CN101974514A - Single nucleotide polymorphism (SNP) related to sheep eye muscle property and application thereof - Google Patents
Single nucleotide polymorphism (SNP) related to sheep eye muscle property and application thereof Download PDFInfo
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- CN101974514A CN101974514A CN 201010277415 CN201010277415A CN101974514A CN 101974514 A CN101974514 A CN 101974514A CN 201010277415 CN201010277415 CN 201010277415 CN 201010277415 A CN201010277415 A CN 201010277415A CN 101974514 A CN101974514 A CN 101974514A
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Abstract
The invention relates to the field of biological engineering, in particular to a method for predicting a sheep eye muscle property by single nucleotide polymorphism (SNP). Polymerase chain reaction (PCR) amplification is performed on the total deoxyribonucleic acid (DNA) of sheep by primers shown as SEQ ID No.2 and SEQ ID No.3, the SNP of a PCR amplification product is detected, the 73rd basic group at the 5' end of SEQ ID No.1 in a sequence table is determined as G or A and an individual with a G allelic gene has a large eye muscle area and a large eye muscle width. Due to the detection of a polymorphic site, a new material is provided for molecular breeding and scientific basis is provided for the marker-assisted selection of the sheep eye muscle property.
Description
Technical field
The present invention relates to a kind of method of utilizing single nucleotide polymorphism prediction sheep eye muscle 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 73rd bit base from 5 ' of sequence shown in the SEQID No 1 is G or A.
The present invention also provides above-mentioned sheep single nucleotide polymorphism to be marked at sheep eye muscle proterties Application in Prediction.
The purpose of this invention is to provide the primer that utilizes single nucleotide polymorphism to detect sheep eye muscle proterties, 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 eye muscle 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 73rd bit base from SEQ ID No 1 is G or A, have the allelic individuality of G and have bigger eye muscle area and eye muscle width, also be the eye muscle area of GG, the genotypic individuality of GA and width significance greater than the AA genotype.
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 Nsi I for use.
The present invention also provides the test kit that is used for single nucleotide polymorphism prediction sheep eye muscle 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, dNTPs, sterilization distilled water.This test kit also can comprise restriction enzyme Nsi I 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 association analysis is carried out in the SNP site of the g.G73A of the CAST-S1 of CAST gene and the production traits of sheep, the g.G73A sudden change that shows CAST-S 1 is related with eye muscle thickness and eye muscle area existence, and wherein the eye muscle thickness utmost point of GG type and AG type is significantly higher than AA type (P＜0.01); The eye muscle area of GG type and AG type is significantly higher than AA type (P＜0.05).Detecting of this polymorphic site, not only for molecular breeding provides new material, the marker assisted selection for sheep eye muscle proterties provides scientific basis simultaneously.
Description of drawings
Fig. 1 is the SSCP result of CAST-S1 amplified fragments.
Fig. 2 is the order-checking peak figure of CAST-S1 amplified fragments.
Fig. 3 is the RFLP result of CAST-S1 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.
Embodiment 1
(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 336 in sample altogether, purebred individual 36 of the cold sheep of little tail, individual 229 of Du's pool sheep * sheep hybridization, individual 71 of Tao Saite sheep * 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; Two hybrid flock of sheep bodies are used for the PCR-RFLP polymorphism and detect on a large scale.
Embodiment 2
1, pcr amplification
The PCR primer of PCR-SSCP is as shown in the table.
5 pairs of specific primer sequences of table 1, annealing temperature and purpose fragment length
PCR reaction amplification system is 25 μ l, wherein 10 * contain Mg
2+PCR damping fluid 2.5 μ l, dNTPs 2 μ l (each 2.5mmol/L), each 0.4 μ l (20 μ mol/L) of upstream primer and downstream primer, 0.3 μ lTaq enzyme (5U/ μ l), template DNA (cDNA) 2 μ l, ultrapure water polishing to 25 μ l.
The PCR reaction conditions is: behind pre-94 ℃ of 3min of sex change; Enter following circulation: 94 ℃ of sex change 30sec, each sees the above table the primer annealing temperature, annealing 30sec, 72 ℃ are extended 60sec, after 35 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations.Get PCR product electrophoresis in 1.5% sepharose of 6 μ l, detect the quality of PCR product.
The result shows that the PCR product of 5 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.
The polyacrylamide of 12% (perhaps other concentration) that (2) 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 lPCR 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.
Because fragment length is in different size, adjust the concentration of gel according to clip size.CAST-S4 adopts 8% polyacrylamide gel, and CAST-S1, CAST-S2, CAST-S3 and CAST-S5 adopt 10% polyacrylamide gel.By the SSCP electrophorogram is analyzed, the amplified fragments of finding primer CAST-S2, CAST-S3, CAST-S4 and CAST-S5 does not have polymorphism, the amplified fragments of primer CAST-S1 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-S1 amplified production (referring to Fig. 2) 4 mutational sites, wherein there is the sudden change (Fig. 2 A) of A-G in 73 site of amplified production, there is the disappearance (Fig. 2 B) of CA in the 105-106 site, there is the sudden change (Fig. 2 C) of G-A in 210 site, and there is the sudden change (Fig. 2 D) of G-A in 219 site.
Embodiment 3
According to 229 first filial generations of embodiment 2 described methods to Du Boyang * sheep, 71 total DNA of sub-monobasic of Tao Saite sheep * sheep carry out pcr amplification.Because Nsi I enzyme can be discerned the sudden change of 73 site, with Nsi I enzyme PCR is carried out enzyme and cut back somatotype (Fig. 3).
Endonuclease reaction totally be 20 μ l, wherein contain 10 μ l PCR products, 2 μ l10 * Buffer (restriction enzyme enzyme spcificity damping fluid), 4～5U 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 μ-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-field-effect; e
Ijklmn-random error effect.
The CAST gene sees Table 2 in the gene frequency and the genotype frequency of the g.G73A of CAST-S1 site.The result shows.In Du Boyang * sheep and two filial generation colonies of Tao Saite sheep * sheep, all detect three kinds of genotype GG, AG and AA.Through suitability χ
2Check shows that two filial generation colonies of Du Boyang * sheep and Tao Saite sheep * sheep all are in Hardy-Weinberg nonequilibrium situations (P＜0.01) in this site.Heterozygosity, effective number of allele and the polymorphism information content of this polymorphic site of CAST gene in colony is referring to table 3.This site, two colonies of Du Boyang * sheep and Tao Saite sheep * sheep all belong to moderate polymorphic (0.25＜PIC＜0.5).
The genotype and the gene frequency of two sheep colonies of g.G73A site of table 2CAST-S1
Annotate: df=2, X
0.05 2=5.9, X
0.01 2=9.21
G.G73A site heterozygosity, effective number of allele and the polymorphism information content of table 3CAST-S1
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 associated effect of g.G73A sudden change three kinds of genotype GG, AG, AA and 2 the sheep population growth proterties of CAST-S1 is as shown in table 4.The result shows: eye muscle thickness difference heteropole remarkable (P＜0.01) between 3 kinds of genotype in the g.G73A site of CAST-S1, the GG type and the AG type utmost point are significantly higher than the AA type, GG type and AG type difference are not remarkable, illustrate that having the allelic individuality of G has bigger eye muscle width; While 3 kinds of genotype eye muscle area significant differences (P＜0.05), GG type and AG type are significantly higher than AA type (P＜0.05), and GG type and AG type difference is remarkable (P＞0.05) not, illustrates that having the allelic individuality of G has bigger eye muscle area.
The g.G73A site different genotype of table 4CAST-S1 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, holding the 73rd bit base from 5 ' of sequence shown in the SEQ ID No 1 is G or A.
2. the described single nucleotide polymorphism of claim 1 is marked at sheep eye muscle 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 73rd bit base from SEQ ID No 1 is G or A, have the allelic individuality of G bigger eye muscle area and eye muscle width are arranged.
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 Nsi I.
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, dNTPs, sterilization distilled water.
9. test kit according to claim 8 is characterized in that, also comprises restriction enzyme Nsi I and damping fluid thereof.
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Cited By (2)
| 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 |
| CN104120123A (en) * | 2014-07-03 | 2014-10-29 | 中国农业科学院北京畜牧兽医研究所 | SNP marker related to sheep callipyge trait and application thereof |
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| CN1824807A (en) * | 2005-12-22 | 2006-08-30 | 上海交通大学 | Process of calpain inhibiting protein gene 475 site SNP label screening swine |
| CN1824806A (en) * | 2005-12-22 | 2006-08-30 | 上海交通大学 | Process of calpain inhibiting protein gene 1836 site SNP label screening swine |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1824807A (en) * | 2005-12-22 | 2006-08-30 | 上海交通大学 | Process of calpain inhibiting protein gene 475 site SNP label screening swine |
| CN1824806A (en) * | 2005-12-22 | 2006-08-30 | 上海交通大学 | Process of calpain inhibiting protein gene 1836 site SNP label screening swine |
Non-Patent Citations (2)
| Title |
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| 《Animal Biotechnology 》 20000430 Palmer, BR et al Single nucleotide polymorphisms in an intron of the ovine calpastatin gene 63-67 1-9 第11卷, 第1期 2 * |
| 《中国优秀硕士学位论文全文数据库;农业科技辑》 20091015 张菊 绵羊CAST、MC4R和BTG1基因的克隆、组织表达和遗传多态性分析 D050-112 1－9 , 第 10 期 2 * |
Cited By (2)
| 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 |
| CN104120123A (en) * | 2014-07-03 | 2014-10-29 | 中国农业科学院北京畜牧兽医研究所 | SNP marker related to sheep callipyge trait and application thereof |
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Open date: 20110216 |