CN106048048B - A kind of LAMP rapid detection method of wheat high-molecular-weight glutelin subunit 1Dx5 - Google Patents
A kind of LAMP rapid detection method of wheat high-molecular-weight glutelin subunit 1Dx5 Download PDFInfo
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Abstract
The present invention provides the LAMP rapid detection methods of wheat high-molecular-weight glutelin subunit 1Dx5 a kind of, the present invention designs LAMP primer using the wheat 1Dx5 gene order that GenBank is announced, the LAMP detection architecture of wheat high-molecular-weight glutelin subunit 1Dx5 is established, and reaction condition is optimized.LAMP is in primers F IP, under the 25 μ L systems that BIP, F3, B3 ratio are 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, 60 DEG C of reaction 60min are optimum reaction condition, and LAMP primer can distinguish wheat high-molecular-weight glutelin subunit 1Dx5 from 38 parts of wheat lines for examination.The result shows that the LAMP detection architecture of foundation is simple, quick, sensitivity is good, specific height, have a high potential in wheat high-molecular-weight glutelin subunit 1Dx5 context of detection.
Description
Technical field
The present invention relates to agricultural and technical field of plant quarantine, and in particular to a kind of wheat high-molecular-weight glutelin subunit
The LAMP detection method of 1Dx5 gene.
Technical background
Wheat (Triticum aestivum L.) is cereal crops important in the world, can be processed into bread, steamed bun,
The different food such as noodles.Protein content and quality are to influence one of the most important index of wheat quality in wheat, special
It is not glutenin and gliadin, is content highest in wheat gluten, two hatching egg maximum to wheat processing food effect
White matter.Glutenin is divided into high-molecular-weight glutelin subunit and low-molecular-weight glutenin subunit again, encodes high-molecular-weight glutelin
The gene of subunit is located at the homologous group of the first chromosome, and theoretically, there are six high-molecular wheat glutelin subunit genes for common wheat.So
And, it is generally the case that the gluten subunit expressed in wheat is 3-5.Allelic variation is generally each high molecular weight
Gluten subunit, the especially site Glu-D1 are affected to dough characters, are important Quality Gene, for example, 1Dx5+
1Dy10 subunit group credit union significantly improves the elasticity of dough.
Effectively identifying and filtering out the wheat lines containing excellent protein protomer is to carry out field good quality wheat breeding material
The important prerequisite of breeding, and carry out the important evidence of wheat food processing.Currently, high-molecular-weight glutelin subunit in wheat
Identification mainly uses the methods of protein electrophoresis, regular-PCR method.These methods generally existing used time is longer, needs special instrument
Device, is unable to satisfy the needs of field quick detection at the problems such as cannot achieve on-site test.
Wheat Grain Protein is mainly made of alcohol soluble protein and glutenin, and alcohol soluble protein assigns dough extensibility, and
Glutenin makes dough have elasticity.Wheat glutenin subunit accounts for 35% of mucedin or so, mainly by high molecular weight 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 wheat.Wheat high-molecular-weight glutelin subunit 1Dx5 has wheat quality can not
The effect of ignorance.The new method for establishing Rapid identification wheat high-molecular-weight glutelin subunit 1Dx5 can be the excellent wheat breeding in field
Material screening provides important evidence.Currently, domestic there is not yet screening wheat high-molecular-weight glutelin subunit 1Dx5 with LAMP technology
Report, the wheat high-molecular-weight glutelin subunit 1Dx5 of accurate Fast Practical identification have important potential using value.
Summary of the invention
It cannot achieve that execute-in-place, quick, accurate, sensitive wheat high-molecular-weight glutelin is sub- to solve the prior art
The defect of base 1Dx5, the present invention provides the LAMP rapid detection method of wheat high-molecular-weight glutelin subunit 1Dx5 a kind of, mesh
Be the LAMP of quick, accurate, sensitive wheat high-molecular-weight glutelin subunit 1Dx5 for facilitating execute-in-place a kind of is provided
Detection method.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of LAMP detection method of wheat high-molecular-weight glutelin subunit 1Dx5, includes the following steps:
A) design primer, primer sequence are as follows:
B) extraction of Wheat volatiles: field takes wheat seedling 3-5g, cleans, utilizes after being put into liquid nitrogen frozen
Cwbiotech plant genome DNA extracts kit extracts wheat seedling genome.Ultraviolet specrophotometer Nanodrop 2000
It detects DNA and extracts quality and concentration, choose the DNA sample that OD260/OD280 detected value is 1.7-1.9 and expanded for LAMP, -20
It DEG C saves backup.
C) LAMP is expanded: carrying out LAMP amplification to the genome that step b) is obtained, reaction temperature gradient is arranged in LAMP system
It is 60 DEG C, 61 DEG C and 62 DEG C；25 μ L of total system: 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)；Setting reaction time gradient is 40min or 60min.
D) it analyzes: 2% agarose gel electrophoresis being carried out to the amplified production that step c) is obtained or development process is analyzed.2% fine jade
It is the sample containing wheat high-molecular-weight glutelin subunit 1Dx5 that sepharose electrophoresis, which goes out belt-like strip, blank be without containing
The sample of wheat high-molecular-weight glutelin subunit 1Dx5；Having fluorescence in development process analysis reaction is containing wheat high-molecular-weight paddy
The sample of protein protomer 1Dx5, not having fluorescence is the sample without containing wheat high-molecular-weight glutelin subunit 1Dx5.
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.
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 beneficial effects of the present invention are: wheat 1Dx5 (the GenBank accession that the present invention utilizes GenBank to announce
Number X12928) gene order design LAMP (loop-mediated isothermal amplification) primer, it builds
The LAMP detection architecture of wheat high-molecular-weight glutelin subunit 1Dx5 has been found, and reaction condition has been optimized.LAMP is drawing
Object FIP (SEQ ID NO:3), BIP (SEQ ID NO:4), F3 (SEQ ID NO:1), B3 (SEQ ID NO:2) ratio are 0.8 μ
Under M:0.8 μM: 0.1 μM: 0.1 μM of 25 μ L systems, 60 DEG C of reaction 60min are optimum reaction condition, and LAMP primer can be from confession
Wheat high-molecular-weight glutelin subunit 1Dx5 is distinguished in 38 parts of wheat lines of examination.The result shows that the LAMP detection architecture of foundation
Simply, quickly, sensitivity is good, specific height, have a high potential in wheat high-molecular-weight glutelin subunit 1Dx5 context of detection.
Detailed description of the invention
The location drawing of Fig. 1 1Dx5 (X12928) partial sequence and LAMP primer in the sequence；
In figure: blackening 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 sensitivity detection figure；
In Fig. 2: M, marker；1,2,3,4,5 and 6 swimming bands respectively represent Wheat volatiles 200ng, 150ng, 100ng,
50ng, 30ng and 20ng；
Fig. 2 explanation: there is belt-like strip in 1,2,3,4 and 5 swimming band, shows to detect wheat high-molecular-weight glutelin subunit
1Dx5 gene.6 swimming band blank, show not detect wheat high-molecular-weight glutelin subunit 1Dx5 gene.Therefore, lowest detection
It is limited to 30ng/ system；
The LAMP of 1Dx5F+1Dx5R (Ma, 2003) detection and 1Dx5 that Fig. 3 is 1Dx5 detects figure；
Wherein: 1Dx5F+1Dx5R (Ma, the 2003) detection that A figure is 1Dx5；The LAMP detection that B figure is 1Dx5.
Fig. 3 explanation: A, which desires to make money or profit, examines 38 parts of wheat lines with the 1Dx5F+1Dx5R primer (Ma, 2003) of 1Dx5
It surveys, band occur shows that, containing wheat high-molecular-weight glutelin subunit 1Dx5, blank shows without containing wheat high-molecular-weight
Gluten subunit 1Dx5.B, which desires to make money or profit, detects 38 parts of wheat lines with the LAMP primer of 1Dx5, the table of scalariform band occurs
It is bright containing wheat high-molecular-weight glutelin subunit 1Dx5, blank shows without containing wheat high-molecular-weight glutelin subunit 1Dx5.
Fig. 4 is the LAMP color developing detection figure of specific detection and 1Dx5；
Fig. 4 explanation: having fluorescence in display reaction is the wheat samples containing wheat high-molecular-weight glutelin subunit 1Dx5,
Not having fluorescence is the wheat samples without containing wheat high-molecular-weight glutelin subunit 1Dx5.
Specific embodiment
Below by specific implementation case, technical solution of the present invention is further illustrated.It should be appreciated that this
The embodiment of invention is not limited by the following examples, and the accommodation in any form made to the present invention and/or changed all
The scope of the present invention will be fallen into.
Method in following embodiments is unless otherwise instructed the conventional method of this field.
LAMP method Bst DNA polymerase by New England Biolabs company provide, Wheat volatiles DNA by
Plant Genome extracts kit (health is century, Beijing), agarose Agarose H (the raw work in Shanghai), Betaine is by Sigma-
Aldrich provides.
It is set using wheat 1Dx5 (GenBank accession number X12928) gene order that GenBank is announced
Count LAMP (loop-mediated isothermal amplification) primer.1Dx5 (X12928) partial sequence and LAMP
Position of the primer in sequence is as shown in Figure 1, blacken and underscore part is LAMP primer sequence.Wherein, FIP sequence is F2+
F1c reverse complementary sequence, BIP sequence are B2+B1c reverse complementary sequence.
Embodiment 1
A kind of LAMP rapid detection method of wheat high-molecular-weight glutelin subunit 1Dx5, includes the following steps:
A) design primer, primer sequence are as follows:
B) extraction of Wheat volatiles: field takes wheat seedling 3-5g, cleans, is put into liquid nitrogen frozen, utilizes after grinding
Cwbiotech plant genome DNA extracts kit extracts wheat seedling genome.Ultraviolet specrophotometer Nanodrop 2000
It detects DNA and extracts quality and concentration, DNA sample of the OD260/OD280 detected value between 1.7-1.9, -20 DEG C save backup.
C) LAMP is expanded: the genome that step b) is obtained carries out LAMP amplification, and LAMP system setting reaction temperature gradient is
60 DEG C, 25 μ L of total system: 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)；Setting reaction time gradient is 40min.
D) analyze: the amplified production that step c) is obtained carries out 2% agarose gel electrophoresis or development process analysis.
It is the sample containing wheat high-molecular-weight glutelin subunit 1Dx5 that 2% agarose gel electrophoresis, which goes out belt-like strip,
Blank is the sample without containing wheat high-molecular-weight glutelin subunit 1Dx5；Having fluorescence in chromogenic reaction is containing wheat height
The sample of molecular weight gluten subunit 1Dx5, not having fluorescence is the sample without containing wheat high-molecular-weight glutelin subunit 1Dx5.
In this step, 2% agarose gel electrophoresis and development process analysis be can choose first, can qualification result.
Embodiment 2
A kind of LAMP rapid detection method of wheat high-molecular-weight glutelin subunit 1Dx5, includes the following steps:
A) design primer, primer sequence are as follows:
B) extraction of Wheat volatiles: field takes wheat seedling 3-5g, cleans, is put into liquid nitrogen frozen, utilizes after grinding
Cwbiotech plant genome DNA extracts kit extracts wheat seedling genome.Ultraviolet specrophotometer Nanodrop 2000
It detects DNA and extracts quality and concentration, DNA sample of the OD260/OD280 detected value between 1.7-1.9, -20 DEG C save backup.
C) LAMP is expanded: the genome that step b) is obtained carries out LAMP amplification, and LAMP system setting reaction temperature gradient is
61 DEG C, 25 μ L of total system: 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)；Setting reaction time gradient is 60min.
D) analyze: the amplified production that step c) is obtained carries out 2% agarose gel electrophoresis and development process analysis.
It is the sample containing wheat high-molecular-weight glutelin subunit 1Dx5 that 2% agarose gel electrophoresis, which goes out belt-like strip,
Blank is the sample without containing wheat high-molecular-weight glutelin subunit 1Dx5.Having fluorescence in chromogenic reaction is containing wheat height
The sample of molecular weight gluten subunit 1Dx5, not having fluorescence is the sample without containing wheat high-molecular-weight glutelin subunit 1Dx5.
In this step, 2% agarose gel electrophoresis and development process analysis be can choose first, can qualification result.
Embodiment 3
A kind of LAMP rapid detection method of wheat high-molecular-weight glutelin subunit 1Dx5, includes the following steps:
A) design primer, primer sequence are as follows:
B) extraction of Wheat volatiles: field takes wheat seedling 3-5g, cleans, utilizes after being put into liquid nitrogen frozen
Cwbiotech plant genome DNA extracts kit extracts wheat seedling genome.Ultraviolet specrophotometer Nanodrop 2000
It detects DNA and extracts quality and concentration, DNA sample of the OD260/OD280 detected value between 1.7-1.9, -20 DEG C save backup.
C) LAMP is expanded: the genome that step b) is obtained carries out LAMP amplification, and LAMP system setting reaction temperature gradient is
62 DEG C, 25 μ L of total system: 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)；Setting reaction time gradient is 60min.
D) analyze: the amplified production that step c) is obtained carries out 2% agarose gel electrophoresis and development process analysis.
It is the sample containing wheat high-molecular-weight glutelin subunit 1Dx5 that 2% agarose gel electrophoresis, which goes out belt-like strip,
Blank is the sample without containing wheat high-molecular-weight glutelin subunit 1Dx5.Having fluorescence in display reaction is containing wheat height
The sample of molecular weight gluten subunit 1Dx5, not having fluorescence is the sample without containing wheat high-molecular-weight glutelin subunit 1Dx5.
Sensitivity detection: it will be diluted using the Wheat volatiles DNA that step b) of the present invention is obtained, to guarantee each system
(25 μ L) interior wheat templet gene composition is not 200ng, 150ng, 100ng, 50ng, 30ng and 20ng, verifying LAMP optimization
System is directed to the detection effect of wheat high-molecular-weight glutelin subunit 1Dx5.LAMP result is as shown in Figure 2: LAMP detection architecture spirit
Sensitivity can reach 30ng/ system to 1Dx5, can satisfy the quick detection of wheat high-molecular-weight glutelin subunit 1Dx5
Demand.
Specific detection: wheat seedling genome is extracted using plant genome DNA extracts kit (Cwbiotech).It is purple
Outer spectrophotometer Nanodrop 2000 detects DNA and extracts quality and concentration, takes and carries out according to DNA template concentration 30ng/ system
LAMP and PCR amplification verify the specificity of LAMP optimization system.It can be seen that from electrophoretogram and chromogenic reaction containing wheat height
The amplification sample liquid of molecular weight gluten subunit 1Dx5 (Fig. 3 A.B) can run out of clearly PCR and gradient LAMP band, also, root
1Dx5 (Fig. 4) can be obviously distinguished according to band and chromogenic reaction.The result shows that the present invention establish for wheat high-molecular-weight paddy
The LAMP detection method high specificity of protein protomer 1Dx5.
38 parts of wheat lines and its high-molecular-weight glutelin subunit are shown in Table 1.
38 parts of wheat lines used in invention are that agricultural college, Shandong Agricultural University Kong Lingrang professor laboratory mentions herein
For.38 parts of wheat lines carry different wheat high-molecular-weight glutelin subunits respectively, the spy for LAMP primer of the invention
Opposite sex verifying and practical function monitoring.
1 wheat lines of table and its high-molecular-weight glutelin subunit form
Claims (1)
1. a kind of LAMP detection method of wheat high-molecular-weight glutelin subunit 1Dx5, which is characterized in that steps are as follows:
A) design primer, primer sequence are as follows:
B) extraction of Wheat volatiles: field takes wheat seedling 3-5g, cleans, and is planted after being put into liquid nitrogen frozen using Cwbiotech
Object genome DNA extracting reagent kit extracts wheat seedling genome；Ultraviolet specrophotometer Nanodrop 2000 detects DNA and extracts
Quality and concentration are chosen the DNA sample that OD260/OD280 detected value is 1.7-1.9 and are expanded for LAMP；
C) LAMP is expanded: carrying out LAMP amplification to the genome that step b) obtains, LAMP system reaction temperature is 60 DEG C；It is overall
The ratio for being 25 μ L: wherein primers F IP, BIP, F3, B3 is 0.8 μM: 0.8 μM: 0.1 μM: 0.1 μM, 4 mM
MgSO4, 1.6 mM dNTPs, 0.8 M betaine, 1 μL BstDNA polymerase；Reaction time 60 is set
min；
D) it analyzes: 2% agarose gel electrophoresis being carried out to the amplified production that step c) obtains or development process is analyzed；2% agarose is solidifying
It is the sample containing wheat high-molecular-weight glutelin subunit 1Dx5 that gel electrophoresis, which goes out belt-like strip, and blank is without containing wheat height
The sample of molecular weight gluten subunit 1Dx5；Having fluorescence in development process analysis reaction is containing wheat high-molecular-weight glutelin Asia
The sample of base 1Dx5, not having fluorescence is the sample without containing wheat high-molecular-weight glutelin subunit 1Dx5.
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| CN106520981B (en) * | 2016-11-29 | 2020-03-31 | 山东农业大学 | Method for rapidly detecting LOX transgenic wheat and kit applying same |
| CN110205398B (en) * | 2019-06-11 | 2022-07-12 | 四川农业大学 | KASP marker primer for detecting wheat high molecular weight glutelin Dy10-m619SN subunit and application |
| 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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