CN108410993A - With ox three way cross cattle CAPN 1 Gene A 316G mutational site detection methods and its application - Google Patents
With ox three way cross cattle CAPN 1 Gene A 316G mutational site detection methods and its application Download PDFInfo
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Abstract
The present invention provides a kind of and ox three way cross cattle CAPN 1 Gene A 316G mutational site detection methods and application.Detection method, using T ARMS PCR primer combinations, passes through PCR amplification cattle CAPN 1 gene segment using ox complete genome DNA to be measured as template；The genotype in CAPN1 Polymorphisms site is identified according to agarose gel electrophoresis result.The T ARMS PCR primer combinations include the site-specific primer of sequence composition shown in the specific outer primer that sequence shown in SEQ ID No.1 and SEQ ID No.2 forms and SEQ ID No.2 and SEQ ID No.3.The present invention is directed to and tetra-primer ARMS-PCR PCR (T ARMS PCR) detection method of ox three way cross cattle CAPN 1 Gene A 316G mutation, it is identified again through agarose gel electrophoresis after PCR amplification by the primer of special designing, it is capable of the polymorphism of mononucleotide that is simple, quick, inexpensive, accurately detecting the site, to be conducive to assist the early screening of high-quality hybrid ox, auxiliary to improve excellent and the selection of ox Three-way cross cattle the accuracy of Meat Quality.
Description
Technical field
The invention belongs to animal breeding molecular biosciences detection fields, are related to the detection of gene mononucleotide polymorphism (SNP),
More particularly to a kind of single nucleotide polymorphism of detection and 1 gene C of ox Three-way cross cattle calpain/G transversion
(SNP) tetra-primer ARMS-PCR PCR (T-ARMS-PCR) method.
Background technology
It is existing research shows that using Japan Black Cattle do terminal male, with the miscellaneous ox (sharp wood is praised to be hybridized with western Shandong ox) of profit,
Western miscellaneous ox (Simmental hybridizes with western Shandong ox), summer miscellaneous ox (Xia Luolai hybridizes with western Shandong ox) be hybridization of female parent and Niu Sanyuan it is miscellaneous
Hand over ox both to remain cow group and easily raise, adaptable speciality, and the meat marbling for inheriting male parent and ox it is apparent,
Feature with good meat quality (equal Japan Black Cattle three way cross bullock Experiment On Fattening preliminary study Chinese Cattle industry science 2016 is built at sea,
42(3)：1-3；21).
Meat Quality is stocker important economical trait, includes mainly Meat Tenderness, pH value, intramuscular fat content, is water
Power etc..Meat has significant impact to the price of meat product and the economic benefit of production.In order to improve meat, scientific research personnel and
The producer has carried out a large amount of research.Calpain (CAPN) is a member in calpain hydrolysis system family, calpain
Be one of the Major Enzymes of fribrillin of degrading, the adjusting of the enzymatic activity is related with the variation of meat tenderness degree.In vitro test table
Bright, calpain can degrade fribrillin, after beef cattle is butchered, with the consumption of ATP, be put aside in sarcoplasmic reticulum vesicle body
Calcium ion is released, and has activated calpain, decomposes the tenderization that fribrillin promotes meat.Currently, numerous studies table
Bright CAPN1 genes are related to beef cattle postmortem muscle maturation tenderization, and the transversion being especially located between the G/C of exon 9 causes
The 316th amino acids change between glycine and alanine in protein and peptide chain；The other is positioned at exons 14
Conversion between A/G, corresponding 530th amino acids have two kinds of possibilities of isoleucine and valine, gene C APN316 and
When the nucleotide of CAPN530 labels is C and G respectively, the tenderness of meat is high.There are AvaIII restriction enzyme sites, A/ due to CAPN530
G mutation cause restriction enzyme site and cannot generate cutting segment, can be distinguished by the method for PCR-RFLP to realize.
Tetra-primer ARMS-PCR PCR (T-ARMS-PCR) is on amplification Refracting Mutation system (ARMS) basis
On grow up and be a kind of single dedicated for detection the advantages of combine four primer PCRs (tetra-primer PCR) technology
The deriving technology of nucleotide polymorphisms.The technology to single nucleotide mutation detection have quickly, it is easy, allele can be distinguished
Whether it is homozygous and low-cost the features such as.
The technical principle of T-ARMS-PCR is to lack 3 ' -5 ' 5 prime excision enzyme activities using Taq archaeal dna polymerases, therefore for 3 '
The primer of terminal mismatch, the speed to match primer less than normal end extends, when the number of base mismatch reaches a certain level
When, 3 ' terminal bases cannot then extend because phosphodiester bond forms difficulty, and reaction terminating not will produce specific amplification item
Band is mutated to show that template DNA is not corresponding with 3 ' end of primer；If PCR results can obtain the amplification of special length
Band shows there is be mutated corresponding with 3 ' end of primer on template DNA.
T-ARMS-PCR relies on primer 3 ' and most end base is held to realize abrupt climatic change, to amplification sensitivity and accuracy requirement
It is very high.Touch down PCR (TD-PCR) are that each (or n) cycle annealing temperature gradually reduces 1 DEG C, until reaching
One lower annealing temperature.The raising of TD-PCR temperature early period improves the specificity of PCR amplification, but also improves primer knot
The difficulty of conjunction, reduces the efficiency of amplification, therefore is initially expanded with high temperature, ensures the preciseness of amplification, waits for target gene
Abundance rise after, reduce the temperature of amplification, improve the efficiency of amplification to improve the specificity and amplification efficiency of amplification.
The mutation of CAPN1 Gene A A types is significantly correlated with meat shearing force, can improve the tenderness of meat, auxiliary in molecular labeling
It helps in selection and use and applies.By identifying the genotype of CAPN1 genes in ox germ plasm resource or its breeding population, can be improved pair
The efficiency of selection of CAPN1-316Ala genotype is conducive to the early screening for assisting high-quality hybrid ox.
But currently without the relevant report about the abrupt climatic change with ox three way cross cattle CAPN 1 Gene A 316G.
Invention content
The present invention in view of the deficiencies of the prior art, in conjunction with T-ARMS-PCR and TD-PCR technologies realize pair and ox three way cross
The abrupt climatic change of cattle CAPN 1 gene A316G realizes fast and accurately testing goal.And it detects cheap, is suitble in meat
It is promoted and applied on a large scale in ox seed selection.
One aspect of the present invention provides and the mutational sites ox three way cross cattle CAPN 1 Gene A 316G detection method, including following
Step：
Using ox complete genome DNA to be measured as template, is combined using T-ARMS-PCR primers, cattle CAPN 1 is expanded by PCR
Genetic fragment；The genotype in CAPN1 Polymorphisms site is identified according to agarose gel electrophoresis result.
The T-ARMS-PCR primers combination includes the special of the composition of sequence shown in SEQ ID No.1 and SEQ ID No.2
Property outer primer and SEQ ID No.2 and SEQ ID No.3 shown in sequence composition site-specific primer.
The site-specific primer is the specific primer for the sites CAPN316.
1 CAPN1 gene T-ARMS-PCR primers of table combine
The system of the PCR amplification is：The overall reaction system of 20 μ l, wherein genomic DNA >=20ng/ μ l, specificity are outer
Each 0.2 μ l of primer, 0.3 μ l of locus specificity sense primer, locus specificity downstream primer 0.35 μ l, the Premix of 10 μ L
Taq, remaining uses ddH2O polishings are to 20 μ l.
The program of pcr amplification reaction is：
94 DEG C of pre-degeneration 3min；
94 DEG C of denaturation 30s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s；Each cycle annealing temperature reduces by 1 DEG C successively later,
Totally 10 cycles；
94 DEG C of denaturation 30s, 55 DEG C of renaturation 30s, 72 DEG C of extension 30s；Totally 25 cycles；
72 DEG C of extension 5min.
Judged according to agarose gel electrophoresis result：If electrophoresis result has 433bp and 295bp, two characteristic bands are
316Ala homozygotes (AA types)；If it is the homozygote (BB types) of 316Gly that electrophoresis result, which has 433bp and two bands of 177bp,；If
Electrophoresis result exists simultaneously three bands of 433bp, 295bp and 177bp, then is the heterozygote of 316Ala-Gly (AB types).
It is described the present invention also provides for detecting and the kit in the mutational sites ox three way cross cattle CAPN 1 Gene A 316G
Kit includes the T-ARMS-PCR primers combination.Further include 10 × PCR amplification buffer solution, MgCl2, dNTPs, DNA polymerization
Enzyme and related reagent for PCR amplification.
The present invention also provides application of the T-ARMS-PCR primers combination in the early screening of auxiliary high-quality hybrid ox.
Beneficial effects of the present invention are embodied in：
The present invention provides for four primer amplification Refracting Mutations being mutated with ox three way cross cattle CAPN 1 Gene A 316G
System PCR (T-ARMS-PCR) detection method is reflected through agarose gel electrophoresis by the primer of special designing after PCR amplification again
It is fixed, it is capable of the polymorphism of mononucleotide that is simple, quick, inexpensive, accurately detecting the site, it is high-quality to be conducive to assist
The early screening of catalo, auxiliary improve excellent and the selection of ox Three-way cross cattle the accuracy of Meat Quality.
Description of the drawings
Fig. 1 is and ox three way cross cattle CAPN 1 gene PCR expands electrophoresis result and the-ARMS-PCR amplifications of A316G mutation Ts
Product electrophoresis result, including AA, AB and theory T T genotype as a result, M is 100bp marker.
Fig. 2 be and ox three way cross cattle CAPN 1 gene PCR amplified production sequencer map；a：Genotype is AA, b：Genotype
For AB, the part that black box marks indicates mutant nucleotide sequence：c.947-948C/G.
Specific implementation mode
It elaborates with reference to the accompanying drawings and examples to the present invention, it is described to be explanation of the invention rather than limit
It is fixed.
Unless otherwise specified, the conventional hand that the technological means employed in embodiment is well known to those skilled in the art
Section, is referred to《Molecular Cloning:A Laboratory guide》(works such as J. Pehanorm Brookers, Huang Peitang etc. are translated, the third edition, Science Press)
Or Related product carries out, used reagent and product are also available commercial.The various processes be not described in detail and
Method is conventional method as known in the art, the source of agents useful for same, trade name and it is necessary to list its constituent person,
It indicates on the first appearance, identical reagent used is unless otherwise specified, identical with the content indicated for the first time thereafter.
Used enzyme preparation is purchased from TaKaRa treasured bioengineering (Dalian) Co., Ltd, poba gene group DNA extraction kit
Purchased from TIANGEN Biotech (Beijing) Co., Ltd..
Embodiment 1
The present invention is using PCR amplification method pair and ox three way cross cattle CAPN 1 gene in c.947-948C/G site mutation
Issuable single nucleotide polymorphism is detected, and carries out the accuracy of sanger sequence verification genotyping results, to make
For the detection means of CAPN1A316G Genotypings, it is applied to and is selected with ox Three-way cross cattle group, be conducive to assist excellent
The early screening of matter and ox Three-way cross cattle, auxiliary improve excellent and the selection of ox Three-way cross cattle the accuracy of Meat Quality.
(1) bovine blood acquires
The sample source of the present invention in ox Three-way cross cattle, totally 50 parts of samples.Take oxtail venous blood collection mode：By ox
Binding sterilizes blood sampling site, then at 10 centimetres from oxtail root or so, the 4th to fifth coccygeal vertebra bone boundary midpoint recess
It holds disposable blood taking device to be vertically pierced into about 0.5 centimetre of depths of oxtail veutro position of center line blood was collected, wine is used after the completion of blood sampling
Stop blooding in smart cotton balls pressing hole.The blood of acquisition directly can be detected or be placed in -20 DEG C of freezen protectives of anticoagulant tube (EDTA).
(2) design of the CAPN1 genes in c.947-948C/G site PCR primer
According to CAPN1 gene orders (Gene ID:It AC_000186), and can using 5.0 designs of Primer Premier
Four primers for expanding the Partial Fragment (segment includes c.947-948C/G site) of CAPN1 genes intron8-exon10 expand
Increase Refracting Mutation system PCR primer, primer sequence is shown in Table 1.
According to 1 primer of table, cow genome group is expanded using tetra-primer ARMS-PCR PCR, can expand and include
The different genotype segments of the CAPN1 genes c.947-948C/G SNP in site.Theoretically, when the site is CG homozygotes (AA
Type) when, PCR product is two band lines of 433bp and 295bp sizes after agarose gel electrophoresis detection；When the site is
When CG/GC heterozygous mutations (AB types), PCR products are 433bp, 295bp and 177bp after agarose gel electrophoresis detection
Three band lines of size.When the site is GC homozygous mutations (BB type), PCR product is after agarose gel electrophoresis detection
Two band line of 433bp and 177bp.
(3) T-ARMS-PCR expands to be measured and ox Three-way cross cattle CAPN1 genetic fragments
1) amplification system
The overall reaction system of 20 μ l, wherein genomic DNA >=20ng/ μ l, each 0.2 μ l of upstream and downstream outer primer draw in upstream
0.3 μ l of object, downstream inner primer 0.35 μ l, the Premix Taq of 10 μ L, remaining uses ddH2O polishings to 20 μ l.
2) program of pcr amplification reaction
94 DEG C of pre-degeneration 3min；
94 DEG C of denaturation 30s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s；Each cycle annealing temperature reduces by 1 DEG C successively later,
Totally 10 cycles；
94 DEG C of denaturation 30s, 55 DEG C of renaturation 30s, 72 DEG C of extension 30s；Totally 25 cycles；
72 DEG C of extension 5min.
3) PCR product agarose gel electrophoresis is analyzed
Take 5 μ L PCR products into row agarose gel electrophoresis, gel strength 1.5%.Electrophoresis result is referring to Fig. 1 and figure
2, it is two band of 433bp and 295bp that specific product, which only has a 433bp meal band, AA types, electrophoresis result,；AB type electrophoresis is examined
It is three band lines of 433bp, 295bp and 177bp size after surveying.After BB type PCR products are detected through agarose gel electrophoresis
It is two band line of 433bp and 177bp, as shown in Figure 1.
It randomly selects different genotype individual and carries out sanger sequence verification accuracy in detection, the results show that CAPN1 bases
Because the T-ARMS-PCR partings in the sites A316G are consistent with sanger sequencing results, as shown in Fig. 2, illustrate that genotyping result is correct.
SEQUENCE LISTING
<110>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricul
<120>With ox three way cross cattle CAPN 1 Gene A 316G mutational site detection methods and application
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
taagataacc cctgggactg g 21
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ttgcggaacc tctggctctt 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
agctcctcgg agtggaacgg 20
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence
<400> 4
ccgcatgtaa gggtccacc 19
Claims (9)
1. a kind of and mutational sites ox three way cross cattle CAPN 1 Gene A 316G detection method, characterized in that include the following steps：
Using ox complete genome DNA to be measured as template, is combined using T-ARMS-PCR primers, pass through PCR amplification cattle CAPN 1 gene segment；
The genotype in CAPN1 Polymorphisms site is identified according to agarose gel electrophoresis result；
The T-ARMS-PCR primers combination includes that the specificity of the composition of sequence shown in SEQ ID No.1 and SEQ ID No.2 is outer
The site-specific primer of sequence composition shown in primer and SEQ ID No.2 and SEQ ID No.3.
2. the as described in claim 1 and mutational sites ox three way cross cattle CAPN 1 Gene A 316G detection method, characterized in that
The site-specific primer is the specific primer for the sites CAPN316.
3. the as described in claim 1 and mutational sites ox three way cross cattle CAPN 1 Gene A 316G detection method, characterized in that
Judged according to agarose gel electrophoresis result：If it is 316Ala homozygosis that electrophoresis result, which has two characteristic bands of 433bp and 295bp,
Body AA types；If it is the homozygote BB types of 316Gly that electrophoresis result, which has 433bp and two bands of 177bp,；If electrophoresis result is deposited simultaneously
Then it is the heterozygote AB types of 316Ala-Gly in three bands of 433bp, 295bp and 177bp.
4. the as described in claim 1 and mutational sites ox three way cross cattle CAPN 1 Gene A 316G detection method, characterized in that
The system of the PCR amplification is：Genomic DNA >=20ng/ μ l, specific each 0.2 μ l of outer primer, locus specificity sense primer
0.3 μ l, locus specificity downstream primer 0.35 μ l, Premix Taq10 μ L, ddH2O polishings are to 20 μ l.
5. the as described in claim 1 and mutational sites ox three way cross cattle CAPN 1 Gene A 316G detection method, characterized in that
The program of the pcr amplification reaction is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s；With
Each cycle annealing temperature reduces by 1 DEG C successively afterwards, totally 10 cycles；94 DEG C of denaturation 30s, 55 DEG C of renaturation 30s, 72 DEG C of extension 30s；
Totally 25 cycles；72 DEG C of extension 5min.
6. for detecting and the kit in the mutational sites ox three way cross cattle CAPN 1 Gene A 316G, characterized in that the reagent
Box includes the T-ARMS-PCR primers combination.
7. as claimed in claim 6 for detect with the kit in the mutational sites ox three way cross cattle CAPN 1 Gene A 316G,
It is characterized in, further includes 10 × PCR amplification buffer solution, MgCl2, dNTPs, archaeal dna polymerase and the related reagent for PCR amplification.
8. application of the T-ARMS-PCR primers combination in the early screening of auxiliary high-quality hybrid ox described in claim 1.
9. being assisted with the kit in the mutational sites ox three way cross cattle CAPN 1 Gene A 316G described in claim 6 for detecting
Application in the early screening of high-quality hybrid ox.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109943649A (en) * | 2019-05-08 | 2019-06-28 | 刘学峰 | Sheep intramuscular fat content molecular labeling and its naked eyed test cultivation in apply |
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| KR101390536B1 (en) * | 2013-03-28 | 2014-05-07 | 전라북도 임실군(임실군 농업기술센터장) | Mass producing method for high meat quality korean native cattle by in vitro fertilization technique |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109943649A (en) * | 2019-05-08 | 2019-06-28 | 刘学峰 | Sheep intramuscular fat content molecular labeling and its naked eyed test cultivation in apply |
| CN109943649B (en) * | 2019-05-08 | 2019-12-03 | 黑龙江省农业科学院畜牧兽医分院 | Sheep intramuscular fat content molecular labeling and its naked eyed test cultivation in apply |
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