CN109161601A - A kind of PLAGL1 gene SNP label auxiliary quickly detects the method and its application of ox growth traits - Google Patents
A kind of PLAGL1 gene SNP label auxiliary quickly detects the method and its application of ox growth traits Download PDFInfo
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- CN109161601A CN109161601A CN201811184564.XA CN201811184564A CN109161601A CN 109161601 A CN109161601 A CN 109161601A CN 201811184564 A CN201811184564 A CN 201811184564A CN 109161601 A CN109161601 A CN 109161601A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses the method and its application that a kind of PLAGL1 gene SNP label auxiliary quickly detects ox growth traits.Using ox genomic DNA as template, ox PLAGL1 Gene Partial segment is obtained by PCR amplification；By PCR product after restriction enzyme Nde1 digestion, agarose gel electrophoresis is carried out；Ox PLAGL1 gene the 95141st SNP is identified according to electrophoresis result.The genotype of the SNP site of present invention ox PLAGL1 gene detected, can be used as the molecular genetic marker closely related with ox growth traits, for the molecular marker assisted selection of ox, good breed of cattle be pushed to breed process.
Description
Technical field
The invention belongs to molecular genetics fields, are related to ox gene mononucleotide polymorphism (SNP) as molecular genetic
The screening and detection of label, in particular to a kind of method and its application for detecting ox PLAGL1 gene mononucleotide polymorphism.
Background technique
The label of genetic polymorphism on molecular genetic marker, that is, DNA level, it can directly reflect on DNA sequence dna
Hereditary variation.Because it derives from DNA sequence dna difference, so being called molecular labeling, there is genetic diversity in biocenose
High, stability is strong and small feature affected by environment.Molecular marker assisted selection breeding is one of modem animal molecular breeding
Main research, because it selects the genotype of character directly on DNA level, so making the accuracy of seed selection significantly
It improves, compensates for the defect of conventional animal breeding method.
Single nucleotide polymorphism (SNP) is a kind of important molecular genetic marker, is primarily referred to as due to some in DNA sequence dna
Sequence polymorphism in genomic level caused by the change of nucleotide, form are mainly conversion and the top of single nucleotide acid
It changes.SNP compared with other molecular labelings, have covering gene group range is big, density is high, high resolution, inheritance stability and easily realize
The advantages that analysis automated.
After Botestein etc. proposes the related notion of DNA restriction fragment length polymorphism (RFLP) for the first time,
PCR-RFLP method is just widely used in the detection of SNP.The premise of adopting said method is that the site of SNP must be containing suitable
Restriction enzyme enzyme recognition site.Target fragment is cut using restriction enzyme, then carries out gel electrophoresis analysis,
The genotype of SNP site can accurately just be identified.
Studies have shown that PLAGL1 is related with the cell cycle, it is a kind of growth suppressor gene, participates in Apoptosis, cell week
Phase stagnates.Meanwhile it is proved to be a kind of transcription regulaton factor of 1 type pituitary adenylyl cyclase activating polypeptide receptor, it may
Participate in the adjusting grown for pancreas.Currently, focusing primarily upon itself and new life to the research of PLAGL1 gene pleiomorphism both at home and abroad
The association Journal of Sex Research of youngster's diabetes, tumour etc., and not yet appear in the newspapers for the research of domestic animal PLAGL1 gene mononucleotide polymorphism
Road.
Summary of the invention
The purpose of the present invention is to provide the sides that a kind of PLAGL1 gene SNP label auxiliary quickly detects ox growth traits
Method and its application.
In order to achieve the above objectives, the invention adopts the following technical scheme:
A kind of detection method of ox PLAGL1 gene SNP, comprising the following steps:
Using ox genomic DNA as template, using primer pair P as primer, PCR amplification ox PLAGL1 Gene Partial segment,
By pcr amplification product after restriction enzyme NdeI digestion, agarose gel electrophoresis is carried out, ox is identified according to electrophoresis result
The genotype of the 95141st (reference sequences NC_037336.1) SNP site of PLAGL1 gene；
Preferably, the sequence of the primer pair P are as follows:
Upstream primer F:5'-CAGAGGCAAGTCCTCAAGCA-3'；
Downstream primer R:5'-CTCACCGGTGTGCTTACTGT-3'.
Preferably, the response procedures of the PCR amplification are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing
30s, 72 DEG C of extension 50s, totally 32 recycle；72 DEG C of extension 5min.
Preferably, the agarose gel electrophoresis uses the Ago-Gel of mass concentration 3%.
Preferably, the electrophoresis result of the genotype of the 95141st SNP site of ox PLAGL1 gene are as follows: TT gene
Type shows as mono- band of 821bp；TC genotypic expression is tri- band of 821bp, 742bp and 79bp；CC genotypic expression is
Two band of 742bp and 79bp.
Application of the detection method of above-mentioned ox PLAGL1 gene SNP in ox molecular marker assisted selection breeding.
Application of the detection method of above-mentioned ox PLAGL1 gene SNP in auxiliary detection ox growth traits.
Preferably, the growth traits is that point of the buttocks is wide or buttocks is long.
Preferably, the ox is Qinchuan Cattle or growth traits in Jiaxian red cattle.
A kind of detection kit of ox PLAGL1 gene SNP, including it is above-mentioned for carrying out ox PLAGL1 gene the
The PCR amplification primer pair P of 95141 SNP site partings.
The beneficial effects of the present invention are embodied in:
The present invention is directed to ox PLAGL1 gene the 95141st SNP, by design primer, PCR amplification target fragment,
Then it is identified with digestion with restriction enzyme, it being capable of the mononucleotide simple, quickly, inexpensive, accuracy is high that identify gene
The genotype of polymorphic site.The present invention has carried out detection to the genotype of above-mentioned SNP site and gene frequency is analyzed, and should
The growth traits of site and two yellow cattle breeds is associated analysis.The result shows that the ox PLAGL1 gene that detection obtains
The genotype in 95141 single nucleotide polymorphism (T > C) sites, can be used as the genetic marker of molecular marker assisted selection breeding.
The present invention not only can provide basic data for the molecular marker assisted selection breeding of ox, but also can promote the germplasm of ox
Resource improves process.
Detailed description of the invention
Fig. 1 is the sequencer map of ox PLAGL1 gene the 95141st (NC_037336.1): outlining position indicates mutation position
Point (T95141C).
Fig. 2 is the electrophoretogram of ox PLAGL1 gene PCR amplified fragments.
Fig. 3 is the 95141st different genotype (TT, TC, CC) pcr amplification product of ox PLAGL1 gene through NdeI digestion
Rear electrophoresis result, in which: M D2000, respectively 2000bp, 1000bp, 750bp, 500bp, 300bp, 200bp.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples, the embodiment be explanation of the invention and
It is not to limit.
The present invention is with the genomic DNA pond of 2 kinds of yellow cattle breeds respectively according to ox PLAGL1 gene order design primer
Template, carries out PCR amplification, and product is sequenced to obtain the partial sequence of ox PLAGL1 gene.With the reference sequences announced on NCBI
It compares, in ox PLAGL1 gene the 95141st, there are the mutation of T > C for discovery, using PCR-RFLP method to the mutational site
It is detected.The finally single nucleotide polymorphism and the association analysis of ox growth traits by ox PLAGL1 gene in the site,
Prove that it can provide foundation for ox molecular breeding.
One, the clone of ox PLAGL1 Gene Partial sequence and its polymorphic detection
1. sample collection and extracting genome DNA
(1) acquisition of blood sample
Present invention employs 2 yellow cattle breeds, amount to 342 individuals and are used as test object, the acquisition method of blood is that neck is quiet
Arteries and veins blood sampling.The data information situation of the collecting region of blood sample and each kind is as shown in table 1.
1. experimental animal situation of table
(2) extraction of blood sample genomic DNA
1. freezing blood sample (predominantly haemocyte) thaw at RT, 500 μ L blood are drawn in 1.5mL centrifuge tube, be added etc.
The phosphate buffer (PBS) of volume mixes, mild to shake, and 4 DEG C, 12000rpm is centrifuged 5min, discards supernatant liquid, repeats above-mentioned step
Suddenly transparent to supernatant.
2. 500 μ L of DNA extraction buffer is added in centrifuge tube, gently blow and beat, haemocyte precipitating made to be detached from centrifugation tube wall,
37 DEG C of water-bath 1h.
3. plus Proteinase K is to 3 μ L (20mg/mL), and mixes, digested in 55 DEG C of water-baths overnight (16h or so) to cotton-shaped heavy
Shallow lake loses, solution clarification, not yet clear, can add the mixing of 1 μ L Proteinase K and continue digestion until clarification.
4. taking out the 6mol/L NaCl that 200 μ L are added in sample, mouth bottom, which is shaken 15 times, mixes well it, at 4 DEG C
12000rpm is centrifuged 10min, takes supernatant into 2.0mL centrifuge tube.
5. plus Tris- saturated phenol 1mL, placement mildly shake 20min on ice, mix well it；4 DEG C, 12000rpm from
Upper strata aqueous phase is transferred in another sterilizing 2.0mL centrifuge tube by heart 10min with pipettor.
6. the Tris- saturated phenol of 0.5mL and the chloroform of 0.5mL is added, 20min is mildly shaken on ice；4 DEG C,
12000rpm is centrifuged 10min；Upper strata aqueous phase is moved on in another sterilizing 2.0mL centrifuge tube with pipettor.
7. the chloroform of 1mL is added, places and mildly shake 20min on ice；4 DEG C, 12000rpm is centrifuged 10min；By upper water
Mutually moved on in another sterilizing 1.5mL centrifuge tube with pipettor.
8. 1mL pre-cooling dehydrated alcohol (- 20 DEG C) is added, gently mouth bottom, which is rocked, is repeatedly precipitated to DNA, then -20 DEG C of placements
30min；After taking-up, 4 DEG C, 12000rpm is centrifuged 10min, discards ethyl alcohol.
9. 70% ethyl alcohol 1mL is added, 10min is mildly shaken；Then 4 DEG C, 12000rpm is centrifuged 10min, discards ethyl alcohol, weight
It is multiple to rinse primary (tube bottom residual alcohol is sucked out with pipettor).
10. room temperature lower open mouth places 15min, being put into 60 DEG C of baking oven 30s later keeps ethyl alcohol volatilization clean；50 μ of ultrapure water is added
L, 4 DEG C of preservations to DNA are completely dissolved, after spectrophotometric determination concentration, -80 DEG C of preservations.
(3) building in the pond DNA
With OD value and its DNA content of the ultraviolet light spectrophotometric determination DNA sample at 260nm, 280nm.If
OD260/OD280Ratio illustrates then be purified in sample containing more protein or phenol less than 1.6；If ratio is greater than
1.8, then it should consider to remove RNA purifying.After DNA is detected, takes out a certain amount and be diluted to 10ng/ μ L, then from 2 Huangs
The sample after 50 dilutions is randomly choosed in cows body, each sample takes 2.5 μ L to be uniformly mixed the building pond DNA.
2. amplimer designs (design of primers deadline in September, 2017)
It is reference with the ox PLAGL1 gene order (NC_037336.1) that NCBI is announced, is set using 7.0 software of Oligo
Meter can expand the PCR primer comprising the 8th exon region of ox PLAGL1 gene to P, and primer sequence is as follows:
Upstream primer F:5'-CAGAGGCAAGTCCTCAAGCA-3'(20nt)
Downstream primer R:5'-CTCACCGGTGTGCTTACTGT-3'(20nt)
3.PCR amplification
(1) PCR is shown in reaction system such as table 2.
Table 2.PCR reaction system
(2) PCR response procedures are as shown in table 3.
Table 3.PCR response procedures
The sequencing of 4.PCR product
It will send Beijing AudioCodes prosperous Biotechnology Co., Ltd with the PCR product that the pond DNA mixed above is template amplification
It is sequenced.Ox PLAGL1 gene target fragment sequencing result is compared with reference sequences, is found in the 8th exon 1
There are the mutation of a T > C (Fig. 1, NC_037336.1:T95141C) in domain.
The digestion of 5.PCR product and RFLP detection
PCR reaction system: reference table 2, using the genomic DNA that blood sample extracts individual individually as template.
PCR response procedures: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 50s amount to 32
A circulation；72 DEG C of extension 5min.
Pcr amplification product is as shown in Fig. 2, amplified fragments size is about 821bp.
Restriction enzyme NdeI digestion is carried out for pcr amplification product respectively first, is then determined according to electrophoresis result
Its SNP polymorphism.
The NdeI digestion system of 10 μ L are as follows: 5 μ L PCR products, 10 × Buffer Tango 1 μ L, NdeI (10U/ μ L) 1 μ
L, 3 μ L sterilizing distilled water.Digestion system digests 12~16h in 37 DEG C of constant incubators, 3% Ago-Gel in 110V,
55mA and under room temperature electrophoresis 40min are imaged with Bio-RAD gel imager.According to the image of electrophoretic band, gene is judged
Type.
Referring to Fig. 3, since the amplified production of primer pair P is free of others NdeI restriction enzyme site, the PCR containing " C " expands
Volume increase object segment can be cut into two segments of 742bp and 79bp；PCR product containing " T " cannot be identified by NdeI, therefore still be one
A 821bp segment.Therefore, TT genotype electrophoresis result shows as mono- band of 821bp；TC genotypic expression be 821bp,
Tri- band of 742bp and 79bp, CC genotypic expression are two band of 742bp and 79bp.But due to 79bp is too short and in electrophoresis
It is not seen in figure, therefore in actual electrophoretic analysis, it can only at most observe two bands, but do not influence parting.
Two, the frequency statistics of ox PLAGL1 gene SNP site and its association analysis with growth traits
1. gene and genotype frequency
Genotype frequency refers to the ratio in a group between the various genotype of a certain character.Calculation formula is as follows:
PYY=NYY/N
Wherein PYYRepresent the YY genotype frequency in a certain site；NYYIndicate the number of individuals in group with YY genotype；N
For the individual total quantity for detecting group.
Gene frequency refers to the relative ratios of a certain its allele of gene pairs in a group.Calculation formula can be write as:
Py=(2Nyy+Nyy1+Nyy2+Nyy3+Nyy4+……+Nyyn)/2N
In formula, PyIndicate allele y frequency, NyyIndicate the individual amount in group with yy genotype, NyyiIndicate group
There is yy in bodyiGenotype individuals quantity, y1~ynFor the n mutually different multiple alleles of allele y.
The statistical result of gene frequency and genotype frequency in each yellow cattle breed is as shown in table 4.
The genotype and gene frequency of PLAGL1 gene in 4. two yellow cattle breeds of table
2. correlation analysis statistical model
Utilize the correlation of SPSS (18.0) software analysis gene loci and growth traits.First unite to being described property of data
Meter analysis, it is determined whether there are outliers to be analyzed according to data characteristics using t analysis, variance analysis or multivariate linear model
Genotype effects.In data handling, different according to the factor for influencing the Growth and development indexes such as body ruler and weight, it is contemplated that environment
Effect, age, genotype effects and relevant reciprocal effects, are analyzed using fixed model, meanwhile, according to the actual situation into
Row is accepted or rejected.Complete model is as follows:
Yijk=μ+Gj+Eijk
Wherein: YijkFor individual phenotypic record；μ is group's mean value；GjFor the genotype effects in each site；EijkFor with chance error
Difference.
Each yellow cattle breed group related data is counted using the above method, Qinchuan Cattle and growth traits in Jiaxian red cattle related data
Statistical result such as table 5 and table 6.
The association analysis of table 5. Qinchuan Cattle PLAGL1 gene different genotype and growth traits
Note: there is same letter to indicate that difference is not significant (P>0.05), letter is different to indicate significant difference (P<0.05)
The association analysis of table 6. growth traits in Jiaxian red cattle PLAGL1 gene different genotype and growth traits
Note: there is same letter to indicate that difference is not significant (P>0.05), letter is different to indicate significant difference (P<0.05)
The result shows that the different bases on the 95141st mononucleotide polymorphism site of ox PLAGL1 gene order
Because type and the wide equal Growth and development indexes association analysis of the buttocks of Qinchuan Cattle and Jiaxian County ox length and point of the buttocks show: the mononucleotide polymorphic
Property site is wide on point of the buttocks and buttocks is long influence it is significant.In Qinchuan Cattle, CC genotype and the point of the buttocks of the individual of TC genotype are wide
It is significantly narrower than the individual of TT type, the long individual for being considerably longer than TT type of the individual buttocks of CC genotype and TC genotype.In growth traits in Jiaxian red
The point of the buttocks width of the individual of Niu Zhong, TC genotype is significantly narrower than the individual of TT genotype, and the buttocks of the individual of TC genotype is long significant
It is longer than the individual of TT genotype.As can be seen that allele C and the growth traits of Qinchuan Cattle and growth traits in Jiaxian red cattle are (for example, point of the buttocks
Wide and buttocks is long) it is closely related, corresponding genotype can be used as the wide molecular genetic marker with the long Seedling selection of buttocks of ox point of the buttocks.
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>a kind of PLAGL1 gene SNP label auxiliary quickly detects the method and its application of ox growth traits
<160> 2
<210> 1
<211> 20
<212> DNA
<213>artificial synthesized
<400> 1
cagaggcaag tcctcaagca 20
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized
<400> 2
ctcaccggtg tgcttactgt 20
Claims (10)
1. a kind of detection method of ox PLAGL1 gene SNP, it is characterised in that: the following steps are included:
Using ox genomic DNA as template, PCR amplification ox PLAGL1 Gene Partial segment, the segment that PCR amplification is obtained is passed through
Restriction enzyme NdeI digests laggard row agarose gel electrophoresis, identifies ox PLAGL1 according to agarose gel electrophoresis results
The genotype of the 95141st SNP site of gene.
2. a kind of detection method of ox PLAGL1 gene SNP according to claim 1, it is characterised in that: the PCR amplification
The primer pair of use are as follows:
Upstream primer F:5'-CAGAGGCAAGTCCTCAAGCA-3'
Downstream primer R:5'-CTCACCGGTGTGCTTACTGT-3'.
3. a kind of detection method of ox PLAGL1 gene SNP according to claim 1, it is characterised in that: the PCR amplification
Response procedures are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 50s, totally 32 recycle；72
DEG C extend 5min.
4. a kind of detection method of ox PLAGL1 gene SNP according to claim 1, it is characterised in that: the agarose
Gel electrophoresis uses the Ago-Gel of mass concentration 3%.
5. a kind of detection method of ox PLAGL1 gene SNP according to claim 1, it is characterised in that: the SNP site
Genotype electrophoresis result are as follows: TT genotypic expression be mono- band of 821bp；TC genotypic expression be 821bp, 742bp and
Tri- band of 79bp；CC genotypic expression is two band of 742bp and 79bp.
6. the detection method of ox PLAGL1 gene SNP as described in claim 1 is in ox molecular marker assisted selection breeding
In application.
7. the detection method of ox PLAGL1 gene SNP as described in claim 1 is in auxiliary detection ox growth traits
Using.
8. application according to claim 7, it is characterised in that: the growth traits is wide selected from point of the buttocks or buttocks is long.
9. application according to claim 6 or 7, it is characterised in that: the ox is selected from Qinchuan Cattle or growth traits in Jiaxian red cattle.
10. a kind of detection kit of ox PLAGL1 gene SNP, it is characterised in that: including for carrying out ox PLAGL1 base
Because of the PCR amplification primer pair of the 95141st SNP site parting.
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| CN107988385A (en) * | 2017-12-04 | 2018-05-04 | 西北农林科技大学 | A kind of method and its dedicated kit for detecting beef cattle PLAG1 genes Indel marks |
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Non-Patent Citations (4)
| Title |
|---|
| ALMAS R JUMA等: "Emerging role of PLAG1 as a regulator of growth and reproduction", 《JOURNAL OF ENDOCRINOLOGY》 * |
| 佚名: "rs110111117", 《ENSENMBL》 * |
| 佚名: "rs110111117", 《UCSC》 * |
| 阎建宇: "中国三个黄牛品种ASB-3基因多态性及其遗传效应与mRNA表达研究", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 * |
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