CN101481739B - Separated nucleic acid sequence of pork quality trait related gene TRIM63 - Google Patents

Abstract

The invention relates to a separated nucleic acid sequence of a pork quality trait related gene TRIM63 of a pig in the technical field of genetic engineering. The separated nucleic acid sequence is provided with a sequence shown by an SEQ ID No:1, and a sequence shown by an SED ID No:4, the length of which is 1462bp; the nucleic acid sequence shows two pairs of allele-specific nucleic acid primers the lengths of which are 20bp; the two pairs of nucleic acid primers are specifically hybridized; and the sequence shown by the SEQ ID No:1 containing a third exon and a third intron of the TRIM63 gene of the pig and the sequence shown by the SEQ ID No:4 containing a fifth intron and a sixth intron of the TRIM63 gene of the pig are amplified. The implementation and the application of the separated nucleic acid sequence can be used for detecting and judging the quality of partial meat quality traits of the pig; the molecular marker is applied to conduct a marker assisted selection during breeding of the pig, thereby correcting the defect of required indirect selection of the meat quality traits; and the separated nucleic acid sequence has important practical significance.

Description

The separated nucleic acid sequence of pork quality trait related gene TRIM 63

Technical field

What the present invention relates to is the separated nucleic acid sequence of the nucleotide sequence of a kind of pig of gene engineering technology field, particularly a kind of pork quality trait related gene TRIM 63.

Background technology

China is the first in the world pork producing country, and according to the data that the Ministry of Agriculture announces, China's pork output was 5,200 ten thousand tons in 2006, accounted for 53% of global pork output.Though output is huge, the pork production of China is still failed self-sufficient fully, and the supply and demand anxiety causes the very long in the past following period of time of pig industry of China only to pay attention to quantity, and lower to specification of quality.Along with the raising of standard of living and health care consciousness, very big variation has taken place in people's diet formula, more and more pays attention to the dietary ingredient relation healthy with it, and food has been had higher requirement, and especially the requirement to livestock product improves constantly.Pork is the emphasis that people improve in recent years as integral part important in people's meals.High protein, lower fat, low-cholesterol, not only thin but also tender, the delicious pork of succulence is favored by the human consumer quite on market.Therefore, meat quality has become raise pigs key subjects of the expert of boundary common concern research of the world today.Meat quality has different notions in different countries, the different markets of same country.What accept extensively is the definition of Hofmann (1994), be that meat quality is the comprehensive embodiment of fresh meat physical property and chemical property, mainly be meant organoleptic quality, technology processing quality, nutritive value and hygienic quality 4 aspects such as (toxicity or food safety aspects).Concrete pH value, color, be that indexs such as waterpower, tender degree, intramuscular fat content, local flavor weigh calmly with muscle.The factor that influences meat quality is a lot, comprises heredity, nutrition, raising battalion reason, butchers pre-treatment and butcher technology etc., and wherein heredity is most important factor, and therefore, the breeding of high-quality pork boar is the important channel of improving meat quality.The needed time of conventional breeding is long, and marker assisted selection (Marker assisted selection, MAs) be that Modern Molecular Biotechnology is combined with the conventional breeding method, select a certain site to change the process of the gene frequency in this site by molecule marker, also claim the molecule assisted Selection.Promptly by to the selection of genetic marker, realize indirectly the quantitative trait locus (QTL) of controlling certain proterties or the selection of gene, thereby reach the purpose that this proterties is selected.Therefore, performance is particularly outstanding on the proterties that just shows in sexlimited character and the later stage of growing with respect to the superiority of Phenotypic Selection of marker assisted selection.Livestock and poultry species be can accelerate to improve, animal production efficiency and economic benefit improved.

In recent years, the research of molecular level obtains develop rapidly, and discovery and location along with some major genes and the QTL of animal have formed a brand-new field of molecular breeding.Tool influence be halothane (Hal) Lan Niding acceptor I (RYRI) gene just.Halothane for the influence of meat be it can cause pig stress syndromes (porcine stress syndrome, PSS). stress syndromes be to cause PSE (Pale, soft, exudative) reason of meat.In 1843 base of RYRI gene by the sudden change of C to T, cause pig stress the time skeletal muscle calcium ionic improper release, cause muscle gray, quality softness, tool is exudative and water-based.Intramuscular fat (IMF) content is relevant with the organoleptics property of pork, closely related with eating qualities such as the tender degree of meat and local flavors, and the content of intramuscular fat has very big difference between the kind of each pig, and heart fat acid binding protein gene (Heart Fatty AcidBinding Protein H-FABP) is the important factor that influences intramuscular fat deposition.Pleomorphism site is arranged on the H-FABP gene that Gerben et al (1997) has reported pig, and (Hinfl), the IMF of different genotype correspondence is different with the thickness of back fat for HaeIII, MspI.In addition, relevant with pig flesh characters also have sour meat gene, melanocyte cortical hormone receptor-4 gene, leptin receptor gene, peroxidase are bred incitant, calpain inhibiting protein gene, adipocyte decision and differentiation factor 1 gene or the like.

The TRIM gene family is a protein family that more and more comes into one's own in recent years, this family is structurally conservative, distinguishing feature is to have 3 structural domains (tripartite motif), it is Zinc finger domain (RING finger), 1 or 2 B-box, 1 coiled coil (coiled-coil) structural domain successively that these 3 structural domains are held C end from N, wherein, B-box is the feature structure territory of TRIM family, and the title of TRIM is also come therefrom.Had been found that the albumen member of more than 60 TRIM family at present the mankind.TRIM63 is the member of this subfamily of family, is called muscle specific again and expresses the factor.The existing TRIM63 gene of discovering plays a role in the process of intramuscular sarcomere assembling.The genome total length 16324bp of human TRIM63 gene comprises 9 exons and 8 intron parts, the long 1764bp of mRNA, and CDS is long for 1062bp, is positioned at chromosomal p34-p33 district No. 1.The research of TRIM63 gene in pig does not have report as yet, relation between itself and pig flesh characters is still indeterminate, and the polymorphism of research pig TRIM63 gene mutation site in colony, and carry out the proterties association analysis be research this gene function very strong means, therefore this gene has been carried out polymorphic research and association analysis, be used for the pig flesh characters improvement in the hope of accessing the molecule marker relevant with pig flesh characters.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art, a kind of separated nucleic acid sequence of pork quality trait related gene TRIM 63 is provided.Clone the portion gene group dna sequence dna of pig TRIM63 gene, in the breeding of pig, use and carry out assisted Selection.

The present invention is achieved by the following technical solutions:

Separated nucleic acid sequence of the present invention has the sequence shown in the SEQ ID No:1, wherein comprises pig TRIM63 Gene Partial exon 3, the sequence of the exon 4 of part and complete introne 3; Another has the sequence shown in the SEQ ID No:4, long is 1462bp, wherein comprises pig TRIM63 Gene Partial exon 5, the exon 7 of part, the sequence of complete exon 6 and complete intron 5 and complete intron 6; There is a base mutation (92A-92T) at the 92nd bit base place of sequence table SEQ ID No:1, causes NheI-RFLP (Restriction FragmentLength Polymorphism) polymorphism; This base mutation is positioned on the 3rd exon, and causes the change (halfcystine becomes Serine) of coded amino acid; There is a base transversion (435CT-435TC) at the 435th bit base place, causes the EcoRV-RFLP polymorphism, and this sudden change is arranged in the 3rd intron; There is a base mutation (977A-977G) at the 977th bit base place of sequence table SEQ ID No:4, causes the RsaI-RFLP polymorphism, and this sudden change is arranged in the 6th exon, and causes the change (Serine becomes l-asparagine) of coded amino acid.

The present invention separates the gene order of pig TRIM63 gene the 3rd exon, the 3rd intron and the 6th intron pleomorphism site, and said " separation " is meant on the genomic dna level, utilizes primer to coming the gene order in amplification polymorphism site.By searching to the mutational site of pig TRIM63 gene, found 3 mutational sites, be respectively that the 92nd of sequence shown in the SEQ ID No:1 exists T → A polymorphism, and cause that amino acid becomes Serine by halfcystine; The 435th of sequence the exists CT → TC polymorphism shown in the SEQ ID No:1; The 977th of sequence the polymorphism that has T → A shown in the SEQ ID No:4.And by finding that with the association analysis of meat proterties the 435th site of sequence shown in the SEQ ID No:1, the 977th site of sequence shown in the SEQ ID No:4 exist related with part meat proterties respectively.

Shown that at described nucleotide sequence length is 2 pairs of allele specific nucleic acid primers of 20bp, and hybridization specifically, and amplify sequence shown in the SEQ ID No:1 that contains pig TRIM63 gene the 3rd exon and the 3rd intron and amplify sequence shown in the SEQ ID No:4 that contains pig TRIM63 gene the 6th intron.

Of the present invention practicing can be judged the quality of pig part meat proterties in order to detection, and uses this molecule marker and carry out marker assisted selection in the breeding of pig, thereby overcomes the meat proterties deficiency of selection indirectly, has important and practical meanings.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.Experimental technique in the following example is according to normal condition, and the experimental technique that indicates actual conditions is according to dated condition:

It is specific as follows that the following example indicates condition:

In commercially available Ningxiang pig, plum mountain pig, the white pig of Pudong, U.S.A is random choose 2 individualities separately in Large White and the genealogy of law Large White, carries out pcr amplification with 2 pairs of primers in the invention, and the PCR product reclaims the rear clone order-checking; Biological software is compared in twos to the sequence of same primer amplification, finds 3 mutational sites, and the base substitution that wherein is positioned at the 3rd exon has caused amino acid whose change (halfcystine becomes Serine);

The a variety of technological methods of the following example can be used for detecting whether 3 described sites of pig TRIM63 gene exist single nucleotide polymorphism among the present invention.These technology comprise (being not limited to): dna sequencing, sequencing by hybridization, restriction fragment length polymorphism, oligonucleotide arrays (or claiming the DNA chip), sex change high performance liquid chromatography (DHPLC) or the like.

The following example adopts the mutational site of PCR-RFLP technology for detection pig TRIM63 gene, uses the GLM program of SAS (8.02 editions) and carries out the association analysis of mutational site and pig flesh characters.The present invention provides 3 new molecule markers for the seed selection of pig flesh characters, for the function of studying pig TRIM63 gene lays the foundation.

The following example is used to seek the mutational site, comprises that commercially available Ningxiang pig (2), U.S.A are Da Bai (2), plum mountain pig (2), the white pig of Pudong (2) and genealogy of law Da Bai (2); Being used for the analyzing molecules mark test swinery related with pig flesh characters is the too pig (113) of reviving, meat proterties, DNA sample extract by present embodiment, DNA is stored in-20 ℃, described DNA sample is not particularly limited, for detecting the mutational site, can be the DNA that extracting goes out from samples such as ear tissue, blood.

The intestinal bacteria Dh5 α that the following example relates to is disclosed technical intelligence, can be by commercially available purchase, as: available from sky root biochemical technology company limited.

On above-mentioned basis, the following example adopts the distribution situation of conventional 3 mutational sites of PCR-RFLP technology for detection in the pig test colony too of reviving, and carries out the mark property association analysis.

Embodiment 1

The acquisition of the portion gene group sequence of pig TRIM63 gene

(1) personnel selection TRIM63 gene cDNA (the GenBank number of including: NM_032588) be the information probe, utilize the BLAST instrument among the NCBI in GenBank pig est database, to do the homologous sequence screening, obtain a series of homologys and be the ESTs (fragment length is greater than 100bp) more than 90%, the number of including of these ESTs is inquired about corresponding sequence with ENTREZ (http://www.ncbi.nlm.nih.gov/Web/Search/index.html) in NCBI, use the Seqman program construction pig EST-contig among the DNAStar then.With EST splice sequence and human TRIM63 gene genome sequence (the GenBank number of including: NC_000001) compare, confirm the exon joint, right according to EST splicing sequences Design be used to the to increase primer 2 of portion gene group sequence at last:

1PL:5 '-GCTGAGCCAGAAATTTGATG-3 ' (sequence table SEQ ID No:2)

1PR:5 '-CTCAGGGTGTCTGCTATGTG-3 ' (sequence table SEQ ID No:3)

2PL:5 '-GGAGCACGAAGATGAGAAAA-3 ' (sequence table SEQ ID No:5)

2PR:5 '-CACCAGCATGGAGATAAAGA-3 ' (sequence table SEQ ID No:6)

Pcr amplification condition: (remove ddH ₂Outside O and the dna profiling, agents useful for same provides by sky root biochemical technology company limited)

The reaction system cumulative volume is 20 μ l, wherein

DdH ₂The O14 μ l distilled water of sterilizing

10×buffer2μl

Each 0.4 μ l of the forward and reverse primer of 10 μ M

10mM?dNTP?0.4μl

2.5U Taq archaeal dna polymerase 0.4 μ l

50 ng pig DNA templates, 2 μ l

(2) purifying of PCR product, clone and order-checking

The purifying of PCR product: under ultraviolet lamp, contain the segmental gel of purpose, put into the 1.5mlEpendorff pipe, with PCR product purification test kit (Hua Shun) purifying from the sepharose cutting-out.

Ligation: the PCR product of purifying is connected with the pMDl8-T carrier, and the ligation cumulative volume is 10 μ l, comprising 5 μ l solution I, and the T carrier of 0.5 μ l, the purified pcr product of 2.5 μ l adds 2 μ l aqua sterilisas at last and puts 4 ℃ of water-baths and spend the night.

Transform: get 100～120 μ l competent cells under the sterile state in 1.5ml Ependorff pipe, the connection product of 5 μ l is added mixing, place 30min on ice, 42 ℃ of heat shock 90s do not shake the Ependorff pipe therebetween, take out back ice bath 3～4min, the LB liquid nutrient medium that adds 400 μ l antibiotic-frees, shaking culture 45min gets 100 μ l and coats on the agar plate, and 37 ℃ keep flat to be inverted behind the 1h and cultivate.Single Dh5 α intestinal bacteria on the picking flat board joined in the centrifuge tube that 800 μ lLB liquid nutrient mediums (being added with microbiotic) are housed shaking culture 8 hours.

Bacterium colony PCR identifies: with the LB nutrient solution of the single bacterium colony of Dh5 α intestinal bacteria in the above-mentioned steps template as pcr amplification, increase according to primer among the embodiment 1 (1) and pcr amplification condition, detect the bacterium liquid order-checking that amplified band is identical with PCR product size among the embodiment 1 (2) with agarose gel electrophoresis.Sequence is respectively 942bp and 1462bp after splicing.

(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 that order-checking back is obtained and the GenBank database carries out sequence homology relatively, with evaluation with obtain the function information of this dna sequence dna.Found that the partial dna sequence of the pig TRIM63 gene of being cloned and people's TRIM63 gene have high homology, is 93%.

Embodiment 2

The searching in mutational site in the pig TRIM63 genome sequence

By Different Individual DNA is carried out pcr amplification with two pairs of primers among the embodiment 1, the PCR product reclaims, cloning and sequencing; Use bioanalysis software that the sequence of same primer amplification is compared, the 92nd of sequence has A and two kinds of allelotrope of T shown in the SEQ IDNo:1 of discovery TRIM63 gene, the 435th has CT and two kinds of allelotrope of TC, and the 977th of sequence shown in the SEQ IDNo:4 has A and two kinds of allelotrope of G.

Embodiment 3

The detection method in pig TRIM63 mutational site

Sequence 1

(1) primer sequence (with embodiment 1)

1PL:5 '-GCTGAGCCAGAAATTTGATG-3 ' (sequence table SEQ ID No:2)

1PR:5 '-CTCAGGGTGTCTGCTATGTG-3 ' (sequence table SEQ ID No:3)

(2) pcr amplification condition

PCR reaction cumulative volume is 20 μ l, and wherein the about 100ng of pig genomic dna contains 1 * buffer (Promega), 1.5mmol/L MgCl ₂, the dNTP final concentration is 150 μ mol/L, the primer final concentration is 0.2 μ mol/L, 2U TaqDNA polysaccharase (Promega).The pcr amplification program is: 94 ℃ of 4min, and the 94 ℃ of 20s that circulate then 35 times, 56 ℃ of 20s, 72 ℃ of 30s, last 72 ℃ are extended 10min.The PCR reaction product detects with 1.5% agarose gel electrophoresis.

(3) RFLP testing conditions

PCR product endonuclease reaction volume is 10 μ l, 1 * buffer, 1 μ l wherein, and PCR product 3 ~ 5 μ l, restriction enzyme NheI or EcoRV are 0.5 μ l (5U), use H ₂O supplies 10 μ l, and with centrifugal behind the sample mixing, enzyme is cut 8h.

(4) RFLP somatotype

Detect enzyme with 2% agarose gel electrophoresis and cut the result, the record genotype.

The NheI restriction enzyme site (G ↓ CTAGC), controlled by two allelotrope, and T allelotrope has only fragment of 942bp, and A allelotrope has two fragments of 89bp+853bp by this locus.These two allelotrope can be formed three kinds of frequency of genotypes AA, AT, TT.

There is 1 EcoRV restriction enzyme site in the 435bp place, and (GAT ↓ ATC), this locus is controlled by two allelotrope, and CT allelotrope has only fragment of 942bp, and TC allelotrope has two fragments of 433bp+509bp.These two allelotrope can be formed three kinds of genotype CT/CT, CT/TC, TC/TC.

Sequence 2

(1) primer sequence (with embodiment 1)

2PL:5 '-GGAGCACGAAGATGAGAAAA-3 ' (sequence table SEQ ID No:5)

2PR:5 '-CACCAGCATGGAGATAAAGA-3 ' (sequence table SEQ ID No:6)

(2) pcr amplification condition

PCR reaction cumulative volume is 20 μ l, and wherein the about 100ng of pig genomic dna contains 1 * buffer (Promega), 1.5mmol/L MgCl ₂, the dNTP final concentration is 150 μ mol/L, the primer final concentration is 0.2 μ mol/L, 2U TaqDNA polysaccharase (Promega).The pcr amplification program is: 94 ℃ of 4min, and the 94 ℃ of 30s that circulate then 35 times, 56 ℃ of 30s, 72 ℃ of 30s, last 72 ℃ are extended 10min.The PCR reaction product detects with 1.5% agarose gel electrophoresis.

(3) RFLP testing conditions

PCR product endonuclease reaction volume is 10 μ l, 1 * buffer, 1 μ l wherein, and PCR product 3 ~ 5 μ l, restriction enzyme RsaI are 0.5 μ l (5U), use H ₂O supplies 10 μ l, and with centrifugal behind the sample mixing, enzyme is cut 8h.

(4) RFLP somatotype

Detect enzyme with 2% agarose gel electrophoresis and cut the result, the record genotype.

There is 1 RsaI restriction enzyme site in the 978bp place, and (GT ↓ AC), this locus is controlled by two allelotrope, and G allelotrope has only fragment of 1462bp, and A allelotrope has two fragments of 978bp+484bp.These two allelotrope can be formed three kinds of genotype GG, GA, AA.

Embodiment 4

The mark property association analysis

Utilize test swinery (the too pig of reviving) to do the proterties association analysis, 121 DNA samples are used for genotype detection.

The proterties of being analyzed is the meat proterties, comprises yellowish pink, pH1 (killing the pH value of back 45 ~ 60min), pH2 (killing the pH value of back 24h), intramuscular fat, drip loss, shearing force, intramuscular protein content.Set up following least square model:

y _ijk＝μ+GENOTYPE _i+SEX _j+BREED _k+e _ijk

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

Test very much the distribution situation that detects 4 pleomorphism sites in the swinery in Soviet Union.

Test very much in 113 individualities of swinery in Soviet Union, for the NheI-RFLP pleomorphism site, TT genotype individuality has 105, and the AT genotype has 8 individualities, does not have TT genotype individuality.

Test very much in 113 individualities of swinery in Soviet Union, for the EcoRV-RFLP pleomorphism site, CT/CT genotype individuality has 33, and the CT/TC genotype has 65 individualities, and the TC/TC genotype has 15 individualities.

Test very much in 113 individualities of swinery in Soviet Union, for the RsaI-RFLP pleomorphism site, GG genotype individuality has 101, and the AG genotype has 12 individualities, does not find AA genotype individuality.

Adopt SAS (8.02) software that data are carried out statistical study.The result shows:

(1) there be related (table 1) in pig TRIM63 gene EcoRV-RFLP pleomorphism site with pH1 and drip loss;

(2) there be related (table 2) in pig TRIM63 gene RsaI-RFLP pleomorphism site with intramuscular fat content.

The different genotype of table 1:TRIM63 gene the 3rd intron EcoRV-RFLP and the association analysis of meat proterties

Genotype	pH1	Drip loss
			CT/TC CT/CT TC/TC	6.145±0.084 a 6.030±0.184 ab 5.882±0.075 b	0.206±0.021 ab 0.309±0.046 a 0.188±0.019 b

Annotate: the alphabetical identical table differential of same row different not remarkable (P＜0.05)

The different genotype of table 2:TRIM63 gene the 6th intron RsaI-RFLP and the association analysis of meat proterties

Proterties	AG	GG
			IMF	0.037±0.003 a	0.029±0.001 b

Annotate: with delegation's letter identical table differential different not remarkable (P＜0.05)

Sequence table

＜110〉Shanghai Communications University

＜120〉separated nucleic acid sequence of pork quality trait related gene TRIM 63

<160>6

<210>1

<211>942

<212>DNA

＜213〉pig (Sus scrofa)

<220>

<221>gene

<222>(1)...(942)

<223>

<220>

<221>mutation

<222>(92)

＜223〉n=a or t

<220>

<221>mutation

<222>(435)...(436)

＜223〉nn=ct or tc

<220>

<221>exon

<222>(1)...(133)

<223>

<220>

<221>intron

<222>(134)...(908)

<223>

<220>

<221>exon

<222>(909)...(942)

<223>

<400>1

g?gag?cac?gaa?gat?gag?aaa?atc?aac?atc?tac?agc?ctc?acg?tgc?gaa?gtg 49

Glu?His?Glu?Asp?Glu?Lys?Ile?Asn?Ile?Tyr?Ser?Leu?Thr?Cys?Glu?Val

1 5 10 15

ccc?acg?tgc?tcc?atg?tgc?aag?gtg?ttt?ggg?gcc?cac?aag?gct?ngc?gag 97

Pro?Thr?Cys?Ser?Met?Cys?Lys?Val?Phe?Gly?Ala?His?Lys?Ala?Xaa?Glu

20 25 30

gtg?gcc?ccg?ctg?cag?agt?gtc?ttc?cag?gga?cag?aag?gtactgg?cctgagccac 150

Val?Ala?Pro?Leu?Gln?Ser?Val?Phe?Gln?Gly?Gln?Lys

35 40 45

ttctaccccc?cagcctgagc?ctgctgggca?cgaccagatg?attcaggatg?ctgatttgat 210

ccctgtagat?ctgctgagct?gggctttcag?gaatagtccc?cctggagtga?cttcccaggc 270

cacagggaca?acagcccttg?ccttcctcag?ggagatggct?gagctcatgg?gttatactta 330

gagctacttg?agagccacaa?gggagagacc?agatcccagg?gccaggcaac?aaggcccttt 390

caggccacga?cctctgcaac?ccaggaagct?tcggaccctg?gatanntctc?ccatgctggt 450

cacctctccc?caaatccccc?tgcagcagga?ggcagctgag?agccctctcc?tgggtctctg 510

ggcctgtggg?agccccgcca?atctgtacat?gcagtaagct?gtagctcccc?tttctgcact 570

ctgtcctccc?cacctcttgt?tcttctctct?atctcttcta?gaactctcca?aacctgttgc 630

ttttctgggt?ccttttcttc?ctagaaataa?gcctctcctc?tgccagcaca?ttgctctgtg 690

tttttgggca?gctcacttcc?cctctctggg?cctcatctgt?aaaatgaggg?atgcctcatc 750

tcactgagag?tctctgctct?ggaaatgagc?taagcctcag?aaacaggctc?tttctggaag 810

agatgagcat?gagggtgcca?ttttgctgcc?cctcccagag?ggcaggcctt?gagtctatct 870

ttgtcttctg?ctgatccccc?gctccttcct?cccatctag?act?gag?ctg?agt?atc?ttt 927

Thr?Glu?Leu?Ser?Ile?Phe

50

atc?tcc?atg?ctg?gtg 942

Ile?Ser?Met?Leu?Val

55

<210>2

<211>20

<212>DNA

＜213〉pig (Sus scrofa)

<220>

<221>primer_bind

<222>(1)...(20)

<223>

<400>2

gctgagccag?aaatttgatg 20

<210>3

<211>20

<212>DNA

＜213〉pig (Sus scrofa)

<220>

<221>primer_bind

<222>(1)...(20)

<223>

<400>3

ctcagggtgt?ctgctatgtg 20

<210>4

<211>1462

<212>DNA

＜213〉pig (Sus scrofa)

<220>

<221>gene

<222>(1)...(1462)

<223>

<220>

<221>misc-feature

<222>(977)

＜223〉n=a or g

<220>

<221>exon

<222>(1)...(208)

<223>

<220>

<221>exon

<222>(976)...(998)

<223>

<220>

<221>exon

<222>(1358)...(1462)

<223>

<220>

<221>intron

<222>(209)...(975)

<223>

<220>

<221>intron

<222>(999)...(1357)

<223>

<400>4

g?ctg?agc?cag?aaa?ttt?gat?gtg?ctg?tac?gcc?atc?ctg?gac?gag?aag?aag 49

Leu?Ser?Gln?Lys?Phe?Asp?Val?Leu?Tyr?Ala?Ile?Leu?Asp?Glu?Lys?Lys

1 5 10 15

agt?gag?ctg?ctt?cag?cgg?atc?acg?cgg?gag?cag?gag?gag?aag?ctc?agc 97

Ser?Glu?Leu?Leu?Gln?Arg?Ile?Thr?Arg?Glu?Gln?Glu?Glu?Lys?Leu?Ser

20 25 30

ttc?ctc?gag?ggc?ctc?atc?cag?cag?tac?cgg?gag?caa?ttg?gac?aag?tcc 145

Phe?Leu?Glu?Gly?Leu?Ile?Gln?Gln?Tyr?Arg?Glu?Gln?Leu?Asp?Lys?Ser

35 40 45

acc?aag?ctg?gtg?gag?aca?gcc?atc?cag?tcc?ctg?gac?gag?cct?ggt?ggg 193

Thr?Lys?Leu?Val?Glu?Thr?Ala?Ile?Gln?Ser?Leu?Asp?Glu?Pro?Gly?Gly

50 55 60

gcc?atc?ttc?ctc?ttg?gt?gagcaggact?gggagggtgt?gggcatttca 240

Ala?Ile?Phe?Leu?Leu

65

actcaacagc?tctgtctgag?ggtcaagtcg?tgctccatca?ctgctcatca?ctgctcacca 300

gtgagtgtgc?cctggggcaa?ggcacttaaa?gctctcccag?cctcagtttc?ctcatctgta 360

aaatgagcct?gtaactattc?ctgtcttcct?tcctcttaac?tgattttatg?aggatcagat 420

aagaggatgt?ttgtgaaacg?gctttgtaaa?ctgcaaatac?agtgtccatg?gagagactgt 480

taattgagca?cctactatag?gccagccact?gaggtggaga?cacgtatgaa?ggaaaaagag 540

ccctgctcta?gaaaagctgg?ttgggggaga?cagaaaagga?tcagctataa?gattttatga 600

gggaggaatt?cagttcaaca?aaaatttcct?aagtgcatac?tatggagcag?gctttgtgct 660

acatgatggg?tataaaacgg?tgattagaat?gtgattccca?tcttggagat?ttcagggttt 720

aagactttag?gcggatagtc?ctagaatcca?agctgtctgc?caggaccatg?ggagggtcct 780

aggatgatga?ccaagacctg?tgcagactgg?acagagggaa?aaagacccaa?gtttggtttg 840

gctgaagcta?gcaagcaggt?cccaagtggg?ctagagagca?gcgacaagtg?cccatctgta 900

gtccctggtt?tcgctgatct?cctggggcca?ccctttcttc?agtcctgacc?acatgctctt 960

tctctctctc?cacag?ant?gcc?aag?cag?ctc?atc?aga?ag gt?cagtgtctct 1010

Xaa?Ala?Lys?Gln?Leu?Ile?Arg?Ser

70 75

tcagggctat?gatccaaatc?cctaactttc?cccctccacc?accccccctg?cacccctgat 1070

ccccctgggc?ctctgacttt?ttgttcaggg?ggcctcctcc?tttgctttag?atctgctggg 1130

agggctttcc?tggcctttgc?atccctgctt?tcttgaacca?tctcttctgc?ttgtgactga 1190

ggaggaaata?ctctaccctc?tggtggggag?cctcagggta?caaggcctgg?gtgtggacac 1250

aagcatgtgc?actggtttaa?agaggagcta?aaacacctcc?ttttctcact?gaggccaaag 1310

atctcactgt?cccccccacc?accccctttc?ctctctctcc?ctcgcag?t?att?gta?gaa 1367

Ser?Ile?Val

80

gct?tcc?aag?ggt?tgc?cag?ctg?ggg?aag?aca?gag?cag?ggc?ttt?gaa?aac 1415

Glu?Ala?Ser?Lys?Gly?Cys?Gln?Leu?Gly?Lys?Thr?Glu?Gln?Gly?Phe?Glu

85 90 95

atg?gac?tac?ttc?act?ttg?aac?tta?gag?cac?ata?gca?gac?acc?ctg?ag 1462

Asn?Met?Asp?Tyr?Phe?Thr?Leu?Asn?Leu?Glu?His?Ile?Ala?Asp?Thr?Leu

100 105 110

<210>5

<211>20

<212>DNA

＜213〉pig (Sus scrofa)

<220>

<221>primer_bind

<222>(1)...(20)

<223>

ggagcacgaa?gatgagaaaa 20

<210>6

<211>20

<212>DNA

＜213〉pig (Sus scrofa)

<220>

<221>primer_bind

<222>(1)...(20)

<223>

<400>6

caccagcatg?gagataaaga 20

Claims (4)

1. the separated nucleic acid sequence of a pork quality trait related gene TRIM 63 is characterized in that, separated nucleic acid sequence is as SEQ ID №: shown in 1, longly be 942bp.

2. the separated nucleic acid sequence of pork quality trait related gene TRIM 63 according to claim 1, it is characterized in that, at described SEQ ID №: the sequence shown in 1 wherein comprises pig TRIM63 Gene Partial exon 3, the sequence of the exon 4 of part and complete introne 3.

3. the separated nucleic acid sequence of pork quality trait related gene TRIM 63 according to claim 2 is characterized in that, sequence table SEQ ID №: there is a base mutation 92A-92T at 1 the 92nd bit base place, causes the NheI-RFLP polymorphism; This base mutation is positioned on the 3rd exon, and causes that amino acid becomes Serine by halfcystine; There is a base transversion 435CT-435TC at the 435th bit base place, causes the EcoRV-RFLP polymorphism, and this sudden change is arranged in the 3rd intron.

4. the separated nucleic acid sequence of pork quality trait related gene TRIM 63 according to claim 1, it is characterized in that, shown that at described nucleotide sequence length is 2 pairs of allele specific nucleic acid primers of 20bp, and hybridization specifically and amplifies the SEQ ID № that contains pig TRIM63 gene the 3rd exon and the 3rd intron: sequence shown in 1 and amplify the 5th intron that contains pig TRIM63 gene complete and the SEQ ID № of complete the 6th intron: sequence shown in 4.