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Two-dimensional gel electrophoresis separation method of rice total protein under heavy metal stress

A separation method and heavy metal technology, applied in the field of protein separation and purification

Active Publication Date: 2011-09-07
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Rice is a model plant for proteomics research. Many scholars have optimized rice protein extraction and electrophoresis conditions, but they mainly focus on rice leaves. There are no relevant reports on the extraction of rice root protein under mercury stress.

Method used

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  • Two-dimensional gel electrophoresis separation method of rice total protein under heavy metal stress
  • Two-dimensional gel electrophoresis separation method of rice total protein under heavy metal stress

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 reagent formula

[0024] 30% polyacrylamide stock solution

[0025] Acrylamide 150g

[0026] Methylenebisacrylamide 4g

[0027] Double distilled water to 500ml

[0028] After filtering through a 0.45 μm membrane filter, the brown bottle was stored in a refrigerator at 4°C.

[0029] 1.5M Tris base pH8.8

[0030] Tris base 90.75g

[0031] Double distilled water 400ml

[0032] Adjust the pH to 8.8 with 1M HCl, add double distilled water to make the volume to 500ml. Store in refrigerator at 4°C. 10% SDS

[0033] SDS 10g

[0034] Double distilled water to 100ml

[0035] After mixing, store at room temperature.

[0036] 10% AP (ammonium persulfate)

[0037] AP0.1g

[0038] Double distilled water 1ml

[0039] After dissolving, store in the refrigerator at 4°C, and it is best to use and prepare immediately.

[0040] 10× electrophoresis buffer

[0041] (1×buffer=25mM Tris, 192mM glycine, 0.1% SDS, pH8.3)

[0042] Tris base 30g

[0043] Glycine 144g...

Embodiment 2

[0082] Embodiment 2 test material and paddy rice cultivation

[0083] Using rice Zhonghua 11 (Oryza sativa L. subsp. Japonica) as a material, select plump and healthy rice seeds, treat them with 1% dilute nitric acid for 12 hours to break dormancy, and wash them with distilled water. Then sterilized with 0.5% NaClO solution for 15 minutes, washed with distilled water, and placed in a petri dish covered with wet filter paper (all at 30°C). After 3 days, transfer to the grid for one week of hydroponic culture (pH 5.0-5.6), then culture with 1 / 2 rice nutrient solution for one week, then culture with full nutrient solution, and use HgCl when it grows to the 6-leaf stage 2 deal with.

Embodiment 3

[0084] The extraction of embodiment 3 rice root total protein

[0085] Adopt improved trichloroacetic acid (TCA)-acetone precipitation method, concrete steps are as follows:

[0086] (1) Take 2.0-3.0g of rice material and grind it thoroughly in liquid nitrogen (the mortar can be pre-cooled in a -20°C refrigerator in advance, and then pre-cooled with liquid nitrogen) until it becomes white powder.

[0087] (2) First add 25ml-20°C pre-cooled TCA extract (10%TCA, 0.07%DTT), mix well (use a gun tip to smash the powder and wash down the sample on the wall), put it in a -20°C refrigerator Precipitate in medium for 2h.

[0088] (3) Centrifuge at 20,000×g for 15 min at 4°C, discard the supernatant, and collect the precipitate.

[0089] (4) Add 20ml-20°C pre-cooled sample washing solution (acetone containing 0.07% DTT), mix thoroughly (the powder can be crushed with a tenon), and put it in a -20°C refrigerator for 2 hours.

[0090] (5) Centrifuge at 20,000×g for 15 min at 4°C, disca...

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Abstract

The invention discloses a two-dimensional gel electrophoresis separation method of rice total protein under heavy metal stress. The method comprises the following steps: (a) extracting the coarse total protein of the rice root, washing with the mixed solution of ethyl ether and ethanol, performing freeze drying, dissolving in lysate, heating for 0.5-2 hours with a water bath, centrifuging, takingsupernatant to precipitate with acetone, centrifuging to separate the precipitate, and performing freeze drying; (b) dissolving the precipitate in thickened solution, precipitating with cold acetone under the temperature from -20 DEG C to -15 DEG C, centrifuging to separate the precipitate, repeating the step 2-4 times, performing freeze drying to the precipitate; and (c) dissolving the precipitate obtained in the step (b) in thickened solution, and separating by utilizing two-dimensional gel electrophoresis. In the method disclosed by the invention, surface detergents with different strengths such as urea and thiourea and various chaotropic agents are added, then polyvinyl pyrrolidone (PVP), ethanol and ethyl ether are added to wash the product and remove other impurities; and finally purer protein is obtained through stepwise dissolution, thus the complexity of the protein sample can be reduced and high purity total protein of the rice root can be obtained.

Description

technical field [0001] The invention relates to a method for separating and purifying proteins, in particular to a method for separating rice total proteins under heavy metal stress. Background technique [0002] The problem of heavy metal pollution is becoming more and more serious. At present, my country's sewage irrigation and waste have caused large-scale soil pollution to farmland. Mercury is a non-essential element of plants and an environmental pollutant that has great harm to the ecological environment and human health. After bioaccumulation and food chain amplification, mercury will have more serious consequences (Rodriguez et al., 2003; Zhang and Wong, 2007). As the main crop, rice is widely cultivated in 120 countries and regions in the world. More than half of the world's population uses rice as a staple food, but the global average rice yield is only about 200 kilograms per mu (China News, 2009). The research on heavy metal pollution has been highly valued by ...

Claims

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Application Information

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IPC IPC(8): C07K1/26C07K14/415
Inventor 王飞娟朱诚
Owner CHINA JILIANG UNIV
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