CN106755371A - Method and its application using PCR RFLP detection sheep PCNP gene mononucleotide polymorphisms - Google Patents
Method and its application using PCR RFLP detection sheep PCNP gene mononucleotide polymorphisms Download PDFInfo
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Abstract
Method and its application the invention discloses a kind of utilization PCR RFLP detection sheep PCNP gene mononucleotide polymorphisms：With sheep complete genome DNA to be measured as template, with primer pair P as primer, PCR amplification sheep PCNP genetic fragments；After with restriction enzyme SPhI digestion pcr amplification products, then row agarose gel electrophoresis are entered to the amplified fragments after digestion；The nucleotide polymorphisms of sheep PCNP genes the 5019th are identified according to electrophoresis result.Due to the base mutation site and Sheep Reproductive Characters litter size close association, it is in this way a kind of method of detection on DNA level and the closely related molecular genetic marker of ovine growth proterties, can be used for the assisted Selection of sheep and molecular breeding, accelerate sheep stock breeding speed.
Description
Technical field
The invention belongs to molecular genetics field, it is related to make with the SNP of the functional gene of sheep (SNP)
It is molecular genetic marker, the more particularly to SNP and its detection method of sheep PCNP genes.
Background technology
In Sheep Breeding, it is intended that by closely related to reproductive trait, and with quantitative character close linkage
The selection of DNA marker, reaches early stage seed selection and improves the purpose of breeding value accuracy, so as to obtain bigger in Animal Breeding
Genetic progress.
Molecular breeding, i.e. molecular marker assisted selection breeding (Molecular Mark-Assist Selection, MAS),
The technology is that genetic resources or breeding material are selected by DNA molecular marker, and the Comprehensive Traits to livestock and poultry carry out kind
Improvement, it is the method being combined using modern molecular biology and traditional genetic breeding, carries out breeding of new variety.
Gene pleiomorphism refers to the difference of genome sequence between the Different Individual in different plant species or same species, these
Difference be due in chromosome DNA allele nucleotide change cause, mainly the replacement including base, insertion, lack
And the change of repetitive sequence copy number.
SNP (Single Nucleotide Polymorphism, SNP) is by masschusetts, U.S.A science and engineering
The scholar Lander (1996) in the human genome research center of institute as soon as the class genetic marker system for proposing, refers to genome
The polymorphism caused due to the replacement of single nucleotide acid (A/T/C/G) in DNA sequence dna.SNP is wide as new genetic marker
It is general to be applied to the assignment of genes gene mapping, clone, genetic breeding and multifarious research.SNP be a kind of quantity present in genome very
Abundant variant form, accounts for more than 90% of genetic polymorphism in human genome.SNP is different from rare variation, generally exists
This kind of variation of the frequency equal to or less than 1% is referred to as mutation in population, and is just referred to as monokaryon glycosides when only frequency is more than 1%
Sour polymorphism.Its variant form has：Transversion, conversion, insertion and missing etc., mainly by conversion or the transversion institute of single base
Cause.The SNPs of the nucleotide variation with conversion hysteria accounts for 2/3.
According to the position that SNP in genome is produced, following 3 class can be divided into：Gene coding region mononucleotide
Polymorphism (Coding-region SNPs, cSNPs), gene periphery SNP (Perigenic SNPs, pSNPs)
And SNP (Intergenic SNPs, iSNPs) between gene.
Research shows that the cSNP in code area is fewer.CSNP in gene coding region can be divided into 2 kinds again：It is a kind of
It is the synonymous cSNP (Synonymous cSNP) in code area, i.e. the change of coded sequence caused by SNP can't influence it to be turned over
The change of the Amino Acids in Proteins sequence translated；Another kind is the non-synonymous cSNP (Non-Synonymous in code area
CSNP), i.e., the change of base sequence will cause the change of coded amino acid, so as to cause changing for Amino Acids in Proteins sequence
Become, the function of protein may be eventually affected.
Because SNPs is two equipotential gene molecule markers, so, in theory in a diplont colony, SNPs can
Can be made up of 2,3 or 4 allele, but the SNPs of actually 3 or 4 allele is very rare, therefore SNPs is logical
Often it is referred to simply as two equipotential gene molecule markers.At present, mainly SNPs is found using several different routes：That is DNA
Sequencing methods, polymerase chain reaction-single-strand conformation polymorphism (Polymerase Chain Reaction-Single
Strand Conformation Polymorphism, PCR-SSCP) and DNA sequencing combined techniques, allele specific PCR
(Allele Specific PCR, AS-PCR) method, primer extension and oligonucleotides coupled reaction etc..In these SNP detections
In technology, determined dna sequence method is SNP detection method the most accurate, but, its testing cost is expensive, and needs DNA surveys
The large-scale instruments such as sequence instrument, meanwhile, very those skilled in the art and experience are needed in sequencing procedure, so, determined dna sequence
Method is not a kind of preferable SNP detection method for being applied to produce reality；Certainly, examined using PCR-SSCP and DNA sequencing combined techniques
Surveying SNP can suitably reduce testing cost, but, the experimentation of PCR-SSCP is long, and operation is comparatively laborious, and tests
There is false positive issue in journey, so, also and nonideal SNP detection means；AS-PCR methods are examined as a kind of new SNP
Survey method, has boundless prospect in following application field, but, the method needs to design special primer, and
Specific gene loci can only be directed to, meanwhile, also there is the probability of flase drop in detection process, therefore, do not have generally should at present
The characteristics of using；And primer extension and oligonucleotides coupled reaction technology for detection SNP site are, it is necessary to plate reader, gene core
The detection platforms such as piece, micro-sphere array technology and mass spectrograph, exploitativeness is not strong for general Molecular Laboratory.
Polymerase chain reaction-restriction fragment length polymorphism (Restriction Fragment Length
Polymorphism-Polymerase Chain Reaction, RFLP-PCR) method is a kind of effective technology of detection SNP,
Upstream and downstream primer is designed after SNP site is found to be cut with restriction enzyme, then carries out agarose, polyacrylate hydrogel
Electrophoretic analysis, just can exactly differentiate SNP site.RFLP-PCR methods not only have the accuracy of DNA sequencing method, overcome again
Somewhat expensive, troublesome operation, the shortcoming of false positive, and the sequence site for being detected is without particularity requirement.
PCNP (PEST-containing nuclear protein) is that a class is positioned at nucleus and contains PEST sequences
(i.e. Pro-rich (P), glutamic acid (E), four kinds of albumen of amino acid of serine (S) and threonine (T).
Find that the homology of PCNP gene nucleic acid sequences is higher by the sequence for comparing multiple species, it is especially dynamic in lactation
More than 94% is reached in thing, all than more conservative, illustrates that the albumen should have important effect and function in organism.In people
PCNP genes are positioned in 3q12.3 in genoid group, encode 178 amino, show that it is after being analyzed to its physicochemical property
A kind of half-life period for being made up of 18 kinds of amino acid shorter and unstable hydrophilic protein.PCNP is likely in cell cycle regulating
Played a role with tumour generating process, No. 3 chromosomes where encoding the gene of PCNP contain chemokine receptors (CKR) base
Because of cluster and various human cancer related gene locis, crucial tumor suppressor gene such as apoptotic effector RASSF1, cell transposition
Control factor HYAL1, Angiostatin SEMA3B are also in No. 3 chromosomes, marfan's syndrome, porpharia, Von
The hereditary diseases such as Hippel-Lindau syndromes, osteogenesis imperfecta are also related to No. 3 chromosomes.
The research on PCNP at present is also fewer both at home and abroad.Zhu Jiangmu researchs find that the overexpression of PCNP can be remarkably promoted
Mice skeletal cell C2C12 cell growths, PCNP may take part in the cycle regulating of myocyte, have regulation and control to muscle cell growth
Effect, the function to myocyte also has an impact.Sun Yingchuan researchs find that PCNP is in base in rheumatoid arthritis patients peripheral blood
Because the expression of level and protein level is increased, the weight of the rheumatoid arthritis state of an illness and the expression quantity positive correlation of PCNP.In class wind
In wet arthritic morbidity, the expression change of the gene is closely related with the state of an illness of patient, and PCNP is likely to become clinical diagnosis
The Testing index of rheumatoid arthritis, also also can be probably potential molecule as disease condition development, the reference index of prognosis
Therapy target.The expression difference fragment GW360093 for belonging to PCNP genes is obtained by mRNA differential display techniques, in difference
There is expression difference in the ovary tissue of reproduction goat, real influence Goat Reproduction Traits change is that the gene expression is produced
The number of thing.
PCNP is accurately positioned onto No. 1 chromosome on sheep, including 5 extrons.Accession number (the NC_ of Genomic
019458.1), predicted mRNA accession number XM_012189341.1, mRNA total lengths are 712bp, the CDS sequences of the gene
Total length is 537bp (7-543), can encode 179 amino acid.Research for sheep PCNP is also little, domestic and international rarely seen report
Road.Agricultural University Of Hebei Xing in 2013 increases happiness and has successfully cloned the promoter of goat PCNP genes and predicted the regulation and control of its core
Area, for the transcription regulation mechanism for studying PCNP genes lays the foundation.
The animals such as people, mouse, pig are more common in research on the variation of animal PCNP gene genetics both at home and abroad, and do not have sheep
PCNP gene genetics become the report of XOR SNP researchs.Due to current China sheep, Small-fat-tail sheep PCNP gene genetics variation field
Research it is deficient, make the functional study of the gene loci and gene genetic variation and economic characters, such as reproductive trait association
Research turns into blank.
The content of the invention
It is an object of the invention to provide a kind of utilization PCR-RFLP detection sheep PCNP gene mononucleotide polymorphisms
Method and its application, may cause encoding proteins conformation to be sent out using PCR-RFLP methods for the missense mutation on its gene loci
The SNP of changing is detected that eliminate the individuality for producing missense mutation in advance, quickening has Quality and economy
The foundation of shape sheep population.
The present invention is achieved through the following technical solutions：
Sheep PCNP genes the 5019th are that the detection method of the SNP of G or A is：With comprising PCNP genes
Sheep to be measured (such as sheep, Small-fat-tail sheep) complete genome DNA be template, with primer pair P as primer, PCR amplification sheep, small
The cold sheep PCNP genes of tail；After with restriction enzyme SPhI digestion pcr amplification products, then the amplified fragments after digestion are entered
Row agarose gel electrophoresis；The SNP of sheep PCNP genes the 5019th is identified according to electrophoresis result；
Described primer pair P is：
Sense primer：5’-AGAATAGCATGGGCAGAA-3’ 18nt；
Anti-sense primer：5’-TCATTTCTGTGGGACACC-3’ 18nt.
Described pcr amplification reaction program is：94 DEG C of predegeneration 5min；94 DEG C of denaturation 30s, 55.3 DEG C of anneal 30s, 72 DEG C
Extend 50s, 35 circulations；72 DEG C of extension 10min.
Described agarose gel electrophoresis is agarose gel electrophoresis that mass concentration is 1%.
It is according to the bit base polymorphism of agarose gel electrophoresis result PCNP genes the 5019th：AA types are showed：377bp and
309bp；GA types are showed：686bp, 377bp and 309bp；GG types are showed：686bp.
Compared with prior art, the present invention has following beneficial technique effect：
The present invention is using RFLP-PCR methods to the mutation on the site of sheep PCNP genes the 5019th there may be coding egg
The SNP that white conformation changes is detected, is the mutation of G → A, the mutation when site sports A by G
Do not cause the change of amino acid, belong to same sense mutation, make the space of the PCNP coded by said gene albumen with important physiological function
2nd, three-level configuration changes, so that the biological function of influence albumen.The nucleotide polymorphisms can be lost as a molecule
Mark is passed, using marker site information and the phenotypic information of quantitative character, the more accurate breeding value for estimating animal individual improves choosing
Efficiency is selected, accelerates Advances in Breeding.
The present invention, being capable of, quick, cost simple with RFLP-PCR methods by designing specific PCR primer amplified fragments
The polymorphism of mononucleotide that is low, accurately detecting above-mentioned PCNP genes.
The present invention has carried out Genotyping and gene frequency analysis to the SNP of PCNP genes, and with sheep, Small-fat-tail sheep
Association analysis has been carried out between growth traits；It is auxiliary that the nucleotide polymorphism site of result display PCNP genes can turn into molecular genetic
Help the mark of breeding.
The detection method that the present invention is provided is laid a good foundation for the SNP of PCNP genes with the foundation of growth traits relation, with
Just it is used for the marker assisted selection of the sheep variety growth traits such as Chinese sheep, Small-fat-tail sheep, quickly sets up genetic resources excellent
Sheep population.
Brief description of the drawings
Fig. 1 is sheep, Small-fat-tail sheep blood sample genome dna electrophoresis detection figure；
Fig. 2 is sheep, the electrophoretogram of the 686bp fragments of Small-fat-tail sheep PCNP gene PCRs amplification；
Fig. 3 is the SPhI restriction enzyme digestion and electrophoresis detection PCNP gene polymorphics of sheep, Small-fat-tail sheep PCNP gene 686bp PCR primers
The electrophoresis result figure of property；Swimming lane 1,2：GG genotype individuals (686bp)；Swimming lane 3,4：AA genotype individuals (377bp, 309bp)；
Swimming lane 5,6：GA genotype individuals (686bp, 377bp and 309bp)；M：Marker (2000bp, 1000bp, 750bp, 250bp,
100bp)；
Fig. 4 is the different genotype sequencing peak figure of sheep, 5019 SNP of Small-fat-tail sheep PCNP genes, wherein, a correspondences GG
Genotype, b corresponding A A genotype, c correspondence GA genotype.
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.
The present invention expands PCNP Exon 2 686bp fragments with PCNP genes conserved sequence design primer, with lake
Sheep, Small-fat-tail sheep genome are template, enter performing PCR amplification, and amplified production finds the mononucleotide of the amplified fragments after sequencing
It is polymorphic；Proterties correlation analysis is carried out for the mononucleotide polymorphic for finding, and its detection method is provided so that PCNP genes
Nucleotide polymorphisms turn into a kind of molecular genetic marker that can quickly, conveniently detect, are that Speed-up Establishment has Quality and economy
The sheep such as sheep, Small-fat-tail sheep of shape population provides foundation.
The detection of a, sheep PCNP gene pleiomorphisms
1st, the collection and treatment of sheep blood sample
Sheep, Small-fat-tail sheep blood sample 10mL are taken, the μ L anti-freezings of EDTA 500 of 0.5mol/L are added, put after slowly overturning 3 times
Enter ice chest, -80 DEG C save backup.
The present invention is specific as shown in table 1 using sheep, Small-fat-tail sheep kind.
The sheep sample source table of table 1
2nd, the extraction of blood sample genomic DNA
(1) blood sample of frost melts in room-temperature water bath；
(2) 1mL whole bloods are transferred in an aseptic 2mL centrifuge tube；
(3) PBS of isometric (1mL) is added, 15-20min is gently shaken；
(4) 4 DEG C of 12000*g are centrifuged 10min；
(5) supernatant is abandoned with liquid-transfering gun, repeat step 3,4 to supernatant is transparent, precipitate white；
(6) DNA extract solution 650-750 μ L are added in centrifuge tube, gentle shake makes cell precipitation suspend；
(7) 37 DEG C of water-bath 1h；(can omit)
(8) the μ L of Proteinase K 3 (final concentration of 60 μ g/mL) is added, is mixed；
(9) in constant water bath box 55 DEG C be incubated overnight (16h or so), digested completely to cell pellet, solution is clear
Clearly；
(10) reaction solution is cooled to room temperature, adds 1 times of Tris saturated phenol of volume (800 μ L-1mL), places gentle on ice
Shake 15min；
(11) 4 DEG C, 12000g centrifugations 10min；
(12) in upper strata aqueous phase liquid-transfering gun being moved on into another sterile centrifugation tube；
(13) 0.5 times of phenol and 0.5 times of chloroform of (0.5mL) of volume (0.5mL) are added, gentle shake on ice is placed
15min；
(14) 4 DEG C, 12000g centrifugations 10min；
(15) in upper strata aqueous phase liquid-transfering gun being moved on into another sterile centrifugation tube；
(16) 1 times of chloroform of volume (1mL) is added, gentle shake 15min on ice is placed；
(17) 4 DEG C, 12000g centrifugations 10min；
(18) in upper strata aqueous phase liquid-transfering gun being moved on into another sterile centrifugation tube；
(19) 2 times of precooling absolute ethyl alcohols (- 20 DEG C) of volume are added, gently mouth bottom is rocked and repeatedly separated out to DNA, then-
20 DEG C of placement 30min；
(20) 4 DEG C, 12000g centrifugations 10min；Abandon supernatant；
(21) 70% ethanol 1mL is added, 10min is gently shaken；
(22) 4 DEG C, 12000g centrifugation 10min abandon ethanol, repeat rinsing once；
(23) it is vacuum dried or ethanol is volatilized totally at room temperature；
(24) according to the amount of DNA, ultra-pure water 100-300 μ L are added, 4 DEG C of preservations to DNA are completely dissolved, spectrophotometric measurement
After determining concentration, -80 DEG C of preservations.
3rd, the structure in DNA ponds
(1) 1% agarose gel electrophoresis is detected
Select part DNA sample enter row agarose gel electrophoresis detect, selection DNA sample band it is homogeneous, without hangover, without degraded
Sample carry out the structure in DNA ponds.
(2) OD values are determined
With OD value of the ultraviolet light photometric determination DNA sample at 260nm, 280nm.Calculate DNA content and OD260/OD280
Ratio.Such as OD260/OD280Ratio is less than 1.6, illustrates to contain more protein or phenol in sample, then should be purified；If
Ratio is more than 1.8, then should consider removal RNA purifying.
DNA concentration (ng/ μ L)=50 × OD260Value × extension rate
(3) structure in kind DNA ponds
After DNA detections are finished, take out a certain amount and be diluted to 50ng/ μ L, be then from sheep, 50 concentration of Small-fat-tail sheep
10 μ L mixing is taken in 50ng/ μ L DNA samples and is built into kind DNA ponds；
Sheep, the testing result of Small-fat-tail sheep blood sample genomic DNA are shown in Fig. 1, as can be seen from the figure sheep, Small-fat-tail sheep
The quality of genomic DNA is very high.
4th, PCR amplifications
With sheep DNA ponds as masterplate, enter performing PCR with the primer pair P of design and expand, PCR overall reactions system is 25.3 μ L, is seen
Table 2；PCR overall reaction programs, are shown in Table 3.
The PCR reaction systems of table 2
Primer pair P：
Sense primer：5’-AGAATAGCATGGGCAGAA-3’ 18nt；
Anti-sense primer：5’-TCATTTCTGTGGGACACC-3’ 18nt
The optimal PCR response procedures of the primer of table 3
5th, PCR primer purifying and sequencing
PCR amplifications enter row agarose gel electrophoresis after completing, and electrophoresis result is as shown in Figure 2, here it is apparent that 686bp
Band；Then gel extraction and the purifying of PCR primer are carried out：Cut containing purpose fragment from Ago-Gel under uviol lamp
Gel, be put into 1.5mL centrifuge tubes, then with PCR primer recovery purifying kit (Beijing Tiangeng biotech firm) purify PCR
Product, operates according to kit specification, comprises the following steps that：
(1) first to 500 μ L equilibrium liquids BL, 12000r/min centrifugation 1min are added in adsorption column, in outwelling collecting pipe
Waste liquid, during adsorption column placed back in into collecting pipe.
(2) single target DNA band is cut from Ago-Gel and is put into clean centrifuge tube, weigh weight.
(3) to isometric solution PC is added in blob of viscose, 10min or so is placed in 60 DEG C of water-baths, constantly leniently upper and lower therebetween
Upset centrifuge tube, to ensure that blob of viscose fully dissolves.
(4) in previous step resulting solution being added into an adsorption column, 12000r/min centrifugation 1min, in outwelling collecting pipe
Waste liquid, adsorption column is reentered into collecting pipe.
(5) to 700 μ L rinsing liquids are added in adsorption column, 12000r/min centrifugation 1min outwell waste liquid, by adsorption column again
It is put into collecting pipe.
(6) to 500 μ L rinsing liquids are added in adsorption column, 12000r/min centrifugation 1min outwell waste liquid, by centrifugal adsorbing column
It is put into collecting pipe, 12000r/min centrifugation 2min remove rinsing liquid as far as possible.Adsorption column is placed in room temperature or 50 DEG C of incubator numbers point
Clock, thoroughly dries.
(7) adsorption column is put into a clean centrifuge tube, appropriate wash-out is vacantly added dropwise to adsorbed film centre position
Buffer solution, room temperature is placed 2 minutes.12000r/min centrifugations 1min collects DNA solution.
(8) in order to improve the yield of DNA, the solution for obtaining can be centrifuged again in add-back centrifugal adsorbing column, repeats to walk
Rapid 7.
The PCR purified products with sheep, Small-fat-tail sheep DNA ponds as template serve marine growth Engineering Co., Ltd carry out it is double
To sequencing.Sheep, Small-fat-tail sheep PCNP gene purpose fragments 686bp sequencing result it is as shown in Figure 4.
Sequencing peak figure is analyzed, wherein there are two different peaks in same site is that there occurs single nucleotide mutation；
Two kinds of testing results of G, A are occurred in that positioned at sheep, the 5019th of Small-fat-tail sheep PCNP genes, it is sheep that as examination is arrived, small
The SNP polymorphisms of the cold sheep PCNP genes of tail, the site is the nucleotide polymorphisms for G or A.
B, sheep PCNP genes G>The RFLP-PCR detections of A mutation polymorphisms
Because the nucleotide polymorphisms that examination is arrived are nature restriction enzyme site, can carry out PCR-RFLP by conventional restriction endonuclease to reflect
It is fixed.When the PCNP genes the 5019th of sheep, Small-fat-tail sheep do not occur G>When A is mutated, G before being as mutated is expanded using primer pair P
The PCNP gene order GCATGC of increasing, are the restriction enzyme enzyme recognition site of SPhI；Can be directly by SPhI to purpose fragment
Digestion carry out Genotyping.
1st, RFLP-PCR design of primers
With reference to primer pair P.
Above-mentioned primer can expand sheep, the exon 6 86bp fragments of Small-fat-tail sheep PCNP genes 2.
2nd, RFLP-PCR reaction conditions
Respectively as described in table 2 and table 3,1% agarose of pcr amplification product coagulates for PCR primer amplification system and reaction condition
Gel electrophoresis collection of illustrative plates is as shown in Figure 2, it can be seen that the primer pair P of design can expand the fragment of 686bp.
3rd, the SPhI digestions of pcr amplification product
(1) digestion system is：It is 0.5 μ L, Buffer B of sphI restriction endonucleases 2 μ L, ddH2μ L, 4 μ L the PCR amplifications of O 13 are produced
Thing, amounts to 20 μ L.
(2) it is digested condition：12~16h is digested in 37 DEG C of constant incubators.
(3) agarose gel electrophoresis analysis after SPhI digestion PCR primer
With 1.0% Ago-Gel, 200V electrophoresis 18min, nucleic acid staining dye detection digestion result uses UVP
Gel imaging system (GelDoc-It TS Imaging System) PHOTOGRAPHIC ANALYSIS, and sentence type, record its genotype；
Due to not including other SPhI digestions recognition sites in the 686bp fragments of PCR-RFLP amplifications, when PCNP genes
5019th there is no G>When A is mutated, after the PCNP gene outcomes of PCR amplifications are recognized by restriction enzyme SPhI,
Amplified fragments are cut to 2 sections by GCATGC/CGTACG to amplified fragments digestion；And work as PCNP genes the 5019th and undergo mutation, no
New restriction enzyme SPhI digestion recognition sites can be formed, amplified fragments can not be digested；
Because sheep is 2 times of body animals, so as generation G>During the mutation of A, 3 kinds of different genotype can be formed, respectively
It is GG, GA, AA, the gel result of its PCR-RFLP detections is as shown in Figure 3：
Wherein, GG genotype is wild type, and its two SNP sites of DNA can not be digested, and show as 686bp
Band；Two SNP sites of chain of the AA genotype after undergoing mutation can show as 377bp and 309bp bars by SPhI digestions
Band；The SNP site of one in two chains of heterozygote GA can identified and another can not be identified, show as 686bp,
377bp and 309bp bands, the size of number and band according to band, determining whether of can will be apparent that there occurs point mutation,
Three kinds of genotype are distinguished, so as to detect its SNP polymorphism.
(4) sequence verification of different genotype individuality PCR primer
Positive and negative two-way survey is carried out respectively to different genotype individuality PCR primer using ABI 5019 and the sequenators of ABI 3730
Sequence；Meanwhile, SNP position analyses are carried out, as a result show the heterozygote GA genotype comprising 686bp, 377bp and 309bp band
Body its 5019th sequencer map is expressed as G or A really, and as illustrated in fig. 4 c, the 5019th is two peaks from left to right；And GG bases
Because type, AA genotype are respectively G, A, respectively as shown in Fig. 4 a, Fig. 4 b.
In c, different Sheep Populations the SNP of PCNP genes the 5019th as molecular labeling application
1st, the detection of colony's SNP
Amount to 604 parts of DNA samples to sheep, Small-fat-tail sheep using above-mentioned SNP pleiomorphism detecting methods, carry out SNP many
The identification of state property；Count the frequency distribution situation of its SNP site.
2nd, the frequency statistics analysis of SNP site
Genotype frequency refers to that certain genotype individuals number of a certain proterties in a colony accounts for the ratio of total individual number.PAA
=NAA/ N, wherein PAARepresent the AA genotype frequencies in a certain site；NAARepresent the number of individuals with AA genotype in colony；N is
Detect the total quantity of colony.
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+……+NAan)/2N.In formula, PARepresent allele A frequencies, NAAIn expression colony
Individual amount with AA genotype, NAaiRepresent in colony that there is Aai genotype individuals quantity, a1-an is the n of allele A
Individual different multiple allele；Statistics is shown in Table 4.
The sheep of table 4, the 5019th SNP Gene frequency distribution table of Small-fat-tail sheep PCNP genes
3rd, the association analysis of gene effect
Genotype data：The genotype (GG, GA, GA) of SPhI identifications
Reproductive trait data：First tire litter size
Relation analysis model：
Using the correlation of SAS (9.2) software analysis gene loci and reproductive trait (litter size).First data are retouched
The property stated statistical analysis, it is determined whether there is outlier, according to data characteristics, using t analyses, variance analysis or multiple linear mould
Type analysis genotype effects.In data handling, it is different according to influence litter size factor, it is contemplated that environmental effect, age, gene
The reciprocal effects of type effect and correlation, are analyzed using fixed model, meanwhile, accepted or rejected according to actual conditions.Complete
Model is as follows：
Yijk=μ+Gj+Eijk
Wherein：YijkFor individual phenotype is recorded；μ is colony's average；GjIt is the genotype effects in each site；EijkIt is with chance error
Difference.
Variance analysis between the PCNP gene mononucleotide polymorphisms of table 5 and Sheep Reproductive Characters
Note：With the not notable (P of difference between same letter shoulder target average value>0.05) it is, flat with different alphabetical shoulder targets
Significant difference (P between average<0.05)
Result shows, the different genotype on the 5019th mononucleotide polymorphic site of sheep PCNP genome sequences
Show with sheep litter size association analysis, the individual litter size of AA genes is significantly higher than GG genotype individuals, is shown in Table 5.According to
This result of study, can set up genotype for the homozygous Sheep Populations of AA by selection, improve its lambing percentage.
Nucleotides sequence list
<110>Lanzhou University
<120>Method and its application using PCR-RFLP detection sheep PCNP gene mononucleotide polymorphisms
<160> 2
<210> 1
<211> 18
<212> DNA
<213>It is artificial synthesized
<400> 1
agaatagcat gggcagaa 18
<210> 2
<211> 18
<212> DNA
<213>It is artificial synthesized
<400> 2
tcatttctgt gggacacc 18
Claims (7)
1. a kind of method that utilization PCR-RFLP detects sheep PCNP gene mononucleotide polymorphisms, it is characterised in that including with
Lower step：
With sheep complete genome DNA to be measured as template, with primer pair P as primer, PCR amplification sheep PCNP genetic fragments；With limit
After property restriction endonuclease SPhI digestion pcr amplification products processed, then row agarose gel electrophoresis are entered to the amplified production after digestion；According to
The genotype of mononucleotide polymorphism site on electrophoresis result identification sheep PCNP genes；
Described primer pair P is：
Sense primer：5’-AGAATAGCATGGGCAGAA-3’；
Anti-sense primer：5’-TCATTTCTGTGGGACACC-3’.
2. the method for detecting sheep PCNP gene mononucleotide polymorphisms using PCR-RFLP as claimed in claim 1, it is special
Levy and be, described pcr amplification reaction program is：94 DEG C of predegeneration 5min；94 DEG C of denaturation 30s, 55.3 DEG C of anneal 30s, 72 DEG C
Extend 50s, 35 circulations；72 DEG C of extension 10min.
3. the method for detecting sheep PCNP gene mononucleotide polymorphisms using PCR-RFLP as claimed in claim 1, it is special
Levy and be, the mass concentration of described Ago-Gel is 1.0%.
4. the method for detecting sheep PCNP gene mononucleotide polymorphisms using PCR-RFLP as claimed in claim 1, it is special
Levy and be, described mononucleotide polymorphism site shows as the SNP of A or G, with tri- kinds of AA, GA and GG
Genotype, electrophoresis result is respectively：AA types show as two bands of 377bp and 309bp；GA types show as 686bp, 377bp and
Tri- bands of 309bp；GG types show as mono- band of 686bp.
5. the method using PCR-RFLP detection sheep PCNP gene mononucleotide polymorphisms as claimed in claim 1 is in sheep
Application in assisted Selection and molecular breeding.
6. application as claimed in claim 5, it is characterised in that：It is AA homozygous Sheep Populations to set up genotype, improves continuous
Sheep lambing percentage.
7. application as claimed in claim 6, it is characterised in that：The sheep is selected from sheep or Small-fat-tail sheep.
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| CN107630027A (en) * | 2017-09-14 | 2018-01-26 | 吴东栋 | A kind of preparation of medicine of overexpression PCNP genes and purposes |
| CN108315445A (en) * | 2018-04-26 | 2018-07-24 | 兰州大学 | It is a kind of detection sheep sry gene single nucleotide polymorphism method and application |
| CN109735630A (en) * | 2019-01-05 | 2019-05-10 | 兰州大学 | The detection method and label application of sheep ZFY gene mononucleotide polymorphism label |
| CN109837352A (en) * | 2019-04-17 | 2019-06-04 | 锡林郭勒职业学院 | Identify the primer and method of sheep FecB gene SNP genotype |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107630027A (en) * | 2017-09-14 | 2018-01-26 | 吴东栋 | A kind of preparation of medicine of overexpression PCNP genes and purposes |
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| CN108315445A (en) * | 2018-04-26 | 2018-07-24 | 兰州大学 | It is a kind of detection sheep sry gene single nucleotide polymorphism method and application |
| CN109735630A (en) * | 2019-01-05 | 2019-05-10 | 兰州大学 | The detection method and label application of sheep ZFY gene mononucleotide polymorphism label |
| CN109837352A (en) * | 2019-04-17 | 2019-06-04 | 锡林郭勒职业学院 | Identify the primer and method of sheep FecB gene SNP genotype |
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