CN1256428C - Gene PSME3 of fat thickness at back of pig as well as preparation method - Google Patents

Abstract

The present invention belongs to the technical field of gene engineering, particularly to a clone of a fat thickness PSME3 gene on the back of a pig and a preparation method thereof. The present invention is characterized in that basic group mutation of three sites is detected from a cDNA sequence and a DNA sequence of the fat thickness PSME3 gene on the back of the pig, wherein the cDNA sequence and the DNA sequence are shown in sequence tables SEQ ID NO1 and SEQ ID NO. 2. The mutation of one of basic groups is relevant to the property of the fat thickness on the back of the pig. The present invention discloses a PSME3 gene sequence of the pig and the basic group mutation of three sites comprising the basic group mutational site influencing the fat thickness on the back of the pig. The present invention also discloses a preparation method obtaining the gene and a primer sequence for detecting the basic group mutation of the three sites. The present invention provides a novel molecular marker for mark auxiliary seed breeding of the pig.

Description

Fat thickness at back of pig PSME3 gene and preparation method thereof

Technical field

The invention belongs to gene engineering technology field, relate in particular to clone of fat thickness at back of pig PSME3 gene and preparation method thereof.

Background technology

Along with people to the continuous growth of animal proteinum food demand and the development of commodity economy, carcass quality evaluation has become the problem that heredity, breeding scholar, the producer, meat manager, human consumer generally are concerned about.Along with the development of the commodity market of domestic and international live hog and pork product and the raising of scientific and technological level, it is popular that the stdn of carcass quality evaluation, normalization etc. have been become research, the carcass quality deliberated index of generally acknowledging in the world specifically has at present: carcass lean meat percentage (lean percentage in the carcass, CLP), cutability (lean content in the carcass, CLC) maybe can sell lean meat block ratio (lean cuts ratio, LCR).In real work, people attempt to seek the method that a kind of easy live body is evaluated carcass quality, butcher the financial loss of bringing with minimizing, and therefore big quantity research is devoted to seek the optimum prediction index that trunk is formed.The genetic correlation analytical results of live body proterties and carcass lean meat percentage shows, the r of the average thickness of backfat of live body and the cut out yield of trunk lean meat _AThe highest, for-0:80, the r of the live body thickness of backfat and carcass lean yield _AFor-0:54, therefore by direct selection to the live body thickness of backfat, be expected to make lean ratio (amount) to obtain correlated response (Peng Zhong town etc.: " genetic improvement of pig ", Beijing, agricultural press, 1991), and the development and the application of dna molecular marker technology at present, be the direct selection of carcass lean meat percentage, the rapid evaluation of particularly early stage selection and carcass quality has been opened up new way.

The gene of having studied at present relevant with fat thickness at back of pig has: (Geldermann H etc. such as (1) Geldermann, Mapping ofquantitative traits loci by means of marker genes in F2 generations of wild boar, Pietrian and Meishan pigs:J Anim Breed Genet, 1996,113:381-387 :), (Knorr C etc., Association of GH gene variants withperformance traits in F such as Knorr ₂Generations of Europesan wild boar, Pietrain and Meishan pigs:AinmGenet, 1997,28:124-128 :) discover that (Growth hormore, GH) gene haplotype is formed proterties with several trunks has remarkable related tethelin different on No. 12 karyomit(e)s of pig.(2) (the Majorhistocompatibility complex of main histocompatibility complex that reports on No. 7 karyomit(e)s of pig is arranged, MHC) relevant (the Peng Zhong town etc. of gene haplotype with proterties such as birth weight, weaning weight, the speed of growth, the thickness of backfats, quantitative character gene and the mark research evolution thereof of pig, external livestock technology, 1999,26 (10): 28-32).(3) (Yu T P etc. such as Yu, Association of PIT1 polymorphisms with growth and carcass traits in pigs:J Anim Sci, 1995,73:1282-1288 :) contain at one and to detect the hypophysis transcription factor in China and the resource family of U.S.'s pig variety " blood " (PIT1) there is significant correlation in Pituitantranscription factor 1 with the average thickness of backfat.(4) (Joen J T etc. such as Jeon, A paternally expressedQTL affecting skeletal and muscle mass in pigs maps to the IGF2 locus:Nature Genetics, 1992,21:157-158 :) and (Nezer C etc. such as Nezer, An imprinted QTL with major effect on muscle mass andfat deposition maps to the IGF2 locus in pigs:Nature Genetics, 1992,21:155-156 :) use two family insulin-like growth factors 2 (Insulin like growth factor II respectively, IGF-II) on location and No. 2 karyomit(e)s of pig, and detect this gene and the thickness of backfat has significant correlation.(5) (M:F:W:tePas etc. such as te Pas, Messenger ribonucleic acid expression of theMyoD gene family in mucle tissue at slaughter in relation to selection for porcine growth rate, J Anim:Sci, 2000,78:69-77) important member's myogenin (Myogenin) gene of having analyzed the MyoD gene family in the hybridization colony of Large White and growth, carcass trait may exist relevantly, and the myogenin gene is positioned on No. 9 karyomit(e)s of pig.(6) melanocyte cortical hormone receptor 4 (Melanocortin-4 receptor, MC4R) be positioned at (Kim K S etc. on No. 1 karyomit(e) of pig, Linkage and physicalmapping of the porcine melanocortin-4 receptor (MC4R) gene:J Anim Sci, 2000a, 78:791-792 :), (Kim K S etc. such as Kim, A missense variant of the porcine melanocortin-4 receptor (MC4R) gene is associationwith fatness, growth, and feed intake traits:Mamm Genome, 2000b, 11:131-135 :) in 5 pig commodity systems, detect variation of MC4R gene genetic and thickness of backfat significant correlation.(7) candidate gene of studying relevant with carcass trait with the pig growth also has: with growth, the relevant leptin gene (Leptin of carcass trait possibility, LEP) be ob gene and the myogenic factor 3 (myf3) (Kim KS etc., A missense variant of the porcine melanocortin-4 receptor (MC4R) gene is association withfatness, growth, and feed intake traits:Mamm Genome, 2000b, 11:131-135 :).Myostatin (the Myostatin that the growth of skeletal muscle is had the negative regulation effect, MSTN) gene, this gene claims GDF-8 (Growth differentiation factor 8 again, growth and differentiation factor 8) (Mcpherron A C etc., Double muscling in cattle due to mutation in the myostatin gene:Proc Natl Acad Sci, USA, 1997,94 (23): 12457-12461 :), with the thickness of backfat (the CathepsinB of cathepsin B of significant correlation is arranged, CTSB) gene (Russo V etc., Investigation of candidate genes for meat quality in dry-cured hamproduction:the porcine cathepsin B (CTSB) and cystatin B (CSTB) genes:Anim Genet, 2002,33:123-131 :) etc.

Proteasome activates subunit 3, and (Proteasome activator subunit 3 PSME3) claims proteasome PA28-γ subunit (PA28-γ subunit) again, is a subunit of 20S proteasome incitant PA28 complex body.PA28 complex body (also claiming the 11S regulon), it is the important regulatory factor of 20S proteasome, its effect is the peptidase activity that can increase the 20S proteasome, increase the 20S proteasome and produce the ability that is suitable for the multiple peptide substrate of major histocompatibility complex I quasi-molecule bonded, improve maximum reaction velocity (Albertsen H M etc., A physical map amd candidate genes in the BRCA1 region on chromosome 17q12-21:Nature Genet, 1994,7 (4): 472-479:; Jiang H etc., Sequence and expression of mouse proteasomeactivator PA28 and the related autoantigen Ki:Immunol, 1997,46:93-98:; Murray etc., Identificationand linkage of the proteasome activator complex PA28 subunit genes in Zebrafish:Scand J Immunol, 2000,51 (6): 571-577 :).

The PA28 complex body is made up of three subunits, and two molecular weight are α and the β subunit of 28KDa, and the γ subunit of 30KDa, respectively by PSME1, PSME2 and three genes encodings of PSME3.

At present existing people, mouse, ox and zebra fish (Albertsen H M etc., A physical map amd candidate genes in theBRCAl region on chromosome 17q12-21:Nature Genet, 1994,7 (4): 472-479:; Jiang H etc., Sequenceand expression of mouse proteasome activator PA28 and the related autoantigen Ki:Immunol, 1997,46:93-98:; Murray etc., Identification and linkage of the proteasome activator complex PA28 subunit genesin Zebrafish:Scand J Immunol, 2000,51 (6): 571-577 :) PSME3 gene organization structure, expression and functional study report, the people PSME3 assignment of genes gene mapping is in 17q12-21, cDNA sequence total length 2,894bp.The mouse PSME3 assignment of genes gene mapping on No. 11 karyomit(e), genome sequence 8,733bp has 11 exons.Through the Northern analysis revealed, the PSME3 gene is at the spleen of mouse, expression amount height (Jiang H etc. in thymus gland and the testis tissue, Sequence and expression of mouse proteasome activatorPA28 and the related autoantigen Ki:Immunol, 1997,46:93-98 :), (Kandil E etc. such as Kandil, PA28subunits of the mouse proteasome:primary structures and chromosomal localization of the genes:Immunogenetics, 1997,46 (4): 337-344) and (Kohda K etc. such as Kohda, Characterization of the mouse PA28activator complex gene family:complete organizations of the three member genes and a physical map of the～150-kb region containing the α-and β-subunit genes J Immunology, 1998,160:4923-4935) to the people, mouse, the PSME1 of rat, the comparative analysis result of the aminoacid sequence of PSME2 and three genes of PSME3 shows that these three genes have very high sequence homology, illustrates that these three genes are to be produced by the repetition (Duplication) of an ancestral gene.

The function of PA28-γ subunit is also not fully aware of at present, (Murata S etc. such as Murata, Growth retardation in micelackingthe proteasome activator PA28 γ: J Biol Chem:1999,274:38211-38215 :) adopt the homologous recombination method to obtain the homozygote and the heterozygote mouse of the 2nd to 8 exon region of disappearance PSME3 gene, compare with normal mouse, organizing all that all are studied during these mouse births acted normally, it is slow that but postnatal growth but shows, and shows that the PSME3 gene function may be relevant with the adjusting of the cell proliferation of mouse and bulk-growth.

Although the research of fat thickness at back of pig candidate gene has obtained some impressive progresses, but still exist not enough: the important economical trait of (1) pig is quantitative character normally, the physiological and biochemical procedure that relates to is quite complicated, even same quantitative character, state its 1-2 controlled gene although taken off, still have other new gene to remain to be discovered with big effect.(2) research of at present relevant quantitative character gene is to select for use single candidate gene to analyze basically, has ignored intergenic interaction.One of content of modern molecular breeding is the genome breeding, promptly according to the gene of individual ownership proterties and genotypic weave construction and functional effect carries out integral body or full genome is selected, apolegamy, protect kind and Heterosis Analysis utilization etc., its basis is that complete highdensity gene map is arranged, and fully understand the weave construction, functional expression Regulation Mechanism of all genes and related with proterties, yet the gene and the mark of hereditary location and physical positioning are still very limited in pig at present, and the work of this respect also needs further reinforcement.(3) it is comprehensive inadequately to seek the method with important physiological function gene, detects the limited amount of gene, and efficient is not high, needs innovation, further seeks with the work of pig important economical trait relative new gene extremely urgent.

But it is still blank to the research of pig PSME3 gene both at home and abroad at present.

Summary of the invention

The objective of the invention is to clone pig PSME3 gene, preparation method and application, for marker-assisted breeding provides molecule marker.

The present invention realizes by following technology:

A kind of fat thickness at back of pig PSME3 gene, its cDNA sequence is as described in the sequence table SEQ ID NO:1.Its dna sequence dna is as described in the sequence table SEQ ID NO:3.

The PSME3 gene cDNA sequence length that is obtained is 2592bp, comprises the open reading frame of 762bp in the sequence, the 3 ' non-translational region of 140bp 5 ' non-translational region and 1690bp.

There is a base to replace in the PSME3 gene cDNA sequence that is obtained as the described C659-T659 of sequence table SEQ ID NO:1.

One section deletion mutantion from the common 147bp of 218 bases of the 72nd base to the is arranged among the sequence table SEQ ID NO:3.

There is the base of 1 A619-G619 to replace among the sequence table SEQ ID NO:3.

There is the base of 1 C1425-T1425 to replace among the sequence table SEQ ID NO:3.

Be used for detecting as two primer sequences of sequence table SEQ ID NO:3 72bp place's deletion mutantion as described in sequence table SEQ ID NO:4 and the SEQ ID NO:5.

Be used for detecting as two primer sequences of sequence table SEQ IDNO:3 619bp place base mutation as described in sequence table SEQ ID NO:6 and the SEQ ID NO:7.

Be used for detecting as two primer sequences of sequence table SEQ ID NO:3 1425bp place base mutation as described in sequence table SEQ ID NO:8 and the SEQ ID NO:9.

A kind of preparation is as the method for the cDNA of the described fat thickness at back of pig PSME3 of sequence table SEQ ID NO:1 gene, according to following steps:

(1) personnel selection PSME3 gene cDNA is the information probe, does the homologous sequence screening, obtains the expressed sequence tag of homology more than 80%; Splice pig EST-contig then; According to two pairs of primers of EST-contig design, amplification obtains two bar segment; Be the 3rd pair of primer of stencil design with people PSME3 gene cDNA and pig EST-contig respectively again, amplification obtains three bar segment; Extracting total RNA with the spleen tissue of the fragrant pig that grows up is template, makes RT-PCR amplification and 3 ' RACE, and PCR product purification and cloning and sequencing carry out sequential analysis, and acquisition is as the described cDNA sequence of sequence table SEQ ID NO:1.

(2) extracting DNA from the pig blood genome, is template design primer with pig PSME3 gene cDNA sequence, carries out pcr amplification, and PCR product purification and cloning and sequencing obtain as dna sequence dna as described in the sequence table SEQ ID NO:3.

Use the polymorphism that PCR and PCR-RFLP method detect pig PSME3 gene (for the marker-assisted breeding of pig provides molecule marker).

More detailed technical scheme is by the following stated:

1.PSME3 the clone of gene:

(1) design of primers:

(the GenBank number of including: NM005789) be the information probe of personnel selection PSME3 gene cDNA, 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 80%, 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 ASSEMBLY program construction pig EST-contig among the GeneTool then.According to two couples of primer PS-A of EST splicing sequences Design ₂, PS-B ₁And PS-A ₃, PS-B ₃, amplification obtains two bar segment A ₂B ₁And A ₃B ₃For obtaining the complete coding region sequence of pig PSME3 gene, with people PSME3 gene cDNA and pig EST splicing sequence be respectively

The 3rd couple of primer PS-A of stencil design ₁And PS-B ₁, amplified fragments is labeled as A ₁B ₁

Table 1 is used for the primer sequence of PSME3 gene cDNA clone

Primer label Primer symbol	Primer sequence Primer sequences (5 '-3 ') (Forward and Reverse)	Annealing temperature Anneal Temp:	Expection PCR product length Expected PCR product size (bp)	The sequence Primer index sequence of design of primers institute foundation
					PS-A 1 PS-B 1 PS-A 2 PS-B 3 PS-A 3 PS-B 3 3’RACE	CATGGCCTCGTTGCTGAAG TCAATCTCTGTCACGGTGCG GGAGCAAGTGAGCAGTGAGT CAAFGTCGGTCTTGAGCCAG ATGTGGAGGACTATCGGCG CAATGTCGGTCTTGAGCCAG GCAGAGACACTGTACTGAGGCCAG	57℃ 58℃ 58℃ 58℃	About 600 about 960 about 220 about 1200	XM032767 EST contig EST contig EST contig EST contig

(2) reverse transcription PCR amplified reaction:

Utilize TRIzoL test kit (U.S. GIBCO company) to extract total RNA from the fragrant pig spleen tissue of growing up, concrete operations are carried out according to the test kit specification sheets.

CDNA first chain synthetic: the reaction cumulative volume is 50 μ l, at first with total RNA of 2 μ g and oligo d (T) ₁₁Be mixed in the Ependorff pipe, 70 ℃ of incubation 5min are to remove the secondary structure of RNA, place cooled on ice to avoid regenerating of secondary structure immediately, add all the other components according to the application of sample condition of table 2 through of short duration after centrifugal, behind 37 ℃ of incubation 1h, temperature is risen to 95 ℃ of deactivation ThermoScript II, place-20 ℃ of preservations standby.

The PCR reaction: the reaction cumulative volume is 20 μ l, and the application of sample volume and the final concentration of each component are as described in Table 3.The pcr amplification program is 94 ℃ of 3min, 94 ℃ of 30s, and annealing 45s, 72 ℃ of 1min circulate 35 times, and last 72 ℃ are extended 5min, and annealing temperature sees Table 1.The PCR reaction product detects with 2% agarose gel electrophoresis.

Table 2 reverse transcription reaction diagram of system 3 PCR reaction systems

Table2 Protocol of RT Table3 Protocol of PCR

Component Reagents	Volume Volumn	Final concentration Final concentration
			Total RNA oligo d (T) 11 DEPC H 2O 5 * PCR buffer DNTP RNAsin M-MLV cumulative volume	10μl 5μl 20μl 10μl 2.5μl 1μl 1.5μl 50μl	2μg 1μmol/L 5× 500μmol/L 40U 300U

Component Reagents	Volume Volumn	Final concentration Final concentration
			dd H 2O 10×PCR buffer MgCl 2Upstream primer downstream primer dNTP TaqDNA polysaccharase cDNA cumulative volume	15.3μl 2μl 1.2μl 0.4μl 0.4μl 0.3μl 0.4μl 1μl 20μl	10× 1：5mmol/L 0：2μmol/L 0：2μmol/L 150μmol/L 2U ＞100ng

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

Under ultraviolet lamp, contain the segmental gel of purpose from the cutting-out of low melting-point agarose gel, 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, mixing 20s in the gel that per 300 μ l melt, with the Resin/DNA mixture syringe of packing into, slurries are extruded by Minicolumn.In syringe, add 80% Virahol 2ml again, touching piston makes Virahol extrude by Minicolumn, take off Minicolumn and pack in the 1.5ml Ependorff pipe, 10, the centrifugal 2min of 000g is with dry Resin, Minicolumn is packed in another clean 1.5ml Ependorff pipe, add 30～50 μ l aqua sterilisas, leave standstill 1min, 10, the centrifugal 20s of 000g is stored in the Ependorff pipe with eluted dna.

With being connected of purified pcr product and pGEM-T carrier, the 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 0.5 μ l, the T of 0.5 μ l ₄Ligase enzyme adds 1 μ l aqua sterilisa at last and puts 4 ℃ of water-baths and spend the night.

The preparation of competent cell be from 37 ℃ of fresh flat boards of having cultivated 16～20h the single colony inoculation of DH5 α of picking in 2ml LB, in 37 ℃ of shaking culture 3h, switching 1ml bacterium liquid continues to treat OD at 37 ℃ of about 4h of shaking culture in the saline bottle that contains 30ml LB ₆₀₀Reach at 0.3～0.4 o'clock with saline bottle and take out from shaking table and put ice bath cooling 10～15min, then bacterium liquid is changed in the centrifuge tube in 4 ℃ 4, the centrifugal 10min of 000g is with collecting cell, centrifuge tube is inverted abandoning clean nutrient solution, with the CaCl of the 0.1mol/L of 10ml ice precooling ₂Resuspended precipitation, ice bath 30min repeats 4 ℃ 4, and the centrifugal 10min of 000g once ices the CaCl of the 0.1mol/L of precooling with 4ml ₂Resuspended precipitation, it is standby to put 4 ℃ of preservations.

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, add the LB liquid nutrient medium of 400 μ l antibiotic-frees, 37 ℃ of shaking culture 45min.Get 100 μ l and coat in advance that 4h has been coated with on the agar plate of IPTG (Isopropylthio-β-D-galactoside, isopropylthio-) and X-gal, be inverted cultivation after keeping flat 1h for 37 ℃.

The sequencing strategy is that each fragment all adopts PCR product directly order-checking and two kinds of methods of cloning and sequencing simultaneously.Cloning and sequencing is that single clone's of picking is used for order-checking, and sequencing is finished by Shanghai Bo Ya biotech company, and each gene fragment is surveyed two clone's at least.

(4) the dna sequence dna homology search is identified:

By the American National biotechnology (NCBI of information center, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) BLAST of website (Basic Local Alignment Search 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.

2. physical positioning:

(1) primer sequence that is used for physical positioning is

PSME3-P：PL 5′-GCGAAGGTTGGATGAGTGTG-3′

PR 5′-CCTGTGGCATGTGCAAGATC-3′

This primer amplification fragment length is 700bp.

(2) be used for the experiment material of physical positioning

With pig * rodents somatic cell hybrid plate (Pig * rodent somatic cell hybrid panel, SCHP) carry out the chromosomal region location, with common pig radiation hybrid panel (the INRA-Minnesota porcine radiation hybrid panel that makes up of U.S. Minnesota university, IMpRH) carrying out karyomit(e) accurately locatees, two cover somatic cell hybrid plates are by by French Matin doctor Yerle (Laboratoire de G é n é tiqueCellulaire, INRA, Castanet-Tolosan, France) favour increases.

Wherein SCHP comprises 27 individual cells hybrid cells systems, and 1～No. 19 is pig * hamster somatic cell hybrid clone, and 20～No. 27 is pig * mouse somatic cell hybrid clone, and with hamster, mouse and pig genomic dna as positive control.Identify that through cytogenetics this hybrid plate has kept whole 18 euchromosomes and the X chromosome except that Y chromosome of pig, wherein contain 127 non-overlapped chromosomal regions, pig karyomit(e) that is comprised in each clone and chromosome segment information can obtain from WWW (http://www.toulouse.inra.fr/lgc/lgc:html/).

The radiation dose that IMpRH uses is 7,000-rad.IMpRH comprises 118 pigs * hamster radiation hybrid cell line, and hamster and pig genomic dna positive control, qualification result with 757 marks shows that the average mark rate of retaining among the IMpRH is 29.3%, include 128 linkage groups, 18 pairs of euchromosomes and X chromosome have been covered, be used to estimate that the kb/cR ratio of distance between mark is～70kb/cR (1Ray=100cR) that theoretical resolution is 145kb.

(3) PCR somatotype condition

Carrying out amplification PCR reaction cumulative volume is 15 μ l, and wherein template DNA is 20ng, contains 1 * buffer (Promega), 1.5mmol/LMgCl ₂, the dNTP final concentration is 150 μ mol/L, the primer final concentration is 0.4 μ mol/L, 2U Taq archaeal dna polymerase (Promega).The pcr amplification program is: 94 ℃ of 3min, and the 94 ℃ of 30s that circulate 35 times, 60 ℃ of 40s, 72 ℃ of 1min then, last 72 ℃ are extended 5min.The PCR reaction product detects with 2% agarose gel electrophoresis.

3.PCR-RFLP diagnostic method is set up

(1) primer sequence

PSME3CATS-1 PL 5′-GCGAAGGTTGGATGAGTGTG-3′

PR 5′-ATCAACAGCCGGATCTCAGG-3′

This primer amplification fragment length 919bp.

PSME3CATS-2 PL 5′-AGAGGTTGGAGGTGGCTAC-3′

PR 5′-GGGAATCAGGAGCTGTACC-3′

This primer amplification fragment length 835bp.

PSME3CATS-3 PL 5′-GGAGGTGATGTTGGAGTTGG-3′

PR 5′-CTGATATGATGAGCCGAAGG-3′

This primer amplification fragment length 772bp.

(2) pcr amplification condition

PCR reaction cumulative volume 20 μ l, 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 0.2umol/L, 2U Taq archaeal dna polymerase (Promega).The pcr amplification program is: 94 ℃ of 4min, and the 94 ℃ of 45s that circulate 5 times, 62 ℃ of 45s, 72 ℃ of 1min, and then the 94 ℃ of 45s that circulate 30 times, 57 ℃ of 45s, 72 ℃ of 1min, last 72 ℃ are extended 5min.The PCR reaction product detects with 2% agarose gel electrophoresis and takes pictures.

(3) RFLP testing conditions

PCR product endonuclease reaction volume is 15 μ l, 1 * buffer, 1.5 μ l wherein, and PCR product 3～5 μ l, restriction enzyme A lul are 0.5 μ l (5U), use H ₂O supplies 15 μ l, and with centrifugal behind the sample mixing, 37 ℃ of water-bath 4h detect enzyme with 2% agarose gel electrophoresis and cut the result, and the record genotype is taken pictures under ultraviolet lamp.

4. mark property association analysis

The test swinery is a Large White group, with the PCR-RFLP diagnostic method of setting up 201 DNA samples is carried out genotype detection.

Effect of the present invention is:

1. the clone result of pig PSME3 gene

Extracting total RNA reverse transcription synthetic cDNA with the spleen tissue of the fragrant pig that grows up is template, uses three couples of listed primer PS-A of table 1 respectively ₁And PS-B ₁, PS-A ₂And PS-B ₁And PS-A ₃And PS-B ₃Carry out pcr amplification, the PCR product is reclaimed the order-checking of purifying rear clone, sequencing result shows states A ₁B ₁Fragment length is 630bp, A ₂B ₁And A ₃B ₃Clip size is respectively 957bp and 225bp.

With three Segment A ₁B ₁, A ₂B ₁And A ₃B ₃Splice the cDNA integration sequence that to obtain a length be 2592bp with the ASSEMBLY program in the GeneTooll:0 software.This section cDNA sequence is carried out homology search in GenBank, result for retrieval should

(the GenBank number of including: homology NM005789) reaches 96%, and sequential analysis shows that this cDNA sequence has the open reading frame of 762bp (nt 141-902), the protein of being made up of 254 amino acid of encoding for sequence and people PSME3 gene cDNA.

2. the positioning result of pig PSME3 gene

Somatotype result in SCHP shows with the PSME3-P primer, in 19 pig * Chinese hamster somatic cell hybrid clones (1～No. 19), the purpose fragment that occurs the 700bp consistent for 15-16 number with the amplification of pig genomic dna positive control, and in 8 pigs * mouse somatic cell hybrid clone (20～No. 27), 20-27 hybrid cell system all increases and obtains the purpose fragment of 700bp.The PCR somatotype data of above-mentioned actual observation are submitted to HybWeb (http://www.toulouse.inra.fr/lgc/lgc.html/) carry out statistical study to obtain zone location information, the data analysis result is that the PSME3 assignment of genes gene mapping is on No. 12 karyomit(e)s of pig (P=1.000), further the zone location result is SSC12 p11-(2/3) p13 (P=0.8901, and the relation conefficient between this zone marker is 1.000, P＜0.1%).

Be 0,000,000,000 0,100,000,110 0,000,001,000 00,000,000,000,000,001,000 1,001,000,001 0,000,001,010 1,000,101,000 0,000,100,001 0,000,000,101 0,010,000,001 10111100 in IMpRH somatotype result (wherein 0 and 1 explain respectively amplification negative and positive) with the PSME3-P primer.Statistic analysis result, the GH gene close linkage on No. 12 karyomit(e)s of PSME3 gene and pig, the LOD value is 6.52, the RH map distance is 0.58Ray.Confirm that further this gene is positioned on No. 12 karyomit(e)s of pig.

3.PCR-RFLP diagnostic method is set up

Is 919bp with PSME3 CATS-1 primer at the fragment length that the painted face in Beijing opera pig genomic dna that is used for the PSME3 gene isolation increases, and two ends are respectively that the length of inferring is the partial sequence of the 5th and the 6th exon of 35bp and 97bp.With this PSME3 CATS-1 primer augmentation detection in several pig variety genomic dnas, found that to have length polymorphism.Sequencing result analysis revealed, the reason that causes length polymorphism are because in the 5th intron zone one segment length to be arranged be the fragment deletion (as shown in Figure 2) of 147bp.The 1066bp pcr amplified fragment that will include the insertion of 147bp fragment is decided to be allelotrope 1, and the 919bp fragment is decided to be allelotrope 2.

With primer PSME3 CATS-2 amplified production length in the pig genome is 835bp, comprises the partial sequence and intron 6 sequences of exon 6 and part exon 7 sequence and intron 5.Enzyme is cut detection and is found that a HhaI restriction enzyme site in intron 5 has polymorphism, respectively two the homozygous sequencing results in this site are shown, if when the segmental 257bp of this 835bp A257 of place, then do not have the HhaI restriction enzyme site, detected result has only a 835bp fragment (being decided to be allelotrope A); But there is the replacement of A257 → G257 in this site in colony, and its result causes the generation of a HhaI restriction enzyme site, obtains two fragments, and length is respectively 578bp and 257bp (being decided to be allelotrope G), three kinds of frequency of genotypes AA, and AG, GG are as shown in Figure 3.

Obtain 772bp specific amplified fragment with PSME3 CATS-3 amplification pig genomic dna, comprise exon 7,8,9 and the partial sequence of part exons 10 sequence and intron 6 and the sequence of intron 7,8,9.There is four AluI restriction enzyme sites (AG ↓ CT) in found that in this 772bp fragment of order-checking, wherein the 404bp place is the polymorphism point of contact, be arranged in exon 8, respectively two the homozygous sequencing results in this site are shown again and state, when the 406bp position is C406, then this AluI restriction enzyme site does not exist, and the AluI enzyme is cut the back detected result and had only 4 fragments, and length is 502bp, 123bp, 83bp and 64bp (being decided to be allelotrope B); But when having the replacement of C406 → T406, its result causes the generation of the AluI restriction enzyme site in 404bp place, obtains 5 fragments, length is respectively 404bp, 123bp, 98bp, 83bp and 64bp (being decided to be allelotrope A), three kinds of frequency of genotypes AA, AB, BB are as described in Figure 4.

4. mark property association analysis

Pig PSME3 gene the 8th exon AluI-RFLP pleomorphism site and part producing proterties are carried out association analysis, and the proterties of being analyzed is birth weight, weaning weight and the 170 ages in days thickness of backfat when butchering.

Genotype detection result shows that accounting for most is the AA genotype in 201 individualities, there are 182, the AB genotype has 18 individualities, the simple mean of proterties and standard deviation analytical results are summarized in table 4 between AA and AB genotype, analytical results shows, AA and the AB genotype thickness of backfat when 170 ages in days are butchered is significant difference (P＜0.05), and there is relevant trend (P=0.11) in this site with birth weight.

Table 4: the association analysis of different PSME3 gene A luI-RFLP genotype and part producing proterties

Genotype Genotypes	Number of individuals N	Birth weight Birth weight (kg)	Weaning weight Weaning weight (kg)	The thickness of backfat 1 Backfat thickness 1 (mm)
					AA AB P value P-value	182 18	1.5±0.25 1.43±0.23 0.11	10.55±1.52 10.74±1.72 0.5	11.09±1.97 11.98±2.12 0.047

¹Thickness of backfat when 170 ages in days are butchered

Sequence table, accompanying drawing and explanation thereof:

Sequence table SEQ ID NO:1 is the cDNA sequence of the present invention's fat thickness at back of pig PSME3 gene of cloning;

Sequence table SEQ ID NO:2 is the dna sequence dna of the present invention's fat thickness at back of pig PSME3 gene of cloning;

Sequence table SEQ ID NO:3 is that the present invention detects the forward primer sequence as 72bp place deletion mutantion among the sequence table SEQ ID NO:2;

Sequence table SEQ ID NO:4 is that the present invention detects the reverse primer sequence as 72bp place deletion mutantion among the sequence table SEQ ID NO:2;

Sequence table SEQ ID NO:5 is that the present invention detects the forward primer sequence as 619bp place base mutation among the sequence table SEQ ID NO:2;

Sequence table SEQ ID NO:6 is that the present invention detects the reverse primer sequence as 619bp place base mutation among the sequence table SEQ ID NO:2;

Sequence table SEQ ID NO:7 is that the present invention detects the forward primer sequence as 1425bp place base mutation among the sequence table SEQ ID NO:2;

Sequence table SEQ ID NO:8 is that the present invention detects the reverse primer sequence as 1425bp place base mutation among the sequence table SEQ ID NO:2.

Fig. 1: be the schema of the present invention about the preparation of PSME3 gene;

Fig. 2: the sequencing result who is PSME3 gene 72bp place deletion fragment among the present invention, represent exon and the intron zone inferred with capitalization and lowercase respectively among the figure, 5 '-and two conservative Nucleotide (GT/AG) of 3 ' splicing site mark with double underline, the deletion fragment sequence of 147bp represents that with underscore what show in the square frame is primer sequence.

Fig. 3: three kinds of genotype (the AA AG GG) electrophoresis result that is the HhaI-RFLPs at pig PSME3 gene 619bp place among the present invention.M:DNA molecular weight standard (100bp ladder)

Fig. 4: three kinds of genotype (the AA AB BB) electrophoresis result that is the AluI-RFLPs at pig PSME3 gene 1425bp place among the present invention.M:DNA molecular weight standard (100bp ladder)

Embodiment

Embodiment 1: the association analysis of pig mark property

In the commercial group of Large White pig PSME3 gene the 8th exon AluI-RFLP pleomorphism site and part producing proterties are carried out association analysis, the proterties of being analyzed is birth weight, weaning weight and the 170 ages in days thickness of backfat when butchering.

Genotype detection result shows that accounting for most is the AA genotype in 201 individualities, there are 182, the AB genotype has 18 individualities, the simple mean of proterties and standard deviation analytical results are summarized in table 5 between AA and AB genotype, analytical results shows, AA and the AB genotype thickness of backfat when 170 ages in days are butchered is significant difference (P＜0.05), and there is relevant trend (P=0.11) in this site with birth weight.

Table 5: the association analysis of different PSME3 gene A luI-RFLP genotype and part producing proterties

Genotype	Number of individuals	Birth weight (kg)	Weaning weight (kg)	The thickness of backfat 1 (mm)
					AA AB P value P-value	182 18	1.5±0.25 1.43±0.23 0.11	10.55±1.52 10.74±1.72 0.5	11.09±1.97 11.98±2.12 0.047

¹Thickness of backfat when 170 ages in days are butchered

Embodiment 2: the association analysis of pig mark property

In Tongcheng swinery pig PSME3 gene the 8th exon AluI-RFLP pleomorphism site and part producing proterties are carried out association analysis, the proterties of being analyzed is birth weight and reaches 90 kilograms of thickness of backfats when butchering.Analytical results shows that the genotypic thickness of backfat of BB and AB is significant difference (P＜0.05) (table 6)

Table 6: the association analysis of different PSME3 gene A luI-RFLP genotype and part producing proterties

Genotype Genotypes	Number of individuals N	Birth weight Birth weight (kg)	Thickness of backfat Backfat thickness 1 (mm)
				BB AB P value P-value	90 18	1.1±0.27 1.21±0.31 0.16	12.18±2.11 11.07±1.86 0.04

The distribution situation of embodiment 3:PCR-RFLP-AluI polymorphism in each pig variety

In 6 pig varieties, detect pig PSME3 gene PCR-RFLP-AluI polymorphism, detected result is as described in Table 7, the data analysis of table 7 shows to be stated, in these several pig varieties that detected, the frequency of dominant allelotrope A is respectively 0.8475 and 0.6406 in the fragrant pig in Du Luoke and Guizhou, and the frequency of advantage allelotrope B is respectively 0.9688,0.7884 and 0.6539 in painted face in Beijing opera, Tongcheng and the Large White, the frequency of equipotential gene A and allelotrope B is approaching in hiding pig, is respectively 0.4255 and 0.5745.

The genotype and the gene frequency of PSME3 gene A luI polymorphism in the several pig varieties of table 7

Kind Breeds	Number of individuals No:of animals	Genotype Genotype			Gene frequency Allele frequency
							AA	AB	BB	A	B
		Du Luoke Duroc hides pig Tibet painted face in Beijing opera Erhualian Tongcheng Tongcheng Guizhou Xiang pig Guizhou Xiangzhu greatly from Large white	36 27 32 26 32 13	28 6 0 1 14 4	5 11 2 9 13 1	3 10 30 16 5 8						0.8475 0.4255 0.0312 0.2116 0.6406 0.3461	0.1525 0.5745 0.9688 0.7884 0.3594 0.6539

The distributional difference of pig PSME3 gene A luI-RFLP pleomorphism site gene frequency in different varieties tested, and significance of difference result shows that there is difference largely in this locus gene frequency in these six pig varieties that detected.The distribution situation of embodiment 4:PCR-RFLP-HhaI polymorphism in each pig variety

Detect pig PSME3 gene PCR-RFLP-HhaI polymorphism in 5 pig varieties, detected result is as described in Table 8, and the data analysis of table 8 shows to be stated, and in these several pig varieties that detected, dominant is allelotrope G.

The genotype and the gene frequency of PSME3 gene HhaI polymorphism in the several pig varieties of table 8

Kind Breeds	Number of individuals No:of animals	Genotype Genotype			Gene frequency Allele frequency
							AA	AG	GG	A	G
		Du Luoke Duroc hides the peaceful Qingping plum mountain Meishan of pig Tibet painted face in Beijing opera Erhualian	22 22 22 27 15	0 1 5 0 0	0 8 10 13 1	22 13 7 14 14						0 0.227 0.455 0.241 0.033	1 0.773 0.545 0.759 0.967

Embodiment 5: the detected result of length polymorphism in the different swinerys

In the Large White group of 6 pig varieties and Dutch animal science and the establishment of health institute, detect the deletion mutantion of pig PSME3 gene the 5th intron, detected result is as shown in table 9, the data analysis of table 9 shows, in these several pig varieties that detected, the frequency of dominant allelotrope 1 is respectively 0.8475 and 0.6406 in Du Luoke and the fragrant pig, and painted face in Beijing opera, the frequency of advantage allelotrope 2 is respectively 0.9688 in Tongcheng and the Large White, 0.7884 and 0.6539, the frequency of allelotrope 1 and allelotrope 2 is approaching in hiding pig, be respectively 0.4255 and 0.5745, and the frequency of dominant allelotrope 1 is very high in the Large White group of Dutch animal science and the establishment of health institute, reaches 0.9545.

The distributional difference of pig PSME3 gene the 5th intron deletion mutantion locus gene frequency in different varieties carried out the t check, and analytical results shows that there is difference largely in this locus gene frequency in these 7 swinerys that detected.

Table 9: the genotype and the gene frequency of the deletion mutantion polymorphism of different swinery PSME3 gene the 5th introns

Table9.Genotypes and allele frequencies of length polymorphism caused by fragment deletion withinintron 5 for the porcine PSME3 gene in different populations

Kind Breeds	Number of individuals No.of animals	Genotype Genotype			Gene frequency Allele frequency
							11	12	22	1 (1066bp)	2 (919bp)
		Du Luoke Duroc hides the fragrant pig Xiangzhu of pig Tibet painted face in Beijing opera Erhualian Tongcheng Tongcheng Da Bai Large white Holland Large White group Large white (Netherlands)	36 27 32 26 32 13 187	28 6 0 1 14 4 171	5 11 2 9 13 1 15	3 10 30 16 5 8 1						0.8472 0.4259 0.0312 0.2116 0.6406 0.3461 0.9545	0.1528 0.5741 0.9688 0.7884 0.3594 0.6539 0.0455

Sequence table (SEQUENCE LISTING)

＜110〉Hua Zhong Agriculture University

＜120〉fat thickness at back of pig PSME3 gene and preparation method thereof

<130>

<141>2003-08-25

<150>02139236.6

<151>2002-11-01

<160>9

<170>PatentIn version 3.1

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＜213〉pig (Sus scrofa)

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ggagcaagtg agcagtgagt gggccagcga gagcggccgc gtcccgagag cagccgagat 60

tcctcccgtc cctctgaccc tctccccgga gcccacagcg cctccggtct ccagcggagc 120

ggacacggcc cggcccggcc atg gcc tcg ggg cgg gag gtg gat cag gaa gta 173

Met Ala Ser Gly Arg Glu Val Asp Gln Glu Val

1 5 10

aag ctc aag gtt gat tct ttc agg gag cgg att aca agt gag gca gaa 221

Lys Leu Lys Val Asp Ser Phe Arg Glu Arg Ile Thr Ser Glu Ala Glu

15 20 25

gac ttg gtg gca aat ttt ttc cca aag aaa ttg tta gaa ctt gat agt 269

Asp Leu Val Ala Asn Phe Phe Pro Lys Lys Leu Leu Glu Leu Asp Ser

30 35 40

ttt ttg aag gag cca atc cta aac atc cat gac cta act cag atc cac 317

Phe Leu Lys Glu Pro Ile Leu Asn Ile His Asp Leu Thr Gln Ile His

45 50 55

tca gac atg aat ctc cca gtc cct gac ccc att ctt ctc acc aat agc 365

Ser Asp Met Asn Leu Pro Val Pro Asp Pro Ile Leu Leu Thr Asn Ser

60 65 70 75

cat gat gga ctg gac ggt ccc act tay aag aag cga agg ttg gat gaa 413

His Asp Gly Leu Asp Gly Pro Thr Tyr Lys Lys Arg Arg Leu Asp Glu

80 85 90

tgt gaa gag gcc ttc caa gga aec aag gtg ttt gtg atg ccc aat ggg 461

Cys Glu Glu Ala Phe Gln Gly Thr Lys Val Phe Val Met Pro Asn Gly

95 100 105

atg cta aaa agc aac cag cag ctg gtg gac att att gag aaa gtg aag 509

Met Leu Lys Ser Asn Gln Gln Leu Val Asp Ile Ile Glu Lys Val Lys

110 115 120

cct gag att cgg ctg ctg atc gag aaa tgc aac acg gtc aaa atg tgg 557

Pro Glu Ile Arg Leu Leu Ile Glu Lys Cys Asn Thr Val Lys Met Trp

125 130 135

gta caa ctc ctg att cct agg ata gaa gat ggg aac aac ttt ggg gtg 605

Val Gln Leu Leu Ile Pro Arg Ile Glu Asp Gly Asn Asn Phe Gly Val

140 145 150 155

tcc att cag gaa gag aca gtt gca gaa tta aga act gtt gag agt gaa 653

Ser Ile Glu Glu Glu Thr Val Ala Glu Leu Arg Thr Val Glu Ser Glu

160 165 170

gca gcc tct tat ctg gac cag att tct aga tat tat att aca aga gcc 701

Ala Ala Ser Tyr Leu Asp Gln Ile Ser Arg Tyr Tyr Ile Thr Arg Ala

175 180 185

aaa ttg gtt tct aaa ata gct aaa tac ccc cat gtg gag gac tat cgg 749

Lys Leu Val Ser Lys Ile Ala Lys Tyr Pro His Val Glu Asp Tyr Arg

190 195 200

cgc acc gtg aca gag att gat gag aaa gaa tat atc agc ctt cgg ctc 797

Arg Thr Val Thr Glu Ile Asp Glu Lys Glu Tyr Ile Ser Leu Arg Leu

205 210 215

atc ata tca gag cta agg aat cag tat gtc act cta cat gac atg atc 845

Ile Ile Ser Glu Leu Arg Asn Gln Tyr Val Thr Leu His Asp Met Ile

220 225 230 235

ctg aaa aac atc gag aag atc aaa cgg ccc cgg agc agc aat gca gag 893

Leu Lys Asn Ile Glu Lys Ile Lys Arg Pro Arg Ser Ser Asn Ala Glu

240 245 250

aca ctg tac tgaggccagg gccagggcca ggggactctg tgagtctggc 942

Thr Leu Tyr

tcaagaccga cattgccttg gtttgttaca tgactatcgt gatggggaaa ctggctggaa 1002

atagtaatca cacctctctc tgtttttagt tagagtctaa tgaaactctc atgtagttct 1062

gtgacgtgtt tacctctttt ttcaggcctc aggaactctt ctatttcctt ccctcatacc 1122

ccagaccccc gtcctaattt ctggagtact ccaggtttgt gtgcgcagga tgttggcaca 1182

agtttacttg tgttttctct ccccacttcc cttctgtgtg ttgggcttta tgttttcttc 1242

cctttgatgt tcagttggtt agaagttgag ggaacttgaa ggaaagtgct aggtattttg 1302

taggaactgg ggctggatgg gggaaceagc ttctctcttc ctgatggcag gttagtatct 1362

cttgcctctg tgggacactg gatcctccct gccacagttg ccctggtgat gacttagggt 1422

ttcccatcca catacatgac accttgaacc tgatcacgac aagaaacacc ttagaccttc 1482

tgccaagtcc ctagctcctt cagatatttt tataagtagg attttcacat acacaaatgt 1542

gcatctcaca cacacacaca cacacgtaca tgcgcgccct ccgactctgt tacttgatgc 1602

ctcccccact ctgccccacc cacagacctt gtctgtcagt ctgtctgtgt ctcacacaca 1662

catacaccgt tcagaaggtg atagttgttt aagtccttat ctgctgggac ccattgtctc 1722

ctgacattgt agcctcttgg tgcttgaggc tggtgggttg gaagacagct gagagatgag 1782

aagcagaggg agtggggaag gccccttcct ctgtgactca ggtccttttg gacgagttga 1842

aattggtgtg ggggtgacca gaagctgctg ctcctactgg gctttcccgt tgggaaaaat 1902

gtttctctta tttatagtgt tgtgcttcca gggaaagcag tcacttgtgt gtgtatgtgt 1962

gtgcatgcac acgtaggcat gtacacgttg tgtgtatgtg gacatctgtt gggcaagtca 2022

aaaccataga agagttgcct cttatctctc gaatcgtctg gagatatcac ttgttacagc 2082

ttttgtgtta accctcatca acccccactt ttttattctc accactggag agagagtctg 2142

ttggtgactc tctcggtgtc taattctgac ttttattctt tgtcctgtct ctgtcttcca 2202

acctccaatc atctttccac ctggatgcat catctttact cccagtgcgc tgtagagaag 2262

gagttcgggt gtggtcttcc cgtgaatagt actcagtaac aaaccaattg cattttagtt 2322

gggcagtgct cctacccacc ctcagacccc tttcaacgaa aaccctccct tcctcccaac 2382

atgtgtttct cagtttccct ttttgtttgt tggattgttc cagtgcccct gctccccacc 2442

ttaccaccct tggatcgtaa tgtaaaattc ttttaccatg tcaagaaatt attaaaaaca 2502

caggtacttt gacctctttc taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaacca 2562

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2592

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20 25 30

Phe Phe Pro Lys Lys Leu Leu Glu Leu Asp Ser Phe Leu Lys Glu Pro

35 40 45

Ile Leu Asn Ile His Asp Leu Thr Gln Ile His Ser Asp Met Asn Leu

50 55 60

Pro Val Pro Asp Pro Ile Leu Leu Thr Asn Ser His Asp Gly Leu Asp

65 70 75 80

Gly Pro Thr Tyr Lys Lys Arg Arg Leu Asp Glu Cys Glu Glu Ala Phe

85 90 95

Gln Gly Thr Lys Val Phe Val Met Pro Asn Gly Met Leu Lys Ser Asn

100 105 110

Gln Gln Leu Val Asp Ile Ile Glu Lys Val Lys Pro Glu Ile Arg Leu

115 120 125

Leu Ile Glu Lys Cys Asn Thr Val Lys Met Trp Val Gln Leu Leu Ile

130 135 140

Pro Arg Ile Glu Asp Gly Asn Asn Phe Gly Val Ser Ile Gln Glu Glu

145 150 155 160

Thr Val Ala Glu Leu Arg Thr Val Glu Ser Glu Ala Ala Ser Tyr Leu

165 170 175

Asp Gln Ile Ser Arg Tyr Tyr Ile Thr Arg Ala Lys Leu Val Ser Lys

180 185 190

Ile Ala Lys Tyr Pro His Val Glu Asp Tyr Arg Arg Thr Val Thr Glu

195 200 205

Ile Asp Glu Lys Glu Tyr Ile Ser Leu Arg Leu Ile Ile Ser Glu Leu

210 215 220

Arg Asn Gln Tyr Val Thr Leu His Asp Met Ile Leu Lys Asn Ile Glu

225 230 235 240

Lys Ile Lys Arg Pro Arg Ser Ser Asn Ala Glu Thr Leu Tyr

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gcg aag gtt gga tga atg tga aga ggc ctt cca ag gtaagagcca 45

Ala Lys Val Gly Met Arg Gly Leu Pro Arg

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ccttcactgt cccctccccc aacccttttc tttatagggc cgcactcacg gcatatggag 105

gttcccaggc taggggtctg atcagagctg ttgctgctgg cctacaccac agtcacagca 165

atgccaggtc tgagccgcat cttcgaccta caccacagct catggcaacg ccagatcctt 225

taacccactg agcaaggcca gggattgaac tcacaacctc atgggtccta gttggattgt 285

ttccactgtg ccaggactgg aactccgtaa ttttccattt aacagggagg gttggactta 345

atgtgaacta ccaagggaga ggttggaggt ggctacacat cacagatttt tcgcaaaacc 405

ccagttcata tcattttttc ctcttcaata atgcttggtg cgttaagtct gtagagcata 465

ttttaatctt gttctttaga ctttccggat gttttagaca ctcatctgtt aatccccata 525

ttctctcttt ttttaattct gttggtggtg tcacggttta tagatagctt tgtgacagat 585

aaaaggcaga agtcataagc ttttccttgt ggcgcagtgg gttaaggatc cggtattgtc 645

actgttgtgg ctcaggtcgc tactgtggag cccaggatct tgcacatgcc acaggcatgg 705

ccaggaacaa aggtagaggt catagtatct cacgggtcta ggaaagagag aaaggtgggg 765

aatgctgcct ccttccaccc agctccagtc atttgacctt cctcgtgtcc ttgccag g 823

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Asn Gln Gly Val Cys Asp Ala Gln Trp Asp Ala Lys Lys Gln Pro Ala

15 20 25

gct ggt gga cat tat tga gaa agt gaa gcc tga gat tcg gct gct gat 919

Ala Gly Gly His Tyr Glu Ser Glu Ala Asp Ser Ala Ala Asp

30 35 40

cga gaa atg caa cac g gtgaggcagg ggctgatgac ctaggggctg accccaaata 975

Arg Glu Met Gln His

45

gcctggcttg agctatgcaa gacaatggat gtggggccag aggcatggag gtgatgttgg 1035

agttggcaga ctaaatatct tgactcctgt gctcctggca tctggcccca gcagcatgag 1095

ttgtatatgt cttggcttaa gtagaaaagg ctaagatcct catatgcata ttttggtccc 1155

ctcacccttc ag gt caa aat gtg ggt aca act cct gat tcc tag gat aga 1205

Gly Gln Asn Val Gly Thr Thr Pro Asp Ser Asp Arg

50 55

aga tgg gaa caa ctt tgg ggt gtc cat tca g gtaatgtgct tgcattggaa 1256

Arg Trp Glu Gln Leu Trp Gly Val His Ser

60 65

atgggagagg aaggtttggg aggtaatctc atctcccctg cttttgtgtt ttccctttct 1316

ctttggtttg ctgtggggtg atgtggaagt ggctttcaac ctttttgttt tgtttttttt 1376

tttcag ga aga gac agt tgc aga att aag aac tgt tga gag tga agc 1423

Gly Arg Asp Ser Cys Arg Ile Lys Asn Cys Glu Ser

70 75

agc ttc tta tct gga cca gat ttc tag gtagggctgc ttggacttgg 1470

Ser Phe Leu Ser Gly Pro Asp Phe

80 85

cccaggaatt gagggctggt gagatggcat gcgctgtctg gggtcccctg cagctttctc 1530

ctcttctct ctc ctt cca gat att ata tta caa gag cca aat tgg ttt cta 1581

Leu Leu Pro Asp Ile Ile Leu Gln Glu Pro Asn Trp Phe Leu

90 95 100

aaa tag cta aat acc ccc atg tg gtaagtaagg gtttgtggcc gagggtggaa 1634

Lys Leu Asn Thr Pro Met Trp

105

gtggtaggac agggaggcat gccttcctgg gaagagctgc gtgggacagt cagccctcga 1694

ggactagcct tttctctgcc cttcgtcag g agg act atc ggc gca ccg tga cag 1748

Arg Thr Ile Gly Ala Pro Gln

110 115

aga ttg atg aga aag aat ata tca gcc ttc ggc tca tca tat cag 1793

Arg Leu Met Arg Lys Asn Ile Ser Ala Phe Gly Ser Ser Tyr Gln

120 125 130

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＜213〉pig (Sus scrofa)

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gcgaaggttg gatgagtgtg 20

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ggaggtgatg ttggagttgg 20

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ctgatatgat gagccgaagg 20

Claims (12)

1, a kind of fat thickness at back of pig PSME3 gene, its cDNA sequence is as described in the sequence table SEQ ID NO:1.

2, gene according to claim 1, its dna sequence dna is as described in the sequence table SEQ ID NO:3.

3, cDNA sequence according to claim 1 is characterized in that: the PSME3 gene cDNA sequence length that is obtained is 2592bp, comprises the open reading frame of 762bp in the sequence, the 3 ' non-translational region of 140bp 5 ' non-translational region and 1690bp.

4, cDNA sequence according to claim 1 is characterized in that: have a base as the described C659-T659 of sequence table SEQ ID NO.1 to replace in the PSME3 gene cDNA sequence that is obtained.

5, gene according to claim 2 is characterized in that: one section deletion mutantion from the common 147bp of 218 bases of the 72nd base to the is arranged among the sequence table SEQ ID NO:3.

6, gene according to claim 2 is characterized in that: have the base of 1 A619-G619 to replace among the sequence table SEQ ID NO.3.

7, gene according to claim 2 is characterized in that: have the base of 1 C1425-T1425 to replace among the sequence table SEQ ID NO:3.

8, gene according to claim 2 is characterized in that: be used for detecting as two primer sequences of sequence table SEQ ID NO:3 72bp place's deletion mutantion as described in sequence table SEQ ID NO:4 and the SEQ ID NO:5.

9, gene according to claim 2 is characterized in that: be used for detecting as two primer sequences of sequence table SEQ ID NO:3 619bp place base mutation as described in sequence table SEQ ID NO.6 and the SEQ ID NO:7.

10, gene according to claim 2 is characterized in that: be used for detecting as two primer sequences of sequence table SEQ ID NO:3 1425bp place base mutation as described in sequence table SEQ ID NO:8 and the SEQ ID NO:9.

11, the method for preparation dna sequence dna as claimed in claim 1 or 2, its feature is according to following steps:

(1) personnel selection PSME3 gene cDNA is the information probe, does the homologous sequence screening, obtains the expressed sequence tag of homology more than 80%; Splice pig EST-contig then; According to two pairs of primers of EST-contig design, amplification obtains two bar segment; Be the 3rd pair of primer of stencil design with people PSME3 gene cDNA and pig EST-contig respectively again, amplification obtains three bar segment; Extracting total RNA with the spleen tissue of the fragrant pig that grows up is template, makes RT-PCR amplification and 3 ' RACE, and PCR product purification and cloning and sequencing carry out sequential analysis, and acquisition is as the described cDNA sequence of sequence table SEQ ID NO:1;

(2) extracting DNA from the pig blood genome, is template design primer with pig PSME3 gene cDNA sequence, carries out pcr amplification, and PCR product purification and cloning and sequencing obtain as dna sequence dna as described in the sequence table SEQ ID NO:3.

12, use PCR and PCR-RFLP method and detect the 619th of pig PSME3 gene, 1425 base replacement and the deletion mutantion of the 72nd 147bp of bit base place.

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