CN103898104A - Molecular marker applied as pig marker assisted selection and related to immune traits and application thereof - Google Patents

Abstract

The invention discloses a molecular marker applied as pig marker assisted selection and related to immune traits and application thereof, and belongs to the technical field of livestock genetic engineering. The molecular marker is obtained by an IP10 gene in a cloning manner; a nucleotide sequence of the molecular marker is shown in a sequence table SEQ ID NO:2; a C119-T119 base mutation is formed at the 119th bit of basic group of the sequence shown in the sequence table SEQ ID NO:2, resulting in RFLP-MspI (Restriction Fragment Length Polymorphism-matrix associated serine protease inhibitor) polymorphism. The invention also discloses a method for preparing the molecular marker and an application of a polymorphism detection method. A new molecular marker is provided for marker assisted selection of the pig.

Description

A kind of molecule marker and the application thereof relevant to immune character as the application of pig marker assisted selection

Technical field

The invention belongs to domestic animal gene engineering technology field, be specifically related to a kind of molecule marker and application thereof relevant to immune character as the application of pig marker assisted selection.

Background technology

In recent years, GENERALIZATION OF MODERN BREEDING TECHNIQUE is being made very large contribution in improvement aspect pig production character, but the genetic improvement of ability to healthy and resist the disease does not really obtain enough attention.The application of some preventive measures, as raise the management level, improve hygienic condition, injectable drug and vaccine etc., in Pig Industry industry, bring into play vital role, but along with the whole world is more and more higher to the cry of minimizing medicine and vaccine use, adopt molecular genetics method more and more to receive publicity from improving in essence livestock and poultry body self resistance against diseases, become the emphasis of research.

Individual immunity power is to weigh the important indicator (Knap etc., 2000) of animal health, and it belongs to quantitative character, wherein relates to genes involved more, and molecular mechanism is very complicated.Immunizing power and production performance are usually expressed as phenotype negative correlativing relation, and this has proposed a large difficult problem for breeder, how are keeping and are improving that on the basis of production performance, to improve individual immunity ability be the new problem that breeder studies simultaneously.Therefore, disclosing the physical relationship between the controlling gene system of production performance and immunizing power from genomic level, is the effective way that solves an above-mentioned difficult problem.In breeding for disease resistance, the searching of disease-resistant gene is crucial.

For the candidate gene of the disease-resistant proterties of pig, study to such an extent that be clear that swine escherichia coli K88 and F18 acceptor gene.If research shows shortage intestinal bacteria K88 acceptor or F18 acceptor in animal body, the diarrhoea that will cause corresponding serotype intestinal bacteria produces resistance, the gene of current encoder intestinal bacteria K88 acceptor (K88abR and K88acR) has been positioned at the position (Edfors-Lilja etc. near Transferrins,iron complexes gene on No. 13 karyomit(e)s of pig, 1998),-(1, 2)-trehalose transferring enzyme 1(α (1, 2) fucosyltransferase, FUT1) gene is the candidate gene of F18 acceptor (ECF18R), be positioned at (Vogeli P etc. on No. 6 karyomit(e)s of pig, 1992).Halothane (Hal) is the resistance related gene that another impact is larger, and its responsive allelotrope of fluothane (Haln) can cause the physique of pig and resistibility to decline, and produces violent stress reaction.The Interferon, rabbit of pig is proved to be all has wide spectrum defence and restraining effect to multiple serious infectious diseases viruses such as breeding difficulty and respiratory syndrome virus, foot and mouth disease virus, Pestivirus suis, gastro-enteritis coronavirus, thereby interferon gene, Interferon Receptors gene are the important candidate genes of selecting the non-specific disease resistance of pig.In addition, MHC has been proved to be and antibody replying plurality of antigens, the phagocytosis of bacterium, the proterties such as the susceptibility to spiral rotary caterpillar and malignant melanoma have relevant (Peelman etc., 1996), other gene relevant with disease resistance that has research is MX1(Myxovirus (influenza) resistance 1, myxovirus (influenza) resistance factor 1), NRAMP1(Natural resistance-associated macrophage protein 1, the natural resistance albumen 1 relevant with scavenger cell), IL1(Interleukins 1, interleukin 1), PPARG (Peroxisome proliferator activated receptor gamma, Peroxisome Proliferator-activated Receptors γ) etc. gene.

IP10 is the chemokine that belongs to CXC family, has another name called CXCL10, and being mainly subject to IFN γ by monocyte, scavenger cell, NK cell etc. stimulates rear secretion inducing.IP10 can mediate the inflammatory reaction of Th1 type.IP10, as the chemokine of monocyte and activated T lymphocytes, can stimulate monocyte, the directional migration of activated T lymphocytes, and increase the adhesion (Taub et al., 1993) of T cell Human Umbilical Vein Endothelial Cells.IP10 activates Th1 cell can bring into play the biological action of several aspects: (1) participates in the generation of some delayed type hypersensitivity disease.In the bronchovesicular of active tuberculosis disease, the expression level of IP10 is very high; Separately studies have found that in allotransplantation and use IP10 monoclonal antibody can extend the survival time of graft, be i.e. after explanation blocking-up IP10, can suppress the generation of graft-rejection; (2) generation of participation autoimmune disorder.The IP10 of high level expression not only in SLE patient's serum, detected, and there is scholar to find that it may be inducement (the Li et al. of type i diabetes, 2005), IP10 activates Th1 type cell, thereby activating cytotoxic scavenger cell, cytotoxic T cell and NK cell, attack islet cells and cause B cell destruction to cause type i diabetes; (3) antiviral immunity function.In the time of herpesvirus infection human brain, find that the IP10 of external source can directly support antiviral copying, strengthen the antiviral defensive raction of microglia, and strengthen effect of immune response (Lokensgard et al., 2001); (4) there is the effect of angiogenesis inhibiting.It is as one of angiogenesis supressor in body, can effectively suppress angiogenesis (the Angiolillo et al. that IL8 and bFGF cause, 1995), its effect is mainly manifested in and participates in the adjusting of vascular remodeling and suppress two aspects of tumor growth migration.IP10 is by suppressing the angiogenesis of tumour, reducing tumor blood supplies with, cause tumor ischemia necrosis, it can be by chemotactics such as T lymphocyte, monocyte and NK cells to tumour happening part simultaneously, thereby induction Th1 type inflammatory reaction killing tumor cell, finally brings into play antineoplastic important biomolecule effect.

Up to now, there is not yet the report of comprehensive research pig IP10 gene function, and the polymorphism of research mutational site in colony, and carry out the very strong means that proterties association analysis is research gene function.So the exon of the part of applicant to this gene has carried out polymorphic research and association analysis, to finding its function at immunology.

Summary of the invention

The problem existing for prior art, the object of the invention is to design provides a kind of molecule marker relevant to immune character as the application of pig marker assisted selection and the technical scheme of application thereof.

The present invention is achieved through the following technical solutions:

Applicant clone obtains the DNA fragmentation with pig immune trait genes involved IP10, and its cDNA sequence (comprising total length CDS and part 5 '-UTR and 3 '-UTR) is as described in sequence table SEQ ID NO:1; Its partial dna sequence is as described in sequence table SEQ ID NO:2.

The IP10 gene cDNA of pcr amplification is long is 787bp, comprises CDS total length 315bp and part 5 '-UTR and 3 '-UTR, as described in sequence table SEQ ID NO:1 and the nucleotide sequence as shown in accompanying drawing 2.

The IP10 genome sequence total length of pcr amplification is 497bp, as described in sequence table SEQ ID NO:2 and accompanying drawing 3 as shown in nucleotide sequence.

There is a base mutation (C119-T119) at 119bp place at sequence table SEQ ID NO:2, and this sudden change causes MspI-RFLP polymorphism (Restriction Fragment Length Polymorphism).

The primer of amplification IP10 gene cDNA sequence is as follows:

Forward primer sequence as shown in SEQ ID NO:5,

Reverse primer sequence is as shown in SEQ ID NO:6.

Amplification IP10 gene sequencing to detect C119-T119 place base mutation primer sequence used as follows:

Forward primer sequence as shown in SEQ ID NO:3,

Reverse primer sequence is as shown in SEQ ID NO:4.

The preparation method of molecule marker of the present invention is:

Employment IP10 gene mRNA (the GenBank number of including: NM_001565.3) is information probes, does homologous sequence screening, obtains more than 90% expressed sequence tag (EST) of homology; Then EST is spliced; Compare and design primer with people's homology, extract RNA and carry out RT-PCR and pcr amplification, and clone obtains the cDNA sequence (comprising CDS total length and part 3 '-UTR) as shown in sequence table SEQ ID NO:1; According to roughly drawing the splicing site of exon with the comparison result of the genome full length DNA sequence of pig IP10 gene, design of amplification primers, extract DNA, pcr amplification, PCR product purification and order-checking, obtain the nucleotide sequence as shown in sequence table SEQ ID NO:2.

The method of applying conventional PCR-RFLP detects the 119th bit base sudden change shown in sequence table SEQ ID NO:2, and tentatively carry out the application of the association analysis between its genotype and the Traits of pig, for the molecular marker assisted selection of pig provides a new molecule marker.

More detailed technical scheme is referring to " embodiment ".

Accompanying drawing explanation

Fig. 1: techniqueflow chart of the present invention.

Fig. 2: pig IP10 Gene Partial cDNA Sequence (comprising CDS total length and part 5 '-UTR and 3 '-UTR) in the present invention.

Fig. 3: pig IP10 gene 3 '-UTR partial sequence in the present invention is wherein allelic mutational site in the bracket at the 119th bit base place.

Fig. 4: the electrophoretogram of the extension increasing sequence of pig IP10 gene 3 '-UTR partial sequence in the present invention, clip size is that 497bp(agarose gel concentration is 2%).In figure: M swimming lane is DNA molecular amount standard (DL2000).

Fig. 5: the allelic mutation of the C119-T19 that in the present invention, pig IP10 gene sequencing is found.

Fig. 6: three kinds of genotype of the MspI-RFLP of pig IP10 gene 3 '-UTR (AA AB BB) electrophoretogram in the present invention.M swimming lane is DNA molecular amount standard (DL2000).

Embodiment

Further illustrate the present invention below in conjunction with embodiment.

Embodiment 1

(1) IP10 gene C DS clone and partial dna sequence amplification

(1) design of primers

Employment IP10 gene mRNA (the GenBank number of including: NM_001565.3) is information probes, utilize the BLAST instrument in NCBI in GenBank pig est database, to do homologous sequence screening, obtaining a series of homologys is more than 90% ESTs (fragment length is greater than 100bp), the number of including of these ESTs is used in NCBI to ENTREZ(http: //www.ncbi.nlm.nih.gov/Web/ Search/index.html) inquiry corresponding sequence, then use the SeqMan program construction pig EST-contig in sequence analysis software DNAStar.According to comparing the preliminary initial and terminator codon position of determining with the homology of people IP10 gene mRNA, design primer clone obtains the cDNA sequence (comprising CDS total length and part 5 '-UTR and 3 '-UTR sequence) as shown in sequence table SEQ ID NO:1; According to roughly drawing the splicing site of exon with the comparison result of the genome full length DNA sequence of pig IP10 gene, infer that pig IP10 gene contains 4 exons, design of amplification primers, carry out pcr amplification, product purification and order-checking, obtain the nucleotide sequence as shown in sequence table SEQ ID NO:2.Wherein, by the amplification order-checking comparison to exon IV partial sequence (497bp), find 1 base mutation (C 119-T 119) in 119bp place, this sudden change has been carried out to restriction analysis, found that this sudden change affects the identification of restriction endonuclease MspI just.

The primer of clone IP10 gene cDNA sequence is as follows:

Forward: this sequence of 5'-CGGGGGAGACACTCTTCAACT-3'(is as shown in SEQ ID NO:5),

Reverse: this sequence of 5'-CAGTAGAAGCCCACGGAGTAAAG-3'(is as shown in SEQ ID NO:6).

Amplification IP10 gene sequencing to detect C119-T119 place base mutation primer sequence used as follows

Forward: this sequence of 5'-AAGCAGAACTCAGACTGAAACC-3'(is as shown in SEQ ID NO:3),

Reverse: this sequence of 5'-GTAGAAGCCCACGGAGTAAAG-3'(is as shown in SEQ ID NO:4).

(2) purifying of PCR product, Cloning and sequencing

In the reaction system of pcr amplification condition: 10uL, add DNA profiling 0.5 μ L, 10 ' PCR buffer, 1 μ L, dNTP 0.3 μ L, each 0.2 μ L before and after 10mM primer, Taq enzyme 1U.PCR reaction conditions is: after 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ of extension 30s, 33 circulations, last 72 ℃ are extended 5min.PCR product detects through 1.5% agarose gel electrophoresis.

The purifying of PCR product: cut the gel containing object fragment from low melting-point agarose gel under ultraviolet lamp, put into 1.5 mL centrifuge tubes, being incubated to gel in 65 ℃ melts completely, then use PCR product purification test kit (purchased from TIANGEN company) purified pcr product, operate according to test kit specification sheets, concrete steps are to add 1 mL Resin reagent in the gel melting at every 300 μ L, mix 20 s, Resin/DNA mixture is transferred to the centrifuge tube with adsorption column, the centrifugal liquid of removing.Again to Virahol 2 mL that add 80% in adsorption column, the centrifugal liquid of removing, take off adsorption column and pack in 1.5 mL centrifuge tubes, centrifugal 2 min of 10,000g are to be dried Resin, adsorption column is packed in another 1.5 clean mL centrifuge tubes, add 30 ~ 50 μ L aqua sterilisas, leave standstill 1min, 10, the centrifugal 20s of 000g, is stored in centrifuge tube with eluted dna.

Ligation: purified pcr product is connected with pGEM-T easy carrier (purchased from promega company), ligation cumulative volume is 5 μ L, comprising 2.5 μ L 2 × Buffer, the T carrier of 0.5 μ L, the purified pcr product of 1.5 μ L, the T4 ligase enzyme of 0.5 μ L, puts 16 ℃ of water-baths and spends the night.

The preparation of competent cell: mono-colony inoculation of DH5 α of picking is in 2 mL LB from 37 ℃ of fresh flat boards of cultivating 16-20 h, in 37 ℃ of shaking culture 3 h, transfer 1 mL bacterium liquid in the saline bottle that contains 30 mL LB, continue at 37 ℃ of shaking culture approximately 4 h, in the time that OD600 reaches 0.3-0.4, saline bottle is taken out and puts the cooling 10-15 min of ice bath from shaking table, then bacterium liquid is proceeded in centrifuge tube in 4 ℃ 4, centrifugal 10 min of 000g are with collecting cell, centrifuge tube is inverted to abandon clean nutrient solution, by the resuspended precipitation of CaCl2 of 0.1 mol/L of 10 mL ice precoolings, ice bath 30 min, repeat 4 ℃ 4, centrifugal 10 min of 000g once, by the resuspended precipitation of CaCl2 of 0.1 mol/L of 4 ml ice precoolings, putting 4 ℃ saves backup.

Transform: under sterile state, get 100-120 μ L competent cell in 1.5 mL centrifuge tubes, the connection product of 5 μ L is added and mixed, place 30 min on ice, 42 ℃ of heat shock 90 s, do not shake centrifuge tube therebetween, take out rear ice bath 3-4 min, add the LB liquid nutrient medium of 400 μ L antibiotic-frees, 37 ℃ of shaking culture 45 min.Getting 100 μ L coats 4 h in advance and has been coated with IPTG (English name: Isopropylthio-β-D-galactoside, Chinese: isopropylthio-β-D-galactoside, on agar plate purchased from TIANGEN company and X-gal, 37 ℃ keep flat after 1 h and are inverted and cultivate.

The a small amount of preparation of plasmid: the single bacterium colony on picking flat board, be inoculated in 2-3mL LB, 37 ℃ of 300r/min cultivate 6-8h.With the centrifugal several seconds collection of 1.5mL centrifuge tube 12000r/min thalline.According to plasmid, extracting test kit specification sheets step carries out plasmid preparation (test kit is purchased from OMEGA company) in a small amount.

The enzyme of recombinant plasmid is cut evaluation: get 3 μ L plasmid DNA and appropriate distilled water and mix, making its cumulative volume is 10 μ L, add 5U restriction enzyme EcoR I and the corresponding 10 × restriction enzyme reaction damping fluid of 1 μ L (purchased from Fermetas company), flicking tube wall mixes also centrifugal, put 37 ℃ of water-bath 1-2 hour, get 2-3 μ L reaction solution and detect in agarose gel electrophoresis, enzyme is cut result and is estimated identical person, is object recombinant plasmid.Recombinant plasmid adopts two deoxidation end cessation method to check order on automatic dna sequencer, and sequencing is completed by Sangon Biotech (Shanghai) Co., Ltd..

In the present embodiment, pcr amplification product shows and is special PCR product through 1.5% agarose gel electrophoresis detected result.PCR product is reclaimed to the order-checking of purifying rear clone, and sequencing result shows that the cDNA sequence length that PCR obtains is 787bp, comprises that CDS total length 315bp and part 5 '-UTR and 3 '-UTR(are as shown in Fig. 2 and sequence table SEQ ID NO:1); The DNA sequence dna total length that PCR obtains is that 497bp(is as shown in Fig. 3 and sequence table SEQ ID NO:2), sequencing result shows to exist at the 119bp place of this DNA sequence dna C119-T119 sudden change, and sequencing result is as shown in Figure 5.

(3) DNA sequence dna homology search is identified

By the American National biotechnology (NCBI of information center, http://www.ncbi.nlm.nih.gov) the BLAST(Basic Local Alignment Search Tool of website) software, the known physiological function gene of announcing in the DNA sequence dna obtaining after order-checking and GenBank database is carried out to sequence homology comparison, to identify and to obtain the function information of this DNA sequence dna.

(2) PCR-RFLP diagnostic method is set up

RFLP detects: by PCR product 3 μ L, 10 × Buffer, 1 μ L, restriction endonuclease MspI is 0.2 μ L (2U), adding distilled water mends to 10 μ L, by centrifugal after sample blending, 37 ℃ of incubators are placed 12h, detect enzyme cut result with 1.5% agarose gel electrophoresis, record genotype, under ultraviolet lamp, take pictures.

Obtained 497bp specific amplification fragment (referring to Fig. 4) with primer amplification pig genomic dna, the sequencing results shows to exist at 119bp place C119-T119 sudden change, and causes MspI polymorphism.This gene mutation site is by two allelotrope controls, and wherein T is the allelotrope that does not form restriction enzyme site, and C is the allelotrope that forms restriction enzyme site.These two allelotrope can form three kinds of genotype wherein TT type be that homozygous (the only having DNA band of 497bp when electrophoresis detection) that enzyme cuts do not occur, CC type is that homozygous (the occurring two DNA bands of 379bp and 118bp when electrophoresis detection) that enzyme is cut occurs, and TC is heterozygous (occurring tri-DNA bands of 497bp, 379bp and 118bp when electrophoresis detection).

(3) application of the detection of the distribution situation of PCR-MspI-RFLP polymorphism in each pig variety

Utilize PCR-MspI-RFLP to detect six pig kinds: the pig kind that wherein belongs to place of china blood relationship is respectively Jinhua Pigs, Jiaxing Black Pig, Shengxian County flower pig and Chalu Black Pig, the pig kind that belongs to external for example American-European blood relationship is respectively Large White and duroc.Genotype and the gene frequency of this mutational site in different pig kinds is as shown in table 1, and result is presented at allelotrope T and C frequency no significant difference in local pig breed, and in pig kind, allele C is preponderated abroad.

Table 1: genotype and the gene frequency of IP10 gene 11 9 loci polymorphisms in several pig varieties

χ ²assay demonstration, between domestic pig kind, gene frequency difference is not remarkable, and domestic pig kind is shown in that with external pig kind gene frequency difference is remarkable.

Embodiment 2:

Experiment swinery is from totally 394 of Du Luoke × painted face in Beijing opera F2 resource colonies on Guangdong Hua Nongwenshi herding limited-liability company water platform original seed pig farm, in F0 generation, is made up of Duroc boars and 18 Erhualian sows of 8 consanguinity-less relations, and 13 boars of F1 generation and 38 sows avoid close relative's (full sibs or half sibs) mating.Gather nascent, 20 ages in days, 33 ages in days and 80 age in days anticoagulations and non-anticoagulation, adopt ordinary method to detect routine blood test and antibody horizontal.

The proterties of analyzing comprises the level of birth weight, 20 ages in days, 33 ages in days, 80 age in days body weight, five classification routine blood test indexs, IgG, interferon alpha, interferon-gamma, cytokine-8, interleukin 10, and 80 age in days antibody against swine fever virus levels and pig breeding and breathing syndrome virus antibody horizontal etc.

According to the group structure of collected specimens, applicant uses mixture model to come the genotype effect in statistical study IP10 gene SNP s site and the relation with immune indexes thereof:

Y = Xβ+Z b+ε

Wherein Y is proterties observation vector; X is fixed effect incidence matrix; β is the parameter vector of fixed effect, comprises the genotype effect (Genotypes) of candidate gene, sex-effects (Sex); Z is the incidence matrix of stochastic effect; B is the parameter vector of stochastic effect, comprises male animal effect (Sire), the dam (Dam (Sire)) in male animal; ε is random error effect vector.

Pig IP10 gene extron IV pleomorphism site genotype detection result is shown, in 394 individualities, after somatotype, find 40 of TT genotype, 142 of TC genotype, 212 of CC genotype.Analytical results shows, this SNP site and 20 age in days lymphocyte percentages (LY%), and 80 age in days pcvs (HCT), mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) exist significantly associated; There is extremely significantly associated (in table 2) with 80 age in days content of hemoglobin (HGB).

The association analysis result of table 2:IP10 gene extron IV pleomorphism site genotype and Traits

As shown in Table 2: TT genotype is preponderated at 20 age in days LY%, 80 age in days MO%, and with significantly (p<0.05) of CC genotypic difference, T allelotrope is advantage allelotrope in the Immune interrelation such as lymphocyte, monocyte proterties; CC genotype is preponderated at 80 age in days HGB, HCT, MCV and MCH, and with significantly (p<0.05) of TT genotypic difference, C allelotrope is advantage allelotrope in red corpuscle correlated character.

SEQUENCE LISTING

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Claims (4)

1. as a molecule marker relevant to immune character for pig marker assisted selection application, it is characterized in that this nucleotide sequence is as shown in SEQ ID NO:2.

2. a kind of molecule marker relevant to immune character as the application of pig marker assisted selection as claimed in claim 1, is characterized in that in this sequence, the 119th bit base place has the base mutation of a C119-T119 to cause RFLP- mspIpolymorphism.

3. the primer that detects base mutation as claimed in claim 2, is characterized in that forward primer sequence is as shown in SEQ ID NO:3, and reverse primer sequence is as shown in SEQ ID NO:4.

4. the application of the molecule marker described in claim 1 or 2 in pig molecule mark assisted Selection.

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