CN106048048A - LAMP rapid detection method for high molecular weight glutelin subunit 1Dx5 of triticum aestivum L. - Google Patents
LAMP rapid detection method for high molecular weight glutelin subunit 1Dx5 of triticum aestivum L. Download PDFInfo
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Abstract
The invention provides an LAMP rapid detection method for a high molecular weight glutelin subunit 1Dx5 of triticum aestivum L.. A 1Dx5 gene sequence of the triticum aestivum L. published by GenBank is utilized to design an LAMP primer, an LAMP detection system for the high molecular weight glutelin subunit 1Dx5 of the triticum aestivum L. is created, and the reaction condition is optimized. Under the condition that the ratios of the LAMP primers FIP, BIP, F3 and B3 are respectively 0.8[mu]m:0.8[mu]m:0.1[mu]m:0.1[mu]m in a 25[mu]L system, the reaction time of 60 minutes and the reaction temperature of 60 DEG C are the optimal reaction conditions, and the LAMP primer can distinguish the high molecular weight glutelin subunit 1Dx5 of the triticum aestivum L. from the 38 tested triticum aestivum L. materials. The result shows that the created LAMP detection system is simple and rapid, good in sensibility and high in specificity, and the potential on the aspect of detection of the high molecular weight glutelin subunit 1Dx5 of the triticum aestivum L. is huge.
Description
Technical field
The present invention relates to agricultural and technical field of plant quarantine, be specifically related to a kind of wheat high-molecular-weight glutelin subunit
The LAMP detection method of 1Dx5 gene.
Technical background
Semen Tritici aestivi (Triticum aestivum L.) is the most important cereal crops, can be processed into bread, steamed bread,
The food that noodles etc. are different.In Semen Tritici aestivi, protein content and quality are one of most important indexs affecting wheat quality, special
It is not glutenin and gliadin, is that in wheat protein, content is the highest, two hatching egg maximum to wheat processing food effect
White matter.Glutenin is divided into again high-molecular-weight glutelin subunit and low-molecular-weight glutenin subunit, encodes high-molecular-weight glutelin
The gene of subunit is positioned at the first chromosome homology group, and in theory, common wheat has six high-molecular wheat glutelin subunit genes.So
And, it is generally the case that the gluten subunit expressed in Semen Tritici aestivi is 3-5.Allelic variation is generally each high molecular
Gluten subunit, particularly Glu-D1 site is relatively big on the impact of dough characters, is important Quality Gene, such as, 1Dx5+
1Dy10 subunit combinations can significantly improve the elasticity of dough.
Effectively identify and filter out the wheat lines containing excellent protein protomer and be by field good quality wheat breeding material
The important prerequisite of selection-breeding, is also by the important evidence of wheat food processing.At present, high-molecular-weight glutelin subunit in Semen Tritici aestivi
Identify and mainly use the method such as protein electrophoresis, regular-PCR method.It is longer generally to there is the used time in these methods, needs special instrument
Device, it is impossible to realize the problems such as Site Detection, it is impossible to meet the needs of field quick detection.
Wheat Grain Protein is mainly made up of alcohol soluble protein and glutenin, and alcohol soluble protein gives dough extensibility, and
Glutenin makes dough have elasticity.Wheat glutenin subunit accounts for about the 35% of mucedin, mainly by high molecular wheat
Gluten subunit and low molecular weight glutenin subunit composition.Hmw glutenin subunit plays important work to gluten characteristic
With, closely related with the baking properties of Semen Tritici aestivi.Wheat quality is had by wheat high-molecular-weight glutelin subunit 1Dx5 can not
The effect ignored.The new method setting up Rapid identification wheat high-molecular-weight glutelin subunit 1Dx5 can be field excellence wheat breeding
Material screening provides important evidence.At present, domestic there is not yet screens wheat high-molecular-weight glutelin subunit 1Dx5 by LAMP technology
Report, the qualification of the wheat high-molecular-weight glutelin subunit 1Dx5 of accurate Fast Practical has important potential using value.
Summary of the invention
Execute-in-place, quick, accurate, sensitive wheat high-molecular-weight glutelin Asia cannot be realized to solve prior art
The defect of base 1Dx5, the invention provides the LAMP method for quick of a kind of wheat high-molecular-weight glutelin subunit 1Dx5, mesh
Be to provide the LAMP of a kind of quick, accurate, sensitive wheat high-molecular-weight glutelin subunit 1Dx5 facilitating execute-in-place
Detection method.
To achieve these goals, the present invention is by the following technical solutions:
The LAMP detection method of a kind of wheat high-molecular-weight glutelin subunit 1Dx5, comprises the steps:
A) design primer, primer sequence is:
B) extraction of Wheat volatiles: field takes wheat seedling 3-5g, cleans, and utilizes after putting into liquid nitrogen freezing
Cwbiotech plant genome DNA extracts test kit and extracts Caulis et Folium Tritici aestivi genome.Ultraviolet spectrophotometer Nanodrop 2000
Detection DNA extraction quality and concentration, choose the DNA sample that OD260/OD280 detected value is 1.7-1.9 and expand for LAMP, and-20
DEG C save backup.
C) LAMP amplification: the genome obtaining step b) carries out LAMP amplification, and LAMP system arranges reaction temperature gradient
It is 60 DEG C, 61 DEG C and 62 DEG C；Total system 25 μ L: wherein primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ
ID NO:1), B3 (SEQ ID NO:2) ratio is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM or 0.6 μM: 0.6 μM: 0.1 μM: 0.1 μ
M, 4mM MgSO4,1.6mM dNTPs,0.8M betaine(Sigma-Aldrich,Tokyo,Japan),1μL Bst DNA
polymerase(New England Biolabs,Beijing,China)；Arranging response time gradient is 40min or 60min.
D) analyze: the amplified production obtaining step c) carries out 2% agarose gel electrophoresis or development process analysis.2% fine jade
Sepharose electrophoresis go out belt-like strip for sample containing wheat high-molecular-weight glutelin subunit 1Dx5, blank for not containing
The sample of wheat high-molecular-weight glutelin subunit 1Dx5；Have fluorescence in development process analytical reactions is containing wheat high-molecular-weight paddy
The sample of protein protomer 1Dx5, do not have fluorescence is the sample not containing wheat high-molecular-weight glutelin subunit 1Dx5.
As preferably, step c) primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1),
B3 (SEQ ID NO:2) ratio is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, and the amplified reaction time is 40min or 60min, LAMP
Amplified reaction temperature is 60 DEG C or 61 DEG C.
As preferably, step c) primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1),
B3 (SEQ ID NO:2) ratio is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, and the amplified reaction time is 60min, LAMP amplified reaction
Temperature is 60 DEG C.
The invention has the beneficial effects as follows: Semen Tritici aestivi 1Dx5 (the GenBank accession that the present invention utilizes GenBank to announce
Number X12928) gene order design LAMP (loop-mediated isothermal amplification) primer, build
Found the LAMP detection system of wheat high-molecular-weight glutelin subunit 1Dx5, and reaction condition has been optimized.LAMP is drawing
Thing FIP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1), B3 (SEQ ID NO:2) ratio is 0.8 μ
Under the 25 μ L systems of M:0.8 μM: 0.1 μM: 0.1 μM, 60 DEG C of reaction 60min are optimum reaction condition, and LAMP primer can be from confession
38 parts of wheat lines of examination are distinguished wheat high-molecular-weight glutelin subunit 1Dx5.Result shows, the LAMP detection system of foundation
Simply, quickly, sensitivity is good, specificity is high, have a high potential in wheat high-molecular-weight glutelin subunit 1Dx5 context of detection.
Accompanying drawing explanation
Fig. 1 1Dx5 (X12928) partial sequence and the LAMP primer location drawing in this sequence；
In figure: darken and underscore part is LAMP primer sequence；Wherein, FIP sequence is F2+F1c reverse complementary sequence,
BIP sequence is B2+B1c reverse complementary sequence；
Fig. 2 is susceptiveness detection figure；
In Fig. 2: M, marker；1,2,3,4,5 and 6 swimming bands represent Wheat volatiles 200ng, 150ng, 100ng respectively,
50ng, 30ng and 20ng；
Fig. 2 illustrates: belt-like strip occurs in 1,2,3,4 and 5 swimming band, shows wheat high-molecular-weight glutelin subunit to be detected
1Dx5 gene.6 swimming bands are blank, show to be not detected by wheat high-molecular-weight glutelin subunit 1Dx5 gene.Therefore, lowest detection
It is limited to 30ng/ system；
Fig. 3 is 1Dx5F+1Dx5R (Ma, 2003) detection and the LAMP detection figure of 1Dx5 of 1Dx5；
Wherein: A figure is 1Dx5F+1Dx5R (Ma, the 2003) detection of 1Dx5；B figure is the LAMP detection of 1Dx5.
Fig. 3 illustrates: A desires to make money or profit and examined 38 parts of wheat lines with the 1Dx5F+1Dx5R primer (Ma, 2003) of 1Dx5
Surveying, band occur shows containing wheat high-molecular-weight glutelin subunit 1Dx5, and blank showing does not contains wheat high-molecular-weight
Gluten subunit 1Dx5.B desires to make money or profit and is detected 38 parts of wheat lines by the LAMP primer of 1Dx5, the table of scalariform band occurs
Bright containing wheat high-molecular-weight glutelin subunit 1Dx5, blank showing does not contains wheat high-molecular-weight glutelin subunit 1Dx5.
Fig. 4 is the LAMP color developing detection figure of specific detection and 1Dx5；
Fig. 4 illustrates: have fluorescence in display reaction is the wheat samples containing wheat high-molecular-weight glutelin subunit 1Dx5,
Do not have fluorescence is the wheat samples not containing wheat high-molecular-weight glutelin subunit 1Dx5.
Detailed description of the invention
Below by being embodied as case, technical scheme is further illustrated.Should be appreciated that this
Inventive embodiment is not limited to the following examples, and any pro forma accommodation being made the present invention and/or change are all
Scope will be fallen into.
Method in following embodiment, if no special instructions, is the conventional method of this area.
LAMP method Bst DNA polymerase is provided by New England Biolabs company, Wheat volatiles DNA by
Plant Genome extracts test kit (health is century, Beijing), and agarose Agarose H (the raw work in Shanghai), Betaine is by Sigma-
Aldrich provides.
Semen Tritici aestivi 1Dx5 (the GenBank accession number X12928) gene order utilizing GenBank to announce sets
Meter LAMP (loop-mediated isothermal amplification) primer.1Dx5 (X12928) partial sequence and LAMP
Primer position in sequence is as it is shown in figure 1, darken and underscore part is LAMP primer sequence.Wherein, FIP sequence is F2+
F1c reverse complementary sequence, BIP sequence is B2+B1c reverse complementary sequence.
Embodiment 1
The LAMP method for quick of a kind of wheat high-molecular-weight glutelin subunit 1Dx5, comprises the steps:
A) design primer, primer sequence is:
B) extraction of Wheat volatiles: field takes wheat seedling 3-5g, cleans, puts into liquid nitrogen freezing, utilizes after grinding
Cwbiotech plant genome DNA extracts test kit and extracts Caulis et Folium Tritici aestivi genome.Ultraviolet spectrophotometer Nanodrop 2000
Detection DNA extraction quality and concentration, OD260/OD280 detected value is the DNA sample between 1.7-1.9, and-20 DEG C save backup.
C) LAMP amplification: the genome that step b) obtains carries out LAMP amplification, and LAMP system arranges reaction temperature gradient and is
60 DEG C, total system 25 μ L: wherein primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1), B3
(SEQ ID NO:2) ratio is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, 4mM MgSO4,1.6mM dNTPs, 0.8M betaine
(Sigma-Aldrich,Tokyo,Japan),1μL Bst DNA polymerase(New England Biolabs,
Beijing,China)；Arranging response time gradient is 40min.
D) analyze: the amplified production that step c) obtains carries out 2% agarose gel electrophoresis or development process analysis.
What 2% agarose gel electrophoresis went out belt-like strip is the sample containing wheat high-molecular-weight glutelin subunit 1Dx5,
Blank is the sample not containing wheat high-molecular-weight glutelin subunit 1Dx5；Have fluorescence in chromogenic reaction is high containing Semen Tritici aestivi
The sample of molecular weight gluten subunit 1Dx5, do not have fluorescence is the sample not containing wheat high-molecular-weight glutelin subunit 1Dx5.
In this step, 2% agarose gel electrophoresis and development process analysis can select one, so that it may qualification result.
Embodiment 2
The LAMP method for quick of a kind of wheat high-molecular-weight glutelin subunit 1Dx5, comprises the steps:
A) design primer, primer sequence is:
B) extraction of Wheat volatiles: field takes wheat seedling 3-5g, cleans, puts into liquid nitrogen freezing, utilizes after grinding
Cwbiotech plant genome DNA extracts test kit and extracts Caulis et Folium Tritici aestivi genome.Ultraviolet spectrophotometer Nanodrop 2000
Detection DNA extraction quality and concentration, OD260/OD280 detected value is the DNA sample between 1.7-1.9, and-20 DEG C save backup.
C) LAMP amplification: the genome that step b) obtains carries out LAMP amplification, and LAMP system arranges reaction temperature gradient and is
61 DEG C, total system 25 μ L: wherein primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1), B3
(SEQ ID NO:2) ratio is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, 4mM MgSO4,1.6mM dNTPs, 0.8M betaine
(Sigma-Aldrich,Tokyo,Japan),1μL Bst DNA polymerase(New England Biolabs,
Beijing,China)；Arranging response time gradient is 60min.
D) analyze: the amplified production that step c) obtains carries out 2% agarose gel electrophoresis and development process analysis.
What 2% agarose gel electrophoresis went out belt-like strip is the sample containing wheat high-molecular-weight glutelin subunit 1Dx5,
Blank is the sample not containing wheat high-molecular-weight glutelin subunit 1Dx5.Have fluorescence in chromogenic reaction is high containing Semen Tritici aestivi
The sample of molecular weight gluten subunit 1Dx5, do not have fluorescence is the sample not containing wheat high-molecular-weight glutelin subunit 1Dx5.
In this step, 2% agarose gel electrophoresis and development process analysis can select one, so that it may qualification result.
Embodiment 3
The LAMP method for quick of a kind of wheat high-molecular-weight glutelin subunit 1Dx5, comprises the steps:
A) design primer, primer sequence is:
B) extraction of Wheat volatiles: field takes wheat seedling 3-5g, cleans, and utilizes after putting into liquid nitrogen freezing
Cwbiotech plant genome DNA extracts test kit and extracts Caulis et Folium Tritici aestivi genome.Ultraviolet spectrophotometer Nanodrop 2000
Detection DNA extraction quality and concentration, OD260/OD280 detected value is the DNA sample between 1.7-1.9, and-20 DEG C save backup.
C) LAMP amplification: the genome that step b) obtains carries out LAMP amplification, and LAMP system arranges reaction temperature gradient and is
62 DEG C, total system 25 μ L: wherein primers F IP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1), B3
(SEQ ID NO:2) ratio is 0.6 μM: 0.6 μM: 0.1 μM: 0.1 μM, 4mM MgSO4,1.6mM dNTPs, 0.8M betaine
(Sigma-Aldrich,Tokyo,Japan),1μL Bst DNA polymerase(New England Biolabs,
Beijing,China)；Arranging response time gradient is 60min.
D) analyze: the amplified production that step c) obtains carries out 2% agarose gel electrophoresis and development process analysis.
What 2% agarose gel electrophoresis went out belt-like strip is the sample containing wheat high-molecular-weight glutelin subunit 1Dx5,
Blank is the sample not containing wheat high-molecular-weight glutelin subunit 1Dx5.Have fluorescence in display reaction is high containing Semen Tritici aestivi
The sample of molecular weight gluten subunit 1Dx5, do not have fluorescence is the sample not containing wheat high-molecular-weight glutelin subunit 1Dx5.
Susceptiveness detects: will utilize the Wheat volatiles DNA dilution that step b) of the present invention obtains, to ensure every individual system
(25 μ L) interior Semen Tritici aestivi templet gene composition not Wei 200ng, 150ng, 100ng, 50ng, 30ng and 20ng, checking LAMP optimize
System is for the Detection results of wheat high-molecular-weight glutelin subunit 1Dx5.LAMP result is as shown in Figure 2: the spirit of LAMP detection system
Sensitivity can reach 30ng/ system to 1Dx5, it is possible to meets the quick detection of wheat high-molecular-weight glutelin subunit 1Dx5
Demand.
Specific detection: utilize plant genome DNA to extract test kit (Cwbiotech) and extract Caulis et Folium Tritici aestivi genome.Purple
Outer spectrophotometer Nanodrop 2000 detects DNA extraction quality and concentration, takes and carries out according to DNA profiling concentration 30ng/ system
LAMP and PCR expands, the specificity of checking LAMP optimization system.From electrophoretogram and chromogenic reaction it can be seen that contain Semen Tritici aestivi height
The amplification sample liquid of molecular weight gluten subunit 1Dx5 (Fig. 3 A.B) can run out of PCR and gradient LAMP band clearly, and, root
1Dx5 (Fig. 4) can be substantially distinguished according to band and chromogenic reaction.Result shows, the present invention set up for wheat high-molecular-weight paddy
The LAMP detection method high specificity of protein protomer 1Dx5.
38 parts of wheat lines and high-molecular-weight glutelin subunit thereof are shown in Table 1.
The 38 parts of wheat lines used in invention herein are agricultural college of Shandong Agricultural University Kong Lingrang professor's laboratory and carry
Supply.38 parts of wheat lines carry different wheat high-molecular-weight glutelin subunits respectively, for the spy of the LAMP primer of the present invention
Opposite sex checking and practical function monitoring.
Table 1 wheat lines and its high-molecular-weight glutelin subunit form
Claims (3)
1. the LAMP detection method of a wheat high-molecular-weight glutelin subunit 1Dx5, it is characterised in that step is as follows:
A) design primer, primer sequence is:
B) extraction of Wheat volatiles: field takes wheat seedling 3-5g, cleans, utilizes Cwbiotech to plant after putting into liquid nitrogen freezing
Thing genome DNA extracting reagent kit extracts Caulis et Folium Tritici aestivi genome；Ultraviolet spectrophotometer Nanodrop 2000 detects DNA extraction
Quality and concentration, choose the DNA sample that OD260/OD280 detected value is 1.7-1.9 and expand for LAMP；
C) LAMP amplification: the genome obtaining step b) carries out LAMP amplification, and it is 60 that LAMP system arranges reaction temperature gradient
DEG C, 61 DEG C and 62 DEG C；Total system 25 μ L: wherein primers F IP, the ratio of BIP, F3, B3 is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM
Or 0.6 μM: 0.6 μM: 0.1 μM: 0.1 μM, 4mM MgSO4,1.6mM dNTPs,0.8M betaine,1μL Bst DNA
polymerase；Arranging response time gradient is 40min or 60min；
D) analyze: the amplified production obtaining step c) carries out 2% agarose gel electrophoresis or development process analysis；2% agarose
Gel electrophoresis go out belt-like strip for sample containing wheat high-molecular-weight glutelin subunit 1Dx5, blank for not containing Semen Tritici aestivi
The sample of high-molecular-weight glutelin subunit 1Dx5；Have fluorescence in development process analytical reactions is containing wheat high-molecular-weight glutelin
The sample of subunit 1Dx5, do not have fluorescence is the sample not containing wheat high-molecular-weight glutelin subunit 1Dx5.
The LAMP detection method of a kind of wheat high-molecular-weight glutelin subunit 1Dx5 the most as claimed in claim 1, its feature exists
Primers F IP in described step c), the ratio of BIP, F3, B3 is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, and the amplified reaction time is
40min or 60min, LAMP amplified reaction temperature is 60 DEG C or 61 DEG C.
The LAMP detection method of a kind of wheat high-molecular-weight glutelin subunit 1Dx5 the most as claimed in claim 1, its feature exists
Primers F IP in described step c), the ratio of BIP, F3, B3 is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, and the amplified reaction time is
60min, LAMP amplified reaction temperature is 60 DEG C.
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| WO2020248892A1 (en) * | 2019-06-11 | 2020-12-17 | 四川农业大学 | Kasp labeled primer for detecting high molecular weight glutenin subunit dy10-m619sn of wheat, and application thereof |
| CN117044616A (en) * | 2023-05-23 | 2023-11-14 | 贵州师范大学 | Method for improving wheat quality by using aegilops tauschii high-molecular gluten |
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