CN101781685B - Codominant labeling method for identifying wheat polymer glutenin Dx5 and Dx2 subunits - Google Patents
Codominant labeling method for identifying wheat polymer glutenin Dx5 and Dx2 subunits Download PDFInfo
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- CN101781685B CN101781685B CN 201010107848 CN201010107848A CN101781685B CN 101781685 B CN101781685 B CN 101781685B CN 201010107848 CN201010107848 CN 201010107848 CN 201010107848 A CN201010107848 A CN 201010107848A CN 101781685 B CN101781685 B CN 101781685B
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Abstract
The invention discloses a codominant labeling method for identifying wheat polymer glutenin Dx5 and Dx2 subunits. Based on the characteristics of codominant labeling, PCR amplification primers (upstream primer P1: 5'-CCAGCACAAGTGCAACAA-3', downstream primer P2: 5'-TGTCCTAGCTGCAACGGAG -3') are designed, and the PCR amplification primers are used to identify wheat polymer glutenin Dx5 and Dx2 subunits. The method has the advantages of simplicity, stability, easy implementation and capability of accurately identifying Dx5 high-quality subunits, Dx2 subunits, as well as heterozygous types of the two.
Description
Technical field
The present invention is a kind of pcr amplification primer of designing based on codominant marker's characteristics and the method that is used for differentiating wheat high score gluten Dx5 and Dx2 subunit thereof, belongs to the agricultural biotechnology engineering field.
Background technology
Storage Proteins in Wheat is the important factor that determines wheat quality, wherein prolamine extensibility that formation is rolled into a ball plays a major role, and glutenin is to affect the elastic important factor of dough, determine Bread Baking Quality, especially high-molecular-weight glutelin subunit (HMW-Glu) is more close with the relation of wheat bread baking properties, and Common Wheat Varieties only contains 3～5 HMW gluten subunits usually.The encoding gene of HMW-GS is positioned at common wheat first group of homeologous chromosome 1A, 1B, the long-armed Glu-1 site of 1D, respectively called after Glu-A1, Glu-B1 and Glu-D1.The Glu-D1 site is unanimously to be acknowledged as maximum site (Payne, 1981 of wheat quality contribution; Wang Rui, 1995), this site has widely variation type, and its name is generally carried out " numeral name " (Payne, 1980) according to the SDS-PAGE collection of illustrative plates of HMW-GS.Relatively common are Dx5, Dx2, wherein the Dx5 subunit becomes positive correlation with Bread Baking Quality, is the high quality subunit type of generally acknowledging, Dx2 and Bread Baking Quality are negative correlation.
Be used at present identifying that the ordinary method of wheat high-molecular-weight glutelin subunit is the sex change polypropylene amine gel electrophoresis (SDS-PAGE) of seed storage protein.The shortcoming of this method is accurate not, the 2nd, and experimental procedure is loaded down with trivial details, electrophoresis time is long, is unfavorable for the scale evaluation, and the 3rd, must wait until that seed results of future generation just can detect and screening operation later, have affected the speed of breeding greatly.Afterwards along with the announcement of multiple high-molecular-weight glutelin subunit dna sequence dna, development in recent years has gone out a kind of method of the Allele specific PCR based on the SNPs mark, the method is once once popular, and be considered to a kind of than the more accurate more efficiently method of SDS-PAGE, overcome some shortcomings of SDS-PAGE, it can identify high quality subunit Dx5 by the design special primer.But still there is following shortcoming in this method: the one, and the flexibility ratio of the design of primers of Allele specific PCR is little, can only be according to the Position Design primer in SNPs site; The 2nd, Allele specific PCR reaction system and amplification program are relatively harsher, under general environment, be difficult to strict control, increased difficulty and cost to specific amplification, this is because only there is the difference of a Nucleotide in the loci of equipotential Auele Specific Primer and its opposition; The 3rd, the Allele specific PCR mark only is a kind of dominant marker of high quality subunit Dx5, and this is a very large shortcoming, because it can not distinguish heterozygosis and homozygous genotype, this has brought inconvenience to the MAS breeding work.
Summary of the invention
The present invention is exactly for the problems referred to above, invents out easily row of a kind of simple and stable, the method and the primer thereof that are used for differentiating wheat high score gluten Dx5 and Dx2 subunit with codominant marker's characteristics.
In order to achieve the above object, the present invention has adopted following technical scheme:
The present invention is different according to the middle iteron repetition number of Dx5 subunit and Dx2 subunit dna sequence dna, find relatively that by sequence there is the disappearance of several tumor-necrosis factor glycoproteinss in Dx2 subunit sequence in the iteron of nearly C-end, and at its two ends design primer, so that Dx5 has the PCR product of different sizes with the Dx2 subunit, thereby reach the purpose of differentiating Dx2 and Dx5 heterozygous genes type.
Be used for differentiating that wheat polymer glutenin Dx5 and the used PCR primer of Dx2 subunit are:
Upstream primer P1:5 '-CCAGCACAAGTGCAACAA-3 '
Downstream primer P2:5 '-TGTCCTAGCTGCAACGGAG-3 '
This primer produces the band of 340bp when the Dx5 subunit is increased, produce the band of 320bp when the Dx2 subunit is increased, and the fragment of two sizes need to use PAGE glue that it is made a distinction; When Dx2 and Dx5 heterozygous genes type, can produce simultaneously the band of 320bp and 340bp.
The polymerase chain amplification reaction system that the present invention uses is conventional PCR system, without any need for special PCR reaction instrument and special reagent, the reagent that the PCR reaction instrument of the production of any company and any biological reagent company produce all can use and reach purpose of the present invention.
The designed primer of the present invention is positioned at the central iteron of the nearly C-end of polymer glutenin subunit, in view of the primer in the design of central iteron forms a plurality of primer binding sites easily and causes forming a lot of assorted bands in whole central iteron, therefore of the present invention one large characteristics find primer binding specificity position exactly in the iteron, the target section is accurately increased out, do not contain assorted band, thereby reach aobvious altogether purpose.For the primer that designs in the iteron, the present invention uses higher PCR annealing temperature, to eliminate some assorted bands.
The method of differentiating wheat polymer glutenin Dx5 and Dx2 subunit comprises the following steps:
A. from wheat, extract its genomic dna with CTAB trace extraction method;
B. with primer as claimed in claim 1 genomic dna among the step a is carried out pcr amplification,
PCR reaction conditions, amplification system are as follows: amplification system is the 20ul system:
1 * PCR damping fluid
Template 1.5ul
The magnesium ion 1.2ul of 25mM concentration, system Mg
2+Concentration is 1.5mM
2.5mM concentration dNTP adds 1.2ul, the 0.2mM of system dNTP
0.3ul+0.3ul≤10uM primer≤0.4ul+0.4ul, system primer concentration are 0.2uM
5U/ul rTaq enzyme 0.16ul, system rTaq concentration is 1U/25ul,
Add aseptic deionized water or ddH
2O water is to 20ul
The PCR program is:
1) 94 ℃ of denaturation 5min
2) touchdown PCR program totally 8 each 0.5 ℃ of reductions that circulate of circulation
94 ℃, 45 seconds
66 ℃, 45 seconds
72 ℃, 45 seconds
3) 32 circulations of conventional PCR program
94 ℃, 45 seconds
61.5 ℃, 45 seconds
72 ℃, 45 seconds
4) last amplification: 72 ℃, 10 minutes
C. the amplified production with step b gained carries out electrophoresis detection:
The amplified production of step b gained adopted 6% denaturing polyacrylamide gel carry out electrophoretic analysis, wherein acrylamide: methylene diacrylamide=39: 1,6% denaturing polyacrylamide gel applied sample amount is 5-8ul; Voltage is 50 volts during the beginning electrophoresis, and voltage changes 150 volts into after sample enters gel, totally 60 minutes;
The gained gel adopts 0.1% Silver Nitrate silver to dye 4 minutes, then with 2% NaOH and the development of 1.5% formaldehyde;
D. take a picture, analyze.
Among the present invention, use higher annealing temperature and touchdown PCR program to avoid primer to produce a plurality of non-specific bindings site, this is to have the sequence about general consistent with the tumor-necrosis factor glycoproteins of central iteron in the designed primer, and then the author also advocates and is used less applied sample amount and short silver dyes the time, can eliminate so assorted band to the interference of interpretation of result.
The present invention can differentiate common wheat Dx2 and Dx5 subunit, and using simultaneously this primer can will distinguish simultaneously from Triticum tauschii Dtx5 and Triticum tauschii Dtx2 and common wheat Dx2.
The present invention is simple, efficient, can more accurately differentiate efficiently wheat high score gluten Dx5 and Dx2 subunit.
Description of drawings
Fig. 1 is primer P1+P2 specification figure
Fig. 2 is the PAGE figure of the P1+P2 primer extension product of embodiment
Among Fig. 2: swimming lane 1-48 is river wheat 38, river wheat 42, and the F of river wheat 38 and 42 hybridization of river wheat
2The electrophoresis banding pattern of plant; Wherein: swimming lane 14 is the Dx2 subunit electrophoresis banding pattern of river wheat 38; Swimming lane 14 is the Dx5 subunit electrophoresis banding pattern of river wheat 42; Swimming lane 1,4,6,12,13,17,18,27-29,37,39,40,44-48 are the Dx2 subunit electrophoresis banding pattern that isozygotys; Swimming lane 3,5,20,24,25,35,43 is the Dx5 subunit electrophoresis banding pattern that isozygotys; Swimming lane 2,7,9-11,16,19,21-23,26,30-34,36,38,41,42 are the heterozygosis subunit electrophoresis banding pattern of Dx2 and Dx5; Swimming lane 49 is dna molecular amount size Marker (M).
Embodiment
1. material:
River wheat 38 (containing the Dx2 subunit) and river wheat 42 (containing the Dx5 subunit) hybridization F
2Generation 100 strains
2. method:
2.1 extract its genomic dna with CTAB trace extraction method
2.2 utilize primer P1+P2 that it is increased, amplification system and program are as follows:
Amplification system is the 20ul system:
1 * PCR damping fluid
Template 1.5ul
The magnesium ion 1.2ul of 25mM concentration, system Mg
2+Concentration is 1.5mM
2.5mM concentration dNTP adds 1.2ul, the 0.2mM of system dNTP
0.3ul+0.3ul≤10uM primer≤0.4ul+0.4ul, system primer concentration are 0.2uM
5U/ul rTaq enzyme 0.16ul, system rTaq concentration is 1U/25ul,
Add aseptic deionized water or ddH
2O water is to 20ul
The PCR program is:
1) 94 ℃ of denaturation 5min
2) touchdown PCR program totally 8 each 0.5 ℃ of reductions that circulate of circulation
94 ℃, 45 seconds
66 ℃, 45 seconds
72 ℃, 45 seconds
3) 32 circulations of conventional PCR program
94 ℃, 45 seconds
61.5 ℃, 45 seconds
72 ℃, 45 seconds
4) last amplification: 72 ℃, 10 minutes
The amplified production of above-mentioned steps gained adopted 6% denaturing polyacrylamide gel carry out electrophoretic analysis, wherein acrylamide: methylene diacrylamide=39: 1,6% denaturing polyacrylamide gel applied sample amount is 5-8ul; Voltage is 50 volts during the beginning electrophoresis, and voltage changes 150 volts into after sample enters gel, totally 60 minutes;
The gained gel adopts 0.1% Silver Nitrate silver to dye 4 minutes, then with 2% NaOH and the development of 1.5% formaldehyde; Take a picture, analyze.
3. result:
Utilize primer P1+P2 to amplify clearly target stripe and see Fig. 2, we can distinguish the plant of Dx5, Dx2 heterozygosis and the plant of isozygotying from figure, the plant that only has the 320bp fragment contains the Dx2 subunit that isozygotys, the plant that only has the 340bp fragment contains the Dx5 subunit that isozygotys, the plant that has simultaneously two bands then is heterozygous, and the chi square test result meets (Dx2: Dx5: Dx2/Dx5=1: 1: 2).The method can identify the Dx5 high quality subunit accurately, also can identify Dx2 subunit and both heterozygous simultaneously; And the special AS-PCR primer of classical Dx5 high quality subunit of at present widespread use can only detect having or not of Dx5 high quality subunit, the fubaritic heterozygous that goes out Dx5 and Dx2 subunit.
Claims (4)
1. primer of be used for distinguishing wheat polymer glutenin Dx5 and Dx2 subunit, it is characterized in that: its sequence of described primer is as follows:
Upstream primer P1:5 '-CCAGCACAAGTGCAACAA-3 '
Downstream primer P2:5 '-TGTCCTAGCTGCAACGGAG-3 '.
2. method of distinguishing wheat polymer glutenin Dx5 and Dx2 subunit, it is characterized in that: different according to the middle iteron repetition number of Dx5 subunit and Dx2 subunit dna sequence dna, find relatively that by sequence there is the disappearance of several tumor-necrosis factor glycoproteinss in Dx2 subunit sequence in the iteron of nearly C-end, and at its two ends design primer, so that Dx5 has different big or small PCR products with the Dx2 subunit, thereby distinguish Dx2 and Dx5 heterozygous genes type, its sequence of described primer is as follows: upstream primer P1:5 '-CCAGCACAAGTGCAACAA-3 ' downstream primer P2:5 '-TGTCCTAGCTGCAACGGAG-3 '.
3. method according to claim 2 is characterized in that: comprise the following steps:
A. from wheat, extract its genomic dna with CTAB trace extraction method;
B. with described primer the genomic dna among the step a is carried out pcr amplification,
PCR reaction conditions, amplification system are as follows: amplification system is the 20ul system:
1 * PCR damping fluid
Template 1.5ul
The magnesium ion 1.2ul of 25mM concentration, system Mg
2+Concentration is 1.5mM
2.5mM concentration dNTP adds 1.2ul,
0.3ul+0.3ul≤10uM primer≤0.4ul+0.4ul,
5U/ul rTaq enzyme 0.16ul, system rTaq concentration is 1U/25ul,
Add aseptic deionized water or ddH
2O water to 20 ul
The PCR program is:
1) 94 ℃ of denaturation 5min
2) touchdown PCR program totally 8 each 0.5 ℃ of reductions that circulate of circulation
94 ℃, 45 seconds
66 ℃, 45 seconds
72 ℃, 45 seconds
3) 32 circulations of conventional PCR program
94 ℃, 45 seconds
61.5 ℃, 45 seconds
72 ℃, 45 seconds
4) final step amplification: 72 ℃, 10 minutes
C. the amplified production with step b gained carries out electrophoresis detection:
The amplified production of step b gained adopted 6% denaturing polyacrylamide gel carry out electrophoretic analysis, wherein acrylamide: methylene diacrylamide=39:1,6% denaturing polyacrylamide gel applied sample amount is 5-8 ul; Voltage is 50 volts during the beginning electrophoresis, and voltage changes 150 volts into after sample enters gel, totally 60 minutes;
The gained gel adopts 0.1% Silver Nitrate silver to dye 4 minutes, then with 2% NaOH and the development of 1.5% formaldehyde;
D. take a picture, analyze.
4. according to claim 2 or the method for 3 described differentiation wheat polymer glutenin Dx5 and Dx2 subunit, it is characterized in that: pass through pcr amplification reaction, this primer produces the band of 340bp when the Dx5 subunit is increased, produce the band of 320bp when the Dx2 subunit is increased, the fragment of two sizes need to use PAGE glue that it is made a distinction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911453A (en) * | 2014-04-14 | 2014-07-09 | 安徽农业大学 | Molecular identification method for common wheat glutenin subunit Dx2.2 gene and special primer thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048048B (en) * | 2016-07-18 | 2019-07-16 | 山东农业大学 | A kind of LAMP rapid detection method of wheat high-molecular-weight glutelin subunit 1Dx5 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410433A (en) * | 2002-09-12 | 2003-04-16 | 复旦大学 | Specific augmentation primer and its method of labelling wheat macromolecular glutelin subunit |
| CN101303292A (en) * | 2008-06-17 | 2008-11-12 | 南京农业大学 | Method for measuring wheat high molecular weight gluten subunit |
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2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410433A (en) * | 2002-09-12 | 2003-04-16 | 复旦大学 | Specific augmentation primer and its method of labelling wheat macromolecular glutelin subunit |
| CN101303292A (en) * | 2008-06-17 | 2008-11-12 | 南京农业大学 | Method for measuring wheat high molecular weight gluten subunit |
Non-Patent Citations (3)
| Title |
|---|
| R.D Ovidio等.PCR analysis to distinguish between alleles of a member of a multigene family correlated with wheat bread-making quality.《Theor Appl Genet》.1994,(第88期),第759－763页. * |
| 杨恩年.六倍体普通小麦高分子量谷蛋白亚基Glu-A1和Glu-B1共同缺失材料研究初报.《西南农业学报》.2007,第20卷(第2期),第293-295页. * |
| 魏会廷等.节节麦DNA指纹关系所揭示的古代中国与西方农业技术交流.《自然科学进展》.2008,第18卷(第9期),第987－993页. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911453A (en) * | 2014-04-14 | 2014-07-09 | 安徽农业大学 | Molecular identification method for common wheat glutenin subunit Dx2.2 gene and special primer thereof |
| CN103911453B (en) * | 2014-04-14 | 2015-10-28 | 安徽农业大学 | The molecular assay method of common wheat glutenin subunit Dx2.2 gene and primer special |
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