CN1300313C - Clone of gene MAC30 related to pig backfat thickness and its use in mark-assisted selection - Google Patents

Abstract

The present invention discloses a method for detecting a site of the mononucleotide polymorphism of a gene MAG30 which is relevant to the back-fat thickness of a pig, and also discloses the application of the method, which is used as molecular mark for auxiliary selection in genetic breeding of the pig. The DNA sequence of a gene of a partial genome cloned from the gene MAG30 relevant to the back-fat thickness of a pig is shown in a sequence table SEQ ID No. 1, and the length of the sequence is 970 bp, wherein the gene comprises partial exon 2 sequences and partial exon 3 sequences which are disclosed in the specification 2. The 848 bp position of the sequence has one base mutation (848G-848A) which results in that the coded amino acid of the base mutation is changed into isoleucine from valine (GTC-ATC). A user respectively and relatively detects piglets which are native to China and piglets with European lineage by using the present invention, and confirms the effect of the present invention, which is used for genetic auxiliary selection of the pig. The present invention also provides a method for preparing the gene and detecting application.

Description

The clone of fat thickness at back of pig genes involved MAC30 and the application on marker assisted selection thereof

Technical field

The invention belongs to the domestic animal gene engineering technology field, relate in particular to the clone of fat thickness at back of pig genes involved MAC30 and the application on marker assisted selection thereof.

Background technology

China is the individual big country of raising pigs, and also is the abundantest country of pig kind resource, begins very early wild boar is domesticated for tame pig.Select through long-term, formed many local variety that differ from one another, what only " Chinese Pigs kind will " (opening secondary Pueraria lobota, Science and Technology of Shanghai press, 1986) put down in writing just has 48.Compare with external market pig, the place of china kind has many outstanding advantages: meat is good, and litter size height, good stress resistance and adaptability are strong or the like, but two common shortcomings are arranged, and that is exactly that lean ratio is low and weightening finish is slow.Therefore, increase the major objective that the carcass lean meat percentage and the raising speed of growth are the genetic improvement work of China man pig always.

Carcass trait mainly comprises following two aspects: be the metric of each component of trunk on the one hand, heavily wait as back-fat thickness, eye muscle area, trunk length, small intestine length and various internal organ; Be each weight percent of forming on the other hand, as dressing percentage, leg stern ratio, leg stern meat bone rate, leaf fat rate and lactones rate etc.

Many carcass traits belong to quantitative character, by controlled by multiple genes and there is key-gene (major gene) effect.Because carcass trait performance evening and inconvenient somatometry, it is long to adopt the conventional breeding method that it is carried out selection cycle, produces effects slowly.The development of Protocols in Molecular Biology, make people can seek the key-gene or the molecule marker closely linked of control carcass trait at dna level with it, in breeding process, be used for marker assisted selection (marker assisted selection, MAS), select progress so that improve, improve carcass quality better, satisfy people's needs, obtain bigger economic benefit simultaneously.

By the candidate gene method, found some to influence the gene of the carcass quality of pig.Be positioned at growth hormone gene (the Growth Hormon on No. 12 karyomit(e)s of pig, GH) be the core gene of regulating and control animal growth in the neurosecretion in the growth axis, its product tethelin has the effects such as metabolic, that promotion is grown of regulating, be the main candidate relevant with carcass trait with the growth of pig, Chinese and overseas scholars has carried out extensive studies to its genotype and the relation of the production traits.(Knorr C etc. such as Knorr, Associations of GHgene variants with performance traits in F2 generations of European wild boar, Pietrain and Meishan pigs.Anim Genet, 1997,28:124-128) find F2 that plum mountain pig and Pietrain pigs hybridize, different GH genotype and 8 carcass trait significant correlations in the colony.(Li Jiaqi etc. such as Li Jiaqi, the IFG-1 gene pairs is grown the hereditary effect analysis of white * blue pool pig resource population production performance, Acta Genetica Sinica, 2003,30 (9): 835-839) find long white * resource colony that blue pool pig makes up in insulin-like growth factor 1 (Insulin-like growth factor I, IGF-I) different genotype has remarkably influenced to proterties such as daily postweaning gain, bone rate, carcass lean yield and sebum rates.(Liu Guilan etc. such as Liu Guilan, the association analysis of IGF2 gene PCR-RFLP polymorphism and fatty deposits correlated character, Acta Genetica Sinica, 2003,30 (12): 1107-1112) analyzed insulin-like growth factor 2 (Insulin-like growth factor II, IGF-II) polymorphism of two NciI restriction enzyme sites of gene the 8th intron is found B site B in the polymorphism distribution situation of F2 in generation that Large White * plum mountain pig constitutes ¹B ¹Genotypic individuality is significantly than B ²B ²Genotypic individual back fat approaches 18.28% (P＜0.01), lean ratio high by 8.71% (P＜0.01).Hypophysis transcription factor (Pituitan transcription factor 1, PIT1) be tethelin, the regulatory factor that prolactin and thyrotropin are important, (Yu TP etc. such as Yu, Association of PIT1 polymorphisms with growth and carcass traits inpigs.J Anim Sci, 1995,73:1282-1288) reported once that there were significant correlation in the PIT1 and the average thickness of backfat, (Kuryl J etc. such as Kuryland in recent years, Association of POU1F1/RsaI genotypes with carcass traits in pigs.J Appl Genet, 2001,42:309-316) and (Brunsch C etc. such as Brunsch, Analysis of associations of PITl genotypes with growth, meatquality and carcass composition traits in pigs.J Appl Genet, 2002, it is relevant with several carcass traits 43:85-91) to detect PIT1 respectively in different resource familys.Other candidate gene relevant with carcass trait also comprises somatoliberin (GHRH), leptin (Leptin), growth hormone (Ghrelin), myogenin (Myogenin), Somat (somatostatin, SS), melanocyte cortical hormone receptor 4 (Melanocortin-4, MC4R) and growth hormone receptor (growth hormone receptor, GHR) (XiaD etc., Developmental patterns of GHr and SS mRNA expression in porcine gastric tissue.World JGastroenterol, 2003,9:1058-1062), MSTN (Jiang YL etc., Identification of three SNPs in the porcinemyostatin gene (MSTN) .Anim Biotechnol, 2002,13:173-178) wait gene.Being positioned at No. 18 leptin genes (Leptin) on the karyomit(e) is considered to and growth, gene (the Sasaki S etc. that carcass trait is relevant, Assignment of the porcine obese (leptin) gene to chromosome 18 by linkage analysis of a new PCR-based polymorphism.Mamm Genome, 1996,7:471-472), but (Genetic polymorphisms in the leptin gene and their associationwith fatness in four pig breeds.Mamm Genome such as Jiang ZH such as Jiang, 1999, research 10:191-193) is not but found significantly related.The myogenin (Myogenin) that is positioned on No. 9 karyomit(e)s of pig is the member of MyoD gene family, and major function is to be adjusted to the myocyte to be divided into myofiber.(te Pas MF etc. such as Te Pas, Influences of myogenin genotypes on birth weight, growth rate, carcass weight, backfat thickness, and lean weight of pigs.J Anim Sci, 1999,77:2352-2356) in two Large White groups, detected the polymorphism of this gene, analyzed that the individuality of finding different genotype is heavy in birth, there were significant differences on the proterties such as the speed of growth and cutability.The MyoD gene family member mRNA expression level that two selections are muscle is relatively found myogenin in the F-system (the selection speed of growth), myf-5, the mRNA expression level of MyoD1 is than L-system (selection lean ratio) height, in F-system, the thickness of backfat and myoblastic negative correlation (the te Pas MF etc. that are expressed as, Messenger ribonucleic acid expression of the MyoDgene family in muscle tissue at slaughter in relation to selection for porcine growth rate.J Anim Sci, 2000,78:69-77).(Kim KS etc. such as Kim, A missense variant of the porcine melanocortin-4receptor (MC4R) geneis associated with fatness, growth, and feed intake traits.Mamm Genome, 2000,11:131-135) in 5 pig commodity systems, detect variation of MC4R gene genetic and thickness of backfat significant correlation.(Liu Guilan etc. such as Liu Guilan, pig resource family MC4R genescan and with the correlation analysis of fatty character, Acta Genetica Sinica, 2002,29 (6): 497-501) to the F2 of Da Bai * Mei Shan generation 174 individual the detection find that fat thickness (P＜0.05), buttocks fat thickness (P＜0.02), the average thickness of backfat (P＜0.04), eye muscle width (P＜0.003), eye muscle area (P＜0.05), skin rate (P＜0.02) are remarkable relevant between MC4R gene pleiomorphisms and pig chest lumbar vertebrae.

Adopt the genome scanning method, investigators have also located some influences the quantitative trait locus (QTL) of carcass trait.The key-gene that influences the thickness of backfat is located in (de Koning etc. on the 2nd and No. 7 karyomit(e), Detection of quantitative trait loci for backfatthickness and intramuscular fat content in pigs (Sus scrofa) .Genetics, 1999,152:1679-1690).(Paszek AA etc. such as Paszek, Interval mapping of carcass and meat quality traits in a divergent swine cross.AnimBiotechnol, 2001,12:155-165) utilize 119 molecule markers that the trunk and the meat proterties of 116 F2 individualities are analyzed the QTL that the back is found to exist influences a plurality of proterties such as trunk length, the 10th rib thickness of backfat, the average thickness of backfat, leaf fat rate, eye muscle area and intramuscular fat content on No. 12 karyomit(e)s of pig.Recently, (Clop A such as Clop, Deng, Detection of QTL affecting fatty acidcomposition in the pig.Mamm Genome, 2003,14:650-656) F2 to Iberian pig and landrace structure carries out genome scanning, finds to have the QTL that influences unsaturated fatty acids (linolenic acid) content on No. 12 karyomit(e)s.(Yue G etc. such as Yue, Linkageand QTL mapping for Sus scrofa chromosome 12.Journal of Animal Breeding and Genetics, 2003,120:95-102) three F2 colonies that constitute with wild boar, plum mountain pig and Pietrain are research object, have found that on No. 12 karyomit(e)s several influence the QTL of proterties such as the intercostal thickness of backfat of 13-14, lean meat cutting rate.According to statistics, except SSC16 and SSC17, all found to influence the QTL (Bidanel and Rothschild, 2002) of the thickness of backfat on all karyomit(e)s of pig.

The applicant is devoted to the isolation identification work of critical function gene on No. 12 karyomit(e)s of pig for a long time, and certain achievement (Yu M etc. have been obtained, Isolation, physical mapping and polymorphism of the porcine ferredoxin reductase (FDXR) gene.Anim Genet, 2002,33:394-395; Yu M etc., Physical mapping of the rod cGMP-phosphodiesterase-subunit (PDE6G) gene to pig chromosome 12.Anim Genet, 2003,34:76-77).

MAC30 (meningioma associate protein 30, MAC30) gene is one of gene of the overexpression at first found in the meningioma cell, it is one of conjugated protein family member of insulin-like growth factor, participation is to adjusting (the Murphy M etc. of insulin-like growth factor (IGF), Identification and characterization of genes differentially expressed in meningiomas.Cell Growth Differ, 1993,4:715-722).The genome research result of people and mouse is positioned the MAC30 gene for people's 17 (17q11) number karyomit(e) and No. 11 karyomit(e)s of mouse respectively.The increase genomic DNA fragment of MAC30 of pig of applicant, and utilize RH clone plate that it is positioned at (12q13 on No. 12 karyomit(e)s of pig, do not deliver), result (the GoureauA etc. that meet comparative genomics research, Human and porcine correspondence of chromosome segments using bidirectional chromosomepainting.Genomics, 1996,36:252-262).(Malhotra K etc. such as Malhotra, Identification of differentiallyexpressed mRNAs in human fetal liver across gestation.Nucleic Acids Res, 1999,27:839-847) find that by differential display technique (DDRT-PCR) the MAC30 gene has different expression patterns in the liver cell of embryonic liver cell and adult, hint that it may play an important role in the growth of body and atomization.But up to the present, still do not see the report of comprehensive research MAC30 gene function.The polymorphism of research mutational site in colony, and carry out the very strong means that the proterties association analysis is the research gene function.So the applicant has carried out polymorphic research and association analysis to the exon of the part of this gene, in the hope of finding its function.

Summary of the invention

The portion gene group dna sequence dna that the objective of the invention is to clone pig MAC30 gene, the detection method of searching gene mutation site and polymorphism, the relevant molecule marker of screening fat thickness at back of pig proterties.

The present invention is achieved through the following technical solutions:

A kind of fat thickness at back of pig genes involved MAC30, its dna sequence dna is as described in the sequence table SEQ ID NO:1.

The MAC30 genome sequence total length of pcr amplification is 970bp, wherein comprises the exon 3 sequence as accompanying drawing 2 described part exon 2s and part.

There is a base mutation (848G-848A) at 848bp place at sequence table SEQ ID NO:1.

The dna sequence dna of the primer that clone MAC30 gene is used is as follows:

5 '-the ATACCCAGGAGTTCAAAGACACT-3 forward), '

5 '-GAGAGGGATGAGAAAGTAGGGGA-3 ' (oppositely);

The dna sequence dna of the forward and reverse primer of detection 848G-848A base place's sudden change is as follows:

5 '-GGGAGGAGTCTCTGTGTCGTGTGT-3 ' (forward primer)

5 '-GAGAGGGATGAGAAAGTAGGGGA-3 ' (reverse primer)

The dna sequence dna that is used for the direct primer that checks order of PCR product is as follows:

5 '-TAGTCGTTCGTGGAAAGTCGTAGGTC-3 ' (reverse primer)

A kind of screening is applicable to the molecular marker method of fat thickness at back of pig proterties, prepares according to following steps:

Personnel selection MAC30 gene cDNA is the information probe, does the homologous sequence screening, obtains the expressed sequence tag (EST) of homology more than 90%; Then EST is spliced; Roughly draw the splicing site of exon, design of amplification primers according to comparison result with the genome full length DNA sequence of people MAC30 gene; Extract DNA from the pig blood genome, pcr amplification, PCR product purification and cloning and sequencing obtain the nucleotide sequence shown in sequence table SEQ ID NO:1.

The method that application PCR product directly checks order detects the polymorphism in pig MAC30 gene 848G-848A site, the association analysis between the part producing proterties of go forward side by side Xingqi genotype and pig.The present invention provides a new genetic marker for the molecular breeding of pig.

According to the present invention, fat thickness at back of pig genes involved MAC30 can be used for the pig marker assisted selection.

Detailed technology details of the present invention is as described below:

1.MAC30 the clone of Gene Partial dna sequence dna

(1) design of primers:

(the GenBank number of including: XM_031536) be the information probe of personnel selection MAC30 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 90%, the number of including of these ESTs is inquired about corresponding sequence with ENTREZ (document is seen http://www.ncbi.nlm.nih.gov/Web/Search/index.html) in NCBI, use the SeqMan program construction pig EST-contig among the sequence analysis software DNAStar then.According to EST splicing sequences Design amplimer.Sequence is as follows:

5 '-ATACCCAGGAGTTCAAAGACACT-3 ' (forward),

5 '-GAGAGGGATGAGAAAGTAGGGGA-3 ' (oppositely)

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

The purifying of PCR product: under ultraviolet lamp, contain the segmental gel of purpose from the cutting-out of low melting-point agarose gel, put into the 1.5mlEpendorff pipe, being incubated to gel in 70 ℃ melts fully, use PCR product purification test kit (Promega) purified pcr product then, operate according to the test kit specification sheets, concrete steps are to add 1ml Resin reagent in the gel that per 300 μ l melt, mixing 20s, with the Resin/DNA mixture syringe of packing into, slurries are extruded by the Minicolumn in the test kit.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.

Ligation: purifying RACE product is connected with pGEM-T easy carrier (available from promega company), and the ligation cumulative volume is 5 μ l, comprising 2.5 μ l, 2 * buffer, 0.5 the T carrier of μ l, 1.5 the purified pcr product of μ l, the T4 ligase enzyme of 0.5 μ l is put 16 ℃ of water-baths and is spent the night.

The preparation of competent cell: the single colony inoculation of DH5 α of picking is in 2ml LB from 37 ℃ of fresh flat boards of having cultivated 16～20h, in 37 ℃ of shaking culture 3h, switching 1ml bacterium liquid is in the saline bottle that contains 30ml LB, continuation is at 37 ℃ of about 4h of shaking culture, treat that OD600 reaches at 0.3～0.4 o'clock saline bottle is put ice bath cooling 10～15min from the shaking table taking-up, 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 to abandon clean nutrient solution, is iced the CaCl of the 0.1mol/L of precooling with 10ml 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.

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, 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 a small amount of preparation of plasmid: the single bacterium colony on the picking flat board is inoculated among the 2-3ml LB 37 ℃ of 300r/min overnight incubation.With the centrifugal several seconds collection of 1.5mlEP pipe 12000r/min thalline.Every pipe adds the ice-cold solution I of 100 μ l [50mM glucose, 25mM Tris.Cl (pH8.0), 10mM EDTA (pH8.0)], and vortex vibrates to thalline and fully suspends.Solution II [the 0.2M NaOH that adds new preparation, 1%SDS] 200 μ l, put upside down mixing fast, ice bath 5min adds solution III [the 5M potassium acetate of precooling then, glacial acetic acid 11.5ml, H2O28.5ml] 150 μ l, ice bath 5min behind the mixing, the centrifugal 5min of 12000r/min, supernatant is gone in another EP pipe, add phenol: chloroform: primary isoamyl alcohol 500 μ l, vortex vibration, the careful upper strata water of drawing in centrifugal back, the dehydrated alcohol that adds 2 times of volumes,-20 ℃ of precipitation 30min, the centrifugal 5min of 12000r/min, precipitation is with 70% washing with alcohol 2 times, drain, add the TE 20 μ l that contain the RNA enzyme.

The enzyme of recombinant plasmid is cut evaluation: get 3 μ l plasmid DNA and an amount of distilled water mixing, making its cumulative volume is 10 μ l, add 5U restricted endoenzyme EcoR I and the corresponding 10 * restriction enzyme reaction damping fluid of 1 μ l, flick tube wall mixing and centrifugal, put 37 ℃ of water-bath 1-2 hours, get 2-3 μ l reaction solution and detect in agarose gel electrophoresis, enzyme is cut the result and is estimated identical person, is the purpose recombinant plasmid.Recombinant plasmid adopts the terminal cessation method of two deoxidations to check order on automatic dna sequencer, and sequencing is finished by Shanghai Bo Ya Bioisystech Co., Ltd.

(3) 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, mark property association analysis

The test colony (containing 55 pure breeding Tongcheng pigs, 26 long white ♂ * (Da Bai * Tongcheng) ♀ and 21 Da Bai ♂ * (in vain long * Tongcheng) ♀ three way cross combination pigs) that utilizes applicant and the stud farm cooperation of Chinese Hubei Province Tongcheng County bureau of animal husbandry to set up carries out the proterties association analysis for experimental subjects, and 102 DNA samples are used for genotype detection.

The proterties of being analyzed has the part growth traits, part carcass trait and part meat proterties.The applicant has set up following least square model:

y _ij＝μ+GENOTYPE _i+SEX _j+ε _ij，

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

Effect of the present invention

1, the clone of pig MAC30 Gene Partial dna sequence dna

Pcr amplification product is special PCR product through the demonstration of 2% agarose gel electrophoresis detected result.The PCR product is reclaimed the order-checking of purifying rear clone, and sequencing result shows that the length of PCR product is 970bp.Sequence (shown in Fig. 2, SEQ ID NO:1) comprising part exon 2 and exon 3 partly.Related complete intron 2 sequences meet the GT-AG rule.

Sequencing result shows and has two allelotrope of A, G encode respectively Isoleucine (Ile) and Xie Ansuan (Val) at this segmental 848bp place that order-checking peak figure sees Fig. 4.

2, mark property association analysis

To the carrying out between pig MAC30 gene 848bp place (SEQ ID NO:1) polymorphism and the part producing proterties association analysis.Test colony constitutes by three, contains 55 pure breeding Tongcheng pigs, 26 long white ♂ * (Da Bai * Tongcheng) ♀ and 21 the Da Bai ♂ * combination of (in vain long * Tongcheng) ♀ three way cross pig, 102 individualities altogether.The result shows to pig MAC30 gene sequencing, and the genotypic individuality of GG has 87 in the colony of all detections, and the AG genotype has 15 individualities, no AA genotype individuality.Because A allelotrope only occurs in the swinery body of Tongcheng, when carrying out with the Part Traits association analysis, only 55 Tongcheng pigs is analyzed, wherein the GG genotype has 40 individualities, and the AG genotype has 15 individualities.The result of the proterties significant difference of proterties between the different genotype (least square mean and standard error analysis) is summarized in table 1, and other proterties does not have significant difference between different genotype.

As seen from table, the genotypic average thickness of backfat of GG, 6-7 rib place back-fat thickness all the utmost point be markedly inferior to the genotypic pig of AG (P＜0.01).Analyzing A allelotrope according to applicant as a result may be the synergy gene that influences the thickness of backfat, Da Bai, long market pig such as white are owing to be subjected to the influence of strong selective pressure, cause the A allelic loss, only there is G allelotrope, and the selective pressure that Tongcheng pig is subjected to is less relatively, also have a small amount of A allelotrope, the A allelotrope of long white ♂ * (Da Bai * Tongcheng) ♀ and Da Bai ♂ * (in vain long * Tongcheng) ♀ three way cross combination pig is few (not detecting).It also may be the gene close linkage of this gene and certain control thickness of backfat.

Table 1: the association analysis of pig Mac30 gene 848 site different genotype and thickness of backfat proterties

Proterties	Genotype		The P value
				AG(15)	GG(40)
	The average thickness of backfat (mm)-7 rib place's thickness of backfat (mm)	4.55±0.15 4.70±0.19				3.95±0.09 4.07±0.11	0.0009 ** 0.0054 **

Sequence table, description of drawings

Sequence table SEQ ID NO:1 is the dna sequence dna of the fat thickness at back of pig trait related gene MAC30 that clones of the present invention;

Fig. 1: be techniqueflow chart of the present invention.

Fig. 2: be pig MAC30 Gene Partial dna sequence dna.Capitalization is respectively exon 2, and 3.Lowercase is an intron 2.5 '-and two conservative Nucleotide (GT/AG) of 3 '-splicing site mark with black matrix, initiator codon ATG marks with boldface type, the A/G of 848bp place polymorphic site marks expression with parenthesis ().The position of primer shows with square frame.

Fig. 3: be to be used for the directly PCR product agarose gel electrophoresis figure of order-checking of PCR product.

Agarose gel concentration is 2%.The 1-5 swimming lane is the PCR product, and length is 766bp; M swimming lane: dna molecular amount mark (100-1000bp ladder)

Fig. 4: the color peak figure of PCR product order-checking, polymorphic site that arrow refers to.

Fig. 5: be the aminoacid sequence that the partial dna sequence of the fat thickness at back of pig trait related gene MAC30 that clones of the present invention is derived.

Embodiment

Embodiment 1: the association analysis of pig mark property

In Tongcheng swinery MAC30 gene the 3rd exon A848G pleomorphism site and part producing proterties are carried out association analysis, the proterties of being analyzed is part carcass trait and part meat proterties.

Genotype detection result shows that accounting for most is the GG genotype in 55 individualities, have 40, and the AG genotype has 15 individualities.The result of the proterties significant difference of proterties between the different genotype (least square mean and standard error analysis) is summarized in table 2, analytical results shows, the genotypic average thickness of backfat of GG, 6-7 rib place the back-fat thickness all utmost point are markedly inferior to the genotypic pig of AG (P＜0.01), and other proterties does not have significant difference between different genotype.

Table 2: the association analysis of pig Mac30 gene 848 site different genotype and thickness of backfat proterties

Proterties	Genotype		The P value
				AG(15)	GG(40)
	The average thickness of backfat (mm) 6-7 rib place's thickness of backfat (mm)	4.55±0.15 4.70±0.19				3.95±0.09 4.07±0.11	0.0009 ** 0.0054 **

^*It is extremely remarkable to be illustrated on 0.01 level difference.

Embodiment 2: the polymorphic distribution situation in each pig variety in pig MAC30 gene 848 sites

In 6 pig varieties, detect the polymorphism in pig MAC30 gene the 848th site, detected result is as described in Table 3, the data analysis of table 3 shows to be stated, in these several pig varieties that detected, the frequency that all is allelotrope G is preponderated, and the market pig kind is grown among white and the Du Luoke abroad, does not detect A allelotrope and exists.

Kind	Number of individuals	Genotype			Gene frequency
							AA	AG	GG	A	G
		The long Bai Duluoke of peaceful Tibetan, Xiao Mei mountain pig	22 44 25 23 17	0 0 0 0 0	4 20 7 0 0	18 24 18 23 17						0.091 0.227 0.140 0 0	0.909 0.773 0.860 1 1

The distributional difference of pig MAC30 gene 848 loci polymorphism gene frequencies 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.

Sequence table

＜110〉Hua Zhong Agriculture University

＜120〉clone of fat thickness at back of pig genes involved MAC30 and the application on marker assisted selection thereof

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<141>2004-06-07

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<170>PatentIn version 3.1

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＜213〉pig (Sus scrofa)

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<222>(848)..(848)

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<400>1

ata ccc agg agt tca aag aca ctc tgc tcc aga gcc ccc cag cgt ggt 48

Ile Pro Arg Ser Ser Lys Thr Leu Cys Ser Arg Ala Pro Gln Arg Gly

1 5 10 15

tta agg cct tcc tgt ttt gcg agc ttg tgt ttc agc tgc ctt tct ttc 96

Leu Arg Pro Ser Cys Phe Ala Ser Leu Cys Phe Ser Cys Leu Ser Phe

20 25 30

cct ttg caa cat atg cct ttt tca aag gttggtaaat ggtggggaaa 143

Pro Leu Gln His Met Pro Phe Ser Lys

35 40

tcccattttt gctctgaaac caggtcccag ggatcttttg ggaaagtttc accaacagag 203

agggaggagt ctctgtgtcg tgtgtgaggc gatacaggac ctggtgggcc gggctgggag 263

agatcttgcc aggaaacatt gatgtggccg agcctggtgt ctgtggtcgg ctggacgtcc 323

ctcgtacaca agaaatctga ggctgcttta aagattggac ttgctccttt cggtttcccc 383

cacagaattt ttgtgggttg ccttttgttt ttctgctttg ggtcgtgtca tagtggctta 443

taagcggcag catcaagctg attcctgaag cttaactttc aagtgcattt tagaccagcc 503

ggtttaatgg gtaatggcca ttttactgcc ctcgagtttt cctggattaa gttccgcccc 563

ccaccctgct cttcagcagc ccctgcgctg gggctccaac tgtcacgtgc gtgtgcacac 623

tggacactgt taatcacacc aagttttcca tttgtttcca cctgggaaag gaggcagaat 683

gaccgagcca gagggagccc gcagaggggg tggaggctct tggggagctc ggagcagatc 743

taacccttgt ctcttttcct cccctgacct cag gag gct gca agt gga tcc gca 797

Glu Ala Ala Ser Gly Ser Ala

45

ccc ctg caa tca tct act cag ttc aca cca tga cta ctc tga ttc caa 845

Pro Leu Gln Ser Ser Thr Gln Phe Thr Pro Leu Leu Phe Gln

50 55 60

tca tct cta cat ttc tgt tcg agg att tct cca aag cca gtt gtt tca 893

Ser Ser Leu His Phe Cys Ser Arg Ile Ser Pro Lys Pro Val Val Ser

65 70 75

aag gac aag gac cta cga ctt tcc acg aac gac tac tcc ttc tgt ccg 941

Lys Asp Lys Asp Leu Arg Leu Ser Thr Asn Asp Tyr Ser Phe Cys Pro

80 85 90

ttt aca tcc cct act ttc tca tcc ctc tc 970

Phe Thr Ser Pro Thr Phe Ser Ser Leu

95 100

Claims (6)

1, a kind of fat thickness at back of pig genes involved MAC30, its dna sequence dna is as described in the sequence table SEQ ID NO:1.

2, a kind of fat thickness at back of pig genes involved MAC30 according to claim 1, it is characterized in that: there is the base mutation of a 848G-848A at the 848bp place of sequence table SEQ ID NO:1.

3, a kind of fat thickness at back of pig genes involved MAC30 according to claim 1, the dna sequence dna of wherein cloning the used primer of MAC30 gene is as follows:

5 '-ATACCCAGGAGTTCAAAGACACT-3 ' (forward),

5 '-GAGAGGGATGAGAAAGTAGGGGA-3 ' (oppositely).

4, the described a kind of fat thickness at back of pig genes involved MAC30 of claim 1, the dna sequence dna of forward and reverse primer that wherein detects 848G-848A base place sudden change is as follows:

5 '-GGGAGGAGTCTCTGTGTCGTGTGT-3 ' (forward primer),

5 '-GAGAGGGATGAGAAAGTAGGGGA-3 ' (reverse primer);

The dna sequence dna that is used for the direct primer that checks order of PCR product is as follows:

5 '-TAGTCGTTCGTGGAAAGTCGTAGGTC-3 ' (reverse primer).

5, each described fat thickness at back of pig genes involved MAC30 application in the pig marker assisted selection of claim 1-4.

6, a kind of screening is applicable to the molecular marker method of fat thickness at back of pig proterties, according to following steps:

Personnel selection MAC30 gene cDNA is the information probe, does the homologous sequence screening, obtains the expressed sequence tag (EST) of homology more than 90%; Then EST is spliced; Roughly draw the splicing site of exon, design of amplification primers according to comparison result with the genome full length DNA sequence of people MAC30 gene; Extract DNA from the pig blood genome, pcr amplification, PCR product purification and cloning and sequencing obtain the nucleotide sequence shown in sequence table SEQ ID NO:1.

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