CN1537942A - 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
A bone morphogenetic protein 15 (BMP 15) gene and the PCR-RFLP method for detecting its single nucleotide polymorphism (SNP) are disclosed. Its steps include extracting hog blood genom DNA, designing primer PCR amplifying, direct sequencing to amplified product, comparing and analyzing the sequence, and detecting SNP. It provides new marker for the marker aided breeding of pig.
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 of litter 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 IDNo.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 ID No.11, sequence table SEQ ID No.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 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.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 gene pleiomorphism branch of different pig varieties Analyse, and the research gene is Yu the relation of Zhu reproductive performance.
Embodiment
The order-checking of embodiment 1 BMP15 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 (94C 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
Genotype frequency (%) gene frequency (%)
The kind sample number
AA AB BB A B
Painted face in Beijing opera 37 100--100
Plum mountain pig 12 33.3 66.7-66.7 33.3
DIV 47 - - 100 - 100
Large White 37--100-100
Landrace 12--100-100
Duroc 13--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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Met?Val?Leu?Leu?Ser?Ile?Ile?Arg?Thr?Leu?Leu?Leu?Trp?Gly?Leu?Val
1 5 10 15
ctt?ttt?atg?gaa?cac?agg?gtc?caa?atg?acc?cag?gta?ggg?caa?ccc?tct 96
Leu?Phe?Met?Glu?His?Arg?Val?Gln?Met?Thr?Gln?Val?Gly?Gln?Pro?Ser
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gtg?gcc?ctc?ctg?cct?gag?gcc?tgt?acc?ttg?ccc?ctg?att?agg?gag?ctg 144
Val?Ala?Leu?Leu?Pro?Glu?Ala?Cys?Thr?Leu?Pro?Leu?Ile?Arg?Glu?Leu
35 40 45
cta?gag?gaa?gcc?cct?ggc?aaa?cag?cag?agg?aag?cca?cag?gtc?ctg?ggg 192
Leu?Glu?Glu?Ala?Pro?Gly?Lys?Gln?Gln?Arg?Lys?Pro?Gln?Val?Leu?Gly
50 55 60
cat?ccc?ttg?cga?tat?atg?ctg?gag?ttg?tac?cag?cgt?tca?gcc?gac?gca 240
His?Pro?Leu?Arg?Tyr?Met?Leu?Glu?Leu?Tyr?Gln?Arg?Ser?Ala?Asp?Ala
65 70 75 80
cgt?ggg?cac?cct?agg?gag?aac?cgc?acc?att?ggg?gcc?acc?atg?gtg?agg 288
Arg?Gly?His?Pro?Arg?Glu?Asn?Arg?Thr?Ile?Gly?Ala?Thr?Met?Val?Arg
85 90 95
ctg?gtg?agg?cca?ttg?gtt?aat?gga?gca?agg?cct?ctc?aga?ggg?ccc?tgg 336
Leu?Val?Arg?Pro?Leu?Val?Asn?Gly?Ala?Arg?Pro?Leu?Arg?Gly?Pro?Trp
100 105 110
cat?ata?cag?acc?ttg?gac?ttt?cct?ctg?aga?cca?aac?cgg?gta?gcc?tac 384
His?Ile?Gln?Thr?Leu?Asp?Phe?Pro?Leu?Arg?Pro?Asn?Arg?Val?Ala?Tyr
115 120 125
caa?ctt?gtc?aga?gcc?act?gtg?gtt?tat?cgc?cat?caa?ctt?cac?cta?gct 432
Gln?Leu?Val?Arg?Ala?Thr?Val?Val?Tyr?Arg?His?Gln?Leu?His?Leu?Ala
130 135 140
ccc?ttc?cac?ctc?tcc?tgc?cat?gtg?gag?ccc?tgg?atc?cag?aaa?agc?aca 480
Pro?Phe?His?Leu?Ser?Cys?His?Val?Glu?Pro?Trp?Ile?Gln?Lys?Ser?Thr
145 150 155 160
acc?agt?cac?ttt?cct?tcc?tca?gga?aga?ggc?tcc?tta?aag?cct?tcc?ctg 528
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
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Cys?Gln?Gln?Gln?Asn?Gly?Ser?Glu?Ile?Leu?Glu?Phe?Arg?Gly?Arg?Gly
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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
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Leu?Gly?Trp?Asp?His?Trp?Ile?Ile?Ala?Pro?His?Phe?Tyr?Thr?Pro?Asn
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Pro?Asn?His?Ala?Ile?Ile?Gln?Asn?Leu?Val?Asn?Glu?Leu?Val?Asp?Gln
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Ser?Val?Pro?Gln?Pro?Ser?Cys?Val?Pro?Tyr?Lys?Tyr?Val?Pro?Ile?Ser
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Ile?Leu?Leu?Ile?Glu?Ala?Asn?Gly?Ser?Ile?Leu?Tyr?Lys?Glu?Tyr?Glu
370 375 380
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Asp?Met?Ile?Ala?Gln?Pro?Cys?Thr?Cys?Arg
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Met?Val?Leu?Leu?Ser?Ile?Ile?Arg?Thr?Leu?Leu?Leu?Trp?Gly?Leu?Val
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Leu?Phe?Met?Glu?His?Arg?Val?Gln?Met?Thr?Gln?Val?Gly?Gln?Pro?Ser
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Val?Ala?Leu?Leu?Pro?Glu?Ala?Cys?Thr?Leu?Pro?Leu?Ile?Arg?Glu?Leu
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Leu?Glu?Glu?Ala?Pro?Gly?Lys?Gln?Gln?Arg?Lys?Pro?Gln?Val?Leu?Gly
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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
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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>3
<211>1185
<212>DNA
＜213〉pig (Sus scrofa)
<220>
<221>CDS
<222>(1)..(1185)
<223>
<400>3
atg?gtc?ctc?ctc?agc?atc?att?aga?acc?ctt?ctt?ctt?tgg?gga?ctg?gtg 48
Met?Val?Leu?Leu?Ser?Ile?Ile?Arg?Thr?Leu?Leu?Leu?Trp?Gly?Leu?Val
1 5 10 15
ctt?ttt?atg?gaa?cac?agg?gtc?caa?atg?acc?cag?gta?ggg?caa?ccc?tct 96
Leu?Phe?Met?Glu?His?Arg?Val?Gln?Met?Thr?Gln?Val?Gly?Gln?Pro?Ser
20 25 30
gtg?gcc?ctc?ctg?cct?gag?gcc?tgt?acc?ttg?ccc?ctg?att?agg?gag?ctg 144
Val?Ala?Leu?Leu?Pro?Glu?Ala?Cys?Thr?Leu?Pro?Leu?Ile?Arg?Glu?Leu
35 40 45
cta?gag?gaa?gcc?cct?ggc?aaa?cag?cag?agg?aag?cca?cag?gtc?ctg?ggg 192
Leu?Glu?Glu?Ala?Pro?Gly?Lys?Gln?Gln?Arg?Lys?Pro?Gln?Val?Leu?Gly
50 55 60
cat?ccc?ttg?cga?tat?atg?ctg?gag?ttg?tac?cag?cgt?tca?gcc?gac?gca 240
His?Pro?Leu?Arg?Tyr?Met?Leu?Glu?Leu?Tyr?Gln?Arg?Ser?Ala?Asp?Ala
65 70 75 80
cgt?ggg?cac?cct?agg?gag?aac?cgc?acc?att?ggg?gcc?acc?atg?gtg?agg 288
Arg?Gly?His?Pro?Arg?Glu?Asn?Arg?Thr?Ile?Gly?Ala?Thr?Met?Val?Arg
85 90 95
ctg?gtg?agg?cca?ttg?gtt?aat?gga?gca?agg?cct?ctc?aga?ggg?ccc?tgg 336
Leu?Val?Arg?Pro?Leu?Val?Asn?Gly?Ala?Arg?Pro?Leu?Arg?Gly?Pro?Trp
100 105 110
cat?ata?cag?acc?ttg?gac?ttt?cct?ctg?aga?cca?aac?cgg?gta?gcc?tac 384
His?Ile?Gln?Thr?Leu?Asp?Phe?Pro?Leu?Arg?Pro?Asn?Arg?Val?Ala?Tyr
115 120 125
caa?cta?gtc?aga?gcc?act?gtg?gtt?tat?cgc?cat?caa?ctt?cac?cta?gct 432
Gln?Leu?Val?Arg?Ala?Thr?Val?Val?Tyr?Arg?His?Gln?Leu?His?Leu?Ala
130 135 140
ccc?ttc?cac?ctc?tcc?tgc?cat?gtg?gag?ccc?tgg?atc?cag?aaa?agc?aca 480
Pro?Phe?His?Leu?Ser?Cys?His?Val?Glu?Pro?Trp?Ile?Gln?Lys?Ser?Thr
145 150 155 160
acc?agt?cac?ttt?cct?tcc?tca?gga?aga?ggc?tcc?tta?aag?cct?tcc?ctg 528
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 (8)
1, (its coding region nucleotide sequence is shown in 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.
2, the described gene order of claim 1 is characterized in that: 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 ID No.11, sequence table SEQ ID No.12.
3, the described gene order of claim 1, (single nucleotide polymorphism SNP) is characterised in that 390 in the Nucleotide of sequence table 1 described gene coding region to its single nucleotide polymorphism.
4, the method for 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.
5, the described method of claim 3, 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.
6, claim 3 or 5 described methods is characterized in that: set up PCR-restriction fragment length polymorphism (RFLP) detection method of BMP15 gene pleiomorphism, described restriction enzyme is Spe I.
7, claim 4 or 6 described methods, 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).
8, claim 6 or 7 described methods, wherein said polymorphism is 226bp and 277bp restriction fragment length polymorphism.
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| CNB031189601A CN100348721C (en) | 2003-04-16 | 2003-04-16 | Method for detecting PCR-RFLP of pig bone morphogenetic protein 15 gene polymorphism |
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| CNB031189601A CN100348721C (en) | 2003-04-16 | 2003-04-16 | Method for detecting PCR-RFLP of pig bone morphogenetic protein 15 gene polymorphism |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005040400A2 (en) * | 2003-10-24 | 2005-05-06 | Mmi Genomics, Inc. | Methods and systems for inferring traits to manage non-beef livestock |
| US8026064B2 (en) | 2002-12-31 | 2011-09-27 | Metamorphix, Inc. | Compositions, methods and systems for inferring bovine breed |
| CN108950009A (en) * | 2018-06-27 | 2018-12-07 | 华南农业大学 | A kind of breeding method for the multiple gene polymerization effect analysis improving kidding number |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010041743A1 (en) * | 2000-01-12 | 2001-11-15 | Steven Offenbacher | 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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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8026064B2 (en) | 2002-12-31 | 2011-09-27 | Metamorphix, Inc. | Compositions, methods and systems for inferring bovine breed |
| US8450064B2 (en) | 2002-12-31 | 2013-05-28 | Cargill Incorporated | Methods and systems for inferring bovine traits |
| US8669056B2 (en) | 2002-12-31 | 2014-03-11 | Cargill Incorporated | Compositions, methods, and systems for inferring bovine breed |
| US9206478B2 (en) | 2002-12-31 | 2015-12-08 | Branhaven LLC | Methods and systems for inferring bovine traits |
| US9982311B2 (en) | 2002-12-31 | 2018-05-29 | Branhaven LLC | Compositions, methods, and systems for inferring bovine breed |
| US10190167B2 (en) | 2002-12-31 | 2019-01-29 | Branhaven LLC | Methods and systems for inferring bovine traits |
| US11053547B2 (en) | 2002-12-31 | 2021-07-06 | Branhaven LLC | Methods and systems for inferring bovine traits |
| WO2005040400A2 (en) * | 2003-10-24 | 2005-05-06 | Mmi Genomics, Inc. | Methods and systems for inferring traits to manage non-beef livestock |
| WO2005040400A3 (en) * | 2003-10-24 | 2008-03-20 | Mmi Genomics Inc | Methods and systems for inferring traits to manage non-beef livestock |
| CN108950009A (en) * | 2018-06-27 | 2018-12-07 | 华南农业大学 | A kind of breeding method for the multiple gene polymerization effect analysis improving kidding number |
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| Publication number | Publication date |
|---|---|
| CN100348721C (en) | 2007-11-14 |
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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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