CN103760210A - Analysis method of pulp proteome in cucumis melo.L - Google Patents

Abstract

The invention provides an analysis method of pulp proteome in a cucumis melo.L. The method comprises the following steps: extracting pulp protein in the cucumis melo.L, firstly performing isoelectric focusing electrophoresis, and then performing SDS-PAGE gel vertical electrophoresis. The invention provides an economic, simple, easily-implemented and fast method suitable for the extraction and dimensional electrophoresis of total protein in the pulp of the cucumis melo.L. The protein is extracted through a Tris/phenol extraction-methanol/ammonium acetate precipitation method, the protein in the pulp of the cucumis melo.L can be effectively extracted, and the influences from mass sugar and other organic matters are reduced; at present, the repeated experimental results show that the experimental sample is comprehensive in preparation, the 2-DE electrophoresis operation is simple, the graph is clear, the repeatability is good, the reagent is low in toxicity and the result is reliable, a technical example is provided for the research of proteomes of the cucurbitaceae horticultural plants, and the method is suitable for analysis of the pulp proteome in the cucumis melo.L by the dimensional electrophoresis technology.

Description

A method for the analysis of pulp proteome in muskmelon

technical field

The invention belongs to the field of proteome analysis of horticultural crops, and in particular relates to a method for proteome analysis of pulp in muskmelon with thin skin.

Background technique

Melon ( Cucumis melo . L ) has sweet fruit, rich in sugar, starch, and a small amount of protein, minerals and other vitamins. As a high-quality and high-yield horticultural crop, it has become more and more popular in recent years. However, there are few reports on proteome changes during the growth and development of melon, differential proteomes in different habitats, and proteome changes regulated by exogenous substances. In particular, there is a lack of research on the protein content in the fruit and the effects of various enzymes on different physiological and biochemical reactions in the fruit development process, the protein types, functions and metabolic pathways in the fruit under adverse conditions, and the mechanism of protein phosphorylation. . These are closely related to actual production. Therefore, studying the proteome in melon fruit, combined with the study of metabolome to explore the fruit development model and formulate corresponding regulation measures, will provide a bright prospect for creating high-yield and good agronomic characteristics of melon. .

Contents of the invention

The invention provides a method for analyzing the proteome of the pulp in the thin-skinned muskmelon. Using the method of the invention, the protein with higher purity can be extracted from the pulp of the thin-skinned muskmelon, and a clear and repeatable map can be obtained. More differential protein spots can be detected, which is convenient for mass spectrometry analysis.

The present invention provides a method for analyzing the proteome of the pulp of the thin-skinned melon, which is to extract the total protein in the pulp of the thin-skinned melon, first perform isoelectric focusing electrophoresis, and then conduct SDS-PAGE gel vertical electrophoresis.

Preferably, the protein extraction of the thin-skinned melon is obtained by extracting the pulp of the thin-skinned melon with Tris extract solution/Tris-saturated phenol, and precipitating with a methanol solution containing ammonium acetate.

This method can effectively extract the protein of the pulp in the thin-skinned melon, avoiding the influence of a large amount of sugar and other organic matter.

Further, 3~4 ml Tris extract and 3~4 ml Tris-saturated phenol are needed for every 1g of melon pulp.

Under this ratio, the protein in the sample can be dissolved out to the maximum, reducing the waste of samples and reagents. It is the optimal ratio to extract the total protein in the pulp of melon.

The volume ratio of the Tris extract to Tris-saturated phenol is 1:1.

Preferably, a 24cm gel strip is used in the isoelectric focusing electrophoresis.

The purpose of choosing 24cm gel strips is to better separate proteins, avoid overlapping between protein spots, and maximize the separation of protein spots on the gel.

Further, the loading amount of the isoelectric focusing electrophoresis is 800 μg protein, and the final loading volume is 450 μl.

This loading amount is the optimal loading amount for a 24cm gel strip, so that the protein spots are well focused, without horizontal and vertical stripes, and the separation is thorough, so that a clear protein map can be obtained.

Preferably, after the isoelectric focusing electrophoresis, a gel strip balance step is included, and the gel strip balance is divided into two steps, each step is 20 minutes.

Each step is balanced for 20 minutes, which can ensure that the balance of each step is completely thorough without causing excessive balance.

Preferably, in the vertical electrophoresis of the SDS-PAGE gel, first run at 1W/block for 1h, and after the agarose forms a straight line, adjust to 5W/block and run for 8h.

Select the power of 1W/gel to run the gel within the first 1 hour, so that the protein can be evenly and smoothly transferred from the gel strip to the gel at low power, and then choose a higher power to run the gel to achieve fast and efficient separation of different proteins .

Preferably, after the vertical electrophoresis of the SDS-PAGE gel, the steps of fixation, staining and decolorization are included, and the fixation is washed with pure water, and the staining time is 24 hours.

The invention provides an economical, simple, easy and rapid extraction and two-dimensional electrophoresis method suitable for the total protein in the flesh of the thin-skinned melon. The invention uses the Tris/phenol extraction-methanol ammonium acetate precipitation method to extract protein, which can effectively extract the protein from the flesh of the thin-skinned melon and reduce the influence of a large amount of sugar and other organic matter. At present, repeated experiments have proved that: the experimental sample preparation is complete, the 2-DE electrophoresis operation is simple, the map is clear, the repeatability is good, the toxicity of the reagent is low, and the result is reliable. It can provide a technical example for the proteomics research of cucurbit horticultural crops. A suite of two-dimensional electrophoresis techniques for the analysis of the pulp proteome in melons.

Description of drawings

Figure 1 shows the sample load of 500 μg protein, using gel strips in different pH ranges for isoelectric focusing electrophoresis, and then comparing the spectra after gel vertical electrophoresis; among them, Figure 1A uses pH3-10, 24cm gel strips; 1B uses pH4-7, 24cm strip;

Figure 2 shows the comparison of the spectra after isoelectric focusing electrophoresis and gel vertical electrophoresis with different protein sample amounts when using pH4-7, 24cm gel strips; among them, the sample amount in Figure 2A is 500 μg protein; Figure 2B The loading amount was 800 μg protein.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Unless otherwise specified, "%" is a percentage by mass.

Although thin-skinned melons include many varieties, the components and contents of substances such as sugars, colloids, and phenols in fruits between varieties are similar, so the method of the present invention is applicable to all varieties of thin-skinned muskmelons.

The invention adopts Tris extract solution/Tris saturated phenol extraction-methanol ammonium acetate precipitation method to extract protein. Protein cleavage and quantification. It generally takes 30 hours to perform the first-dimension isoelectric focusing after calculating the sample amount. The first direction is completed, and the rubber strip is balanced. Then start the second dimension SDS-PAGE gel vertical electrophoresis. Finally, the gel was fixed, stained and decolorized to obtain a clear map of protein spots.

Example 1

Thin-skinned muskmelon proteome analysis method of the present invention is specifically as follows:

1. Protein extraction

Weigh about 1g of the flesh of thin-skinned melon (Jingmi No. 11), grind it into powder in liquid nitrogen, and add 3ml of Tris extract to the mortar (Tris extract formula: 50 mmol/L Tris, 50 mmol/L EDTA, 100 mmol/L potassium chloride, 2% DTT, 30% sucrose, 1 mmol/L PMSF, the rest is water), after it melts, continue to grind for 3~4min, transfer to a centrifuge tube, add an equal volume of pH8.0 Tris - Saturated phenol, vortex, centrifuge (10000 × g, 4°C) for 10 min. Take the phenolic layer, add an equal volume of Tris extract to the phenolic layer for extraction twice, add about 5 times the volume of 0.1 mol/L ammonium acetate methanol solution to the phenolic layer, and precipitate at -20°C for more than 2 hours. Centrifuge (15000 × g, 4°C) for 20 min, discard the supernatant, wash the precipitate once with -20°C pre-cooled methanol, then wash twice with acetone (containing 2% DTT, -20°C pre-cooled), and dry at room temperature. The obtained dry powder was stored at -80°C until use.

Sample Lysis

Add 250 μl of lysis solution (7M urea, 2M thiourea, 4% CHAPS, 2% IPG-buffer, 50 mMDTT) to the protein powder of the sample for lysis. After 30KHz ultrasonic solubilization for 20min, stand at room temperature for 2h to fully dissolve the protein. Transfer to a small centrifuge tube and centrifuge (20,000×g) for 20 minutes to further induce dissolution.

2. Protein quantification

Quantification was carried out with the Bradford protein concentration assay kit (Beijing Suolaibao Technology Co., Ltd.).

3. Isoelectric focusing

The 24cm gel strip used in the present invention is pH4-7, the sample volume is 800μg protein, sample solution + hydration solution (7M urea, 2M thiourea, 2% CHAPS, 0.5% IPG buffer, 65 mM DTT) final sample volume for 450 μl. The procedure of isoelectric focusing is shown in Table 1:

Table 1

.

4. Strip balance

The first step is to add 0.2g DTT to 10ml of equilibrium mother solution, pour it into the strip hydration tray after it is completely dissolved, cover it, and balance it on a shaker for 20 minutes.

Rinse the supporting surface of the rubber strip with pure water and perform the second step of balancing. Add 0.3g of iodoacetamide to 10ml of equilibrium mother solution, pour it into the strip hydration tray after completely dissolving, and equilibrate on a shaker for 20min.

5. SDS-PAGE gel vertical electrophoresis

Prepare the gel one day in advance, seal it with plastic wrap, and refrigerate overnight in a refrigerator at 4°C to allow the gel to fully polymerize. After washing the support surface, put the strip on the gel, push it until it contacts the gel surface, inject the agarose sealing solution, insert a hole on one side of the gel, and inject the marker solution into the hole after the agarose solidifies. Put the gel into a vertical electrophoresis tank and add electrode buffer. Put the lid on, run at 1W/block for 1 hour, and adjust to 5W/block for 8 hours after the agarose forms a straight line.

6. Fixation, staining, decolorization

Put the gel in a rectangular box, add 500ml of fixative solution, and fix it on a shaker for 1h. Afterwards, wash with pure water 3 times, each time for 15 minutes. Coomassie brilliant blue staining was performed on a shaker for 24 hours. Decolorize with pure water twice, 10min each time. The three steps of fixation, staining and decolorization are all carried out on a shaker.

After the experiment of the above steps, the obtained two-dimensional electrophoresis pattern has a clear background, no obvious horizontal and vertical stripes and diffuse protein spots, the number of protein spots is large, round, the protein spots are independent, and the separation effect is good. It is suitable for two-way protein in the flesh of melon with thin skin Electrophoretic analysis (Figure 2B).

Example 2

The difference between this example and Example 1 is that the gel strip used in the isoelectric focusing step is a 24 cm strip with a pH of 3-10, the loading amount is 500 μg of protein, and the rest are the same as in Example 1. After isoelectric focusing electrophoresis , see Figure 1A for the spectrum of gel vertical electrophoresis.

Example 3

The difference between this example and Example 1 is that the gel strip used in the isoelectric focusing step is a 24 cm strip with a pH of 4-7, the loading amount is 500 μg of protein, and the rest are the same as in Example 1. After isoelectric focusing electrophoresis , see Figure 1B for the spectrum of gel vertical electrophoresis.

Comparing Example 2 with Example 3, it can be seen that the strip is a 24cm strip with a pH of 4-7, and the protein spots are completely separated, mostly round or oval, without horizontal or vertical lines (see Figure 1).

Comparing Examples 1 and 3, it can be seen that when the sample amount is 800 μg of protein, the spectrum is clear, there are many protein spots and the gray level is high (see Figure 2).

Example 4

The difference between this embodiment and embodiment 1 is:

1. Protein extraction

Weigh about 1g of the flesh of thin-skinned melon (Jingmi No. 10), grind it into powder in liquid nitrogen, and add 4ml of Tris extract to the mortar (Tris extract formula: 50 mmol/L Tris, 50 mmol/L EDTA, 100 mmol/L potassium chloride, 2% DTT, 30% sucrose, 1 mmol/L PMSF, the rest is water), after it melts, continue to grind for 3~4min, transfer to a centrifuge tube, add an equal volume of pH8.0 Tris - Saturated phenol, vortex, centrifuge (10000 × g, 4°C) for 10 min. Take the phenolic layer, add an equal volume of Tris extract to the phenolic layer for extraction twice, add about 5 times the volume of 0.1 mol/L ammonium acetate methanol solution to the phenolic layer, and precipitate at -20°C for more than 2 hours. Centrifuge (15000 × g, 4°C) for 20 min, discard the supernatant, wash the precipitate once with -20°C pre-cooled methanol, then wash twice with acetone (containing 2% DTT, -20°C pre-cooled), and dry at room temperature. The obtained dry powder was stored at -80°C until use.

Sample Lysis

Add 200 μl of lysis solution (7M urea, 2M thiourea, 4% CHAPS, 2% IPG-buffer, 50 mMDTT) to the protein powder of the sample for lysis. After 40KHz ultrasonic solubilization for 20min, stand at room temperature for 2h to fully dissolve the protein. Transfer to a small centrifuge tube and centrifuge (20,000×g) for 20 minutes to further induce dissolution.

All the other steps are the same as in Example 1.

Example 5

The difference between this embodiment and embodiment 1 is:

1. Protein extraction

Weigh about 1g of the flesh of thin-skinned muskmelon (Yumeren), grind it into powder in liquid nitrogen, and add 3.5ml Tris extract to the mortar (Tris extract formula: 50 mmol/L Tris, 50 mmol/L EDTA, 100 mmol/L potassium chloride, 2% DTT, 30% sucrose, 1mmol/L PMSF, the rest is water), after it melts, continue to grind for 3~4min, transfer to a centrifuge tube, add an equal volume of pH8.0 Tris- Saturated phenol, vortex, and centrifuge (10,000 × g, 4°C) for 10 min. Take the phenolic layer, add an equal volume of Tris extract to the phenolic layer for extraction twice, add about 5 times the volume of 0.1 mol/L ammonium acetate methanol solution to the phenolic layer, and precipitate at -20°C for more than 2 hours. Centrifuge (15000 × g, 4°C) for 20 min, discard the supernatant, wash the precipitate once with -20°C pre-cooled methanol, then wash twice with acetone (containing 2% DTT, -20°C pre-cooled), and dry at room temperature. The obtained dry powder was stored at -80°C until use.

Sample Lysis

Add 300 μl of lysis solution (7M urea, 2M thiourea, 4% CHAPS, 2% IPG-buffer, 50 mMDTT) to the protein powder of the sample for lysis. After 20KHz ultrasonic solubilization for 20min, stand at room temperature for 2h to fully dissolve the protein. Transfer to a small centrifuge tube and centrifuge (20,000×g) for 20 minutes to further induce dissolution.

All the other steps are the same as in Example 1.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still The technical solutions recorded in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (9)

1. a pulp proteomic analysis methods in Melon, is characterized in that: be, after pulp gross protein in Melon is extracted, first to carry out isoelectric focusing electrophoresis, then carry out SDS-PAGE gel vertical electrophoresis.

2. method according to claim 1, is characterized in that: described Melon Protein Extraction is by the saturated phenol extraction of the Tris extract/Tris-of pulp in Melon, by the methanol solution precipitation containing ammonium acetate, obtains.

3. method according to claim 2, is characterized in that: pulp needs 3 ~ 4 ml Tris extracts and the saturated phenol of 3 ~ 4 ml Tris-in every 1g Melon.

4. method according to claim 3, is characterized in that: the volume ratio of described Tris extract and the saturated phenol of Tris-is 1:1.

5. method according to claim 1, is characterized in that: in described isoelectric focusing electrophoresis, use 24cm adhesive tape.

6. method according to claim 4, is characterized in that: in described isoelectric focusing electrophoresis, applied sample amount is 800 μ g protein, and whole loading volume is 450 μ l.

7. method according to claim 1, is characterized in that: after described isoelectric focusing electrophoresis, comprise the step of adhesive tape balance, described adhesive tape balance in two steps, every step 20min.

8. method according to claim 1, is characterized in that: in described SDS-PAGE gel vertical electrophoresis, first 1W/piece runs 1h, and agarose forms after straight line, and furnishing 5W/ piece runs 8h.

9. method according to claim 1, is characterized in that: after described SDS-PAGE gel vertical electrophoresis, comprise step fixing, that dye, decolour, after fixing, with pure water, clean, described dyeing time is 24 hours.

Images