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Acidic capillary electrophoresis identification method for high molecular weight glutelin subunit of wheat

A technology of capillary electrophoresis and gluten subunit, which is applied in the direction of measuring device, material separation, analysis material, etc., can solve the problems of high cost, high analysis cost, labor and time-consuming, etc., and achieves easy application, low sample consumption, high resolution high degree of effect

Inactive Publication Date: 2007-09-12
CAPITAL NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, although the improvement of wheat quality through genetic engineering and molecular marker-assisted selection has shown great application prospects, these methods are often expensive, high in analysis costs, labor-intensive and time-consuming, and the genetic engineering and The molecular marker-assisted selection method is still far from practical application, and it is still difficult to meet the requirements of quality breeding work

Method used

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  • Acidic capillary electrophoresis identification method for high molecular weight glutelin subunit of wheat
  • Acidic capillary electrophoresis identification method for high molecular weight glutelin subunit of wheat
  • Acidic capillary electrophoresis identification method for high molecular weight glutelin subunit of wheat

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1. Material sampling

[0051] The seeds of the wheat variety Hope were used as test materials, and 10-15 mg of the endosperm-free end of the seeds were randomly selected for gluten extraction and capillary electrophoresis.

[0052] 2. Sample Preparation

[0053] High molecular weight glutenin subunit (HMW-GS) extraction method:

[0054] Alternative reagents:

[0055] A. 50% n-propanol

[0056] B. 50% n-propanol + 1% DTT

[0057] C. Acetone (analytical pure)

[0058] D. 20% acetonitrile + 0.1% trifluoroacetic acid (TFA)

[0059] Operation method:

[0060] (1) Take a single seed, cut off the endosperm-free end (about 10-15mg) with a blade, accurately weigh it, crush it into fine powder with sulfuric acid paper and a small hammer, and put it into a 1.5ml centrifuge tube;

[0061] (2) Add 1ml of 50% n-propanol, extract in a water bath at 45°C for 10 minutes, centrifuge at 10000rpm for 5 minutes, and remove the supernatant;

[0062] (3) Repeat the above steps three t...

Embodiment 2

[0109] 1. Material sampling

[0110] The seeds of wheat varieties China Spring, Hope and Durum wheat were used as test materials respectively, and 10-15 mg of the endosperm-free end of the seeds were randomly selected for gluten extraction and capillary electrophoresis.

[0111] 2. Sample Preparation

[0112] High molecular weight glutenin subunit (HMW-GS) extraction method:

[0113] The selected spare reagents and operation methods are the same as in Example 1, and the obtained HMW-GS precipitate can be fully dissolved by adding 20% ​​acetonitrile + 0.1% w / v trifluoroacetic acid (TFA) in proportion (w / v: 1mg+10μl) , Capillary electrophoresis analysis can be performed after centrifugation; the precipitate can also be stored at -20°C, and dissolved when needed for capillary electrophoresis analysis.

[0114] 3. High performance capillary electrophoresis (HPCE)

[0115] equipment

[0116] High-efficiency capillary electrophoresis system (equipped with software for system ope...

Embodiment 3

[0157] 1. Material sampling

[0158] The seeds of club wheat Club20 and bread wheat variety Kontrast were used as test materials respectively, and 10-15 mg of the endosperm-free end of the seeds were randomly selected for gluten extraction and capillary electrophoresis. Then, the samples prepared by mixing the high molecular weight glutenin subunits extracted from the seeds of club wheat Club20 and Kontrast in a ratio of 1:1 were used as test materials.

[0159] 2. Sample Preparation

[0160] High molecular weight glutenin subunit (HMW-GS) extraction method:

[0161] The selected spare reagents and operation method are the same as in Example 1, and the obtained HMW-GS precipitate can be fully dissolved by adding 20% ​​acetonitrile+0.1% w / v trifluoroacetic acid (TFA) in proportion (w / v: 1 mg+10 μl) , Capillary electrophoresis analysis can be performed after centrifugation; the precipitate can also be stored at -20°C, and dissolved when needed for capillary electrophoresis ana...

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Abstract

This invention relates to a determination method for wheat high molecular weight glutelin subunit, especially a method through technique of acid capillary electrophoresis. The key emphasis in current wheat breeding work is to improve the HMW-GS component, especially to improve the frequency of excellent high molecular weight glutelin subunit or subunit pairs The combination of current usual breeding method with label assistance selection techniques is the main means for wheat breeding improvement, wherein the rapid, microscale and accurate determination of excellent protein subunit for generation in the early hybrid time is the key to improve the breeding efficiency. The main task of this invention is to provide the buffer type and additives and condition parameter of acid capillary electrophoresis and clean-up procedures of capillary.

Description

technical field [0001] The invention relates to a method for identifying wheat high-molecular-weight glutenin subunits, in particular to a method for identifying wheat high-molecular-weight glutenin subunits through acid capillary electrophoresis (A-CE) technology. Background technique [0002] Wheat seed storage proteins are mainly composed of gliadins and glutenins, which are highly heterogeneous and complex in composition. The molecular weight of gliadin is between 30,000-80,000 Daltons. Under the condition of acidic gel electrophoresis, a variety can separate 15-30 components, which can be divided into α, β, γ and ω according to their relative mobility. Four gliadins accounted for 25%, 30%, 30% and 15% of the total amount respectively. Glutenin includes high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS), which account for about 10% and 30% of the total protein content of endosperm respectively. [0003] A large number of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447G01N30/06G01N30/28G01N30/38
Inventor 晏月明余建中姜怡安学丽胡英考
Owner CAPITAL NORMAL UNIVERSITY
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