CN101942438A - Sheep back fat trait-related SNP and application thereof - Google Patents
Sheep back fat trait-related SNP and application thereof Download PDFInfo
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- CN101942438A CN101942438A CN 201010277397 CN201010277397A CN101942438A CN 101942438 A CN101942438 A CN 101942438A CN 201010277397 CN201010277397 CN 201010277397 CN 201010277397 A CN201010277397 A CN 201010277397A CN 101942438 A CN101942438 A CN 101942438A
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Abstract
The invention relates to the field of biological engineering, in particular to a method for predicating sheep back fat trait by using single nucleotide polymorphism. The method comprises the following steps of: performing PCR amplification on total DNA of the sheep by using primers in SEQ ID No: 2 and SEQ ID No: 3; and detecting the single nucleotide polymorphism by using PCR amplified products, and determining whether the 162nd position basic group at the 5' end of SEQ ID No: 1 in a sequence list is G or T, wherein the back fat thickness of the GT gene type is obviously thicker than that of the GG gene type. The detected polymorphic loci provide new materials for molecular breeding and provide scientific evidence for marker-assisted selection of the sheep back fat thickness.
Description
Technical field
The present invention relates to a kind of method of utilizing single nucleotide polymorphism prediction sheep back fat proterties in the bioengineering field.
Background technology
According to the known gene order design of a species degenerated primer is a kind of the method that obtains another species unknown gene.Obtain to utilize the information biology means that new gene is carried out function prediction behind the gene, and this gene is carried out the tissue expression analysis, verify by polymorphism analysis by sxemiquantitative RT-PCR and quantitative fluorescent PCR.Calpastatin (Calpastatin, CAST) there is different transcripts in gene at species such as mouse, people, rabbit and oxen, and the expression of different transcripts in different tissues there are differences (Emori et al., 1987, Proc Natl Acad Sci USA 84 (11): 3590-3594; Killefer andKoohmaraie, 1994, J Anim Sci 72 (3): 606-614; Takano et al., 2000, JBiochem 128 (1): 83-92).People such as Zhu find that there is a new hypotype in CAST in testis tissue, have cloned new gene, and infer may with generation and pass (Zhu, H.et al., 2002, the Acta Pharmacol Sin 23 (5): 450-454) of sperm.
(single nucleotide polymorphism is to be proposed by the E.Lander of U.S. MIT in 1996 SNP) to the single nucleotide polymorphism mark, is called as " s-generation DNA genetic marker ".Its ultimate principle is: for a dna segment that has regular length behind pcr amplification, its molecular conformation is determined by base sequence, therefore the change of single base can cause that there is small conformation difference in the mispairing heteroduplex that forms between dna molecular strand or allelotrope under the sex change condition, and these different conformers are distinguished because of ambulant difference in electrophoresis or high performance liquid phase detection.The detection of SNP can be passed through multiple technologies such as PCR-SSCP, PCR-RFLP, order-checking and SNP chip and realize.
There are four kinds of transcripts in the CAST gene of knowing ox at present, but the research of CAST gene is less on sheep, and the sequence on the GenBank can not be known and knows that it belongs to the sort of transcript of CAST gene.The detection of relevant sheep CAST gene SNP and functional verification research is a blank still, brings very big obstacle for marker assisted selection and the breeding of sheep.
Summary of the invention
An object of the present invention is to provide a kind of sheep single nucleotide polymorphism mark, holding the 162nd bit base from 5 ' of sequence shown in the SEQID No 1 is G or T.
The present invention also provides above-mentioned sheep single nucleotide polymorphism to be marked at sheep back fat proterties Application in Prediction.
Another object of the present invention provides the primer that utilizes single nucleotide polymorphism prediction sheep back fat proterties, and its sequence is shown in SEQ ID No 2 and SEQ ID No 3.
Sheep single nucleotide polymorphism provided by the invention is marked at sheep back fat proterties Application in Prediction, specifically be total DNA of sheep to be carried out pcr amplification with above-mentioned primer, and then pcr amplification product is carried out single nucleotide polymorphism detect, determine that 5 ' end the 162nd bit base from SEQ ID No 1 is G or T, the back-fat thickness significance of the genotypic individuality of GT greater than the genotypic individuality of GG.
The method that described single nucleotide polymorphism detects is dna sequencing, polymerase chain reaction-restriction enzyme site length polymorphism, polymerase chain reaction-single strand conformation polymorphism or allele specific oligonucleotide oligonucleotide hybridization.Be preferably polymerase chain reaction-restriction enzyme site length polymorphism.Wherein restriction enzyme is selected HinfI for use.
The present invention also provides the test kit that is used for single nucleotide polymorphism prediction sheep back fat proterties that contains SEQ ID No 2 and SEQ ID No 3 described primers.This test kit also comprises PCR damping fluid, Taq archaeal dna polymerase, dNPTs, sterilization distilled water.This test kit also can comprise restriction enzyme HinfI and damping fluid thereof.
The present invention utilizes the SNP mutational site of the CAST gene of sheep to carry out the genotype fast typing.Different sheep colonies exists genotype frequency and the gene frequency on these polymorphic sites to have significant difference.Utilize general linear model that the g.G162T of the CAST-S3 of CAST gene and the production traits of sheep are carried out association analysis, carrying out linear model analysis by SASv8.02 software finds, exist relatedly between the g.G162T of CAST-S3 sudden change and back-fat thickness, the back-fat thickness of GT type is significantly higher than GG type (P＜0.05).Detecting of this polymorphic site, not only for molecular breeding provides new material, the marker assisted selection for the sheep back-fat thickness provides scientific basis simultaneously.
Description of drawings
Fig. 1 is the SSCP result of CAST-S3 amplified fragments.
Fig. 2 is the order-checking peak figure of CAST-S3 amplified fragments.
Fig. 3 is the RFLP result of CAST-S3 amplified fragments.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
(1) cuts with ophthalmologic operation about 0.3g muscle tissue piece is fully shredded, place the 1.5ml centrifuge tube, dry 20min.
(2) in above-mentioned centrifuge tube, add 500 μ l tissue DNA extracting solutions.
(3) adding RNA enzyme solution to final concentration in above-mentioned centrifuge tube is 20 μ g/ml, abundant mixing, and 37 ℃ digested 1～2 hour.
(4) adding Proteinase K to final concentration in above-mentioned centrifuge tube is 150 μ g/ml, abundant mixing, and 55 ℃ digested 12～24 hours.
(5) add the saturated phenol of isopyknic Tris in above-mentioned centrifuge tube, slowly put upside down 4 ℃ of the abundant mixings of two-phase that centrifuge tube made solution in 10 minutes, centrifugal 10 minutes of 12000rpm shifts in supernatant liquor to the clean centrifuge tube then.Repeat once.
(6) add again with the saturated phenol of isopyknic Tris: chloroform: primary isoamyl alcohol (25: 24: 1), mixing, 4 ℃, centrifugal 10 minutes of 12000rpm.
(7) take out supernatant liquor, be transferred in the new centrifuge tube, add isopyknic chloroform: primary isoamyl alcohol (24: 1), 4 ℃, centrifugal 10 minutes of 12000rpm.
(8) get supernatant, add the 3M NaAc (pH5.2) of 1/10 volume and the freezing dehydrated alcohol deposit D NA of 2 times of volumes.
(9) DNA precipitation is chosen, placed new clean 1.5ml centrifuge tube, with twice of 90% washing with alcohol.
(10) DNA is dried naturally after, add an amount of TE or the sterilization distilled water dissolve-20 ℃ of preservations.
Detect 676 in sample altogether, purebred individual 36 of the cold sheep of little tail, individual 450 of Du's pool sheep * sheep hybridization, individual 145 of Tao Saite sheep * sheep hybridization, individual 45 of Suffolk * Xinjiang fine-wool sheep hybridization.The cold purebred individuality of sheep of its medium and small tail is used for PCR-SSCP to be analyzed, and detects polymorphic site; Three hybrid flock of sheep bodies are used for the PCR-RFLP polymorphism and detect on a large scale.
Embodiment 2
The PCR primer of PCR-SSCP is as shown in the table.
1, pcr amplification
5 pairs of specific primer sequences of table 1, annealing temperature and purpose fragment length
In the PCR pipe, add:
Template cDNA 1.0 μ l
Primer 1 (20pmol/ μ l) 0.3 μ l
Primer 2 (20pmol/ μ l) 0.3 μ l
10mM?dNTP?Mix?2.0μl
10 * PCR damping fluid, 2.5 μ l
Taq archaeal dna polymerase 0.30 μ l
DdH
2O mends to 25 μ l
The setting of pcr amplification temperature: pre-95 ℃ of 3min of sex change
35 circulations: 95 ℃ of 25sec of sex change; Annealing temperature sees the above table, annealing 25sec; Extend 72 ℃ of 20sec;
Extend 72 ℃ of 10min
Be cooled to 4 ℃ of preservations
Use 2% sepharose, electrophoresis detection result.The result shows that the PCR product of 3 pairs of designed primers is a specific amplification, and fragment length is consistent with the expection size, do not have the non-specific amplification band, so the PCR product can directly carry out sscp analysis.
2, the PCR product is carried out sscp analysis.
(1) before the experiment, puts into the glue frame after electrophoresis plate cleaned airing with distilled water.
(2) 12% the polyacrylamide that will now join is poured in the sheet glass, inserts comb.
(3) treat that gelling is solid after, pull out comb, use distilled water flushing point sample hole at once, with thieving paper the water in the hole is blotted only then, sheet glass is put on the electrophoresis chamber, pour an amount of 1 * TBE electrophoretic buffer into.
(4) 300V is about 60mA prerunning 10min.
(5) get 2 μ l PCR products, add 8 μ l sex change damping fluids, instantaneous centrifugal back was in 98 ℃ of sex change 10 minutes.
(6) take out centrifuge tube, place point sample after at least 5 minutes on ice immediately, at first, 200V electrophoresis 20min.
(7) 4 ℃, 150V left and right sides electrophoresis spends the night, about 10 hours of time.
(8) take out gel, behind distilled water flushing, outwell distilled water rapidly.
(9) fix 2 times with 20% ethanol, each 15 minutes, each fixing afterwards clean with distilled water flushing.
(10) with after the distilled water rinsing, added 1% nitric acid oxidation 3 minutes, gel is with twice of distilled water rinsing (nitric acid needs recovery) afterwards.
(11) in gel, add 0.1% silver nitrate solution, place dyeing 30 minutes or 20 minutes (Silver Nitrate needs to reclaim) on the shaking table under the room temperature.
(12) gel distilled water rinsing adds colour developing liquid then and develops the color.
(13) treat that band is clear after, outwell colour developing liquid, add 4% acetate submergence glue face, color development stopping.
(14) judge banding pattern after, take a picture to stay with gel imaging system and do analytic statistics usefulness.
(15) individuality special to banding pattern, cloning and sequencing is determined the mutational site.
By the SSCP electrophorogram is analyzed, the amplified fragments of finding primer CAST-S1 and CAST-S2 does not have polymorphism, the amplified fragments of primer CAST-S3 has polymorphism (Fig. 1), may there be several mutational sites in the amplified fragments, but because banding pattern more complicated, so adopt the method for cloning and sequencing, finally determine the mutational site.By the definite individuality that may have sudden change of PCR-SSCP, carry out cloning and sequencing.Cloning and sequencing finds to exist in the CAST-S3 amplified production 4 mutational sites, the disappearance that wherein has T in 80 site of amplified production, there is the sudden change of A-G in 83 site, and find that the disappearance of T and the sudden change of A-G are that (Fig. 2 A) takes place simultaneously in the individuality of being surveyed, there is the sudden change of G-A in 158 site, and there is the sudden change (Fig. 2 B) of G-T in 162 site.
Embodiment 3
According to embodiment 2 described methods total DNA of 45 filial generations of 450 filial generations of Du Boyang * sheep, 145 filial generations of Tao Saite sheep * sheep, Suffolk * Xinjiang fine-wool sheep is carried out pcr amplification.For the CAST-S3 amplified production, the HinfI enzyme can be discerned 162 site mutation, with the HinfI enzyme PCR product is carried out enzyme and cuts back somatotype (Fig. 3).
The enzyme system of cutting is 20 μ l, wherein contains 10 μ l PCR products, 2 μ l, 10 * restriction enzyme enzyme spcificity damping fluid, and the 4U restriction enzyme, the autoclaving distilled water is mended to 20 μ l.Mixing the back cuts about 4 hours at 37 ℃ of following enzymes according to the specification sheets of enzyme.Enzyme is cut product and is carried out electrophoresis with 2.0% sepharose, the 5V/cm electrophoresis, and ultraviolet lamp is observed down, and takes pictures.
Adopt the linear model software among the SASv8.02 (Statistical Analysis System) that data are analyzed the different genotype of SNPs polymorphic site and the cognation of the production traits.According to the factor that influences the production traits, adopted following fixed model during analysis:
Y
ijklmn＝μ+Breed
i+Age
j+Genotype
k+Litter
l+Sex
m+Farm
n+e
ijklmn
Wherein: y
Ijklmn-individual production traits record; Colony's average of ì-production traits; Breed
iThe variety effect of-male parent; Age
j-age effect; Genotype
kThe genotype effect of-candidate gene; Litter
l-tire litter size effect; Sex
m-sex effect; Farm
n-farm effect; e
Ijklmn-random error effect.
The CAST gene is in the gene frequency and the genotype frequency (referring to table 2) of the g.G162T of CAST-S3 site.In three filial generation colonies of Du Boyang * sheep, Tao Saite sheep * sheep, Suffolk * Xinjiang fine-wool sheep, all detect two kinds of genotype GG and GT.Through suitability χ
2Check shows that Du Boyang * sheep, Tao Saite sheep * sheep, Suffolk * Xinjiang fine-wool sheep filial generation all are in Hardy-Weinberg equilibrium state (P＞0.05) in this site.Table 3 is depicted as heterozygosity, effective number of allele and the polymorphism information content of this polymorphic site in colony of CAST gene.This site, three filial generation colonies of Du Boyang * sheep, Tao Saite sheep * sheep, Suffolk * Xinjiang fine-wool sheep all belong to low polymorphic (PIC＜0.25).
The genotype and the gene frequency of three sheep colonies of g.G162T site of table 2CAST-S3
Annotate: df=2, X
0.05 2=5.9, X
0.01 2=9.21
Heterozygosity, effective number of allele and the polymorphism information content of three sheep colonies of g.G162T site of table 3CAST-S3
Annotate: PIC＞0.5 is for highly polymorphic, and 0.25＜PIC＜0.5 is that moderate is polymorphic, and PIC＜0.25 is low polymorphic.
Adopt the cognation of the linear model analysis genotype and the production traits.The two kinds of genotype GG of g.G162T sudden change of CAST-S3 and the associated effect of GT and 3 sheep population growth proterties see Table 4.Found that significant difference (P＜0.05) between these two kinds of genotypic back-fat thicknesses, wherein the GT type is significantly higher than the GG type, illustrates that having the allelic individuality of T has thicker back-fat thickness.
The g.G162T site different genotype of table 4CAST-S3 and the association analysis of the production traits
Annotate: above numerical value is least square mean value standard error; In data differences conspicuous level P＜0.01 that different alphabetical A and B are arranged with delegation's acceptance of the bid; Indicate data differences conspicuous level P＜0.05 of different alphabetical a and b.
Claims (9)
1. a sheep single nucleotide polymorphism mark is characterized in that, is G or T in sequence shown in the SEQ ID No 1 from 5 ' end the 162nd bit base.
2. the described single nucleotide polymorphism of claim 1 is marked at sheep back fat proterties Application in Prediction.
3. application according to claim 2, it is characterized in that, with the primer of sequence shown in SEQ IDNo 2 and SEQ ID No 3 total DNA of sheep is carried out pcr amplification, and then pcr amplification product is carried out single nucleotide polymorphism detect, determine that 5 ' end the 162nd bit base from SEQ ID No 1 is G or T, the genotypic back-fat thickness of GT significantly is thicker than the GG genotype.
4. application according to claim 3, it is characterized in that the method that described single nucleotide polymorphism detects is selected from dna sequencing, polymerase chain reaction-restriction enzyme site length polymorphism, polymerase chain reaction-single strand conformation polymorphism or allele specific oligonucleotide oligonucleotide hybridization.
5. application according to claim 4 is characterized in that, the method that described single nucleotide polymorphism detects is polymerase chain reaction-restriction enzyme site length polymorphism.
6. application according to claim 5 is characterized in that, the used restriction endonuclease of described polymerase chain reaction-restriction enzyme site length polymorphism is HinfI.
7. be used for the test kit that test right requires 1 described single nucleotide polymorphism mark, it is characterized in that, comprise the primer of sequence shown in SEQ ID No 2 and SEQ ID No 3.
8. test kit according to claim 7 is characterized in that, also comprises PCR damping fluid, Taq archaeal dna polymerase, dNPTs, sterilization distilled water.
9. test kit according to claim 8 is characterized in that, also comprises restriction enzyme HinfI and damping fluid thereof.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102719532A (en) * | 2012-05-16 | 2012-10-10 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | Method for detecting early stage growth of Poll Dorset by microsatellite marker |
| CN103866003A (en) * | 2014-01-17 | 2014-06-18 | 甘肃农业大学 | Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) detection kit of Gansu alpine fine-wool sheep growth rate related gene ADRB3 and detection method |
| CN108707677A (en) * | 2018-06-20 | 2018-10-26 | 湖北多羔生物育种科技有限公司 | The liquid-phase chip of sheep meat SNP marker and its composition |
| CN113832237A (en) * | 2021-09-18 | 2021-12-24 | 福建省农业科学院畜牧兽医研究所 | SNP molecular marker related to cholesterol metabolism of meat rabbits and application thereof |
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| CN1523119A (en) * | 2003-02-17 | 2004-08-25 | 中国农业科学院畜牧研究所 | Method for predicting the born lamb number and its application |
| CN1680600A (en) * | 2005-02-05 | 2005-10-12 | 江西农业大学 | Polymorphic point of stearyl cozymase A desaturase gene mononucleotide for identifying behavior for back fat thickness of pig and use thereof |
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Patent Citations (2)
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| CN1523119A (en) * | 2003-02-17 | 2004-08-25 | 中国农业科学院畜牧研究所 | Method for predicting the born lamb number and its application |
| CN1680600A (en) * | 2005-02-05 | 2005-10-12 | 江西农业大学 | Polymorphic point of stearyl cozymase A desaturase gene mononucleotide for identifying behavior for back fat thickness of pig and use thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719532A (en) * | 2012-05-16 | 2012-10-10 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | Method for detecting early stage growth of Poll Dorset by microsatellite marker |
| CN103866003A (en) * | 2014-01-17 | 2014-06-18 | 甘肃农业大学 | Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) detection kit of Gansu alpine fine-wool sheep growth rate related gene ADRB3 and detection method |
| CN108707677A (en) * | 2018-06-20 | 2018-10-26 | 湖北多羔生物育种科技有限公司 | The liquid-phase chip of sheep meat SNP marker and its composition |
| CN113832237A (en) * | 2021-09-18 | 2021-12-24 | 福建省农业科学院畜牧兽医研究所 | SNP molecular marker related to cholesterol metabolism of meat rabbits and application thereof |
| CN113832237B (en) * | 2021-09-18 | 2023-07-28 | 福建省农业科学院畜牧兽医研究所 | SNP molecular marker related to cholesterol metabolism of meat rabbits and application thereof |
| CN114438232A (en) * | 2022-03-11 | 2022-05-06 | 浙江省农业科学院 | SNPs molecular marker g.43917A & gtG and application thereof in Hu sheep molecular marker assisted breeding |
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