心10min，去上清；再将沉淀悬浮于90％丙酮中，于4℃下20000×g离心10min，去上清；将沉淀置于冰上风干，即得水稻叶片质膜磷酸化蛋白质。4. extraction method according to claim 3, is characterized in that, the concrete steps of DOC-TCA-acetone precipitation method described in S3 are as follows: add 2%w/v sodium deoxycholate in supernatant and mix, then Add 100% w/v TCA, mix well and place on ice overnight; centrifuge at 18,000 × g for 15 min, remove the supernatant; suspend the precipitate with 25% w/v TCA, centrifuge at 18,000 × g for 15 min at 4°C, and take the precipitate ; Suspend the precipitate in a 0.01 mol/L Tris-HCl solution containing 80% acetone with a pH of 7.5, and place it at -20 °C for more than 30 min; centrifuge at 4 °C for 10 min, remove the supernatant; then suspend the precipitate in 90% In acetone, centrifuge at 20,000 × g for 10 min at 4°C, remove the supernatant, and place the precipitate on ice to air dry to obtain the phosphorylated protein on the plasma membrane of rice leaves. 5.根据权利要求3所述的提取方法，其特征在于，S1和S2中所述相缓冲液包含以下终浓度的各组分：5mmol/L K2HPO4-KH2PO4缓冲液，250mmol/L蔗糖，3mmol/L KCl，1mmol/L DTT；所述相缓冲液的pH值为7.8。5. extraction method according to claim 3 is characterized in that, the phase buffer described in S1 and S2 comprises each component of following final concentration: 5mmol/LK 2 HPO 4 -KH 2 PO 4 buffer solution, 250mmol/ L sucrose, 3 mmol/L KCl, 1 mmol/L DTT; the pH of the phase buffer was 7.8. 6.根据权利要求3所述的提取方法，其特征在于，S2中所述双水相体系包含以下组分：6.3％w/w Dextran T-500/PEG 3350，终浓度250mmol/L的蔗糖，终浓度3mmol/L的KCl，终浓度5mmol/L的KH2PO4；所述双水相体系的pH值为7.8。6. extraction method according to claim 3, is characterized in that, the two-phase system described in S2 comprises following components: 6.3%w/w Dextran T-500/PEG 3350, the sucrose of final concentration 250mmol/L, KCl with a final concentration of 3 mmol/L, KH 2 PO 4 with a final concentration of 5 mmol/L; the pH value of the aqueous two-phase system is 7.8. 7.根据权利要求3所述的提取方法，其特征在于，S2中所述质膜保存液包含以下终浓度的各组分：5mmol/L BTP-MES缓冲液，250mmol/L蔗糖，2mmol/LKCl，1mmol/L DTT；所述质膜保存液的pH值为7.8。7. extraction method according to claim 3, is characterized in that, described in S2, plasma membrane preservation solution comprises each component of following final concentration: 5mmol/L BTP-MES buffer solution, 250mmol/L sucrose, 2mmol/LKCl , 1 mmol/L DTT; the pH value of the plasma membrane preservation solution is 7.8. 8.根据权利要求3所述的提取方法，其特征在于，S3中所述洗脱缓冲液包含以下终浓度的各组分：0.3mol/L焦磷酸钾，8mol/L尿素；所述洗脱缓冲液的pH值为9.0。8. extraction method according to claim 3 is characterized in that, described in S3, the elution buffer comprises each component of following final concentration: 0.3mol/L potassium pyrophosphate, 8mol/L urea; Described wash-out The pH of the buffer is 9.0.
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