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Molecular identification method and special primers of common wheat glutenin subunit bx13 gene

A glutenin and subunit technology, applied in the biological field, can solve the problems of lack of reliable nucleotide sequence, poor repeatability, weak specificity, etc., and achieve the effects of high accuracy, rapid separation and simple method

Inactive Publication Date: 2016-08-03
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

The design of special primers is a key link in PCR technology, but so far, the special primers that can be used to detect HMW-GS include N, Bx17+By18, Bx14+By15, etc. Some special primers for glutenin are weak in specificity and sensitivity. Low, poor repeatability, lack of reliable nucleotide sequence and other reasons, not yet available or still need to be improved

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  • Molecular identification method and special primers of common wheat glutenin subunit bx13 gene
  • Molecular identification method and special primers of common wheat glutenin subunit bx13 gene
  • Molecular identification method and special primers of common wheat glutenin subunit bx13 gene

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Effect test

Embodiment 1

[0031] Embodiment 1, design and synthesis of primers

[0032] Special primers were designed and synthesized based on wheat high molecular weight glutenin subunit Bx13 gene:

[0033] F: CGGGCACGAGACAATACG (sequence 1),

[0034] R: GCCGAGAAGTTGGGAAGTA (sequence 2).

Embodiment 2

[0035] Example 2, Application of Primers in Identification of Wheat High Molecular Weight Glutenin Subunit Bx13 Gene

[0036] 1. Identification of wheat high molecular weight glutenin subunit Bx13 gene by primers

[0037] 1. Genomic DNA extraction

[0038] Wheat variety Wujiangzhuo material Take 3 dry seeds, grind 1-3 wheat dry seeds with a single-grain grinder, put them into a 2ml centrifuge tube, add 900μl SDS extract, bathe in 50-60℃ water for 45-60min, and shake intermittently during the period . Centrifuge at 12000r / min for 10min, absorb 700μl of supernatant, add an equal volume (700μl) of phenol-chloroform-isoamyl alcohol (25:24:1), and invert on ice for 15min. Centrifuge at 12000r / min for 10min, draw 500μl supernatant, add 0.6 times isopropanol (300μl) and 1 / 10 times volume (50μl) NaAc (pH5.2), mix well and let stand on ice for 15min, during Oscillate intermittently. Flocky DNA precipitates appear in the tube, centrifuge at 10000r / min for 10min, discard the supernat...

Embodiment 3

[0055] Embodiment 3, detect the sample to be tested

[0056] Genomic DNA was extracted respectively from wheat Zhongyou 9507, Fengkang No. 2, Changzhi 6406, and Beijing No. 8 according to the method of Example 2, and the upstream primer and downstream primer of the specific molecular marker Bx13 synthesized in Example 1 were used for PCR amplification. The reaction system and reaction procedure are as shown in Example 2-1.

[0057] The result is as figure 1 As shown, a PCR product of 1167bp was obtained in wheat Zhongyou 9507, which proved to contain the Bx13 gene. The PCR product was sent for sequencing, and it was confirmed that the homology with the NCBI system subunit Bx13 gene sequence (FM955452) reached 97%, indicating that the PCR product was Bx13 gene, the method of the present invention is correct;

[0058] A PCR product of 1161bp was obtained in Fengkang No. 2, which proved to contain the Bx13 gene. The PCR product was sent for sequencing, and it was confirmed that...

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Abstract

The invention discloses a molecular identification method of a glutenin subunit Bx13 gene of common wheat and a special primer for the molecular identification method. The invention provides the primer for identification or assisted identification of the glutenin subunit Bx13 gene. The primer is composed of a single-chain DNA shown by sequence 1 in a sequence table and a single-chain DNA shown by sequence 2 in the sequence table. The glutenin subunit Bx13 gene is from common wheat Triticum aestivum L. Proved by an experiment, the invention provides a flux practical testing method of a molecular marker of wheat quality characters and a special primer. The special primer is applied to testing the subunit Bx13 genes of 215 materials in Chinese mini-core collections, and a result shows that the special primer is high in accuracy, good in repeatability, strong in specificity, simple in method, rapid in separation and high in resolution and testing sensitivity without a toxic reagent; and reliable information can be provided for parent selection of wheat quality breeding and excavation and utilization of favorable genes of wheat.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a molecular identification method and special primers for common wheat glutenin subunit Bx13 gene. Background technique [0002] The glutenin content of wheat determines the dough strength and gluten elasticity of wheat, and affects the processing quality of wheat (Figueroa et al. 2009). Studies have shown that the content of high molecular weight glutenin has a more pronounced effect on the rheological properties of bread than gliadin and low molecular weight glutenin. The high and low molecular weight glutenin subunits of wheat form glutenin through disulfide bonds, and its content is closely related to wheat quality. The key has a decisive impact on the good baking quality of wheat (Shewry et al.1989), directly determines the overall quality of bread, and its allelic variation can explain 45%-70% of the variation in bread baking quality of European wheat varieties (Br...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 刘莉马传喜司红起
Owner ANHUI AGRICULTURAL UNIVERSITY
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