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Method for identifying biomass spectrum of wheat macromolecule weight glutelin subunit

A glutenin subunit and high molecular weight technology, applied in the field of plant proteomics, can solve the problems of lack, HMW-GS mobility molecular weight is not exactly the same size, high protein molecular weight, etc., to achieve high accuracy and relative molecular mass range Wide, simple effect of extraction method

Inactive Publication Date: 2008-06-18
CAPITAL NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although SDS-PAGE is a fast and economical method, there is also a phenomenon that the mobility of HMW-GS is not exactly the same as the actual molecular weight (D'Ovidio et al., 1994), and the HMW-GS estimated by SDS-PAGE The molecular weight of the protein deduced from its coding gene sequence is often too high. In practical applications, the molecular weight of different subunits can only be roughly estimated by the SDS-PAGE method. There is still a lack of a method for quickly determining the precise molecular weight of HMW-GS. method

Method used

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  • Method for identifying biomass spectrum of wheat macromolecule weight glutelin subunit
  • Method for identifying biomass spectrum of wheat macromolecule weight glutelin subunit
  • Method for identifying biomass spectrum of wheat macromolecule weight glutelin subunit

Examples

Experimental program
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Effect test

Embodiment 1

[0035] The SDS-PAGE identification of embodiment 1 wheat high molecular weight glutenin subunit

[0036] (1) Plant material: wheat variety "Jing 411".

[0037] (2) Extraction of HMW-GS:

[0038] Crush 10-20mg 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 13000 g for 15 min, and th...

Embodiment 2

[0042] Example 2 HMW-GS biological mass spectrometry identification method

[0043] (1) Plant material: wheat variety "Jing 411".

[0044] (2) Preparation of samples for mass spectrometry analysis

[0045] Crush 10-20mg of seeds into powder and put them into a 1.5ml centrifuge tube, add 100μl of 0.5M NaCl solution, vortex for 1 / 2 to 1 hour, centrifuge at 5000g for 10min, and remove the supernatant. Add ddH 2 O 200μl, vortex for 1 / 2 hour, centrifuge at 5000g for 10min, remove 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 of 50% n-propanol (containing 80mM Tris-HCl [pH8.5] + 1% DTT), shake and mix well, and bathe in water at 65°C for 30min. Take the supernatant, add acetone until the final concentration reaches 40%, precipitate at room temperature for 3 hours, centrifuge at 13,000 g for ...

Embodiment 3

[0061] Example 3 Application of high molecular weight glutenin subunit biological mass spectrometry identification method

[0062] (1) Accurate molecular weight determination

[0063]Determination of the precise molecular weight of wheat high molecular weight glutenin subunits is an important link in further research on its genetic variation, molecular structure and function. The accurate identification of molecular weight can not only accurately distinguish HMW-GS with different functions, but also understand some structural information of protein subunits, laying the foundation for functional research. At present, the determination of the molecular weight of wheat high molecular weight glutenin subunits mainly uses SDS-PAGE gel electrophoresis, but the relative mobility of some proteins in the SDS system does not have a linear relationship with their molecular weight, so the traditional SDS-PAGE can determine the protein molecular weight. There is a large error in the measu...

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Abstract

The invention discloses a mass spectroscopy to examine the high molecular weight glutenin subunit of wheat, and belongs to the technical field of Proteome of life sciences, which in particular relates to accurate examination of molecular weight of the high molecular weight glutelin subunit of wheat and further characterization of the glutenin subunit based on the technology. The invention comprises the following steps: (1) preparation of biologic mass spectrometric analysis specimens of the high molecular weight glutenin subunit of wheat; (2) dissolving the high molecular weight glutenin subunit with dissolving solution; (3) making use of matrix assisted laser desorption-ionization time of flight mass sepectrometry for examination of the high molecular weight glutenin subunit of wheat. The method has the characteristics of use of few specimens, simple method of extraction, no toxic reagent, quick separation, high distinguishability, high detection sensitivity and so on.

Description

technical field [0001] The invention belongs to the technical field of plant proteomics, and in particular relates to a biological mass spectrometry method for accurate identification of wheat high molecular weight glutenin subunits and their molecular weights and further characterization of glutenin subunits based on the method. Background technique [0002] Wheat is the most widely distributed important food crop in the world. Its endosperm contains gluten protein that is lacking in other food crops, so it can be used to make a variety of special foods such as bread, noodles, biscuits, pastries and steamed buns. Studies have shown that the composition and content of seed protein largely determine the quality of flour processing. Wheat seed proteins can be divided into albumin, globulin, gliadin and glutenin according to their dissolution characteristics, and they form glutenin polymers through disulfide bonds, thus determining the viscoelasticity of dough. Studies have sh...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N33/68
Inventor 晏月明张倩王爱丽安学丽张艳贞
Owner CAPITAL NORMAL UNIVERSITY
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