CN100348721C - Method for detecting PCR-RFLP of pig bone morphogenetic protein 15 gene polymorphism - Google Patents
Method for detecting PCR-RFLP of pig bone morphogenetic protein 15 gene polymorphism Download PDFInfo
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Abstract
The present invention relates to the technical field of molecular biology, particularly to the technical field of a bone morphogenetic protein 15 (BMP15) gene and the single nucleotide polymorphism (SNP) detection. The present invention has the steps of pig blood genomic DNA extracting, primer designing (according to human BMP15 genes and pig BMP15 genes), PCR amplifying, direct PCR product sequence testing, sequence comparing and analyzing and SNP detecting. The present invention also discloses a PCR-RFLP detection method of the pig BMP15 gene based on SNP of restriction enzyme Spel. The present invention provides a novel mark for mark assistance seed breeding of pig ovulation rate or parrowed number.
Description
Technical field
The invention belongs to the technical field of molecular biology of pig, specifically the present invention relates to the PCR-RFLP detection technique of pig bone morphogenetic protein 15 gene pleiomorphisms.
Technical background
Litter size is one of most important economic characters in the Swine Production, improves every sow weanling pig and will increase Swine Production person's economy return (Rothschild, 1996) with the extra input of minimum.In general, litter size how much depend on kind (heredity), there are very big-difference in litter size between different varieties (from 2 to 20, average 9 to 10) and embryo survival (15-40%).Litter size phenotype standard deviation is between 2.5 to 3, heritability is 0.10 to 0.15 (Johnson etc., Responses in ovulation rate, embryonal survival, and litter traits in swine to 14generations of selection to increase litter size.J Anim Sci.77 (3): 541-57).China plum mountain pig is than Europe and 4 to 5 piglets of the every nest fecund of american goods pig, manys 7.1 pieces of ovulations than Large White.Plum mountain pig fecund gene imports in Europe and the U.S.'s pig kind and has produced tangible commercial value.According to the literature, as long as litter size of pig is improved 1～1.5, Britain's pig industry can obtain 700,000,000 pounds surplus profit every year, and the annual surplus profit that obtains of whole European Union will be 2,000,000,000 pounds (Clutter, 1995) at least.
Litter size of pig is the sexlimited character that heritability is low, adopts traditional system of selection, and it is very difficult directly selecting litter size and obtaining genetic progress.France has used more than 30 year time to improve this proterties, blank wall as a result; And Denmark has used 50 years just every tire litter size to be improved 1.0.(Ovulatory response such as Johnson, and plasmaconcentrations of luteinizing hormone and progesterone following administration of syntheticmammalian or chicken luteinizing hormone-releasing hormone relative to the first or secondovulation in the sequence of the domestic hen.Biol Reprod.1984,31 (4): 646-55.), Bennett and Leymaster (Integration of ovulation rate, potential embryonic viability and uterine capacity into amodel oflitter size in swine.J Anim Sci.1989,67 (5): the 1230-41.) ovulation rate of giving chapter and verse, embryo survival or uterus capacity are formulated selectivity index and can be obtained big selective reaction than directly selecting litter size, expectation.In people such as Johnson (1999) report continuous 11 generations, selected according to improving ovulation rate and embryo survival index, and next 3 generations are selected litter size.Through the comprehensive selection in 14 generations, to compare with control group (selecting at random), the total litter size of nascent and the young number of living improve 3 and 1.4 respectively.Bibliographical information is also arranged, adopt super voluminous back-and-forth method (hyperprolificacy selection), BLUP (Best LinearUnbiased Prediction, Best Linear Unbiased Estimate) and the breeding hormonal readiness indirect selection, can improve the nest litter size, but make little progress.
(marker assisted selection, MAS) foundation of technology and development provide new method for the genetic improvement of litter size to molecular marker assisted selection.Grow in early days male animal and dam, directly select to influence the gene that litter size is expressed simultaneously, will improve the accuracy and the selective reaction of selection.In order to implement MAS, must differentiate that (quantitative trait loci QTL), and estimates their hereditary effect for the single candidate gene that influences important economical trait and quantitative trait locus position.
Litter size is directly influenced by parent number of eggs ovulated and embryo survival, and ovulation rate is a complex character, influenced by heredity and numerous environmental factors.Present a lot of research directly concentrates ovarian follicle to generate regulation and control, and main direction of studying is the autocrine of identification of ovarian generation or the physiological function of paracrine regulatory factor.Some regulatory factor is synthetic and secretion by ovocyte, and plays the part of the form of controlling ovarian follicular growth and differentiation and have an effect.A large amount of evidences show that ovocyte derives from factor regulation and control ovarian function.Ovocyte derives from the factor growth and differentiation factor-9 (growth differentiation factor-9, GDF9), Delicious peptide 6 (bonemorphogenetic protein 6, BMP6) and Delicious peptide 15 (BMP15), they all belong to maximum extracellular signal protein family transforming growth factor-β (transforming growth factor β, TGF β) superfamily member.Utilization knocks out the GDF9 dna rat and confirms granulosa cell growth and differentiation, and the Oocyte Meiosis ability all needs GDF9 to participate in directly, lacks the female mouse of GDF9 gene and causes early stage ovarian follicle to generate being obstructed, so that can not give birth to.
1998, people such as Dube have cloned mouse and people BMP15 gene, very similar with the GDF9 gene, only expression in ovocyte lacks the 4th (The bone morphogenetic protein 15 gene isX-linked and expressed in oocytes.Mol Endocrinol.12 (12): 1809-17.) in 7 cysteine residues.And 7 typical conserved regions that cysteine residues is more than 40 a TGF beta superfamily member.Because the 4th halfcystine can form the interchain disulfide bond skeleton, therefore, perhaps BMP15 and GDF9 may be monomer or the allos or the homodimer that are connected with non covalent bond, and supposition has complementary action in ovary.Mouse and people BMP15 coding region sequence total length 1176bp contain 2 exons, are separated by 3.5kb and 4.2kb intron respectively; 392 the amino acid propetides of encoding former (wherein 7 amino acid of signalase 11,250 amino acid of propetide and 125 amino acid of mature peptide).Mouse and people's propetide are former, propetide and mature peptide amino acid identity are respectively 63%, 60% and 77%, and nucleotide homology is respectively 76%, 74% and 81%.People BMP15 gene is located in X chromosome P11.2, confirms to guard the colinearity zone with upper being changed to of mouse BMP15 gene place X chromosome.Sheep BMP15 gene is by clone (Galloway etc., 2000, Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulationrate and infertility in a dosage-sensitive manner.Nat Genet.25 (3): 279-83.).Coding region total length 1179bp contains 2 exons, is separated by the 5.4kb intron, 393 amino acid of encoding.Coding region sequence and mouse and people's homology are respectively 78.8% and 82.9%.Sheep BMP15 gene is positioned in X chromosome 10cM zone, with FecXI (voluminous X gene) be the same area.In the reduction division of 78 co-information ewes, do not find the phenotype reorganization of BMP15 and FecXI.In carrying voluminous X gene sudden change Inverdale sheep (FecXI) and Hanna sheep (FecXH), sudden change has taken place in BMP15 mature peptide sequence.BMP15 transgenation heterozygote sheep ovulation rate increases, and causes more twins or triplets.And the sub-sheep of BMP15 gene pure hinders the ovarian follicle generation, can not give birth to the similar GDF9 dna rat phenotype that knocks out.Therefore, the BMP15 gene also is that the dam fertility is necessary, and spontaneous mutation can increase ovulation rate (heterozygous mutation carrier), also can cause sterile phenotype (sudden change homozygote carrier), and the candidate gene that can be used as ovulation rate or litter size is furtherd investigate.
Summary of the invention
One of purpose of the present invention is clone pig BMP15 genes encoding region sequence, obtains single nucleotide polymorphism by sequence comparing analysis; Two of purpose of the present invention is based on the single nucleotide polymorphism of discovery, sets up the PCR-RFLP detection method of BMP15 gene mononucleotide polymorphism, provides the molecule marker of usefulness for the marker assisted selection of pig ovulation rate or litter size.
The present invention is achieved through the following technical solutions:
(its coding region nucleotide sequence is shown in pig sequence table SEQ ID No.1 and sequence table SEQ ID No.2 for bone morphogenetic protein 15, BMP15) gene for pig bone morphogenetic protein 15.
10 kinds of primers of amplification and order-checking coding region nucleotide sequence are respectively shown in sequence table SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, sequence table SEQ IDNo.11, sequence table SEQ IDNo.12.
Sequence table SEQ ID No.3:
F1-1：5’-CTGCCTTTCACTGTTTCCTG-3’
Sequence table SEQ ID No.4:
R1-1：5’-CTCATTTTTCTCCCCTCCAG-3’
Sequence table SEQ ID No.5:
F2-1：5’-ACATCACAGCTCACAGCAAC-3’
Sequence table SEQ ID No.6:
R2-1：5’-ACATGAAGCGGAGTCGTAGA-3’
Sequence table SEQ ID No.7:
F2-2：5’-GTAGCGTCTGCCACCTACAT-3’
Sequence table SEQ ID No.8:
R2-2：5’-CCGGAACTCAAGAATCTCAC-3’
Sequence table SEQ ID No.9:
F2-3：5’-AGAGCCACTGTGGTTTATCG-3’
Sequence table SEQ ID No.10:
R2-3：5’-GAAAGGATGGAGGGAACACT-3’
Sequence table SEQ ID No.11:
F2-4：5’-TAACCAGTGTTCCCTCCATC-3’
Sequence table SEQ ID No.12:
R2-4：5’-CCTGGGAAACCTGATCTAGC-3’
Aforesaid gene order, (single nucleotide polymorphism SNP) is characterised in that 390 in the Nucleotide of BMP15 gene coding region to its single nucleotide polymorphism.
The method of a kind of clone's Huo and order-checking pig BMP15 genes encoding region nucleotide sequence, according to following steps: pig blood extracting genome DNA, design special primer, polymerase chain reaction (PCR) amplification, PCR product purification and directly order-checking, by the nucleotide sequence of sequence comparing analysis acquisition shown in sequence table SEQ ID No.1 and sequence table SEQ ID No.2.
Wherein said primer such as sequence table SEQ ID No.7 and SEQ ID No.8 can amplify the gene fragment that contains pleomorphism site.
Described method also comprises PCR-restriction fragment length polymorphism (RFLP) detection method of having set up the BMP15 gene pleiomorphism, and described restriction enzyme is Spe I.
Described method, the PCR reaction is to contain 50ng genomic dna, 15mmol/L Tri s-HCl, 50mmol/L KCl (pH8.0), 2mmol/L MgCl
2Carry out in the 25 μ l reaction systems of 200 μ mol/L dNTPs, 10 μ mol/L primers, 4% methyl-sulphoxide and 0.5U TaqDNA polysaccharase, reaction conditions is 95 ℃ of 5min, 35 circulations (94 ℃ of 45S, 58 ℃ of 45S and 72 ℃ of 1min 30S), 72 ℃ of prolongation 5min.17 μ l PCR products, digestion product 1.5% agarose gel electrophoresis is spent the night in the 37 ℃ of digestion of restriction enzyme Spe I enzyme that add 0.5 μ l (10U/ μ l).
Wherein said polymorphism is 226bp and 277bp restriction fragment length polymorphism.
Described pig BMP15 gene order, (single nucleotide polymorphism SNP) is characterised in that 390 in the Nucleotide of BMP15 gene coding region to its single nucleotide polymorphism.
Comparative sequences table SEQ ID No.1 and SEQ ID No.2 dna sequence dna (as shown in Figure 1), the SNP site of 390 T → A of discovery BMP15 gene coding region Nucleotide.The described detection technique that is used for pig BMP15 gene SNP somatotype is PCR-RFLP.The primer of pcr amplification is shown in sequence table SEQ ID No.7 and SEQ ID No.8.The PCR reaction is to contain 50ng genomic dna, 15mmol/L Tris-HCl, 50mmol/L KCl (pH8.0), 2mmol/L MgCl
2Carry out in the 25 μ l reaction systems of 200 μ mol/LdNTPs, 10 μ mol/L primers, 4% methyl-sulphoxide and 0.5U Taq archaeal dna polymerase, reaction conditions is 95 ℃ of 5min, 35 circulations (94 ℃ of 45S, 58 ℃ of 45S and 72 ℃ of 1min 30S), 72 ℃ of prolongation 5min.17 μ l PCR products, digestion product 1.5% agarose gel electrophoresis is spent the night in the 37 ℃ of digestion of restriction enzyme Spe I enzyme that add 0.5 μ l (10U/ μ l).Described polymorphism is 226bp and 277bp restriction fragment length polymorphism.
Sequence table and explanation thereof:
1, sequence table SEQ ID No.1: be the nucleotide sequence that derives from " Large White " clone of European descent;
2, sequence table SEQ ID No.2: be the nucleotide sequence that derives from Chinese native pig breed blood lineage " plum mountain pig " clone;
3, sequence table SEQ ID No.3: be the nucleotide sequence of implementing the used special primer of BMP15 gene clone;
4, sequence table SEQ ID No.4: be to implement the used special primer nucleotide sequence of BMP15 gene clone;
5, sequence table SEQ ID No.5: be to implement the used special primer nucleotide sequence of BMP15 gene clone;
6, sequence table SEQ ID No.6: be to implement the used special primer nucleotide sequence of BMP15 gene clone;
7, sequence table SEQ ID No.7: be that enforcement BMP15 gene clone and PCR-RFLP analyze used special primer nucleotide sequence;
8, sequence table SEQ ID No.8: be that enforcement BMP15 gene clone and PCR-RFLP analyze used special primer nucleotide sequence;
9, sequence table SEQ ID No.9: be to implement the used special primer nucleotide sequence of BMP15 gene clone;
10, sequence table SEQ ID No.10: be to implement the used special primer nucleotide sequence of BMP15 gene clone;
11, sequence table SEQ ID No.11: be to implement the used special primer nucleotide sequence of BMP15 gene clone;
12, sequence table SEQ ID No.12: be to implement the used special primer nucleotide sequence of BMP15 gene clone;
Accompanying drawing and explanation thereof:
Fig. 1: Large White and Mei Shan pig BMP15 genes encoding region nucleotide sequence are relatively
The present invention has following effect:
The PCR-RFLP detection technique of genetic marker of the present invention and SNP can be used for the BMP15 Polymorphism Analysis of different pig varieties, and the relation of research gene and swine reproduction performance.
Embodiment
The order-checking of embodiment 1BMP15 gene coding region DNA
Select place of china kind plum mountain pig (Chinese Pigs blood lineage) and adventive Large White (European pig blood lineage) as the typical test material, contain BMP15 gene extron 1 and exon 2 zone according to pig and 5 pairs of primer amplifications of people BMP15 gene DNA sequence design.
F1-1：5’-CTGCCTTTCACTGTTTCCTG-3’
R1-1：5’-CTCATTTTTCTCCCCTCCAG-3’
F2-1：5’-ACATCACAGCTCACAGCAAC-3’
R2-1：5’-ACATGAAGCGGAGTCGTAGA-3’
F2-2：5’-GTAGCGTCTGCCACCTACAT-3’
R2-2：5’-CCGGAACTCAAGAATCTCAC-3’
F2-3：5’-AGAGCCACTGTGGTTTATCG-3’
R2-3：5’-GAAAGGATGGAGGGAACACT-3’
F2-4：5’-TAACCAGTGTTCCCTCCATC-3’
R2-4：5’-CCTGGGAAACCTGATCTAGC-3’
Polymerase chain reaction (PCR) is to contain 50ng genomic dna, 15mmol/L Tris-HCl, 50mmol/L KCl (pH8.0), 2mmol/L MgCl
2Carry out in the 25 μ l reaction systems of 200 μ mol/L dNTPs, 10 μ mol/L primers, 4% methyl-sulphoxide and 0.5U TaqDNA polysaccharase, reaction conditions is 95 ℃ of 5min, 35 circulations (94 ℃ of 45S, 58 ℃ of 45S and 72 ℃ of 1min 30S), 72 ℃ of prolongation 5min, 4 ℃ of preservations.Amplification PCR products is purified the back and directly carries out sequencing with forward primer and reverse primer.Dna sequence dna is arranged with Blast software (http://www.ncbi.nlm.nih.gov/) and compared: Large White and Mei Shan pig BMP15 gene coding region dna sequence dna are seen shown in sequence table SEQ ID No.1 and the sequence table SEQ ID No.2.
The PCR-RFLP detection technique of embodiment 2 BMP15 gene SNPs
Compare Large White and Mei Shan pig BMP15 gene coding region dna sequence dna with Blast software, found 1 sequence change (T → A) in 390 in Nucleotide, be SNP (seeing accompanying drawing 1): plum mountain pig is A, and Large White is T, and has caused the restriction enzyme Spe I (A ↓ CTAGT) change of restriction enzyme site.In view of the above, the PCR-RFLP detection method of having set up based on restriction enzyme Spe I detects SNP.PCR is reflected at and contains 50ng genomic dna, 15mmol/L Tris-HCl, 50mmol/L KCl (pH8.0), 2mmol/L MgCl
2, 200 μ mol/L dNTPs, primers F
2-2And R
2-2Carry out in the 25 μ l reaction systems of 10 μ mol/L, 4% methyl-sulphoxide and 0.5U Taq archaeal dna polymerase, reaction conditions is 95 ℃ of 5min, 35 circulations (94 ℃ of 60S, 58 ℃ of 45S and 72 ℃ of 90S), 72 ℃ of prolongation 10min.The PCR product detects with 1.5% agarose gel electrophoresis: allelotrope A (390 in BMP15 gene coding region Nucleotide is A) restriction fragment length is 226bp and 277bp, and allelotrope B (390 in BMP15 gene coding region Nucleotide is T) restriction fragment length is 503bp; Heterozygous genes type AB restriction fragment length is 503bp, 277bp and 226bp.Adventive Large White, landrace and the duroc BMP15 gene pleiomorphism of Chinese Pigs local variety painted face in Beijing opera, plum mountain pig, employing Tongcheng, Hubei Province pig of Chinese origin being cultivated kind China's lean meat pig new lines (DIV) and European descent have carried out check and analysis, and its genotype and gene frequency are shown in Table 1.As can be seen from Table 1, different varieties BMP15 genotype difference, the adventive pig is the BB type, and the painted face in Beijing opera pig is the AA type, occurs AA type and AB type in the pig of plum mountain, has only the BB type and cultivate kind DIV.Next step studies the relation of BMP15 genotype and ovulation rate or litter size with enlarged sample content.
BMP15 genotype of table 1 different varieties pig and gene frequency detected result
| Kind | Sample number | Genotype frequency (%) | Gene frequency (%) | |||
| AA | AB | BB | A | B | ||
| Painted face in Beijing opera plum mountain pig DIV Large White landrace duroc | 37 12 47 37 12 13 | 100 33.3 - - - - | - 66.7 - - - - | - - 100 100 100 100 | 100 66.7 - - - - | 33.3 100 100 100 100 |
SEQUENCE LISTING
＜110〉Hua Zhong Agriculture University
＜120〉the PCR-RFLP detection method of pig bone morphogenetic protein 15 gene pleiomorphisms
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Thr Ser His Phe Pro Ser Ser Gly Arg Gly Ser Leu Lys Pro Ser Leu
165 170 175
ctg ccc caa gct tgg acg gag atg gat gtc acg caa cat gtt gga caa 576
Leu Pro Gln Ala Trp Thr Glu Met Asp Val Thr Gln His Val Gly Gln
180 185 190
aag ctc tgg aat cac aag ggg cgc agg gtt cta cga ctc cgc ttc atg 624
Lys Leu Trp Asn His Lys Gly Arg Arg Val Leu Arg Leu Arg Phe Met
195 200 205
tgt cag cag caa aat ggt agt gag att ctt gag ttc cgg ggg cgt ggc 672
Cys Gln Gln Gln Asn Gly Ser Glu Ile Leu Glu Phe Arg Gly Arg Gly
210 215 220
att tca tcc ctg gac act gcc ttc ttg tta ctc tat ttc aat gac act 720
Ile Ser Ser Leu Asp Thr Ala Phe Leu Leu Leu Tyr Phe Asn Asp Thr
225 230 235 240
cgg agt gtt cag aag gcc aaa ctt ctt ccc aga ggc ctg gaa gag ttt 768
Arg Ser Val Gln Lys Ala Lys Leu Leu Pro Arg Gly Leu Glu Glu Phe
245 250 255
atg gca aga gac cct tct ctt ctt ttg cgg aag gcc cgg caa gca ggc 816
Met Ala Arg Asp Pro Ser Leu Leu Leu Arg Lys Ala Arg Gln Ala Gly
260 265 270
agc atc gca tct gag gtt ctt ggc ccc tcc agg gag cac gat ggg cct 864
Ser Ile Ala Ser Glu Val Leu Gly Pro Ser Arg Glu His Asp Gly Pro
275 280 285
gaa agt aac cag tgt tcc ctc cat cct ttc caa gtc agc ttc cac caa 912
Glu Ser Asn Gln Cys Ser Leu His Pro Phe Gln Val Ser Phe His Gln
290 295 300
ctg ggt tgg gat cat tgg atc att gct ccc cat ttc tat acc cca aac 960
Leu Gly Trp Asp His Trp Ile Ile Ala Pro His Phe Tyr Thr Pro Asn
305 310 315 320
tac tgt aag ggg gtc tgc cct cgg gta cta cac tat ggt ctc aat tcc 1008
Tyr Cys Lys Gly Val Cys Pro Arg Val Leu His Tyr Gly Leu Asn Ser
325 330 335
ccc aat cat gcc atc atc cag aac ctt gtc aat gag ctg gtg gac cag 1056
Pro Asn His Ala Ile Ile Gln Asn Leu Val Asn Glu Leu Val Asp Gln
340 345 350
agt gtc cct cag ccc tcc tgt gtc cct tat aag tat gtg cct att agc 1104
Ser Val Pro Gln Pro Ser Cys Val Pro Tyr Lys Tyr Val Pro Ile Ser
355 360 365
atc ctc ctg att gag gca aat ggg agt atc ttg tac aag gag tat gag 1152
Ile Leu Leu Ile Glu Ala Asn Gly Ser Ile Leu Tyr Lys Glu Tyr Glu
370 375 380
gat atg att gcc cag ccc tgt aca tgc aga tga 1185
Asp Met Ile Ala Gln Pro Cys Thr Cys Arg
385 390
<210>4
<211>394
<212>PRT
＜213〉pig (Sus scrofa)
<400>4
Met Val Leu Leu Ser Ile Ile Arg Thr Leu Leu Leu Trp Gly Leu Val
1 5 10 15
Leu Phe Met Glu His Arg Val Gln Met Thr Gln Val Gly Gln Pro Ser
20 25 30
Val Ala Leu Leu Pro Glu Ala Cys Thr Leu Pro Leu Ile Arg Glu Leu
35 40 45
Leu Glu Glu Ala Pro Gly Lys Gln Gln Arg Lys Pro Gln Val Leu Gly
50 55 60
His Pro Leu Arg Tyr Met Leu Glu Leu Tyr Gln Arg Ser Ala Asp Ala
65 70 75 80
Arg Gly His Pro Arg Glu Asn Arg Thr Ile Gly Ala Thr Met Val Arg
85 90 95
Leu Val Arg Pro Leu Val Asn Gly Ala Arg Pro Leu Arg Gly Pro Trp
100 105 110
His Ile Gln Thr Leu Asp Phe Pro Leu Arg Pro Asn Arg Val Ala Tyr
115 120 125
Gln Leu Val Arg Ala Thr Val Val Tyr Arg His Gln Leu His Leu Ala
130 135 140
Pro Phe His Leu Ser Cys His Val Glu Pro Trp Ile Gln Lys Ser Thr
145 150 155 160
Thr Ser His Phe Pro Ser Ser Gly Arg Gly Ser Leu Lys Pro Ser Leu
165 170 175
Leu Pro Gln Ala Trp Thr Glu Met Asp Val Thr Gln His Val Gly Gln
180 185 190
Lys Leu Trp Asn His Lys Gly Arg Arg Val Leu Arg Leu Arg Phe Met
195 200 205
Cys Gln Gln Gln Asn Gly Ser Glu Ile Leu Glu Phe Arg Gly Arg Gly
210 215 220
Ile Ser Ser Leu Asp Thr Ala Phe Leu Leu Leu Tyr Phe Asn Asp Thr
225 230 235 240
Arg Ser Val Gln Lys Ala Lys Leu Leu Pro Arg Gly Leu Glu Glu Phe
245 250 255
Met Ala Arg Asp Pro Ser Leu Leu Leu Arg Lys Ala Arg Gln Ala Gly
260 265 270
Ser Ile Ala Ser Glu Val Leu Gly Pro Ser Arg Glu His Asp Gly Pro
275 280 285
Glu Ser Asn Gln Cys Ser Leu His Pro Phe Gln Val Ser Phe His Gln
290 295 300
Leu Gly Trp Asp His Trp Ile Ile Ala Pro His Phe Tyr Thr Pro Asn
305 310 315 320
Tyr Cys Lys Gly Val Cys Pro Arg Val Leu His Tyr Gly Leu Asn Ser
325 330 335
Pro Asn His Ala Ile Ile Gln Asn Leu Val Asn Glu Leu Val Asp Gln
340 345 350
Ser Val Pro Gln Pro Ser Cys Val Pro Tyr Lys Tyr Val Pro Ile Ser
355 360 365
Ile Leu Leu Ile Glu Ala Asn Gly Ser Ile Leu Tyr Lys Glu Tyr Glu
370 375 380
Asp Met Ile Ala Gln Pro Cys Thr Cys Arg
385 390
<210>5
<211>20
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>Intron
<222>(1)..(20)
<223>
<400>5
ctgcctttca ctgtttcctg 20
<210>6
<211>20
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>6
ctc att ttt ctc ccc tcc ag 20
Leu Ile Phe Leu Pro Ser
1 5
<210>7
<211>20
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>Intron
<222>(1)..(20)
<223>
<400>7
acatcacagc tcacagcaac 20
<210>8
<211>20
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>8
aca tga agc gga gtc gta ga 20
Thr Ser Gly Val Val
1 5
<210>9
<211>20
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>Intron
<222>(1)..(20)
<223>
<400>9
gtagcgtctg ccacctacat 20
<210>10
<211>20
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>10
ccg gaa ctc aag aat ctc ac 20
Pro Glu Leu Lys Asn Leu
1 5
<210>11
<211>20
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>11
aga gcc act gtg gtt tat cg 20
Arg Ala Thr Val Val Tyr
1 5
<210>12
<211>20
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>12
gaa agg atg gag gga aca ct 20
Glu Arg Met Glu Gly Thr
1 5
<210>13
<211>20
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>13
taa cca gtg ttc cct cca tc 20
Pro Val Phe Pro Pro
1 5
<210>14
<211>20
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>Intron
<222>(1)..(20)
<223>
<400>14
cctgggaaac ctgatctagc 20
Claims (2)
1, a kind of method that detects pig bone morphogenetic protein 15 gene pleiomorphisms of pig, its step is as follows:
(1) according to pig bone morphogenetic protein 15 gene orders design primer, described primer is shown in sequence table SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQID No.9, SEQ ID No.10, SEQID No.11 or SEQ ID No.12;
(2) extract the pig blood genomic dna;
(3) DNA that step (2) is obtained carries out PCR;
(4) to the product purification and the order-checking of step (3);
(5) sequence that step (4) is obtained is carried out sequence comparing analysis, finds out mononucleotide polymorphism site;
(6) polymorphism of step (5) being carried out PCR-RFLP detects.
2, method according to claim 1, wherein said mononucleotide polymorphism site are positioned at 390 in coding region Nucleotide.
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| CN100348721C true CN100348721C (en) | 2007-11-14 |
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| US7468248B2 (en) | 2002-12-31 | 2008-12-23 | Cargill, Incorporated | Methods and systems for inferring bovine traits |
| CA2543786A1 (en) * | 2003-10-24 | 2005-05-06 | Mmi Genomics, Inc. | Methods and systems for inferring traits to manage non-beef livestock |
| CN108950009B (en) * | 2018-06-27 | 2021-04-27 | 华南农业大学 | Breeding method for polygene polymerization effect analysis for improving goat lambing number |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001051054A2 (en) * | 2000-01-12 | 2001-07-19 | University Of North Carolina - Chapel Hill | Use of cyclopentenone derivatives for bone and periodontal regeneration |
| CN1333295A (en) * | 2000-07-11 | 2002-01-30 | 韩延 | Reconbination human bone morphogenetic protein-2truncated mutant and preparation method thereof |
| WO2003029429A2 (en) * | 2001-10-03 | 2003-04-10 | Selective Genetics, Inc. | Traversal of nucleic acid molecules through a fluid space and expression in repair cells |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001051054A2 (en) * | 2000-01-12 | 2001-07-19 | University Of North Carolina - Chapel Hill | Use of cyclopentenone derivatives for bone and periodontal regeneration |
| CN1333295A (en) * | 2000-07-11 | 2002-01-30 | 韩延 | Reconbination human bone morphogenetic protein-2truncated mutant and preparation method thereof |
| WO2003029429A2 (en) * | 2001-10-03 | 2003-04-10 | Selective Genetics, Inc. | Traversal of nucleic acid molecules through a fluid space and expression in repair cells |
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Assignee: Wuhan Chopper Biology Co., Ltd. Assignor: Huazhong Agricultural University Contract fulfillment period: 2008.11.6 to 2023.11.5 contract change Contract record no.: 2008420010028 Denomination of invention: PCR-RFLP detection method of pig bone morphogenetic protein 15 gene polymorphism Granted publication date: 20071114 License type: Exclusive license Record date: 20081111 |
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