A method for the analysis of pulp proteome in muskmelon

A thin-skinned melon and proteome technology, which is applied in the field of proteome analysis of horticultural crops, can solve problems such as lack of research, and achieve good repeatability, simple operation, and clear maps

Inactive Publication Date: 2016-08-17
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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.

Method used

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  • A method for the analysis of pulp proteome in muskmelon
  • A method for the analysis of pulp proteome in muskmelon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Thin-skinned muskmelon proteome analysis method of the present invention is specifically as follows:

[0027] 1. Protein extraction

[0028] Weigh about 1 g of the flesh of thin-skinned melon (Jingmi No. 11), grind it into powder in liquid nitrogen, and add 3 ml 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, 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 (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...

Embodiment 2

[0046] 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 the spectrum of gel vertical electrophoresis figure 1 a.

Embodiment 3

[0048] 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 the spectrum of gel vertical electrophoresis figure 1 b.

[0049] 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 ).

[0050] Comparing Examples 1 and 3, it can be seen that when the loading amount is 800 μg protein, the map is clear, there are many protein spots and the gray level is high (see figure 2 ).

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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

technical field [0001] 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 [0002] Melon (Cucumis melo.L) fruit is sweet, rich in sugar, starch, and a small amount of protein, minerals and other vitamins. As a high-quality and high-yielding 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 phosphoryl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447G01N1/28
Inventor 郝敬虹黄雅芳房克凤杨瑞刘超杰韩莹琰杨柳王建立张卿宋婷婷孙中华于同泉王绍辉范双喜
Owner BEIJING UNIV OF AGRI
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