CN106755422A - A kind of detection method of MEG3 gene SNP related to ox growth traits and its application - Google Patents

A kind of detection method of MEG3 gene SNP related to ox growth traits and its application Download PDF

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CN106755422A
CN106755422A CN201611219653.4A CN201611219653A CN106755422A CN 106755422 A CN106755422 A CN 106755422A CN 201611219653 A CN201611219653 A CN 201611219653A CN 106755422 A CN106755422 A CN 106755422A
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陈宏�
李波
刘梅
白跃宇
蓝贤勇
黄永震
雷初朝
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Northwest A&F University
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Abstract

Detection method and its application the invention discloses a kind of MEG3 gene SNP related to ox growth traits, according to ox MEG3 gene extrons sub-district single nucleotide mutation site position, parting, genotyping result and growth data association analysis are carried out to ox individuality using PCR RFLP, Tetra primer ARMS round pcrs.The detection method that the present invention is provided is detection and the closely related SNP marker of the ox production traits on DNA level, be can be used in Chinese Cattle kind growth traits marker assisted selection, the excellent ox population of Speed-up Establishment.

Description

A kind of detection method of the MEG3 gene SNP related to ox growth traits and its Using
Technical field
The present invention relates to domestic animal molecular Biological Detection field, and in particular to one kind detection Chinese Cattle kind is grown The method of the related MEG3 gene SNPs of proterties and application.
Background technology
SNP (single nucleotide polymorphisms, SNP) refers to because of single nucleotide acid Change and cause the DNA sequence polymorphism in genomic level.1 SNP has single nucleotide acid on certain site of genome Change, it is most to be presented by two kinds of situations：Caused by one is the conversion of single base, i.e., another pyrimidine, or one are changed into a kind of pyrimidine Plant purine and replace with another purine；Another situation is transversion, i.e. purine and pyrimidine is exchanged.According to biological heredity The effect of shape, also can be that encoding histone SNP and non-protein coding SNP, wherein encoding histone SNP are located at transcription sequence by SNP points In, non-protein coding SNP is located at non-transcribed sequences.In encoding histone SNP, if not causing changing for coded amino acid sequence Become, then referred to as synonymous encoding histone SNP, on the contrary referred to as non-synonymous encoding histone SNP, the generally function of influence correspondence protein.
The method of general detection SNPs has：PCR-RFLP methods, Tetra-primer ARMS-PCR, PCR-SSCP method, directly PCR sequencing PCR etc..Direct sequencing is simple, convenient, by the pcr amplified fragment of direct detection Different Individual, finds SNPs sites, But when SNPs is more, sample is big, experiment budget also can be big, or even mistake sometimes is sequenced, causes to increase repetition, further make into This increase.PCR-RFLP is mainly by restriction enzyme, the distinguished sequence of specific recognition DNA, and at particular sequence DNA molecular is cut, that is, the distinguished sequence of restriction fragment occurs, however, this method may be limited on recognition sequence, it is most handy To detect and find pettiness SNPs.Tetra-primer ARMS-PCR technologies, i.e. tetra-primer ARMS-PCR PCR, be It is a kind of growing up on the basis of regular-PCR and dedicated for detect SNP deriving technology.The technology is Grow up and combine the advantage of four primed PCR techniques on the basis of Refracting Mutation system is expanded, single nucleotide mutation is examined Measuring tool have the advantages that it is quick, easy, allelotype and low-cost can be distinguished.But the method is also not suitable for many sites Detected while SNP.PCR-SSCP is single-strand conformation polymorphism, is done based on single stranded DNA conformation differences, the detection of point mutation Method.The difference of single base, single-stranded DNA sequence are totally different, even if fragment is equally long, the speed migrated during its electrophoresis is different, two grades of knots Structure conformation is also different.The detection method has the drawback that and cannot be used for detecting SNPs on a large scale, because when the change of three-dimensional conformation Change during without acting on or act on small, polyacrylamide gel electrophoresis can not be distinguished.
Molecular breeding, i.e. molecular marker assisted selection (molecular mark-assist selection, MAS), the skill Art is that genetic resources or breeding material are selected by DNA molecular marker, and the Comprehensive Traits to livestock and poultry carry out breed improvement. In Animal Breeding, by closely related to growth traits, and with the selection of the DNA marker of quantitative character tight association, reach The purpose of breeding value accuracy is chosen seeds and improved to early stage, so as to obtain bigger genetic progress in Animal Breeding.
Maternal expressing gene 3 (maternally expressed 3, MEG3), is a kind of male parent imprinted gene, also known as Gtl2 genes, length is about 58kb, on No. 21 chromosome of ox, with class mRNA structures, but does not possess coding egg White ability.Have confirmed that MEG3 belongs to long-chain non-coding RNA at present, be also relatively more research at present, compare deep long-chain Non-coding RNA.
MEG3 expression high in mankind's pituitary, but pituitary gland tumour and people in gonadotrophic cell induction Low expression or do not expressed in other kinds of tumor cells systems of class, MEG3 is expressed in tumour is suppressed cell growth, and p53 Expressing quantity rises, and activates some downstream target genes of p53.There are some researches show the mouse for knocking out MEG3 genes is going out Before death will be dead and with serious muscle development defect.In livestock and poultry are studied, it has been found that multiple variant sites are to growth Proterties is made a significant impact, but report is had no in Chinese Cattle.Therefore, gene SNP variation is studied and by itself and China The important growth traits association analysis of yellow cattle breed is most important, can provide theoretical foundation for China's ox molecular breeding.
The content of the invention
It is an object of the invention to provide a kind of MEG3 gene SNP related to ox growth traits detection method and its Using, it is easy to the early stage marker assisted selection of native Chinese cattle growth traits, quickly set up the excellent ox kind of genetic resources Group.
To achieve these goals, present invention employs following technical scheme：
The SNP marker related to ox growth traits in the present invention, refers to be positioned at ox MEG3 gene candidates region Chr21:The mononucleotide polymorphic of AC_000178.1 (67377727-67395997).Specifically, described SNP marker is located at ox In the exon region of MEG3 genes (GenBank Accession No.AC_000178.1).
The present invention includes 2 × Taq PCR for detecting the reagent of the above-mentioned SNP marker related to ox growth traits Master Mix, restriction enzyme A vaI, agarose, TBE, ethidium bromide nucleic acid dye and deionized water.
SNP marker of the present invention can be answered in the outstanding yellow cattle breed of early molecule marker assisted selection growth traits With.
(1) genomic DNA of 3 kinds of Chinese Cattle (Xia Nanniu, Pi Nanniu and Qinchuan Cattle) to be measured is extracted；
(2) with 3 yellow cattle breed genomic DNAs pond to be measured (each kind (colony) selects 50 individual mixing) as mould Version, enters performing PCR and expands using the primer pair in table 2, and amplified production is sequenced.
(3) the MEG3 gene orders for sequencing result being provided with NCBI are compared, determine two SNP (SNP1 and SNP2) site；PCR-based-RFLP, is expanded, digestion and agar to SNP1 sites respectively with the primer pair in table 2-1 Sugared gel electrophoresis parting；Based on Tetra-primer ARMS-PCR, performing PCR is entered to SNP2 sites with the primer pair in table 2-2 Amplification, agarose gel electrophoresis parting.
Enter the amplification system used by performing PCR in step (2) for 50 μ L, wherein：The μ L of 50ng/ μ L template DNAs 2.5, Upstream and downstream primer (F, R) each μ L of 2.5 μ L, 2 × Taq PCR Master Mix 25, the μ L of deionized water 17.5 of 10pmol/L.
Amplification system in step (3) used by SNP1 sites is 10 μ L, wherein：The μ L of 50ng/ μ L template DNAs 0.5, Upstream and downstream primer (F1, R1) each μ L of 0.5 μ L, 2 × Taq PCR Master Mix 5, the μ L of deionized water 3.5 of 10pmol/L.
Amplification system in step (3) used by SNP2 sites is 10 μ L, wherein：The μ L of 50ng/ μ L template DNAs 0.5, Outer primer upstream and downstream primer (F3, R3) of inner primer upstream and downstream primer (F2, R2) each 0.4 the μ L, 10pmol/L of 10pmol/L Each the μ L of 0.2 μ L, 2 × Taq PCR Master Mix 5, the μ L of deionized water 3.3.
Digestion system in step (3) used by SNP1 sites is 10 μ L, wherein：The μ L of PCR primer 5, restriction enzyme 0.3 μ L, restriction enzyme buffer B uffer 1 μ L, the μ L of deionized water 3.7.
Compared with prior art, the invention has the advantages that：
(1) the Chinese Cattle MEG3 gene mononucleotide mutation detection methods that the present invention is provided, are not limited by the age, can For the early stage seed selection of ox, or even just be can be chosen in just birth.
(2) method PCR-based-RFLP, the Tetra-primer ARMS-PCR of detection ox MEG3 gene SNPs variation, accurate True reliable, easy to operate, cost is more cheap.
(3) detection of the SNP of ox MEG3 genes, for the molecular marker assisted selection that ox grows provide science according to According to.
Brief description of the drawings
Fig. 1 is two site sequencer maps of ox MEG3 genes, and inframe show mutational site.
Fig. 2 is the site restriction enzyme digestion and electrophoresis result figure of ox MEG3 gene SNPs 1 in the present invention.
Fig. 3 is the site PCR primer electrophoretogram of ox MEG3 gene SNPs 2 in the present invention.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples, the embodiment is to solution of the invention Release, rather than restriction.
In embodiments of the invention, with ox MEG3 gene candidates region Chr 21:67377727-67395997 is candidate bit Point：
(1) the SNP variation situations using sequencing technologies detection candidate locus in ox colony, using PCR-RFLP and Tetra-primer ARMS-PCR technologies carry out parting to the different SNP sites of Different Individual.
(2) SNP types and ox growth traits are associated analysis using the softwares of SPSS 20.0.
(3) the individual seed selection of the excellent ox of growth traits is carried out according to SNP types.
1. ox sample collection
The present invention is specifically with 3 populations of native Chinese cattle kind, i.e. Shaanxi Qinchuan Cattle (106), heavy Oil Reservoir of Henan Biyang Depression county summer Southern ox (241) and Henan Xinye County Pi Nanniu (372) as detection object, specific collecting sample (acquisition time in August, 2015) It is shown in Table 1.
The collection of the ox sample of table 1.
2. separation, extraction, the purifying of genomic DNA
Bibliography Sambrock et al (2002) method.
3. the amplification and sequencing of target sequence
With ncbi database (http://www.ncbi.nlm.nih.gov/) announce ox MEG3 gene orders (GenBank Accession No.AC_000178.1) is reference sequences, using the design sequencing PCR primers of Primer 5.0, its Primer pair sequence information is as shown in table 2.
(1) genomic DNA of 3 kinds of Chinese Cattle (Xia Nanniu, Qinchuan Cattle, Pi Nanniu) to be measured is extracted；
(2) with the genomic DNA pond (each kind (colony) selects 50 individual mixing) of 3 yellow cattle breeds to be measured as mould Version, enters performing PCR and expands using the primer pair in table 2, and amplified production is sequenced.
Wherein, used by PCR amplification system (50 μ L)：The upstream and downstream primer of 50ng/ μ L template DNAs 2.5 μ L, 10pM (F, R) each μ L of 2.5 μ L, 2 × Taq PCR Master Mix 25, the μ L of deionized water 17.5.
Wherein, the response procedures of PCR amplifications：95 DEG C of predegeneration 3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C are prolonged Stretch 30s, 35 circulations；72 DEG C of extension 10min；4 DEG C of preservations.
Sequencing result is as shown in figure 1, with the poba gene of 3 Chinese Cattles of kind (Xia Nanniu, Qinchuan Cattle, Pi Nanniu) Group DNA ponds are masterplate, with E1-E5 as primer, are expanded by Town-down round pcrs, and amplified production is sequenced, and are sequenced The MEG3 gene orders that result is provided with NCBI determine 2 SNP site (SNP1 after comparing：g.67384378C>T；SNP2： g.67395814G>T)。
Table 2. is sequenced PCR primer design information
The design of primers of 4.SNP sites parting
The site amplimer of SNP1, SNP2 is designed referring to table 2-1, table 2-2：
Table 2-1. parting PCR primer design informations
Table 2-2. parting PCR primer design informations
5.SNP1 sites PCR is expanded and digestions (PCR-RFLP)
1) amplification system used by SNP1 sites is 10 μ L, wherein：The μ L of 50ng/ μ L template DNAs 0.5,10pmol/L's Upstream and downstream primer (F1, R1) each μ L of 0.5 μ L, 2 × Taq PCR Master Mix 5, the μ L of deionized water 3.5.
2) response procedures of PCR amplifications：95 DEG C of predegeneration 3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extensions 30s, 35 circulations；72 DEG C of extension 10min, 4 DEG C of preservations.
Expanding fragment length for SNP1 is 218bp.
3) endonuclease reaction digests system (10 μ L)：5 μ L, 10 × T Buffer of PCR primer 1.0 μ L, AvaI (10U/ μ L) 0.3 μ L, the μ L of deionized water 3.7.
4) it is digested condition：10~12h is digested in 37 DEG C of constant incubators.
6.SNP2 sites PCR expands (Tetra-primer ARMS-PCR)
1) amplification system (10 μ L)：50ng/ μ L template DNAs 0.5 μ L, 10pmol/L inner primer upstream and downstream primer (F2, R2) each μ of 0.2 μ L, 2 × Taq PCR Master Mix 5 of the outer primer upstream and downstream primer (F3, R3) of each 0.4 μ L, 10pmol/L L, the μ L of deionized water 3.3.
2) response procedures of PCR amplifications：95 DEG C of predegeneration 3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extensions 30s, 35 circulations；72 DEG C of extension 10min, 4 DEG C of preservations.
Amplified fragments for SNP2 are as shown in table 3：
The amplified fragments of table 3.SNP2
7.SNP1 sites digestion with restriction enzyme PCR primer and SNP2 sites pcr amplification product agarose gel electrophoresis Analysis
1) 3.0% Ago-Gel (having been added to ethidium bromide nucleic acid dye), 120V electrophoresis after point sample are made 40min；
2) when the different DNA fragmentation of molecular weight is separated clearly, in the gel imaging systems of BIO-RAD Gel Doc 2000 Imaging；
3) according to agarose gel electrophoresis interpretation of result polymorphism.
Two genotyping results of SNP site of ox MEG3 genes are as shown in Figure 2 and Figure 3.
There are three kinds of genotype in SNP1 agarose gel electrophoresis result：CC genotypic expressions are 218bp bands, CT genotype 218bp, 147bp and 71bp band are shown as, TT genotypic expressions are 147bp and 71bp bands；71bp is in agarose electrophoresis point It is difficult to observe in analysis.
There are three kinds of genotype in SNP2 agarose gel electrophoresis result：GG genotypic expressions are 237bp and 161bp bands, GT genotypic expressions are 237bp, 161bp and 132bp band, and TT genotypic expressions are 237bp and 132bp bands.
8. the frequency statistics analysis of ox MEG3 gene SNP sites
1) gene and genotype frequency
Genotype frequency refers to the ratio that certain genotype individuals number accounts for total individual number in a colony.PAA=NAA/ N, its Middle PAARepresent the AA genotype frequencies in a certain site；NAARepresent the number of individuals with AA genotype in colony；N is detection colony Total quantity.
Gene frequency refers to relative ratios of a certain gene number to its allele sum in a colony.The formula of calculating Can be write as：PA=(2NAA+NAa1+NAa2+NAa3+NAa4+……+NAan)/2N, P in formulaARepresent allele A frequencies, NAARepresent Individual amount with AA genotype, N in colonyAaiRepresent in colony that there is Aai genotype individuals quantity, a1-an is equipotential N mutually different multiple allele of Gene A.
In the different SNP of different yellow cattle breed MEG3 genes, genotype frequency is as shown in table 4.
Table 4.MEG3 Polymorphism information
9. the association analysis of ox MEG3SNP locus genes effect
The association analysis result of two SNP of MEG3 genes and ox growth traits as shown in Table 5, Table 6, Table 7.
Relation analysis model：First data are described with analysis, it is determined whether there is outlier；Recycle least square point Analysis is to Data correction；According to data characteristics, using the growth traits effect between each genotype of SPSS20.0 software analysis.To base Fixed model is employed when being analyzed because of type effect：
Yijk=μ+Gj+eijk,
Wherein：YijkIt is character observation value, μ is population mean, GjIt is j-th fixed effect of single SNP marker genotype, eijkIt is random error.
Characteristic index：Body is high, hip cross is high, Body steep length, body long, bust, chest breadth, waist abdominal circumference, chest depth, buttocks long, Guan Wei, seat Epiphysis is wide, hip width, body weight
Analysis result shows that the different SNP genotype of MEG3 are developed to some growth of different yellow cattle breeds and had significantly Influence.Wherein in Xia Nanniu, SNP2 is enclosed in significantly correlated with Body steep length and pipe；In Pi Nanniu, SNP1 is high with hip cross, waist Angular width is significantly correlated；SNP2 is long with Body steep length and buttocks in extremely significantly correlated, with bust in significantly correlated；In Qinchuan Cattle, bust It is in significantly correlated with SNP1 and SNP2, chest depth is significantly correlated with SNP2.Illustrate that two site mutations of MEG3 genes can be as One candidate molecules genetic marker of raising ox growth traits, can be used for Chinese Cattle kind growth traits marker assisted selection In, the excellent ox population of Speed-up Establishment.
The association analysis of the summer of table 5. south ox MEG3 gene SNP site gene effects
Note：Indicate different letters (a, b) and represent significant difference
The association analysis of the skin of table 6. south ox MEG3 gene SNP site gene effects
Note：Indicate different letters (a, b) and represent significant difference, indicate different letters (A, B) and represent that difference is extremely notable.
The association analysis of the Qinchuan Cattle MEG3 gene SNP site gene effects of table 7.
Note：Indicate different letters (a, b) and represent significant difference.
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Claims (9)

1. a kind of detection method of the MEG3 gene SNP related to ox growth traits, it is characterised in that comprise the following steps：
With ox complete genome DNA to be measured as template, with primer pair P as primer, PCR amplification ox MEG3 genetic fragments, then Digestion is carried out to the product obtained according to primer pair P amplifications using restriction enzyme A vaI, then to the product fragment after digestion Enter row agarose gel electrophoresis, mononucleotide polymorphism site SNP1 on ox MEG3 genes is identified according to Gel electrophoresis results Genotype；And/or, with ox complete genome DNA to be measured as template, within primer pair I and outer primer be primer to U, PCR expands Increase ox MEG3 genetic fragments, row agarose gel electrophoresis are entered to amplified production, ox MEG3 is identified according to Gel electrophoresis results The genotype of mononucleotide polymorphism site SNP2 on gene；
Described primer pair P is：
Sense primer F1：5’-CGTCCTTCTAACGTTCTGCCTGT-3’
Anti-sense primer R1：5’-GCCTGATGAAATCGTGGTGTCTC-3’；
Described inner primer is to I：
Sense primer F2：5’-AAGGAGGACTATGTCAGGTTATTATGAG-3’
Anti-sense primer R2：5’-CGTATTTGGATTAATCCTAATGCTTAGA-3’；
Described outer primer is to U：
Sense primer F3：5’-AAAATGGAAAGATGCAAATTAATATGTAA-3’
Anti-sense primer R3：5’-GTTAAAGTTAAATCTCATCTCTGGTTCC-3’.
2. a kind of detection method of the MEG3 gene SNP related to ox growth traits according to claim 1, its feature exists In, the reaction system of the PCR amplifications is 10 μ L, wherein：The P of 50ng/ μ L template DNAs 0.5 μ L, 10pmol/L it is corresponding it is upper, Each μ L of 0.5 μ L, 2 × Taq PCR Master Mix 5 of anti-sense primer, and the μ L of deionized water 3.5.
3. a kind of detection method of the MEG3 gene SNP related to ox growth traits according to claim 1, its feature exists In, the reaction system of the PCR amplifications is 10 μ L, wherein：The inner primer of 50ng/ μ L template DNAs 0.5 μ L, 10pmol/L is to I Upstream and downstream primer each 0.4 μ L, 10pmol/L outer primer to each 0.2 μ L, 2 × Taq PCR of upstream and downstream primer of U The μ L of Master Mix 5, and the μ L of deionized water 3.3.
4. a kind of detection method of the MEG3 gene SNP related to ox growth traits according to claim 1, its feature exists In, the reaction system of the digestion is 10 μ L, wherein：The μ L of PCR primer 5, the μ L of restriction enzyme 0.3, restriction enzyme delay The μ L of fliud flushing 1, and the μ L of deionized water 3.7.
5. a kind of detection method of the MEG3 gene SNP related to ox growth traits according to claim 1, its feature exists In the PCR amplifications use following response procedures：95 DEG C of predegeneration 3min；94 DEG C of denaturation 30s, 60 DEG C of anneal 30s, 72 DEG C Extend 30s, 35 circulations；72 DEG C of extension 10min.
6. a kind of detection method of the MEG3 gene SNP related to ox growth traits according to claim 1, its feature exists In the agarose gel electrophoresis uses the Ago-Gel of mass concentration 3%.
7. a kind of detection method of the MEG3 gene SNP related to ox growth traits according to claim 1, its feature exists In the Gel electrophoresis results of the mononucleotide polymorphism site SNP1 are：CC genotypic expressions are 218bp bands, CT genes Type shows as 218bp, 147bp and 71bp band, and TT genotypic expressions are 147bp and 71bp bands；The mononucleotide polymorphic The Gel electrophoresis results of property site SNP2 are：GG genotypic expressions are 237bp and 161bp bands, and GT genotypic expressions are 237bp, 161bp and 132bp band, TT genotypic expressions are 237bp and 132bp bands.
8. a kind of detection kit of the MEG3 gene SNP related to ox growth traits, it is characterised in that including based on RFLP The primer pair P of SNP parting detections is carried out, and/or carries out drawing in SNP parting detections based on Tetra-primer ARMS-PCR Thing is to I and outer primer to U；
Described primer pair P is：
Sense primer F1：5’-CGTCCTTCTAACGTTCTGCCTGT-3’
Anti-sense primer R1：5’-GCCTGATGAAATCGTGGTGTCTC-3’；
Described inner primer is to I：
Sense primer F2：5’-AAGGAGGACTATGTCAGGTTATTATGAG-3’
Anti-sense primer R2：5’-CGTATTTGGATTAATCCTAATGCTTAGA-3’；
Described outer primer is to U：
Sense primer F3：5’-AAAATGGAAAGATGCAAATTAATATGTAA-3’
Anti-sense primer R3：5’-GTTAAAGTTAAATCTCATCTCTGGTTCC-3’.
9. a kind of detection method of MEG3 gene SNPs related to ox growth traits as claimed in claim 1 is in ox point Application in sub- marker assisted selection.
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WO2024065376A1 (en) * 2022-09-29 2024-04-04 中国农业大学 Use of novel locus of callipyge gene in sheep breeding

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CN101921848B (en) * 2010-07-26 2012-08-29 西北农林科技大学 Method for detecting single nucleotide polymorphism (SNP) of cattle MGAT2 gene
CN103290123B (en) * 2013-05-28 2014-09-10 西北农林科技大学 Detecting method and kit of cattle IGF2 (Insulin-like Growth Factor 2) gene mononucleotide polymorphism
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CN107475400A (en) * 2017-09-07 2017-12-15 西北农林科技大学 A kind of method and its dedicated kit of MYLK4 genes auxiliary detection ox growth traits
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WO2024065376A1 (en) * 2022-09-29 2024-04-04 中国农业大学 Use of novel locus of callipyge gene in sheep breeding

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