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Mass spectrum method for identifying low-molecular-weight glutenin subunit of wheat

A glutenin subunit and low molecular weight technology, applied in the field of life science proteomics, can solve the problems of complicated operation, low resolution of SDS-PAGE, hysteresis, etc., and achieve the effect of simple extraction method, long preparation time and rapid separation

Inactive Publication Date: 2006-07-12
CAPITAL NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, due to the lack of effective separation methods for a long time, compared with HMW-GS, the research on LMW-GS at home and abroad is relatively lagging behind. Due to the low resolution and complicated operation of the conventional separation method SDS-PAGE, the obtained molecular weight is often compared with that of HMW-GS. The actual molecular weight is quite different, so it is urgent to establish a new and more effective separation and identification technology

Method used

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  • Mass spectrum method for identifying low-molecular-weight glutenin subunit of wheat
  • Mass spectrum method for identifying low-molecular-weight glutenin subunit of wheat
  • Mass spectrum method for identifying low-molecular-weight glutenin subunit of wheat

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The SDS-PAGE identification of embodiment 1 wheat low molecular weight glutenin subunit

[0035] (1) Plant material: wheat variety "Jing 411".

[0036] (2) Extraction of LMW-GS

[0037] Crush 20mg of seeds into powder and put them into a 1.5ml centrifuge tube, add 150μl of 70% ethanol, vortex for 30-60min, centrifuge at 10000g for 10min, and remove the supernatant. Add 250 μl of isopropanol, bathe in water at 65°C for 30 minutes, centrifuge at 10,000 g for 10 minutes, remove the supernatant and dry it with filter paper, repeat 3 times. Add 0.1 ml of 50% isopropanol (containing 80 mM Tris-HCl [pH 8.0] + 1% DTT) and vortex to mix, and bathe in water at 65° C. for 30 min. Add 0.1 ml of 50% isopropanol (containing 80 mM Tris-HCl [pH8.0] + 1.4% 4-vinylpyridine) and vortex to mix, bathe in 65° C. for 30 min, and centrifuge at 12000 g for 15 min. The supernatant was precipitated with 40% acetone at room temperature for 3 hours, centrifuged at 12000g for 15min, and the super...

Embodiment 2

[0041] The MALDI-TOF-MS identification of embodiment 2LMW-GS

[0042] (1) Plant material

[0043] Hexaploid bread wheat (Triticum aestivum L., AABBDD): variety "Jing 411".

[0044] Tetraploid wild emmer wheat (Triticum dicoccoides, AABB): YS-5, YS-13

[0045] Diploid Aegilops tauschii (DD): TD121, TD128, TD132

[0046] Diploid cultivation of einkorn wheat (Triticum monococcum L., A m A m ): TM1, TM3

[0047] (2) Preparation of samples for mass spectrometry analysis

[0048] Crush 20mg of seeds into powder and put them into a 1.5ml centrifuge tube, add 100μl of 0.5M NaCl solution, vortex for 30-60min, centrifuge at 5000g for 10min, and remove the supernatant. Add ddH 2 O 200μl, vortex for 30min, centrifuge at 5000g for 10min, remove the supernatant, repeat 3 times. Add 0.4ml of 50% n-propanol, vortex mix, place at room temperature for 30min, centrifuge at 12000g for 5min, remove the supernatant, and suck it up with filter paper. This step is repeated 3 times. Add 0.2ml...

Embodiment 3

[0059] Example 3 Application of low molecular weight glutenin submatrix spectrometry identification method

[0060] (1) LMW-GS characterization and identification of wheat varieties

[0061] Common wheat is an allohexaploid species (2n=6x=42), and there are many rich related germplasm resources, including diploid A. Among these closely related species, LMW-GS is very rich in variation, contains a large number of high-quality candidate genes, and is an important genetic resource for quality improvement of bread wheat. Therefore effective means of identification is the key to make full use of these resources. At the same time, the LMW-GS map can be used as a reliable marker for variety identification, which is of great significance for marker-assisted selection in breeding and protection of variety rights. Using the established LMW-GS mass spectrometry identification method, the low molecular weight glutenin subunits of wheat with different ploidy were identified, and a high-r...

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Abstract

The invention discloses a precise mass spectrum method to identify wheat low-molecular-weight glutelin subunit, which comprises: (1) to prepare the sample, using 35-45% acetone to deposit protein extract and centrifuge, selecting supernatant, and using 75-85% acetone to deposit the glutelin subunit; (2) using dissolution liquid to dissolve the dissolution; wherein, the dissolution liquid comprises 15-25v% acetonitrile, 0.1v% TFA, and water.

Description

technical field [0001] The invention belongs to the technical field of life science proteome, and particularly relates to the accurate identification of the molecular weight of wheat low molecular weight glutenin subunits and the further characterization of glutenin subunits based on the technical method. Background technique [0002] Wheat is an important crop with the largest planting area, the highest yield, and the widest geographical distribution in the world. Its planting area and output account for about 30% of cereal crops, and it is widely used in food processing and livestock breeding. Wheat seeds contain gluten protein that is lacking in other food crops, and can be used to make a variety of special foods such as bread, noodles, biscuits, pastries and steamed buns. Therefore, the composition and content of seed protein largely determine the quality of wheat processing. [0003] Osborne (1907) classified wheat seed proteins into the following four categories accor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N30/06G01N30/02G01N23/00
Inventor 晏月明张倩李巧云安学丽张艳贞王爱丽
Owner CAPITAL NORMAL UNIVERSITY
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