CN100366636C - Pig production and immunity related protein, its coding gene and application - Google Patents

Abstract

The present invention discloses pig production and immunity trait associated protein, a coding gene and the application thereof. The protein has one of the following amino acid residue sequence: 1) SEQ ID No. 9 in a sequence table; 2) protein which is obtained by replacing, deleting or adding one to ten amino acid residues of the amino acid residue sequence of the SEQ ID No. 9 in the sequence table and is associated with the pig production trait and immunity trait. The method for detecting the pig production trait and immunity trait comprises the following steps: a pair of primers composed of nucleotide sequences of SEQ ID No. 2 and SEQ ID No. 3 or SEQ ID No. 5 and SEQ ID No. 6 in the sequence table are used for PCR amplification of genome DNA of a pig to be measured; then, the single nucleotide polymorphism or the restriction fragment length polymorphism of the PCR amplification product is detected. The pig production and immunity trait associated protein and the coding gene thereof of the present invention can perform important functions in pig breeding.

Description

Pig produces and immune character related protein, its encoding gene and application

Technical field

The present invention relates to a kind of protein and encoding gene thereof and application, particularly relate to albumen and the encoding gene thereof that a boar produces and immune character is relevant and utilize the single nucleotide polymorphism of this protein coding gene and the method that restrictive fragment length polymerphism detects pig production, immune character.

Background technology

GENERALIZATION OF MODERN BREEDING TECHNIQUE is significantly improved the production performance of pig, yet along with the raising of living standards of the people, the requirement of meat quality is also improved thereupon.When the emphasis that herding is produced has extended to the growth efficiency that improves lean tissue, improvement meat, improve the resistance of product, thereby reduce the production cost of unit product, and reduce drug residue, promptly when guaranteeing livestock product quantity, improve the quality of livestock product.

Individual immunity power is to weigh an important indicator of animal health condition, it belongs to quantitative character, related genes involved is more, molecular mechanism also complicated (Knapp etc., Relationships between genetics changesand infections disease in domestic livestock, (eds.Hill W G, Bishop S C, McGuirk B.) brit.Soc Anim Sci., Edinburgh Occasional publication no.27,65-80.2000).Part Study shows that immunizing power and production performance are usually expressed as the phenotype negative correlativing relation, and this is big obstacle for breeding work is provided with, and promptly how improving the individual immunity ability on the basis of maintenance and raising production performance is the new research topic in herding breeding field.But it is two-way the relation that studies show that pig immunological competence and production performance on a small quantity also being arranged abroad, and the immunological competence that promptly strengthens pig under certain situation (standard state) can improve main production performance such as its speed of growth, feed efficiency.So far, the mutual relationship between pig immunological competence and production performance is still uncertain, and therefore very crucial from the mutual relationship between molecular level announcement production performance and immunizing power controlling gene, also the breeding for disease resistance work for domestic animal provides new thinking.

When individuality was subjected to pathogenic agent and infects, body can be transferred the defense mechanism of three aspects, and promptly epithelium defense mechanism, nonspecific defense mechanism and specificity defense mechanism are resisted.Pig whether morbidity is depended on infects and the defensive enginery results of interaction, if defensive enginery just shows the nature disease resistance by force.Disease resistance can be divided into special disease resistance and general disease resistance by the hereditary basis difference.Special disease resistance is meant the resistance of pig to certain specified disease or pathogenic agent, and this resistance is controlled by mainly in a key-gene site, also can be subjected to other site (comprising regulator) and such environmental effects to some extent.Studies show that the inherent mechanism of special disease resistance is to exist in the host or lack certain molecule or its variant, this molecule has following effect: 1. determine allosome identification and specificity simplified reaction; 2. determine the special attachments power of pathogenic agent; 3. pathogenic agent is bred after entering in the body in vivo, and can decision cause the host to fall ill.General disease resistance is not limited to anti-a certain pathogenic agent, it is subjected to the combined influence of polygene and environmental factors, the antigenic specificity of pathogenic agent is minimum to general disease resistance influence, even basic not influence, and this disease resistance has embodied the whole defense function of body to disease.Relevant as resistance and susceptibility that the MHC of chicken and Marek, coccidiosis and Luo Si sarcoma etc. are sick, study to such an extent that be clear that swine escherichia coli K88 and F18 acceptor gene.

Disease resistance major histocompatibility complex (Major Histocompatibility complex, MHC) be and disease resistance and the closely-related one group of gene group of immunne response, the MHC of pig (called after SLA) is positioned at (Warner on No. 7 karyomit(e)s of pig, Mapping of C2, Bf, and C4genes to the swine majorhistocompatibility complex.J.Immunology.1987,139:3388-3395), comprise I type and II type gene, wherein I type gene has extremely strong polymorphism.The research of proterties such as pig SLA gene haplotype and pig birth weight, the speed of growth, the thickness of backfat is engaged in the Rothschild laboratory for a long time, they think that there are correlationship (Rothschild MF in SLA gene haplotype and above-mentioned production performance, Identification of quantitative trait lociand interesting candidate genes in the pig:progress and prospects.Proc 6thWCGALP 1998,26:403-409).Mallard etc. find that in high immunne response of selecting through 8 generations to form of pig (H system) and low immunne response (L system) strain H system reaches market weight than the Zao 10d of L system.The above-mentioned SLA that studies show that pig and the multiple production traits all have chain, and get in touch the relation of being proportionate between the SLA of pig and growth, back fat and reproductive trait more.

The subunit of the core 20S proteasome of PSMB10 genes encoding 26S proteasome.The 26S proteasome is the necessary moiety in the proteolysis approach of uiquitin-protease enzyme body, and this approach is the active degradation pathway of body internal consumption ATP.Uiquitin-protease enzyme body hydrolysis approach and the antigen presentation of MHC I quasi-molecule have important relationship, antigen is degraded into little peptide by the proteolytic enzyme complex body earlier, with the combination of MHC I quasi-molecule, be then and pass T cell, stimulate immune response, generation antibody (Klaus Fr ü h, Young Yang. (1999) Antigen presentation by MHC class I andits regulation by interferon.Current Opinion in Immunology.11,76-81).Therefore, the gene in this approach may have certain contact with the immune character of body.PSMB10 can induce generation by IFN-γ, replace an other proteasome gene PS MB7 (Eleuteri AM, Kohanski RA, CardozoC, Orlowski M. (1997) Bovine spleen multicatalytic proteinase complex (proteasome) .Replacement of X, Y, and Z subunits by LMP7, LMP2, and MECL1, and changes in properties and specificity.J Biol Chem.272,11824-11831).

Also comprise some regulation of Cell Cycle factors in the target protein of uiquitin-protease enzyme body hydrolysis approach degraded, so this proteolytic degradation approach and cell growth, differentiation has substantial connection.The PSMB10 gene is the encoding gene of this approach 26S protease hydrolysis complex body active part, to growing of body certain influence may be arranged.People's PSMB10 gene, to its existing report of research (the Cannon MJ of 26S Proteasome Structure and Function, Pate JL. (2003) Expression andregulation of interferon gamma-inducible proteasomal subunits LMP7 and LMP10in the bovine corpus luteum.Biol Reprod.68 (4), 1447-54).The PSMB10 gene is by 8 exons, and 7 introns are formed.People's the PSMB10 assignment of genes gene mapping is at HSA16q22.1 (Larsen F, SolheimJ, Kristensen T, Kolsto AB, Prydz H. (1993) A tight cluster of five unrelatedhuman genes on chromosome 16q22.1.Hum Mol Genet.2 (10), 1589-95.).The PSMB10 gene is the important β subunit in uiquitin-protease enzyme body core, but so far, the research of relevant PSMB10 gene function also is not very thorough, the polymorphism of research mutational site in colony, and carry out the strong means that the proterties association analysis is the research gene function.

(single nucleotide polymorphism SNP), mainly is meant on genomic level by the caused dna sequence polymorphism of the variation of single Nucleotide single nucleotide polymorphism.The polymorphism that SNP showed only relates to the variation of single base, and this variation can be caused by the conversion (transition) or the transversion (transversion) of single base, also can be by due to the insertion or disappearance of base.But usually said SNP does not comprise back two kinds of situations.This variation may be that (C → T then is G → A) on its complementary strand, also may be transversion (C → A, G → T, C → G, A → T) in conversion.The incidence of conversion is always apparently higher than other several variations, and the SNP with conversion hysteria variation accounts for 2/3, and the occurrence probability of other several variations is similar.The SNP detection method often adopts some existing mature technologies, as dna sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), allele specific oligonucleotide oligonucleotide hybridization (ASO) etc.No matter adopt any method, all at first must increase, just can carry out other detection then target sequence.

The PCR-RFLP full name is that (restrictionfragment length Polymorphism RFLP) analyzes polymerase chain reaction,PCR (PCR) restriction fragment length polymorphism.Mainly comprise three links: (1) target gene pcr amplification; (2) Kuo Zeng dna fragmentation restriction map (length polymorphism), this technology is used PCR target gene fragment is increased more than 1,000,000 times, even sample nucleic acid amount is less than the pg level, also can be amplified out, this has not only improved the sensitivity of genetic analysis greatly, and need not mark or radioactive probe crossover process .PCR-RFLP is mainly used in nucleic acid Variability Analysis and comparison, when DNA or RNA molecule since single base mutation or sequence reset, certain restriction enzyme site is increased, reduce or disappearance, cause restriction fragment length to change, just produced restriction fragment length polymorphism, because restriction enzyme has special restriction enzyme site on dna molecular, position and quantity according to its recognition sequence, can be size is similar and the variant DNA of sequence cuts into different fragments passes through nucleic acid electrophoresis, the DNA sheet that is uneven in length can be separated and show its length polymorphism (segmental size) .PCR-RFLP and be used for genetic analysis and have the resolution height, good reproducibility, advantages such as easy quicklook, be mainly used in the somatotype branch hypotype of hiving off of microorganism, oncogene is analyzed, the diagnosis of inherited disease, the analysis of HLA somatotype and lipophorin gene etc.

Summary of the invention

The purpose of this invention is to provide a boar production and immune character related protein and encoding gene thereof.

The albumen that pig provided by the present invention produces and immune character is relevant, name is called PSMB10, derives from pig, and its amino acid residue sequence is shown in SEQ ID NO:9.

SEQ ID NO:9 in the sequence table is made up of 273 amino-acid residues.

Above-mentioned pig produces and the encoding gene (PSMB10) of immune character related protein also belongs to protection scope of the present invention.

Above-mentioned pig produces and the encoding gene of immune character related protein comprises that pig produces and the genomic gene of the cDNA gene of immune character related protein and pig production and immune character related protein.

Its cDNA gene is one of following nucleotide sequence:

1) polynucleotide of SEQ ID NO:8 in the sequence table;

2) polynucleotide of SEQ ID NO:9 protein sequence in the code sequence tabulation.

SEQ ID NO:8 in the sequence table is by 943 based compositions, the encoding sequence of this cDNA sequence be in the sequence table SEQ ID NO:8 from 5 ' end 116-934 bit base.

Its genomic gene is one of following nucleotide sequence:

1) polynucleotide of SEQ ID NO:7 in the sequence table;

2) DNA of SEQ ID NO:9 protein sequence in the code sequence tabulation.

Sequence 7 in the sequence table is by 2663 based compositions, 1-171 bit base from 5 ' end is first exon of this genomic gene, 383-470 bit base from 5 ' end is second exon of this genomic gene, 561-658 bit base from 5 ' end is the 3rd exon of this genomic gene, 753-893 bit base from 5 ' end is the 4th exon of this genomic gene, 1158-1273 bit base from 5 ' end is the 5th exon of this genomic gene, 1384-1442 bit base from 5 ' end is the 6th exon of this genomic gene, from 5 ' the 2273-2424 bit base of end be the 7th exon of this genomic gene, from 5 ' the 2546-2663 bit base held is the 8th exon of this genomic gene; From 5 ' the 116-118 bit base of end be the initiator codon ATG of this genomic gene, from 5 ' the 2655-2657 bit base held is the terminator codon TAA of this genomic gene; 172-382 bit base from 5 ' end is first intron of this genomic gene, 471-560 bit base from 5 ' end is second intron of this genomic gene, 659-752 bit base from 5 ' end is the 3rd intron of this genomic gene, 894-1157 bit base from 5 ' end is the 4th intron of this genomic gene, 1274-1383 bit base from 5 ' end is the 5th intron of this genomic gene, from 5 ' the 1443-2272 bit base of end be the 6th intron of this genomic gene, from 5 ' the 2425-2545 bit base held is the 7th intron of this genomic gene.

The carrier, clone and the host bacterium that contain pig production of the present invention and immune character related protein encoding gene all belong to protection scope of the present invention.

Second purpose of the present invention provides a kind of method that detects pig production, immune character.

Detection pig provided by the present invention produces, the method of immune character, be to use by SEQ ID № in the sequence table: 2 and SEQ ID №: 3 or by SEQ ID № in the sequence table: 5 and SEQ ID №: a pair of primer that 6 nucleotide sequence is formed carries out pcr amplification to the genomic dna of pig to be measured, and then to SEQ ID № in the sequence table: 2 and SEQ ID №: 3 is that the pcr amplification product of primer carries out following 1) and 2) at least a detection or to SEQ ID № in the sequence table: be the pcr amplification product of primer carry out following 3 at 5 and SEQ ID №: 6) and 4) at least a detection:

1) described pcr amplification product is carried out single nucleotide polymorphism and detect, determine that from 5 of SEQ ID NO:1 ' end the 538th bit base be A or G;

2) cut described pcr amplification product with the Eco81I enzyme, detect in the endonuclease bamhi that obtains whether contain " a 642bp band ", " a 535bp band and a 107bp band " or " 642bp band, a 535bp band and a 107bp band ";

3) described pcr amplification product is carried out single nucleotide polymorphism and detect, determine that from 5 of SEQ ID NO:4 ' end the 550th bit base be T or C;

4) cut described pcr amplification product with the Eco72I enzyme, detect in the endonuclease bamhi that obtains whether contain " a 802bp band ", " a 550bp band and a 252bp band " or " 802bp band, a 550bp band and a 252bp band ".

If the single nucleotide polymorphism detected result of pcr amplification product is from 5 of SEQ ID NO:1 ' end the 538th bit base, when promptly 5 of sequence 7 ' end the 538th bit base was A in sequence table, its homozygotic genotype was AA; From 5 of SEQ ID NO:1 ' end the 538th bit base, when promptly 5 of sequence 7 ' end the 538th bit base was G in sequence table, its homozygotic genotype was GG; Their heterozygote genotype is AG.

The Eco81I enzyme is cut described pcr amplification product, if the endonuclease bamhi that obtains is the band of a 642bp, determines that then the genotype of pig to be measured is GG; If the endonuclease bamhi that obtains is two bands of a 535bp and a 107bp, determine that then the genotype of pig to be measured is AA; If the endonuclease bamhi that obtains is the band of a 642bp, three bands of a 535bp and a 107bp determine that then the genotype of pig to be measured is AG.

If the single nucleotide polymorphism detected result of pcr amplification product is from 5 of SEQ ID NO:4 ' end the 538th bit base, when promptly 5 of sequence 7 ' end the 538th bit base was A in sequence table, its homozygotic genotype was AA; From 5 of SEQ ID NO:1 ' end the 538th bit base, when promptly 5 of sequence 7 ' end the 538th bit base was G in sequence table, its homozygotic genotype was GG; Their heterozygote genotype is AG.

The Eco81I enzyme is cut described pcr amplification product, if the endonuclease bamhi that obtains is the band of a 642bp, determines that then the genotype of pig to be measured is GG; If the endonuclease bamhi that obtains is two bands of a 535bp and a 107bp, determine that then the genotype of pig to be measured is AA; If the endonuclease bamhi that obtains is the band of a 642bp, three bands of a 535bp and a 107bp determine that then the genotype of pig to be measured is AG.

If the single nucleotide polymorphism detected result of pcr amplification product is from 5 of SEQ ID NO:4 ' end the 550th bit base, promptly in sequence table 5 of sequence 7 ' when being T, its homozygotic genotype is TT to end the 1170th bit base (5 of sequence 8 ' end the 511st bit base in sequence table); From 5 of SEQ ID NO:4 ' end the 550th bit base, promptly in sequence table 5 of sequence 7 ' end the 1170th bit base (5 of sequence 8 ' end the 511st bit base in sequence table) when being C, its homozygotic genotype is CC; Their heterozygote genotype is TC.

The Eco72I enzyme is cut described pcr amplification product, if the endonuclease bamhi that obtains is the band of a 802bp, determines that then the genotype of pig to be measured is TT; If the endonuclease bamhi that obtains is two bands of a 550bp and a 252bp, determine that then the genotype of pig to be measured is CC; If the endonuclease bamhi that obtains is the band of a 802bp, three bands of a 550bp and a 252bp determine that then the genotype of pig to be measured is TC.

Wherein, the genotypic birth of AA is to finishing ADG (birth is to finishing average daily gain), and dressing percentage all is higher than AG, the GG genotype, and the AG genotype is placed in the middle, and GG is genotypic minimum; The genotypic 6-7 rib of GG BF (the 6-7 intercostal thickness of backfat), average 3 BF (6-7 intercostal, last rib place and waist are recommended the junction from 3 fat thickness mean values of dorsomeson 4cm), yellowish pink, intramuscular fat all is higher than AG, the GG genotype, the AG genotype is placed in the middle, and the AA genotype is minimum; In HCT% (pcv amount), in these 3 immune characters of MCV (fL) (mean corpuscular volume (MCV)) and RDW (%CV) (red cell distribution width), the GG genotype all is higher than AG, the AA genotype, the AG genotype is placed in the middle, the AA genotype is minimum, shows that tentatively the immune character of GG genotype individuality is better.

The genotypic birth of TT is to finishing ADG, dressing percentage, estimation eye muscle area (cm ²) and leg stern ratio all the utmost point be significantly higher than TC, the CC genotype; And reach in market weight (90KG) age in days and these two growth traitss of intramuscular fat nascent, the CC genotype all is higher than TC, the TT genotype.The genotypic birth of TT is significantly higher than TT to finishing ADG with the estimation eye muscle area utmost point, and the TC genotype shows that the speed of growth of TT genotype pig and these two growth indexes of eye muscle area are better; The genotypic MCV of CC (fL) the proterties utmost point is significantly higher than TT genotype (P＜0.01) and TC genotype; And in HCT% and RDW (%CV) two proterties, the genotypic respective value of CC is significantly higher than the genotypic respective value of TT (P＜0.05); In these 3 proterties, the genotypic respective value of TC all is significantly higher than the genotypic respective value of TT (P＜0.05).

Described single nucleotide polymorphism detects and can adopt dna sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), the hybridization of allele specific oligonucleotide oligonucleotide (ASO), the little sequencing according to the DNA array, dynamic allele specific oligonucleotide hybridization, special connection, DNA chip and the TaqMan system etc. of oligonucleotide to detect.

Pig production of the present invention and immune character related protein and encoding gene thereof can be used for detecting immune characters such as swine erythrocyte volume distributed median width, red blood cell hematocrit amount, mean corpuscular volume (MCV), birth is to finishing ADG, and ADG reaches and finishes body weight age in days, dressing percentage, estimation eye muscle area (cm ²) production traits such as leaf fat rate, leg stern ratio, meat, intramuscular fat, for the molecular breeding of pig provides a new genetic marker, will in the breeding of pig, play a significant role.

Description of drawings

Fig. 1 is for detecting the schema of pig PSMB10 partial dna sequence polymorphism

Fig. 2 is the amplification of pig PSMB10 partial dna sequence

Fig. 3 is the agarose gel electrophoresis detected result of pig PSMB10 partial dna sequence Eco81I-RFLP polymorphism

Fig. 4 is the amplification of pig PSMB10 partial dna sequence and the agarose gel electrophoresis detected result of Eco72I-RFLP polymorphism thereof

Embodiment

Used experimental technique is ordinary method if no special instructions among the following embodiment.

The acquisition of embodiment 1, pig PSMB10

1, the acquisition of the genomic gene of pig PSMB10

CDNA sequence (the GenBank number of including: NM_002801) be the information probe of personnel selection PSMB10, utilize the BLAST instrument of NCBI in GenBank pig est database, to do the homologous sequence retrieval, obtain a series of homologys and reach ESTs (fragment length is greater than 100bp) more than 80%, corresponding sequence is inquired about in the number of including of these ESTs in the ENTREZ of NCBI (http://www.ncbi.nlm.nih.gov/Web/Search/index.htmL), use the EST contig of the Seqman program construction pig among the software DNAStar then, at last according to 4 pairs of EST splicing sequences Design amplimers:

1PL：5′-AGTTAAGCTCAGTCGTGCAG-3′

1PR：5′-TGAAGTGGATCTTTTCGCAG-3′

2PL：5′-AGCTGCGAAAAGATCCACTT-3′

2PR：5′-GGTCTTCTAGCACTGCCAGG-3′

3PL：5′-CCTGGCAGTGCTAGAAGACC-3′

3PR：5′-GTCAGCGGCATCACTGTCTG-3′

4PL：5′-ACAAAGCCCACAGAAAGGTA-3′

4PR：5′-CTCAGCTTACTCCACATCCA-3′

In the genomic dna of pig, increase, obtain 4 specific bands, obtain the sequence of a 2663bp with the splicing of the Seqman program among the software DNAStar, nucleotide sequence with sequence 7 in the sequence table, this sequence is the genome sequence of pig PSMB10, the amino acid residue sequence (pig PSMB10) that coding has sequence 9 in the sequence table.1-171 bit base from 5 ' end is first exon of this genomic gene, 383-470 bit base from 5 ' end is second exon of this genomic gene, 561-658 bit base from 5 ' end is the 3rd exon of this genomic gene, 753-893 bit base from 5 ' end is the 4th exon of this genomic gene, 1158-1273 bit base from 5 ' end is the 5th exon of this genomic gene, 1384-1442 bit base from 5 ' end is the 6th exon of this genomic gene, from 5 ' the 2273-2424 bit base of end be the 7th exon of this genomic gene, from 5 ' the 2546-2663 bit base held is the 8th exon of this genomic gene; From 5 ' the 116-118 bit base of end be the initiator codon ATG of this genomic gene, from 5 ' the 2655-2657 bit base held is the terminator codon TAA of this genomic gene; 172-382 bit base from 5 ' end is first intron of this genomic gene, 471-560 bit base from 5 ' end is second intron of this genomic gene, 659-752 bit base from 5 ' end is the 3rd intron of this genomic gene, 894-1157 bit base from 5 ' end is the 4th intron of this genomic gene, 1274-1383 bit base from 5 ' end is the 5th intron of this genomic gene, from 5 ' the 1443-2272 bit base of end be the 6th intron of this genomic gene, from 5 ' the 2425-2545 bit base held is the 7th intron of this genomic gene.

2, the acquisition of the cDNA of pig PSMB10 gene

At last according to 2 pairs of EST splicing sequences Design amplimers:

5PL：5’-AGTTAAGCTCAGTCGTGCAG-3’

5PR：5’-CTCAGCTTACTCCACATCCA-3’

6PL：5’-GTGCTGGTCCTACTCCTCTG-3’

6PR：5’-TAACATGCGAGTGACAGTGG-3’

Amplification obtains two special bands in the cDNA of pig, and product is checked order, and result and people's PSMB10 and pig PSMB10 genome sequence are compared, and is confirmed to be the cDNA sequence of this gene.It has the SEQ ID № in the sequence table: the nucleotide sequence of 8 (by 943 based compositions), the encoding sequence of this cDNA sequence are SEQ ID № in the sequence table: 8 from 5 ' end 116-934 bit base.

3, the location of pig PSMB10 gene

With pig * rodents somatic cell hybrid clone panel RH clone plate (INRA-Minnesota porcine radiationhybrid panel, ImpRH, 118 hybrid cell systems) DNA in is (available from French Academy of Agricultural Sciences, Laboratoirede G é n é tique Cellulaire, INRA) be template, with forward primer: 5 '-AGCTGCGAAAAGATCCACTT-3 ' and reverse primer: 5 '-GGTCTTCTAGCACTGCCAGG-3 ' carries out pcr amplification.Amplification condition is: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 20s, 59 ℃ of annealing 20s, 72 ℃ are extended 20s, 30 circulations.Extend 5min at 72 ℃ at last.Reaction system wherein consists of: PCR reaction cumulative volume is 10 μ l, and wherein the about 25ng of RH clone plate DNA contains 1 * damping fluid (Promega), 2mmol/L MgCl ₂, the dNTP final concentration is 150 μ mol/L, the primer final concentration is 0.2 μ mol/L, 1U Taq archaeal dna polymerase (Promega).The PCR reaction product is carried out the pcr amplified fragment somatotype with 1.5% agarose gel electrophoresis, wherein the 1st, 2, and 3,4,6,12,21,28,29,37,43,48,61,63,68,69,70,73,94,98,99,100,101,102,103,104,105,108,111,112,113,115, obtain positive amplification among the DNA during No. 118 hybrid cell is.PCR somatotype data are submitted to HybWeb (http://imprh.toulouse.inra.fr/) carry out statistical study to obtain zone location information, the data analysis result shows that the genomic gene of pig PSMB10 is positioned on No. 6 karyomit(e)s of pig, with microsatellite marker SW1108 close linkage, the LOD value is 6.14.

The single nucleotide polymorphism of the partial dna sequence of embodiment 2, pig PSMB10 and RFLP polymorphism detect

The flow process that the single nucleotide polymorphism of the partial dna sequence of pig PSMB10 and RFLP polymorphism detect as shown in Figure 1, concrete steps are as follows:

One, the detection of the single nucleotide polymorphism of the partial dna sequence of pig PSMB10

1, design of primers

CDNA sequence (the GenBank number of including: NM_002801) be the information probe of personnel selection PSMB10, utilize the BLAST instrument of NCBI in GenBank pig est database, to do the homologous sequence retrieval, obtain a series of homologys and reach ESTs (fragment length is greater than 100bp) more than 90%, corresponding sequence is inquired about in the number of including of these ESTs in the ENTREZ of NCBI (http://www.ncbi.nlm.nih.gov/Web/Search/index.htmL), use the EST contig of the ASSEMBLY program construction pig among the software GeneTool then, according to EST splicing sequences Design amplimer, primer sequence is as follows at last:

Primer 1 (forward): 5 '-AGTTAAGCTCAGTCGTGCAG-3 ' (sequence 2)

Primer 2 (oppositely): 5 '-TGAAGTGGATCTTTTCGCAG-3 ' (sequence 3)

Primer 3 (forward): 5 '-AGCTGCGAAAAGATCCACTT-3 ' (sequence 5)

Primer 4 (oppositely): 5 '-GGTCTTCTAGCACTGCCAGG-3 ' (sequence 6)

2, the purifying of pcr amplification and amplified production thereof, clone and order-checking

Respectively to greatly enhance 26 of logical combinations (Da Bai * length in vain * Tongcheng pig), grow up and lead to combination (in vain long * Da Bai * Tongcheng pig) 27,15 of Large Whites, 15 of landraces, total DNA of the poba gene group that 15 of Du Luoke and Tongcheng pure breeding pig are 74 is a template, under the guiding of primer 1 and primer 2, primer 3 and primer 4, pcr amplification pig PSMB10 partial dna sequence.PCR reaction cumulative volume is 20 μ l, wherein, and the about 100ng of pig genomic dna, 2 μ l, 10 * PCR damping fluids (Promega), 1.5mmol/L MgCl ₂, dNTP 150 μ mol/L, each 0.2 μ mol/L of primer 1 and primer 2 (or primer 3 and primer 4), 2U Taq archaeal dna polymerase (Promega).The PCR response procedures is: 94 ℃ of 4min of elder generation; 94 ℃ of 30s then, 62 ℃ of 30s, 72 ℃ of 20s, 35 circulations; Last 72 ℃ are extended 5min.The PCR reaction product is carried out 1.5% agarose gel electrophoresis, and the electrophoresis result of primer 1 and primer 2 amplified production shows that the PCR product size of acquisition is 642bp as shown in Figure 2.Among Fig. 2,2 swimming lanes are pcr amplification product, and the M swimming lane is dna molecular amount mark (100-1000bp ladder).The electrophoresis result of primer 3 and primer 4 amplified productions shows that the PCR product size of acquisition is 802bp as shown in Figure 4.Among Fig. 4, the PCR swimming lane is a pcr amplification product, and the M swimming lane is dna molecular amount mark (100-1000bp ladder).

Carry out purifying, clone and the order-checking of PCR product then as follows:

(1) purifying of PCR product: under ultraviolet lamp, contain the segmental gel of purpose from the sepharose cutting-out, put into 1.5ml Ependorff pipe, being incubated to gel in 70 ℃ melts fully, use PCR product purification test kit (Promega) purified pcr product then, according to the operation of test kit specification sheets, concrete steps are to add 1ml resin (Resin), mixing 20s in the gel that per 300 μ l melt, with the Resin/DNA mixture syringe of packing into, make slurries pass through microtrabeculae (Minicolumn) and extrude.In syringe, add 80% Virahol 2ml again, touch piston Virahol is extruded by Minicolumn, take off Minicolumn and pack in the 1.5ml Ependorff pipe 10 into, the centrifugal 2min of 000g is with dry Resin, Minicolumn is packed in another clean 1.5ml Ependorff pipe, add 30-50 μ l sterilized water, leave standstill 1min, 10, the centrifugal 20s of 000g abandons supernatant, and eluted dna is stored in the Ependorff pipe.

(2) ligation: the PCR product and the pGEM-T carrier (Promega) of step (1) purifying are used T ₄Dna ligase connects, and obtains recombinant vectors.Linked system is: 2.5 μ L, 2 * damping fluid, 0.5 μ L pGEM-T carrier, the PCR product of 0.5 μ L purifying, 0.5 μ L T ₄Dna ligase, 1 μ L aqua sterilisa, 16 ℃ of water-bath 12-24h.

(3) preparation of competent cell: with bacillus coli DH 5 alpha (TaKaRa) on the LB solid medium 37 ℃ cultivate 16-20h after, picking list colony inoculation is in 2mL LB liquid nutrient medium, 37 ℃ of shaking culture 3h, getting 1mL bacterium liquid then transfers in containing the shaking in the bottle of 30mL LB liquid nutrient medium, continue the about 4h of shaking culture at 37 ℃, treat bacterium liquid OD ₆₀₀Value will shake bottle when reaching 0.3-0.4 and take out from shaking table, ice bath 10-15min makes it cooling, then bacterium liquid is changed in the centrifuge tube in 4 ℃, 4, and the centrifugal 10min of 000g collects somatic cells, centrifuge tube is inverted to abandon clean nutrient solution, is iced the CaCl of the 0.1mol/L of precooling with 10mL ₂The resuspended precipitation of solution, i.e. somatic cells, behind the ice bath 30min, 4 ℃ 4, the centrifugal 10min of 000g is again with the CaCl of the 0.1mol/L of 4mL ice precooling ₂The resuspended precipitation of solution, it is standby to put 4 ℃ of preservations.

(4) transform: the competent cell of getting 100-120 μ l step (3) preparation under the sterile state is in the 1.5mLEpendorff pipe, add the connection product that 5 μ L steps (2) obtain again, mixing, ice bath 30min, 42 ℃ of heat shock 90s (not shaking the Ependorff pipe in the above-mentioned conversion process), take out back ice bath 3-4min, add 400 μ l again and do not contain antibiotic LB liquid nutrient medium, at 37 ℃ of shaking culture 45min, get 100 μ L bacterium liquid at last and coat the 200mg/mLIPTG (Isopropylthio-β-D-galactoside that contains 4 μ L, isopropylthio-) and on the LB flat board of the 20mg/mL X-gal of 40 μ L, is inverted under the same conditions behind 37 ℃ of pre-1h of cultivation earlier and cultivates 12-24h;

(5) a small amount of of plasmid preparation: the single colony inoculation that grows on the picking agar plate is in 2-3mL LB liquid nutrient medium, and 37 ℃ of 300r/min cultivate 12-24h, collects thalline with the centrifugal several seconds of 1.5mL EP pipe 12000r/min again.Every pipe adds the ice-cold solution I of 100 μ l (50mM glucose, 25mM Tris-HCl (pH8.0), 10mMEDTA (pH8.0)), and vortex vibrates to thalline and fully suspends.Add new preparation solution II (0.2M NaOH, 1%SDS) 200 μ l put upside down mixing fast, ice bath 5min adds solution III (5M potassium acetate, glacial acetic acid 11.5ml, the H of precooling then ₂O 28.5ml) 150 μ l, ice bath 5min behind the mixing, the centrifugal 5min of 12000r/min, supernatant is gone in another EP pipe, add phenol: chloroform: primary isoamyl alcohol 500 μ l, vortex vibration, the careful upper strata water of drawing in centrifugal back, the dehydrated alcohol that adds 2 times of volumes,-20 ℃ of precipitation 30min, the centrifugal 5min of 12000r/min, precipitation is with 70% washing with alcohol 2 times, drain, add the TE 20 μ l that contain the RNA enzyme.

(6) enzyme of recombinant plasmid is cut evaluation: get 3 μ l plasmid DNA and distilled water mixing, making its cumulative volume is 15 μ l, add 2-3U restricted endoenzyme EcoR I and the corresponding 10 * restriction enzyme reaction damping fluid of 2 μ l, flick tube wall mixing and centrifugal, put 37 ℃ of water-bath 1-2 hours, get 2-3 μ l reaction solution and detect, obtain enzyme and cut the result and estimate identical purpose recombinant plasmid in agarose gel electrophoresis.

(7) order-checking: recombinant plasmid adopts the terminal cessation method of two deoxidations to check order on automatic dna sequencer, and sequencing is finished by Shanghai Bo Ya Bioisystech Co., Ltd.Sequencing result shows that the length of primer 1 and primer 2 amplified production is 642bp, nucleotide sequence with SEQ ID NO:1 in the sequence table, wherein, 1-171 bit base from 5 ' end is the partial sequence of genomic first exon of PSMB10, i.e. 5 of sequence 7 ' end 1-171 bit base (5 of sequence 8 ' end 1-171 bit base in sequence table) in sequence table; 172-382 bit base from 5 ' end is genomic first intron complete sequence of this gene, i.e. 5 of sequence 7 ' end 172-382 bit base in sequence table; 383-470 bit base from 5 ' end is the partial sequence of genomic second exon of PSMB10, i.e. 5 of sequence 7 ' end 383-470 bit base (5 of sequence 8 ' end 172-259 bit base in sequence table) in sequence table; 471-562 bit base from 5 ' end is the partial sequence of genomic second intron of PSMB10, i.e. 5 of sequence 7 ' end 471-562 bit base in sequence table; 561-642 bit base from 5 ' end is the partial sequence of the 3rd exon of PSMB10 genomic gene, i.e. 5 of sequence 7 ' end 561-642 bit base (5 of sequence 8 ' end 260-341 bit base in sequence table) in sequence table.From 5 of SEQ ID NO:1 ' end the 538th bit base, promptly there are two allelotrope of A, G in 5 of sequence 7 ' end the 538th bit base place in sequence table, is pleomorphism site.As holding the 538th bit base from 5 of SEQ ID NO:1 ', when promptly 5 of sequence 7 ' end the 538th bit base was A in sequence table, its homozygotic genotype was AA; From 5 of SEQ ID NO:1 ' end the 538th bit base, when promptly 5 of sequence 7 ' end the 538th bit base was G in sequence table, its homozygotic genotype was GG; Their heterozygote genotype is AG.

Sequencing result shows that the length of primer 3 and primer 4 amplified productions is 802bp, nucleotide sequence with SEQ ID NO:4 in the sequence table, wherein, 1-38 bit base from 5 ' end is the partial sequence of genomic the 3rd exon of PSMB10, i.e. 5 of sequence 7 ' end 621-658 bit base (5 of sequence 8 ' end 320-357 bit base in sequence table) in sequence table; 39-132 bit base from 5 ' end is the 3rd an intron complete sequence of this genomic gene, i.e. 5 of sequence 7 ' end 659-752 bit base in sequence table; 133-273 bit base from 5 ' end is the complete sequence of genomic the 4th exon of PSMB10, i.e. 5 of sequence 7 ' end 753-893 bit base (5 of sequence 8 ' end 358-498 bit base in sequence table) in sequence table; 274-537 bit base from 5 ' end is the complete sequence of genomic the 4th intron of PSMB10, i.e. 5 of sequence 7 ' end 894-1157 bit base in sequence table; 538-653 bit base from 5 ' end is the complete sequence of the 5th exon of PSMB10 genomic gene, i.e. 5 of sequence 7 ' end 1158-1273 bit base (5 of sequence 8 ' end 499-614 bit base in sequence table) in sequence table; 654-763 bit base from 5 ' end is the complete sequence of the 5th intron of PSMB10S genomic gene, i.e. 5 of sequence 7 ' end 1274-1383 bit base in sequence table; 764-802 bit base from 5 ' end is the partial sequence of the 6th exon of PSMB10 genomic gene, i.e. 5 of sequence 7 ' end 1384-1422 bit base (5 of sequence 8 ' end 615-653 bit base in sequence table) in sequence table.From 5 of SEQ ID NO:4 ' end the 550th bit base, promptly 5 of sequence 7 ' end the 1170th bit base (5 of sequence 8 ' end the 511st bit base in sequence table) locates to exist two allelotrope of C, T in sequence table, is pleomorphism site.As from 5 of SEQ ID NO:4 ' end the 550th bit base, promptly in sequence table 5 of sequence 7 ' end the 1170th bit base (5 of sequence 8 ' end the 511st bit base in sequence table) when being C, its homozygotic genotype is CC; From 5 of SEQ ID NO:4 ' end the 550th bit base, promptly in sequence table 5 of sequence 7 ' end the 1170th bit base (5 of sequence 8 ' end the 511st bit base in sequence table) when being T, its homozygotic genotype is TT; Their heterozygote genotype is CT.

3, the dna sequence dna homology search is identified

By the American National biotechnology (NCBI of information center, National Center for BiotechnologyInformation, http://www.ncbi.nlm.nih.gov) BLAST of website (Basic Local AlignmentSearch Tool) software, the known physiological function gene of announcing in the dna sequence dna (SEQ ID NO:1 and SEQ ID NO:4) that order-checking back is obtained and the GenBank database carries out sequence homology relatively, to identify and to obtain the function information of this dna sequence dna, the result shows that pig and people's sequence similarity is 86%, the proteasome subunit PSMB10 of this sequence encoding pig.

Two, the detection of the RFLP polymorphism of the partial dna sequence of pig PSMB10

1, the partial dna sequence Eco81I-RFLP polymorphism of pig PSMB10 detects: 15 of Large Whites, 15 of pigs grow up, total DNA of the poba gene group that Du Luoke and Tongcheng pig are 40 is a template, under the guiding of primer 15 '-AGTTAAGCTCAGTCGTGCAG-3 ' (forward) and primer 25 '-TGAAGTGGATCTTTTCGCAG-3 ' (oppositely), carry out pcr amplification, 20 μ l PCR reaction systems are: the about 100ng of pig genomic dna, 2 μ l, 10 * PCR damping fluids (Promega), 1.5mmol/L MgCl ₂, the dNTP final concentration is 150 μ mol/L, each 0.2 μ mol/L of primer 1 and primer 2,2U Taq archaeal dna polymerase (Promega).The PCR reaction conditions is: 94 ℃ of 4min of elder generation; 94 ℃ of 30s then, 58 ℃ of 30s, 72 ℃ of 20s, 35 circulations; Last 72 ℃ are extended 5min.Then with PCR product Eco81I digestion with restriction enzyme, endonuclease reaction system (10 μ l) is, 1 * enzyme cutting buffering liquid, 1 μ l, and PCR product 3-5 μ l, restriction enzyme 0.5 μ l (5U) uses H ₂O supplies 10 μ l.The enzyme tangent condition is: 37 ℃ of water-bath 4h.Detecting enzyme with 2% agarose gel electrophoresis cuts the result and writes down genotype, the result is as shown in Figure 3 (among Fig. 3, swimming lane M is Marker, swimming lane GG is that genotype is the homozygote of GG, swimming lane AA is that genotype is the homozygote of AA, swimming lane AG is that genotype is the heterozygote of AG), show in the AA genotype individuality, have to the band of a 642bp; In the AG genotype individuality, obtain three bands: a 642bp, a 535bp and a 107bp band; In the GG genotype individuality, obtain two bands: a 535bp and a 107bp band.When allelotrope is A, recognition site (the CC ↓ TAAGG) that occurs restriction enzyme Eco81I from 5 of SEQ ID NO:1 ' end 534-540 bit base, if cut this fragment with the Eco81I enzyme, then allelotrope is that fragment of 642bp only appears in G, allelotrope is that 535bp and two fragments of 107bp then can appear in A, owing to this locus is controlled by above-mentioned two allelotrope, thereby can form AA, AG, three kinds of genotype of GG.

2, the partial dna sequence Eco72I-RFLP polymorphism of pig PSMB10 detects

Respectively with 15 of Large Whites, 15 of pigs grow up, total DNA of the poba gene group that 15 of Du Luoke and Tongcheng pig are 40 is a template, carry out pcr amplification at primer 35 under the guiding of '-AGTTAAGCTCAGTCGTGCAG-3 ' (forward) and primer 45 '-GGTCTTCTAGCACTGCCAGG-3 ' (oppositely), 20 μ l PCR reaction systems are: the about 100ng of pig genomic dna, 2 μ l, 10 * PCR damping fluids (Promega), 1.5mmol/L MgCl ₂, the dNTP final concentration is 150 μ mol/L, each 0.2 μ mol/L of primer 1 and primer 2,2U Taq archaeal dna polymerase (Promega).The PCR reaction conditions is: 94 ℃ of 4min of elder generation; 94 ℃ of 30s then, 58 ℃ of 30s, 72 ℃ of 20s, 35 circulations; Last 72 ℃ are extended 5min.Then with PCR product Eco72I digestion with restriction enzyme, endonuclease reaction system (10 μ l) is, 1 * enzyme cutting buffering liquid, 1 μ l, and PCR product 3-5 μ l, restriction enzyme 0.5 μ l (5U) uses H ₂O supplies 10 μ l.The enzyme tangent condition is: 37 ℃ of water-bath 4h.Detecting enzyme with 2% agarose gel electrophoresis cuts the result and writes down genotype, the result is as shown in Figure 4 (among Fig. 4, swimming lane M is Marker, swimming lane TT is that genotype is the homozygote of TT, swimming lane CC is that genotype is the homozygote of CC, swimming lane TC is that genotype is the heterozygote of TC), show in the TT genotype individuality, have to the band of a 802bp; In the TC genotype individuality, obtain three bands: a 802bp, a 550bp and a 252bp band; In the CC genotype individuality, obtain two bands: a 550bp and a 252bp band.When allelotrope is C, recognition site (the CAC ↓ GTG) that occurs restriction enzyme Eco72I from 5 of SEQ ID NO:4 ' end 548-553 bit base, if cut this fragment with the Eco72I enzyme, then fragment of 802bp only appears in T allelotrope, 550bp and two fragments of 252bp then can appear in C allelotrope, owing to this locus is controlled by above-mentioned two allelotrope, thereby can form TT, TC, three kinds of genotype of CC.

Embodiment 3, detection pig immune trait and the production traits

Respectively with 15 of Large Whites, 15 of pigs grow up, 40 of 15 of Du Luoke and Tongcheng pigs totally 85 individualities are experimental subjects detects 85 individualities according to the method for embodiment 2 single nucleotide polymorphism and RFLP polymorphism, determine their genotype, its result as shown in Table 1 and Table 2, table 1 shows that in 4 pig varieties that detected two gene frequencies of Tongcheng pig are more or less the same; In the landrace that detects, do not see the GG genotype, A allelotrope has comparative advantage; The A gene frequency of Large White and duroc is apparently higher than the frequency of G gene.

The distribution of each genotype of table 1 in 4 pig varieties

Kind	Number of individuals	Genotype			Gene frequency
							GG	GA	AA	G	A
		Large White landrace duroc Tongcheng pig	15 15 15 40	1 0 0 11	5 0 6 20	9 15 9 9						0.2322 0 0.2000 0.5250	0.7678 1 0.8000 0.4750

Table 2 shows that T allelotrope all accounts for clear superiority in 4 pig varieties that detected, and does not see the CC genotype in the landrace that is detected.

The distribution of each genotype of table 2 in 4 pig varieties

Kind	Number of individuals	Genotype			Gene frequency
							CC	CT	TT	C	T
		Large White landrace duroc Tongcheng pig	15 15 15 40	1 0 1 8	3 4 5 26	11 11 9 6						0.1667 0.1333 0.2333 0.2500	0.8333 0.8667 0.7667 0.7500

2, mark property association analysis

Method according to embodiment 2 detects following 87 individualities: greatly enhance 26 pigs of logical combination (Da Bai * (in vain long * Tongcheng)), 27 pigs of logical combination (in vain long * (Da Bai * Tongcheng)) grow up, single nucleotide polymorphism and the RFLP polymorphism of 34 pigs of Tongcheng pure breeding group, determine their genotype, and carry out genotype and production, immune character association analysis.

With following least square model analysis immune character (red cell distribution width (RDW (%CV)), red blood cell hematocrit amount (HCT%), mean corpuscular volume (MCV) (MCV (fL)), blood IgG antibody content, NCHC, average content of hemoglobin, total white blood cells and red corpuscle sum) and the production traits (ADG reaches end body weight age in days, dressing percentage, estimation eye muscle area (cm ²), leg stern ratio, leaf fat rate, intramuscular fat, shearing rate, pH value, average daily gain, the thickness of backfat, yellowish pink, marble grain, percentage of water loss and drip loss):

y _ijk＝μ+GENOTYPE _i+SEX _j+COMBINATION _k+ε _ijk，

Wherein, y _IjkBe the character observation value, μ is a population mean, GENOTYPE _iBe genotype effect, SEX _jBe sex effect, COMBINATION _kBe the effect of various combination, ε _IjkBe random error, suppose obey N (0, σ ²) distribute.

The result of the proterties significant difference between the different genotype (least square mean and standard error analysis) is as table 3, table 4, and shown in table 5 and the table 6, other proterties does not have significant difference between different genotype.

The association analysis of table 3 different genotype and part producing proterties

Genotype	Birth is to finishing ADG	Dressing percentage	6-7 rib BF (cm)	Average 3 BF (cm)	Yellowish pink	Intramuscular fat (%)
							AA AG GG	452.775±10.174 407.949±11.747 367.378±19.303	76.282±0.343 74.488±0.396 73.609±0.651	3.808±.138 4.023±0.159 4.566±0.262	3.534±0.111 3.791±0.128 4.308±0.211	3.014±0.066 3.226±0.076 3.250±0.125	2.671±0.207 3.518±0.239 5.224±0.392
AA/AG AA/GG AG/GG	0.005 ** 0.000 **0.076	0.001 ** 0.001 ** 0.252	0.311 0.012 * 0.081	0.133 0.002 ** 0.040 *	0.039 * 0.100 0.870	0.009 ** ****

Annotate: * * represents that difference is extremely remarkable, and * represents significant difference.

Table 3 shows that the genotypic birth of AA is to finishing ADG, and dressing percentage all is higher than AG, the genotypic respective value of GG, and wherein, between the genotypic respective value of AG was placed in the middle, the genotypic respective value of GG was minimum; The genotypic 6-7 rib of GG BF, average 3 BF, yellowish pink, intramuscular fat all is higher than AG, the GG genotype, with AG genotype respective value placed in the middle between, AA genotype respective value is minimum.

The association analysis of table 4 different genotype and partial immunity proterties

Genotype	Red blood cell hematocrit amount HCT%	Mean corpuscular volume (MCV) MCV (fL)	Red cell distribution width RDW (%CV)
				AA AG GG	36.116±1.154 36.658±1.281 42.111±2.176	57.031±0.711 59.423±0.789 63.333±1.341	17.675±0.292 17.785±0.324 19.344±0.551
AA/AG AA/GG AG/GG	0.754 0.018 * 0.034 *	0.028 * 0.000 ** 0.014 *	0.803 0.009 ** 0.017 *

Annotate: * * represents that difference is extremely remarkable, and * represents significant difference.

Table 4 shows, at HCT%, in these 3 immune characters of MCV (fL) and RDW (%CV), the genotypic respective value of GG all is higher than AG, the genotypic respective value of AA, wherein, placed in the middle with the genotypic respective value of AG, the genotypic respective value of AA is minimum, shows that tentatively the immune character of GG genotype individuality is better.

The association analysis of table 5 different genotype and part producing proterties

Genotype	Birth is to finishing ADG	ADG reaches and finishes the body weight age in days	Dressing percentage	Estimation eye muscle area cm 2	Leg stern ratio	Intramuscular fat
							TT TC CC	452.665±10.665 399.263±10.502 360.439±24.629	193.352±3.312 204.393±3.262 208.377±7.649	76.097±0.362 74.548±0.357 72.670±0.836	31.332±1.302 25.673±1.282 20.863±3.006	30.090±0.311 28.845±0.306 27.602±0.718	2.685±0.237 3.822±0.234 4.650±0.548
TT/TC TT/CC TC/CC	0.001 ** 0.001 ** 0.151	0.020 * 0.075 0.633	0.003 ** 0.000 ** 0.042 *	0.003 ** 0.002 ** 0.145	0.006 ** 0.002 ** 0.115	0.001 ** 0.002 ** 0.168

Annotate: * * represents that difference is extremely remarkable, and * represents significant difference.

Table 5 shows that the genotypic birth of TT is to finishing ADG, dressing percentage, estimation eye muscle area (cm ²) and the value of leg stern ratio all the utmost point be significantly higher than TC, the genotypic respective value of CC; And reach in end body weight age in days and these two growth traitss of intramuscular fat at ADG, CC genotype respective value all is higher than TC, the genotypic respective value of TT.Data analysis is the result show, the birth of TT genotype pig is significantly higher than TT to finishing ADG with the estimation eye muscle area utmost point, and the TC genotype illustrates that the speed of growth of TT genotype pig and these two growth indexes of eye muscle area are better.

The association analysis of table 6 different genotype and partial immunity proterties

Genotype	Red blood cell hematocrit amount HCT%	Mean corpuscular volume (MCV) MCV (fL)	Red cell distribution width RDW (%CV)
				TT TC CC	36.369±1.247 37.093±1.247 42.814±2.539	57.793±0.758 59.276±0.758 63.857±1.543	17.683±0.310 18.059±0.310 19.500±0.630
TT/TC TT/CC TC/CC	0.683 0.026 * 0.047 *	0.171 0.001 ** 0.010 *	0.394 0.012 * 0.044 *

Annotate: * * represents that difference is extremely remarkable, and * represents significant difference.

Table 6 shows that the genotypic MCV of CC (fL) the proterties utmost point is significantly higher than TT genotype (P＜0.01) and TC genotype; And in HCT% and RDW (%CV) two proterties, the genotypic respective value of CC is significantly higher than the genotypic respective value of TT (P＜0.05); In these 3 proterties, the genotypic respective value of TC all is significantly higher than the genotypic respective value of TT (P＜0.05).

Sequence table

<160>9

<210>1

<211>642

<212>DNA

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<220>

<221>misc-feature

<222>(538)

＜223〉n=a or g

<400>1

agttaagctc agtcgtgcag gacaaggaca aaggacaagg ctgtggcctt ccttgcttgg 60

tgctggtcct actcctctga cctccaaacc cctggactgc cctgcccacc tggacatgca 120

gaagatagcg ctagagcccc tagggggctt ttcctttgag aactgccaga ggtgagtggg 180

gggatgattg gtccccagct gcctaggtcc tcagtgaggg tctctgggag tgggagaacc 240

aggatcttta aaagtaagac gacgacttgg gttccgaggc ttacatggag aagtgagaga 300

aacgtggacg gagttggaga atgtgattgg gggaggggga tcccataaga aatgaaacct 360

gagacttttc cctccgttcc agaaatgcat ccttggaacg cgccctccct gggttccggg 420

tccctcacgc actcaagacg gggaccacca ttgcaggtct tgtattccag gtgagcaaga 480

agttgtgact gtggggcttg gaggggcggc cgggaatagg aagtgggcag tgacctangg 540

gtttctcccc tcttctccag gacggggtaa tcctgggcgc ggatacgcgg gccactaacg 600

actcggtcgt ggcggacaag agctgcgaaa agatccactt ca 642

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

agttaagctcagtcgtgcag 20

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

tgaagtggatcttttcgcag 20

<210>4

<211>802

<212>DNA

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<220>

<221>misc-feature

<222>(550)

＜223〉n=t or c

<400>4

agctgcgaaa agatccactt catcgccccc aaaatctagt gagcattcct ctcaagaact 60

gccccttagt tccccgtccc tggtctccgc acaccttagc cccgaccaca tggaccctcc 120

cttcaccctc agctgctgtg gggctggagt agccgcggac gccgagatga ccacgcggat 180

ggcggcatcc aacatagagc tacacgcact atccaccggc cgcgagccgc gcgtggccac 240

tgtcactcgc atgttacgcc agaagctctt ccggtgctgg agctgggcta agacgaccct 300

ggggaggggc agttgcaggg aaggtggggc tgataggagg tgggacaaga ggaaggacag 360

ggctgcgagg tgccgggaga ccggaagagg tcagcctaag aggagttaag aaggcaaccg 420

gaagcagcaa tgtcagtcat ggggctccag accacgggaa ggggcggggc tcgggtgcag 480

gaccatggag gggagggacc tggtttgttt ggctgactgc gcccacttgc ggaaaaggta 540

ccagggccan gtgggcgcgt cgctaatcgt gggcggcgta gacttcacag gaccccagct 600

ctacagcgtg cacccccatg ggtcctacag ccgtctgccc ttcacggccc taggtgagcc 660

cttccatccc ttccccacga atcccacgta tactatcctg gcctcccgtt tctcccgcga 720

ccatgcggcc gcggctgacc tcagataccc ctcctgtcta caggctccgg ccaagatgca 780

gccctggcag tgctagaaga cc 802

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>5

agctgcgaaaagatccactt 20

<210>6

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>6

ggtcttctagcactgccagg 20

<210>7

<211>2663

<212>DNA

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<220>

<221>misc-feature

<222>(538)

＜223〉n=a or g

<220>

<221>misc-feature

<222>(1170)

＜223〉n=c or t

<400>7

agttaagctc agtcgtgcag gacaaggaca aaggacaagg ctgtggcctt ccttgcttgg 60

tgctggtcct actcctctga cctccaaacc cctggactgc cctgcccacc tggacatgca 120

gaagatagcg ctagagcccc tagggggctt ttcctttgag aactgccaga ggtgagtggg 180

gggatgattg gtccccagct gcctaggtcc tcagtgaggg tctctgggag tgggagaacc 240

aggatcttta aaagtaagac gacgacttgg gttccgaggc ttacatggag aagtgagaga 300

aacgtggacg gagttggaga atgtgattgg gggaggggga tcccataaga aatgaaacct 360

gagacttttc cctccgttcc agaaatgcat ccttggaacg cgccctccct gggttccggg 420

tccctcacgc actcaagacg gggaccacca ttgcaggtct tgtattccag gtgagcaaga 480

agttgtgact gtggggcttg gaggggcggc cgggaatagg aagtgggcag tgacctangg 540

gtttctcccc tcttctccag gacggggtaa tcctgggcgc ggatacgcgg gccactaacg 600

actcggtcgt ggcggacaag agctgcgaaa agatccactt catcgccccc aaaatctagt 660

gagcattcct ctcaagaact gccccttagt tccccgtccc tggtctccgc acaccttagc 720

cccgaccaca tggaccctcc cttcaccctc agctgctgtg gggctggagt agccgcggac 780

gccgagatga ccacgcggat ggcggcatcc aacatagagc tacacgcact atccaccggc 840

cgcgagccgc gcgtggccac tgtcactcgc atgttacgcc agaagctctt ccggtgctgg 900

agctgggcta agacgaccct ggggaggggc agttgcaggg aaggtggggc tgataggagg 960

tgggacaaga ggaaggacag ggctgcgagg tgccgggaga ccggaagagg tcagcctaag 1020

aggagttaag aaggcaaccg gaagcagcaa tgtcagtcat ggggctccag accacgggaa 1080

ggggcggggc tcgggtgcag gaccatggag gggagggacc tggtttgttt ggctgactgc 1140

gcccacttgc ggaaaaggta ccagggccan gtgggcgcgt cgctaatcgt gggcggcgta 1200

gacttcacag gaccccagct ctacagcgtg cacccccatg ggtcctacag ccgtctgccc 1260

ttcacggccc taggtgagcc cttccatccc ttccccacga atcccacgta tactatcctg 1320

gcctcccgtt tctcccgcga ccatgcggcc gcggctgacc tcagataccc ctcctgtcta 1380

caggctccgg ccaagatgca gccctggcag tgctagaaga ccggttccag cccaacatga 1440

ctgtgagtga catctgcgcc ccactaggca ttgcattggt gggaagtgca cttgggagct 1500

ggggaaacac agaggtaccc tggcctaatc cggaggtcag gaaaagcttt tcagaggtga 1560

tgactgagta caggaggaga agccagtgga gcgagggtgg agaagtgcaa ttccaacaga 1620

ccactgtcaa tgccaaagct tggactgcat ggagaggtca aaactcagag aggaccattt 1680

agagctaagg gaggagagaa caacaggtgg ggtctggagt ctgggtcttg ttttgaggtg 1740

gatggaaaga gaggcatatg tcacttcata ggtgcctaag gcccagaaag aaaaaggaaa 1800

tgagcctggg attgggggcc aagactacaa tgggtgagtg aataggcccc tggaaccaga 1860

gccacaggtg tgactcagag aagtttgtga agatggggca ttgggaacac cgggtctctg 1920

gagagaggga aactgacacc ccgggaaaag aactttgaag gtggagattg ggagtataag 1980

tggagagcag atggtaaaat agcagtgcct gagttccccg gtcatggctt cagtggtaag 2040

gaacctgact aggatcccat gaaagatgtg ggttcgatcc cctgcccttg ctcagtgggt 2100

gaagaatctg gtgttgccac gagctgcggt ataaattgca gatgcggctc agatttggcg 2160

ttgccgttgc tgtggctgtg gcgtaggcag ggaagagatt agcaatctcc aggctatagt 2220

ctaagggtga taccacagtg tccgtcaggg cttcacacat gcattgttac agctggaggc 2280

agcacagggg ctgctggtgg aagccattac tgcagggatc ctaagtgacc tgggctctgg 2340

gggcaatgtg gatgcatgtg tgatcatggg gaccggtgcc aagctgctgc gaacactgag 2400

ctctcccaca aagcccacag aaaggtaaga gcatggaaga tgggggaaca aaggagagga 2460

tagggtggca gcaagggaga tgggctgcaa agagaggatt gaccacgtga tgggcccatt 2520

ttgagaggtc atctcttctt cccagatcca gccagtatcg ctttgcccct gggaccacag 2580

ctgtcctgtc ccagacagtg atgccgctga ccctggagct ggtggaagaa actgtgcagg 2640

ccatggatgt ggagtaagct gag 2663

<210>8

<211>943

<212>DNA

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<220>

<221>mise-feature

<222>(511)

＜223〉n=c or t

<400>8

agttaagctc agtcgtgcag gacaaggaca aaggacaagg ctgtggcctt ccttgcttgg 60

tgctggtcct actcctctga cctccaaacc cctggactgc cctgcccacc tggacatgca 120

gaagatagcg ctagagcccc tagggggctt ttcctttgag aactgccaga gaaatgcatc 180

cttggaacgc gccctccctg ggttccgggt ccctcacgca ctcaagacgg ggaccaccat 240

tgcaggtctt gtattccagg acggggtaat cctgggcgcg gatacgcggg ccactaacga 300

ctcggtcgtg gcggacaaga gctgcgaaaa gatccacttc atcgccccca aaatctactg 360

ctgtggggct ggagtagccg cggacgccga gatgaccacg cggatggcgg catccaacat 420

agagctacac gcactatcca ccggccgcga gccgcgcgtg gccactgtca ctcgcatgtt 480

acgccagaag ctcttccggt accagggcca ngtgggcgcg tcgctaatcg tgggcggcgt 540

agacttcaca ggaccccagc tctacagcgt gcacccccat gggtcctaca gccgtctgcc 600

cttcacggcc ctaggctccg gccaagatgc agccctggca gtgctagaag accggttcca 660

gcccaacatg actctggagg cagcacaggg gctgctggtg gaagccatta ctgcagggat 720

cctaagtgac ctgggctctg ggggcaatgt ggatgcatgt gtgatcatgg ggaccggtgc 780

caagctgctg cgaacactga gctctcccac aaagcccaca gaaagatcca gccagtatcg 840

ctttgcccct gggaccacag ctgtcctgtc ccagacagtg atgccgctga ccctggagct 900

ggtggaagaa actgtgcagg ccatggatgt ggagtaagct gag 943

<210>9

<211>273

<212>PRT

＜213〉wild boar be born in the year of pig (Sus scrofa domestica Brisson)

<400>9

Met Gln Lys Ile Ala Leu Glu Pro Leu Gly Gly Phe Ser Phe Glu Asn

1 5 10 15

Cys Gln Arg Asn Ala Ser Leu Glu Arg Ala Leu Pro Gly Phe Arg Val

20 25 30

Pro His Ala Leu Lys Thr Gly Thr Thr Ile Ala Gly Leu Val Phe Gln

35 40 45

Asp Gly Val Ile Leu Gly Ala Asp Thr Arg Ala Thr Asn Asp Ser Val

50 55 60

Val Ala Asp Lys Ser Cys Glu Lys Ile His Phe Ile Ala Pro Lys Ile

65 70 75 80

Tyr Cys Cys Gly Ala Gly Val Ala Ala Asp Ala Glu Met Thr Thr Arg

85 90 95

Met Ala Ala Ser Asn Ile Glu Leu His Ala Leu Ser Thr Gly Arg Glu

100 105 110

Pro Arg Val Ala Thr Val Thr Arg Met Leu Arg Gln Lys Leu Phe Arg

115 120 125

Tyr Gln Gly His Val Gly Ala Ser Leu Ile Val Gly Gly Val Asp Phe

130 135 140

Thr Gly Pro Gln Leu Tyr Ser Val His Pro His Gly Ser Tyr Ser Arg

145 150 155 160

Leu Pro Phe Thr Ala Leu Gly Ser Gly Gln Asp Ala Ala Leu Ala Val

165 170 175

Leu Glu Asp Arg Phe Gln Pro Asn Met Thr Leu Glu Ala Ala Gln Gly

180 185 190

Leu Leu Val Glu Ala Ile Thr Ala Gly Ile Leu Ser Asp Leu Gly Ser

195 200 205

Gly Gly Asn Val Asp Ala Cys Val Ile Met Gly Thr Gly Ala Lys Leu

210 215 220

Leu Arg Thr Leu Ser Ser Pro Thr Lys Pro Thr Glu Arg Ser Ser Gln

225 230 235 240

Tyr Arg Phe Ala Pro Gly Thr Thr Ala Val Leu Ser Gln Thr Val Met

245 250 255

Pro Leu Thr Leu Glu Leu Val Glu Glu Thr Val Gln Ala Met Asp Val

260 265 270

Glu

Claims (7)

1. the albumen that a boar produces and immune character is relevant, its amino acid residue sequence is shown in SEQ ID NO:9.

2. the described pig of claim 1 produces and the encoding gene of immune character related protein.

3. encoding gene according to claim 2 is characterized in that: the nucleotide sequence of described encoding gene is selected from down one of group:

1) polynucleotide shown in SEQ ID NO:8 in the sequence table;

2) polynucleotide shown in SEQ ID NO:7 in the sequence table;

3) in the code sequence tabulation shown in SEQ ID NO:9 the polynucleotide of protein sequence.

4. the carrier that contains claim 2 or 3 described encoding genes.

5. the clone that contains claim 2 or 3 described encoding genes.

6. the host bacterium that contains claim 2 or 3 described encoding genes.

7. one kind is detected that pig produces or the method for immune character, be to use a pair of primer formed by SEQ ID NO:2 in the sequence table and SEQ ID NO:3 or by the nucleotide sequence of SEQ ID NO:5 in the sequence table and SEQ ID NO:6 that the genomic dna of pig to be measured is carried out pcr amplification, then to being that the pcr amplification product of primer carries out following 1 with SEQ ID NO:2 and SEQ ID NO:3) and 2) at least a detection or to being that the pcr amplification product of primer carries out following 3 with SEQ ID NO:5 in the sequence table and SEQ ID NO:6) and 4) at least a detection:

1) described pcr amplification product is carried out single nucleotide polymorphism and detect, determine that 5 ' end the 538th bit base of SEQ IDNO:1 in sequence table is A or G;

2) cut described pcr amplification product with the Eco81I enzyme, detect in the endonuclease bamhi that obtains whether contain " a 642bp band ", " a 535bp band and a 107bp band " or " 642bp band, a 535bp band and a 107bp band ";

3) described pcr amplification product is carried out single nucleotide polymorphism and detect, determine that 5 ' end the 550th bit base of SEQ IDNO:4 in sequence table is T or C;

4) cut described pcr amplification product with the Eco72I enzyme, detect in the endonuclease bamhi that obtains whether contain " a 802bp band ", " a 550bp band and a 252bp band " or " 802bp band, a 550bp band and a 252bp band ".

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