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Special primer for identifying which variety of Glu-A3 protein subunit is carried in wheat as well as application thereof

A wheat and protein technology, applied in the fields of application, plant gene improvement, recombinant DNA technology, etc., can solve the problems of lack of simple and effective identification methods, limitations of LMW-GS analysis and utilization, etc., to reduce breeding costs and speed up breeding , the effect of simple operation

Inactive Publication Date: 2009-11-04
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The lack of simple and effective identification methods limits the analysis and utilization of LMW-GS

Method used

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  • Special primer for identifying which variety of Glu-A3 protein subunit is carried in wheat as well as application thereof
  • Special primer for identifying which variety of Glu-A3 protein subunit is carried in wheat as well as application thereof
  • Special primer for identifying which variety of Glu-A3 protein subunit is carried in wheat as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Obtaining the Glu-A3 Site Gene of Common Wheat Low Molecular Weight Glutenin

[0035] The wheat varieties used are listed in Table 1.

[0036] Table 1 Wheat varieties used for cloning Glu-A3 locus genes and analyzing Glu-A3 subunit-specific markers

[0037]

[0038] The seven wheat varieties in Table 1 are all wheat containing the identified Glu-A3 protein subunit (Vawser MJ, Cornish GB, Shepherd KW (2002) Rheological dough properties of Aroonisolines differing in glutenin subunit composition.In: Cereals 2002, Proceedings of the 52nd Australian Cereal Chemistry Conference. Christchurch, New Zealand. (Eds CK Black, JF Panozzo, CW Wrigley, IL Batey and N Larsen) pp.53-58. (Cereal Chemistry Division, Royal Australian Chemical Institute)).

[0039] According to the Glu-A3 site related sequence on GenBank (GENBANK ACCESSION NO.EU189087, AB062868, FJ441117 and EF426565), design 3 pairs of primers as follows:

[0040] Primer pair A: P1 (upstream primer): 5'-AAA...

Embodiment 2

[0054] Example 2. Obtainment of Glu-A3 Subunit Specific Marker of Common Wheat Low Molecular Weight Glutenin

[0055] According to the corresponding relationship between the 3 Glu-A3 genes and Glu-A3 protein subunits obtained above, after sequence alignment, 7 pairs of specific primer pairs for recognizing Glu-A3 protein subunits were designed according to the differences between gene allelic variations , see Table 2.

[0056] Table 2 Specific primer pairs for identifying Glu-A3 protein subunits

[0057]

[0058] The genomic DNA of wheat in Table 1 was amplified by PCR using the above 7 pairs of primers respectively.

[0059] The PCR reaction system was as follows: 50 ng of template DNA, 1 U of Taq enzyme (TaKaRa), upstream and downstream primers (5 μmol L -1 ) each 1.2μl, dNTP (2.5mmol·L -1 , TaKaRa) 0.8 μl, 10×PCR buffer 2 μl, supplement the reaction system with sterile distilled water to 20 μl.

[0060] PCR reaction program: 94°C for 5min; then 94°C for 35s, 60°C for...

Embodiment 3、 7

[0063] Embodiment 3, application and result verification of seven pairs of specific primer pairs

[0064] In order to further verify the practicability of 7 pairs of specific primer pairs developed, 7 pairs of primers were used to detect 121 wheat materials (numbering 8-128) and 7 materials (numbering 1-7) used for cloning genes in Example 1, The results are shown in Table 3. The Glu-A3 site protein subunits of these materials have been analyzed by SDS-PAGE, so the detection results of 7 pairs of specific primer pairs can be verified. Figure 8 The Glu-A3, Glu-B3 and Glu-D3 subunit results of some materials were detected for SDS-PAGE. Figure 8 Medium, 1:21; 2:26; 3:30; 4: Pavón (control); 5:8; 6:11; 7:17; 8:19; 9:31; 10:90; 11:18; 12 : 24; 13: 25; 14: 0pata (control); the above numbers refer to the numbers in Table 3. The control is to provide a reference position of the corresponding band.

[0065] Table 3 Verification of Glu-A3 protein subunit-specific STS labeling

[...

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Abstract

The invention discloses a special primer for identifying which type of Glu-A3 protein subunit is carried in wheat as well as an application thereof. The provided special primer comprises at least one pair of the following seven pairs of primers: the primer pair constituted by nucleotides shown in sequence 18 and 20; the primer pair constituted by the nucleotides shown in sequence 19 and 21; the primer pair constituted by the nucleotides shown in sequence 18 and 22; the primer pair constituted by the nucleotides shown in sequence 19 and 23; the primer pair constituted by the nucleotides shown in sequence 18 and 24; the primer pair constituted by the nucleotides shown in sequence 18 and 25; and the primer pair constituted by the nucleotides shown in sequence 18 and 26. The utilization of the special primer, a reagent box or a method of the invention can screen the variety of the wheat at the early stage of development, thereby optimizing the hybrid combination, accelerating the breeding speed and reducing the breeding cost. The method has simple operation, stability, reliability and low cost, thereby being applicable to popularization and having important significance for researching the related characters of the wheat quality and improving the wheat quality. The invention will play an important role in wheat quality breeding.

Description

technical field [0001] The invention relates to a special primer for identifying which Glu-A3 protein subunit variety is carried by wheat and an application thereof. Background technique [0002] Wheat storage proteins mainly include glutenin and gliadin. The difference between the two is that the former can form a multi-chain structure through intermolecular disulfide bonds, while the latter exists in the form of monomeric subunits. Glutenin accounts for about 35-45% of the seed storage protein and is one of the main components of gluten. The disulfide bonds between the peptide chains of glutenin and the disulfide bonds in the molecule can combine with each other, and its protein is mostly composed of polar amino acids, which can easily form macromolecular aggregates (Polymer), making the dough have a certain degree of elasticity. Good elasticity and extensibility are the basis for making high-quality bread, so selection of high-quality HMW-GS and LMW-GS is one of the mai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11A01H1/04
Inventor 何中虎王林海夏先春
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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