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Aegilops tauschii high molecular weight glutelin 1Dy12.1t gene and its uses

A high-molecular-weight, goat grass technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of small variation of Glu-D1 site, limited HMW glutenin gene resources, etc.

Inactive Publication Date: 2005-01-26
CAPITAL NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although there are different degrees of allelic variation at each locus, the Glu-D1 locus has less variation, and only 6 alleles were detected in bread wheat (Payne and Lawrence, 1983), so the D genome in common wheat HMW gluten gene resources are very limited

Method used

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  • Aegilops tauschii high molecular weight glutelin 1Dy12.1t gene and its uses
  • Aegilops tauschii high molecular weight glutelin 1Dy12.1t gene and its uses
  • Aegilops tauschii high molecular weight glutelin 1Dy12.1t gene and its uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Goatweed TD-159 High Molecular Weight Glutenin HMW-GS12.1 t Identification of subunits

[0022] (1) Plant material Goat grass TD-159;

[0023] (2) HMW-GS extraction:

[0024] ① SDS-PAGE electrophoresis sample preparation: crush 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, discard clear. The above operation of treating flour was repeated 3 times, and the remaining supernatant was sucked off with filter paper to remove the gliadin; add 0.1ml of 55% isopropanol, 0.08M Tris-HCL, pH8.0, containing 1% dithiothreose Alcohol (DTT, freshly added), mixed well, 65 ° C water bath shake well for 30 minutes; add 0.1ml 55% isopropanol, 0.08M Tris-HCL, pH8.0, containing 1.4% freshly mixed 4-vinylpyridine, Mix and shake in a water bath at 65°C for 30 minutes, centrifuge at 12000rpm for 10 minutes; transfer 0.06ml supernatant t...

Embodiment 2

[0032] Example 2 Goatweed TD-159 High Molecular Weight Glutenin 1Dy12.1 t Identification of genes

[0033] (1) Subunit transfer to membrane and N-terminal sequencing

[0034] The protein spots on the two-dimensional electrophoresis gel were excised, recovered and purified, and the purity was checked by capillary electrophoresis, and then electrotransferred to PVDF membrane by Bio-Rad MiniTrans-Blot for protein N-terminal sequencing.

[0035] (2) AS-PCR primer design

[0036] AS-PCR primers were designed according to the N-terminal sequence of HMW glutenin and published conserved sequences of related genes to amplify the coding region and upstream sequence of y-type HMW subunit gene. Primer structure as image 3 As shown, the primer sequences are as follows:

[0037] ①Coding region PCR primers

[0038] P1: 5″-ggCTAgCCgACAATgCgTCg-3″

[0039] P2: 5″-CTATCACTggCTAgCCgACAATg-3″

[0040] ② Upstream PCR primers

[0041] P3: 5″-gAACATAAgAggTTAAACATAggAggAg-3″

[0042] P4: 5″...

Embodiment 3

[0099] Example 3 Goatweed TD-159 High Molecular Weight Glutenin 1Dy12.1 t Gene expression and identification

[0100] (1) Purification and recovery of PCR products

[0101] Using the genomic DNA of Goatweed T151 as a template, amplify 1Dy12.1 t The PCR product of the gene was electrophoresed in 1% agarose gel (steady voltage 50v) for 1 hour, and the DNA band was excised under ultraviolet light, and the DNA gel purification kit (Agarose Gel DNA Purification Kit, Takara) was used Purify and recover the specific amplified bands.

[0102] (2) Double digestion of PCR product and expression vector PET-30a (Novagen)

[0103] Both the recovered PCR product and the PET-30a plasmid were digested with EcoR I (TaKaRa) and Xho I (TaKaRa), and after 2 hours of digestion, the digested product was recovered with Agarose Gel DNA Purification Kit (TaKaRa).

[0104] (3) Connection and identification

[0105] The double digestion product of the fragment to be inserted and the double digestio...

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Abstract

The invention belongs to biological gene engineering field, especially relates to an Aegilops tauschii high molecular weight glutelin 1Dy12.1#+[t] gene and its uses in biological improvement of wheat quality based on the gene. Glutelin gene is mainly separated using library establishment method and PCR method. In the invention, a new high molecular weight glutelin HMW-GS12.1#+[t] and its gene are separated from Aegilops tauschii using allele specific PCR, single-direction or two dimensional gel electrophoresis and capillary electrophoresis. The gene sequence has high homologue with quality gene 1Dy10, can work in wheat quality improvement.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, in particular to a high molecular weight glutenin 1Dy12.1 of goat grass t Gene and the application of improving wheat quality through biotechnology methods based on this gene. Background technique [0002] Wheat is one of the three main 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 a "skeleton" in the dough and endow bread, noodles, and other foods with the viscoelasticity required for production, in which high molecular weight glutenin subunits (HMW-GS) make up the macromolecule Aggregate, which imparts elasticity to dough (Payne, 1987; Shewry et al., 1992). It is now clear that the coding genes of HMW-GS are located on the three sites Glu-A1, Glu-B1 and Glu-D1 on the long arm of the chrom...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/64C12N15/82
Inventor 晏月明郑继刚胡英考姜怡肖英华余建中
Owner CAPITAL NORMAL UNIVERSITY
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