CN104745716B - A kind of detection method of cattle AGPAT6 gene mononucleotide polymorphism - Google Patents
A kind of detection method of cattle AGPAT6 gene mononucleotide polymorphism Download PDFInfo
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Abstract
The invention discloses a kind of detection method of cattle AGPAT6 gene mononucleotide polymorphism, its method is：The first step, carry out complete genome DNA extraction with the whole blood of cattle；Second step, with extract complete genome DNA as template, respectively with primer pair A, B as primer, PCR expand cattle AGPAT6 gene；3rd step, with PCR mix products sequencing, find out SNP site；4th step, with restricted enzyme, enzyme action is carried out to PCR primer；5th step, digestion products are entered row agarose gel electrophoresis, then its polymorphism is judged according to electrophoretic band.Beneficial effect：The present invention utilizes PCR RFLP technology to the T at the 303bp of cattle AGPAT6 exon 1>G at the 299bp of C single nucleotide polymorphism and the 12nd exon>A single nucleotide polymorphism is detected.Provide a kind of examination on DNA level inexpensive, simple, accurate, easy to utilize and detect the genetic marker closely related with cattle Meat Quality and carcass trait, can be used for assisted Selection and the molecular breeding of cattle.
Description
Technical field
The present invention relates to a kind of detection method of gene mononucleotide polymorphism, particularly to a kind of cattle AGPAT6 gene list
The detection method of nucleotide polymorphisms.
Background technology
Single nucleotide polymorphism (singlenucleotidepolymorphisms, SNPs) refers in genomic dna sequence
The variation of single nucleotide acid (A/T/C/G), includes conversion, transversion, disappearance in theory and inserts this 4 kinds of different variant forms,
But actually generation only has two kinds, i.e. conversion and transversion, the likelihood ratio that the two occurs is about 2:1.Wherein conversion refers to pyrimidine
Exchange and pyrimidine between or between purine and purine, and transversion refers to the exchange between pyrimidine and purine.SNP is in GC sequence
Occur the most frequent, and be that C is converted to T mostly, because the C in CG is often methylated, after spontaneous deamination, become T.Theoretical
On, SNP be both probably two equipotential polymorphisms it is also possible to 3 or 4 equipotential polymorphisms, but in fact, latter two is rarely found,
Almost can ignore, therefore we only consider two equipotential polymorphisms when detecting the single nucleotide polymorphism of gene.
The impact to protein amino acid sequence for the single nucleotide polymorphism according to gene is classified, and it includes samesense mutation
And nonsynonymous mutation.The change of base sequence caused by samesense mutation refers to SNP is not result in its Argine Monohydrochloride translated
The change of sequence, this is that is, the degeneracy of codon is led to because same aminoacid may have multiple codons；It is worth
It is to be noted that although samesense mutation will not change protein amino acid sequence, but by same aminoacid difference codon institute
Corresponding tRNA ratio is different, and it still can affect the expression of albumen, and then affects individual character.And nonsynonymous mutation
Then refer to that the change of base leads to coded aminoacid to change, so that the albumen translated changes, affect its function
Play, important function is produced to individual phenotype.With respect to nonsynonymous mutation, samesense mutation is in fact more common, because a kind of
Albumen occurs great change to be likely to make individuality lethal.
At present, the detection for SNPs mainly has following several method：DNA direct Sequencing, single-strand conformation polymorphism technology
, and PCR-RFLP technology etc. (SSCP).Wherein, PCR-RFLP is a kind of method of new effective detection SNP.It is logical first
SNP site is found in the sequencing of overmulling pond, is designing primer for corresponding SNP site, is reusing restricted enzyme and cut,
Finally enter row agarose gel electrophoresis, can accurately SNP site be differentiated according to electrophoretic band typing.PCR-RFLP side
Method is quick, easy, and accurately, expense is low, is well suited for the detection of colony SNP for result.
AGPAT is also called LPAAT, is the key enzyme in triglyceride (TG) building-up process.Glycerol triphosphoric acid in TG
In (glycerol-3-phosphate, G3P) biosynthesis pathway, AGPAT catalysis LPA is changed into PA, completes TGsn-2 position
Acylation.Meanwhile, PA is the main producers synthesizing multiple phosphoglyceride classes, such as PS, PE, PC, PI etc., wherein PS, PE, PC
It is the main phospholipid forming various cell membrane, the change of the LPA and PA ratio that therefore AGPAT activity change is led to, can affect
The distribution of phospholipid on cell membrane, and in cell division, the flexibility of impact film.Up-to-date research shows, AGPAT a total of 11
Middle hypotype, respectively by different gene codes.Test to mice finds, AGPAT1, AGPAT3 table in Various Tissues
Reach, and AGPAT2, the expression of AGPAT4, AGPAT5 has tissue specificity.AGPAT8, AGPAT 9, AGPAT 10, AGPAT
11 have higher expression respectively in different tissues.
AGPAT6 is studied and finds, it is mainly expressed in fatty tissue and galactophore epithelial cell, is only positioned at endoplasmic reticulum, lack
The mice of weary AGPAT6 anorexia can simultaneously avoid heritability fat, galactophore epithelial cell dysplasia, lack in milk diglyceride and
Triglyceride.Up-to-date research shows, the aminoacid sequence of AGPAT6 was both similar to AGPAT family, was also similar to that GPAT,
AGPAT6 has GPAT activity, is a kind of GPAT microgranule, thus is named as GPAT4 again.But regardless of AGPAT family is still
GPAT family, is all the important enzyme of synthetic glycerine three ester.The early stage transcription group result of study of this seminar also indicates that, AGPAT6
Gene may be related to the Meat Quality of cattle.Therefore, the AGPAT6 gene very likely lipid metabolism phase with Chinese Simmental
Close.Animal obtains energy from food, then in the tissue such as liver and fat, excess energy is changed into fat stores.Dynamic
Thing fat over-deposit will affect meat and human health.So, reduce the over-deposit of Animal fat, illustrate its regulation and control
Molecular mechanism and set up corresponding control technique and become the focus of animal genetics research.Thus, AGPAT6 gene is ground
Study carefully significant.At present, the research about AGPAT6 polymorphism is primarily directed to what milch cow was carried out.Zan woods Lignum Rhamnellae lotus this
Smooth milch cow AGPAT6 gene the 3rd intron detects 2 SNP site, and it is closely related with milk yield and butterfat percnetage.Zhang Jialan
Find 1 SNP site Deng in holstein cow intron 2, it is related to milk yield, butterfat percnetage and protein ratio.But it is relevant
AGPAT6 gene there is presently no report with the dependency of beef cattle character.
Content of the invention
Present invention solves the problem in that the method examination cattle AGPAT6 gene being sequenced first with PCR primer mixing is polymorphic
Property site, then enzyme action detection is carried out by restricted enzyme to single sample PCR primer, there is provided a kind of quick, easy, take
Method with low examination and detection cattle AGPAT6 gene pleiomorphism.By between AGPAT6 gene SNP site and obstinacy shape
The analysis of dependency, using function SNP as the labelling of molecular breeding and assisted Selection, accelerates fine-variety breeding speed and improves population
Quality.
The present invention technical scheme as follows：
The single nucleotide polymorphism of cattle AGPAT6 gene, its gene SNP includes：
There is T at the 303bp of cattle AGPAT6 exon 1>The single nucleotide polymorphism of C；
There is G at the 299bp of cattle AGPAT6 gene the 12nd exon>The single nucleotide polymorphism of A.
The detection method of the single nucleotide polymorphism of cattle AGPAT6 gene that the present invention provides, its step is as described below：
The first step, carry out complete genome DNA extraction with the whole blood of cattle；
Second step, with extract complete genome DNA as template, respectively with primer pair A, B as primer, PCR expand cattle
AGPAT6 gene；
Described primer pair A is：
Forward primer：5'TGGCAATGACAGACCTTCAGGAC 3'
Downstream primer：5'CAGGAGGCTGACAATCAGGCTGT 3'
Described primer pair B is：
Forward primer：5'CACCTTGTGTCCCTTTCCGC 3'
Downstream primer：5'AGAACAGCACTCCCCTAGCCCT 3'
3rd step, with PCR mix products sequencing, find out SNP site；
4th step, with restricted enzyme, enzyme action is carried out to PCR primer；
5th step, digestion products are entered row agarose gel electrophoresis, then its polymorphism is judged according to electrophoretic band：
The PCR primer of primer pair A comprises the T at the 303bp of AGPAT6 exon 1>C single nucleotide polymorphism,
After enzyme action, TT genotype individuals show as 139bp, 102bp, 61bp and 48bp band, and CC genotype individuals show as 163bp,
139bp, 61bp and 48bp band, TC genotype individuals show as 163bp, 139bp, 102bp, 61bp and 48bp band；
The PCR primer of primer pair B comprises the G at the 299bp of AGPAT6 gene the 12nd exon>A single nucleotide polymorphism,
After enzyme action, GG genotype individuals show as 119bp, 58bp and 62bp band, and AA genotype individuals show as 177bp, 118bp,
58bp and 62bp band.
The response procedures of the PCR amplification of primer A, B are：95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 61 DEG C of annealing
45s, 72 DEG C of extension 45s, 30 circulations；Extend 72 DEG C of 10min, last 16 DEG C of preservations.
Digestion products agarose gel electrophoresiies adopt 4% agarose gel.
Beneficial effects of the present invention：
The present invention utilizes PCR-RFLP technology to the T at the 303bp of cattle AGPAT6 exon 1>C mononucleotide is many
G at the 299bp of state property and the 12nd exon>A single nucleotide polymorphism is detected.
The present invention has carried out gene type and gene frequency analysis to above-mentioned two SNP of cows body, simultaneously also by SNP position
Point has carried out association analysiss with the Meat Quality of cattle and carcass trait.
The present invention dexterously utilize PCR-RFLP method to detect SNPs it was found that the Meat Quality of two SNP site and cattle and
Dependency between carcass trait.Provide a kind of examination on DNA level inexpensive, simple, accurate, easy to utilize
The genetic marker closely related with cattle Meat Quality and carcass trait with detection, can be used for assisted Selection and the molecular breeding of cattle.
Brief description
The fine jade of Fig. 1 PCR primer 2% to A for the Chinese Simmental AGPAT6 gene primer in the embodiment of the present invention
Sepharose electrophoretogram.
The fine jade of Fig. 2 PCR primer 2% to B for the Chinese Simmental AGPAT6 gene primer in the embodiment of the present invention
Sepharose electrophoretogram.
Fig. 3 is T at the 303bp of Chinese Simmental AGPAT6 exon 1 in the embodiment of the present invention>C position
The mixed pond sequencing result figure of point.
Fig. 4 is G at the 299bp of Chinese Simmental AGPAT6 gene the 12nd exon in the embodiment of the present invention>A position
The mixed pond sequencing result figure of point.
Fig. 5 is the Chinese Simmental AGPAT6 gene E1-303T in the embodiment of the present invention>C site digestion products
Agarose gel electrophoresis figure.
Fig. 6 is the Chinese Simmental AGPAT6 gene E12-299G in the embodiment of the present invention>A site digestion products
Agarose gel electrophoresis figure.
Specific embodiment
First, experiment material and method
1st, experiment material：
1.1. laboratory animal：
With Simmental colony of two China as detection object, it is Inner Mongol Wu Lagai area China Simmental respectively
Cows body and Surrounding Area of Beijing Chinese Simmental colony.
1.2. enzyme and reagent：
DNA extraction kit, 2XTaq PCR MasterMix are purchased from TIANGEN company；Restricted enzyme is purchased from
NEB company；PCR primer is synthesized by Shanghai bio-engineering corporation.
1.3.DNA analysis software：
DNA sequence sequencing result analysis software：Chromas；
PCR primer design software：Primer Premier 5；
Data analysis software：SPSS 13.0；
2. experimental technique：
Carry out complete genome DNA extraction with the whole blood of Chinese Simmental, with the complete genome DNA of extraction as template,
Design primer enters performing PCR amplification Simmental AGPAT6 gene, SNPs site is found in PCR primer sequencing, then produces PCR
Thing carries out enzyme action with restricted enzyme, finally digestion products is entered row agarose gel electrophoresis detection, according to electrophoretic band is
Typing can be carried out to SNP site.Below the present invention is described in further detail, described is explanation of the invention rather than limit
Fixed.
The collection of 2.1 blood samples：
The present invention with Simmental colony of two China as detection object, is respectively specifically：On Inner Mongol Wu Lagai ground
Area gathers 314 samples, gathers 135 samples in Surrounding Area of Beijing；All by the way of jugular vein blood collection.
The extraction of 2.2 blood sample genomic DNAs：
Extract the genome in Simmental blood sample using the TIANamp Blood DNA Kit of TIANGEN company
DNA.
1) in the sample add 2.5 times of volumes cell pyrolysis liquid CL, overturn mix, 10000rpm (11,500 × g) from
Heart 1min, sucks supernatant, stays nucleus to precipitate, and adds 200 μ L buffer GS, vibration in the nucleus precipitation being collected by centrifugation
Mix to thorough.
2) add 20 μ L Proteinase K solution, mix.
3) 200 μ L buffer GB are added, fully reverse mixing, 56 DEG C of placement 10min, period overturns and mixes for several times, solution
Strain limpid, as unchanged in solution limpid, can extend pyrolysis time to solution limpid.
4) plus people 200 μ l dehydrated alcohol, fully vibration mixes 15sec, now it is possible that flocculent deposit.
5) previous step resulting solution and flocculent deposit are all added in an adsorption column CB3 (adsorption column puts into collecting pipe
In), 12,000rpm (13,400 × g) is centrifuged 30sec, outwells waste liquid, adsorption column CB3 is put back in collecting pipe.
6) add in adsorption column CB3 500 μ l buffer GD (using before please first check whether added dehydrated alcohol),
12,000rpm (13,400 × g) is centrifuged 30sec, outwells waste liquid, adsorption column CB3 is put in collecting pipe.
7) add in adsorption column CB3 700 μ l rinsing liquid PW (using before please first check whether added dehydrated alcohol),
12,000rpm (13,400 × g) is centrifuged 30sec, outwells waste liquid, adsorption column CB3 is put in collecting pipe.
8) repetitive operation step 7.
9) 12,000rpm (13,400 × g) centrifugation 2min, outwells waste liquid.Adsorption column CB3 is placed in room temperature and places several points
Clock, thoroughly to dry remaining rinsing liquid in adsorbing material.
10) adsorption column CB3 is proceeded in 1.5ml centrifuge tube, to the hanging Deca in adsorbed film centre position 50～200 μ l eluting
Buffer TB, room temperature places 2～5min, and 12,000rpm (13,400 × g) is centrifuged 2min, and solution is collected in centrifuge tube.
With the quality of the genomic DNA of 0.8% agarose gel electrophoresiies Detection and Extraction, -80 DEG C of preservations.
2.3 amplimer designs：
(1) primer pair A sequence is as follows：
Forward primer：5'TGGCAATGACAGACCTTCAGGAC 3'
Downstream primer：5'CAGGAGGCTGACAATCAGGCTGT 3'
This primer amplification the 1st exon region of Chinese Simmental AGPAT6 gene, expanding fragment length is
411bp.
(2) primer pair B sequence is as follows：
Forward primer：5'CACCTTGTGTCCCTTTCCGC 3'
Downstream primer：5'AGAACAGCACTCCCCTAGCCCT 3'
This primer amplification the 12nd exon region of Chinese Simmental AGPAT6 gene, expanding fragment length is
239bp.
2.4PCR expands Simmental AGPAT6 gene：
Respectively with the DNA of Simmental colony of two China as template, enter performing PCR with the above-mentioned two primer pair of design
Amplification, its reaction system is 25 μ L, is shown in Table one；
Table one, PCR reaction system：
The PCR response procedures of two primer pairs are：95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 61 DEG C of annealing 45s, 72
DEG C extend 45s, 30 circulation；Extend 72 DEG C of 10min, last 16 DEG C of preservations.
The preparation of 2.5 buffer：
50X TAE buffer：Tris 242g, Na2EDTA·2H2O 37.2g, add in beaker about 800ml go from
Sub- water, is sufficiently stirred for dissolving, adds the acetic acid of 57.1ml, be sufficiently stirred for, and finally plus deionized water is settled to 1L, room temperature is protected
Deposit.
2.6PCR product is sequenced：
The pcr amplification product of every 45 different templates is mixed, serves marine growth Engineering Co., Ltd and be sequenced.
After the completion of sequencing, analysis sequencing peak figure, site set peak is to have single nucleotide polymorphism.As Fig. 3, Fig. 4.
3rd, the PCR-RFLP detection of Chinese Simmental AGPAT6 gene mononucleotide polymorphism：
3.1PCR expands Chinese Simmental AGPAT6 gene：
PCR amplification system and reaction condition are as described above, pcr amplification product 2% agarose gel carries out running electrophoresis
Detection, electrophoresis result such as Fig. 1, shown in Fig. 2.
3.2PCR-RFLP detects Chinese Simmental AGPAT6 gene mononucleotide polymorphism：
The pcr amplification product of primer pair A and B is carried out enzyme with restricted enzyme BstNI and HhaI of NEB company respectively
Cut, enzyme action system is shown in Table two；
The enzyme action system of table two restricted enzyme BstNI and HhaI：
Enzyme action condition：BstNI enzyme action system places 30min in 60 DEG C of metal baths, digestion products with 4% agar
Sugared gel, 120V electrophoresis 28min is detected, such as Fig. 5.HhaI enzyme action system is placed on placement 4h in 37 DEG C of metal baths, and enzyme action produces
Thing all with 3% agarose gel, 140V electrophoresis 30min detected, such as Fig. 6.
Use gel imaging system PHOTOGRAPHIC ANALYSIS, and manually carry out sentencing type：
The PCR primer of primer pair A comprises the T at the 303bp of AGPAT6 exon 1>C single nucleotide polymorphism,
When carrying out enzyme action with restricted enzyme BstNI, T allele can digested be opened, and C allele can not be cut open, simultaneously
Because also having the site that others can be cut by restricted enzyme BstNI in PCR primer, TT genotype individuals show as
139bp, 102bp, 61bp and 48bp band, CC genotype individuals show as 163bp, 139bp, 61bp and 48bp band, TC base
Because type individuality shows as 163bp, 139bp, 102bp, 61bp and 48bp band；
The PCR primer of primer pair B comprises the G at the 299bp of AGPAT6 gene the 12nd exon>A single nucleotide polymorphism,
When carrying out enzyme action with restricted enzyme HhaI, G allele can digested be opened, and A allele can not be cut open, simultaneously because
For also having a site that can be cut by restricted enzyme HhaI in PCR primer, GG genotype individuals show as 119bp and 58
(62) bp band, AG genotype individuals show as 177bp, 118bp and 58 (62) bp bands.
4th, the diagnostic application of molecular marker polymorphism in Simmental colony of two China of present invention preparation：
The diagnosis of 4.1 colony's polymorphisms：
Detection method using above-mentioned SNP polymorphism detects to Simmental colony of two China.
The frequency statistics analysis in 4.2SNP site：
Genotypic frequency refers to that certain genotype individuals number of a certain character in a colony accounts for the ratio of total individual number.PAA
=NAA/ N, wherein PAARepresent the AA genotypic frequency in a certain site；NAARepresent the number of individuals in colony with AA genotype；N is
The total quantity of detection colony.
Gene frequency refers to the relative ratios to its allele sum for a certain gene number in a colony.The formula calculating
Can be write as：PA=(2NAA+NAa)/2N.In formula, PARepresent allele A frequency, NAARepresent in colony that there is AA genotype
Individual amount, NAaRepresent in colony that there is Aa genotype individuals quantity.
The genotype frequency of two mononucleotide polymorphism sites of AGPAT6 gene in Simmental colony of two China
Rate and gene frequency are as follows：
In E1-303T>C site, CC type has 243, and TT type has 10, and TC type has 105.CC genotypic frequency is
0.6788, TT genotypic frequency is 0.0279, TC genotypic frequency is 0.8254, T equipotential for 0.2933, C gene frequency
Gene frequency is 0.1746；Therefore, C allele is preponderated, and with homozygous CC type as main genotypes, and meets Hardy's Wenbo
Lattice balance.
In E12-299G>A site, GG type has 336, and AG type has 42, and GG genotypic frequency is 0.8889, AG genotype
Frequency is 0.1111, G gene frequency is 0.0556 for 0.9444, A gene frequency；Therefore, G allele is dominant
Gesture, and with homozygous GG type as main genotypes.It is not detected by AA genotype it may be possible to due to AA base in the colony being surveyed
Because type can not maintain stable hereditary variation to be gradually eliminated in genetic process, or due to detection number of individuals not,
Do not find the presence of this genotype.
Correlation analysiss between two SNP site of AGPAT6 gene and Simmental character：
Table three E1-303T>Correlation analysiss between C site different genotype and Simmental character：
Note：Same character is represented with significant difference (P with different letter in average one hurdle<0.05),
Same character represents the not notable (P of difference with same letter mark in average one hurdle>0.05).
Table four, E12-299G>A site different genotype and the correlation analysiss of Simmental character
Note：Same character is represented with significant difference (P with different letter in average one hurdle<0.05),
Same character represents the not notable (P of difference with same letter mark in average one hurdle>0.05).
Claims (3)
1. a kind of detection method of cattle AGPAT6 gene mononucleotide polymorphism it is characterised in that：Its detection method is as described below：
The first step, carry out complete genome DNA extraction with the whole blood of cattle；
Second step, with extract complete genome DNA as template, respectively with primer pair A, B as primer, PCR expand cattle AGPAT6 base
Cause；
Described primer pair A is：
Forward primer：5'TGGCAATGACAGACCTTCAGGAC 3'
Downstream primer：5'CAGGAGGCTGACAATCAGGCTGT 3'
Described primer pair B is：
Forward primer：5'CACCTTGTGTCCCTTTCCGC 3'
Downstream primer：5'AGAACAGCACTCCCCTAGCCCT 3'
3rd step, with PCR mix products sequencing, find out SNP site；
4th step, with restricted enzyme BstNI, enzyme action is carried out to the PCR primer of primer pair A, with restricted enzyme HhaI pair
The PCR primer of primer pair B carries out enzyme action；
5th step, digestion products are entered row agarose gel electrophoresis, then its polymorphism is judged according to electrophoretic band：
The PCR primer of primer pair A comprises the T at the 303bp of AGPAT6 exon 1>C single nucleotide polymorphism, enzyme action
Afterwards, TT genotype individuals show as 139bp, 102bp, 61bp and 48bp band, and CC genotype individuals show as 163bp,
139bp, 61bp and 48bp band, TC genotype individuals show as 163bp, 139bp, 102bp, 61bp and 48bp band；
The PCR primer of primer pair B comprises the G at the 299bp of AGPAT6 gene the 12nd exon>A single nucleotide polymorphism, with limit
Property restriction endonuclease HhaI processed carries out after enzyme action to the PCR primer of primer pair B, and GG genotype individuals show as 119bp, 58bp and 62bp
Band, AA genotype individuals show as 177bp, 118bp, 58bp, and 62bp band.
2. a kind of cattle AGPAT6 gene mononucleotide polymorphism according to claim 1 detection method it is characterised in that：
The response procedures of the PCR amplification of described primer A, B are：95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 61 DEG C of annealing 45s,
72 DEG C of extension 45s, 30 circulations；Extend 72 DEG C of 10min, last 16 DEG C of preservations.
3. a kind of cattle AGPAT6 gene mononucleotide polymorphism according to claim 1 detection method it is characterised in that：
Described digestion products agarose gel electrophoresiies adopt 4% agarose gel.
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