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Low molecular weight wheat glutelin subunit, coding gene and use thereof

A gluten subunit, low molecular weight technology, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as difficulty in separation and analysis

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

AI Technical Summary

Problems solved by technology

LMW-GS is highly heterogeneous and complex in composition, and it is difficult to separate and analyze it, so related research lags far behind HMW-GS

Method used

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  • Low molecular weight wheat glutelin subunit, coding gene and use thereof
  • Low molecular weight wheat glutelin subunit, coding gene and use thereof
  • Low molecular weight wheat glutelin subunit, coding gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 SDS-PAGE Identification of Cultivated Einkorn Wheat TM-M5 Low Molecular Weight Glutenin Subunit LMW-M5

[0022] (1) Plant materials: Cultivated einkorn wheat TM-M5, and common wheat Chinese spring as a control.

[0023] (2) Extraction and sample preparation of gluten

[0024] After crushing half a seed, put the flour into a 1ml centrifuge tube, then add 0.3ml 55% isopropanol, vortex for 1 minute, shake in a water bath at 65°C for 30 minutes, centrifuge at 10,000rpm for 10 minutes, and discard the supernatant. The above-mentioned operation of treating flour was repeated 3 times, and the residual supernatant was sucked off with filter paper, and the gliadin was removed; sugar alcohol, freshly added), mixed well, 65 ℃ water bath fully shaken for 30 minutes; add 0.1ml 55% isopropanol, 0.08M Tris-HCL, pH8.0, containing 1.4% freshly mixed 4-vinylpyridine, mix Shake in a water bath at 65°C for 30 minutes, and centrifuge at 12,000 rpm for 10 minutes; transfer 0.06ml...

Embodiment 2

[0027] Example 2 MALDI-TOF-MS Identification of Cultivated Einkorn Wheat TM-M5 Low Molecular Weight Glutenin Subunit LMW-M5

[0028] (1) Sample preparation

[0029] Crush a seed into powder and put it into a 1ml centrifuge tube, add 0.5ml 70% ethanol, vortex for 1 / 2 to 1 hour, centrifuge at 13000g for 5min, and remove the supernatant. Add 0.5ml of 55% isopropanol, mix well, bathe in 65°C water for 30min, centrifuge at 13000g for 5min, remove the supernatant and suck it up with filter paper, repeat this step 3 times. Add 0.5ml of 55% isopropanol (containing 0.08M Tris-HCL, pH8.0, and 5% β-mercaptoethanol), and mix well in a 65°C water bath for 30min. Take the supernatant and add -20°C pre-cooled acetone (the concentration of acetone reaches 80%), precipitate for 1-2 hours, centrifuge at 13000g for 5min, remove the supernatant, and dry at room temperature. Add 10 μl of 0.5% TFA+50% acetonitrile solution to dissolve.

[0030] (2) Identification by MALDI-TOF-MS

[0031] Use Sh...

Embodiment 3

[0038] Example 3 Isolation and Identification of Low Molecular Weight Glutenin LMW-M5 Gene of Cultivated Einkorn Wheat TM-M5

[0039] (1) Subunit membrane transfer and N-terminal microsequencing (microsequencing)

[0040] The protein band on the SDS gel electrophoresis was excised, recovered and purified, and the purity was checked by capillary electrophoresis, and then transferred to PVDF membrane by Bio-Rad MiniTrans-Blot electrophoresis, and the N-terminal trace was performed on the ABI cLC 491 protein sequencer sequencing.

[0041] (2) AS-PCR primer design

[0042] AS-PCR primers were designed according to the N-terminal sequence of LMW glutenin and published conserved sequences of related genes to amplify the upstream sequence, coding region and downstream sequence of LMW subunit gene. The primer sequences are as follows:

[0043] LMW-3: 5'-GCCTTTCTTGTTTACGGCTG-3'

[0044] LMW-4: 5'-TCAGATTGACATCCACACAAT-3'

[0045] (3) Leaf and seed genomic DNA extraction

[0046] ...

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Abstract

The invention discloses an einkorn wheat low molecular weight glutenin subunit, its encoding gene and use, wherein the einkorn wheat low molecular weight glutenin subunit is the protein of amino acid residue No.2 sequence in the sequence table, or SEQ ID No.2 derived protein by the substitution, deletion or addition of one or several amino acid residual radicals to the SEQ ID No.2 amino acid residual radical sequence, and having the same activity with the SEQ ID No.2 amino acid residual radical sequence. The encoding gene of the einkorn wheat low molecular weight glutenin subunit is one of the following nucleic acid sequences, (1) DNA sequence of SEQ ID No.1 in the sequence table, (2) the polynucleotide of SEQ ID No.2 in the sequence table, (3) DNA sequence having over 90% homology with DNA sequences defined by SEQ ID No.1 in the sequence table and encoding the same functional protein. The gene can be used in the cultivation of transgenic wheats.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a low-molecular-weight glutenin subunit LMW-M5 of cultivated one-seed wheat, its encoding gene and the application of the gene to improve wheat quality through biotechnological methods. Background technique [0002] Wheat is one of the three major food crops with the largest cultivation area, the highest yield, and the widest geographical distribution in the world. It is widely used in food processing and livestock feed. Endosperm storage proteins (prolamins and glutenins) act as the "backbone" in the dough and impart the viscoelasticity needed for bread, noodle, and other food production. Glutenin is composed of high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS), of which LMW-GS accounts for about 80% of the glutenin content. A large number of studies have shown that LMW-GS is highly correlated with wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N15/82
Inventor 晏月明安学丽张倩李小辉张艳贞王爱丽
Owner CAPITAL NORMAL UNIVERSITY
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