Mass spectrometry method for identifying low molecular weight glutenin subunit allelic variation

A glutenin subunit, low molecular weight technology, applied in the field of life science proteome, can solve the problems of complex, difficult to distinguish, and small molecular weight of Glu-3 allelic variant subunits, and achieve convenient variety identification and genetic breeding, high detection Scope, effect of small sample size

Inactive Publication Date: 2009-10-28
CAPITAL NORMAL UNIVERSITY
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Problems solved by technology

[0007] SDS-PAGE is a routine method for analyzing LMW-GS components, but due to the large copy number of LMW-GS, the allelic variation subunit of Glu-3 is relatively complex and has a small molecular weight, which is often associated with alcohol in the SDS-PAGE profile. The overlapping of dissolved proteins is difficult to distinguish and other reasons, making the related research of LMW-GS far lower than that of HMW-GS

Method used

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  • Mass spectrometry method for identifying low molecular weight glutenin subunit allelic variation
  • Mass spectrometry method for identifying low molecular weight glutenin subunit allelic variation
  • Mass spectrometry method for identifying low molecular weight glutenin subunit allelic variation

Examples

Experimental program
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Embodiment 1

[0077] Example 1, MALDI-TOF-MS identification method and technical system optimization of LMW-GS

[0078] 1. Plant material

[0079] Common wheat (Triticum aestivum L., AABBDD, 2n=6x=42) D1-13(98Y033) (from Adelaide Grain Quality Research Laboratory, Australia, the following examples use this wheat as an example to identify LMW-GS Allelic variation can also be used in other existing wheat varieties).

[0080] 2. Extraction and loading method of LMW-GS

[0081] 1) Put 15 mg of common wheat (Triticum aestivum L., AABBDD, 2n=6x=42) D1-13 (98Y033) flour into a 1 ml centrifuge tube, add 1 ml of 70% ethanol by volume, and vortex at room temperature for 30 minutes, then centrifuged at 10000rpm for 5min, and removed the supernatant;

[0082] 2) Add 1ml of isopropanol solution with a volume percentage of 55% to the precipitate obtained in the above step (1), mix well, incubate at 65°C for 50min, then centrifuge at 10000rpm / min for 5min, remove the supernatant; repeat this step for 3...

Embodiment 2

[0104] Example 2. MALDI-TOF-MS identification of allelic variation at the Glu-3 site of LMW-GS

[0105] 1. Plant materials (see Table 1-5 for details)

[0106] (1) Wheat LMW-GS near-isogenic line material I, a total of 19 copies (Table 2);

[0107] (2) Wheat LMW-GS standard variety II, a total of 22 copies (Table 3);

[0108] (3) Wheat LMW-GS near-isogenic line material III, a total of 76 copies (Table 4);

[0109] (4) Wheat LMW-GS near-isogenic line material IV, a total of 96 copies (Table 5);

[0110] (5) Hexaploid lacking tetrasomy material V of wheat LMW-GS, 28 copies in total (Table 6).

[0111] 2. The extraction and sample loading methods of LMW-GS and the parameter settings of the mass spectrometer are basically the same as those in Example 1. The difference is: the temperature bath condition in the extraction step 2) is 60° C. for 60 minutes; the temperature bath condition for step 3) is 50° C. for 50 minutes; the temperature bath condition for step 4) is 60° C. fo...

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Abstract

The invention discloses a mass spectrometry method for identifying low molecular weight glutenin subunit allelic variation. The method uses matrix-assisted laser desorption-ionization time of flight mass spectrum for identifying the low molecular weight glutenin subunit and determining the allelic variation of the low molecular weight glutenin subunit according to the characteristic peak of protein. The method can be utilized for conducting accurate molecular weight determination on LMW-GS, and the standard mass spectral data of the matrix-assisted laser desorption-ionization time of flight mass spectrum (MALDI-TOF-MS) protein characteristic peak is utilized for identifying the allelic variation of LMW-GS. By the method, the molecular weight of wheat LMW-GS and the composition of LMW-GS allelic variation subunits of different types of wheat can be determined quickly and accurately, which provides powerful technological means for further research on the genetic characteristic and biochemical characteristic of LMW-GS and the contribution of LMW-GS to the processing quality of wheat, thereby laying the foundation for the intensive research on wheat seed proteome.

Description

technical field [0001] The invention relates to the technical field of life science proteome, in particular to a mass spectrometry method for identifying wheat low molecular weight glutenin subunit allelic variation and further characterization of glutenin subunit based on the method. Background technique [0002] Wheat (Triticum aestivum L.) is a major food and feed crop with strong adaptability, wide distribution and multiple uses. It is one of the important crops with the largest cultivated area, highest yield and widest geographical distribution in the world. In China, The status of wheat is second only to rice, and wheat is one of the earliest cultivated plants in the world. Wheat is an important source of plant protein for human beings. It is widely used in food processing and livestock breeding. It is suitable for making bread, noodles, steamed buns, biscuits, cakes and other foods. raw material. The composition and content of seed protein determine the nutritional ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/64G01N1/28G01N1/36
Inventor 晏月明王爱丽高利艳
Owner CAPITAL NORMAL UNIVERSITY
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