CN101979573A - Pork quality character related gene BTG3 and preparation method and application thereof - Google Patents

Abstract

The invention discloses a pork quality character related gene BTG3 and a preparation method and application thereof. The preparation method comprises the following steps of: A, designing a primer, namely performing homologous sequence screening in a GenBank porcine EST database by taking a mRNA sequence of a human BTG3 gene as an information probe and utilizing a basic local alignment search tool (BLAST) tool in a National Center of Biotechnology Information (NCBI) so as to obtain a sequence; B, purifying, cloning and sequencing a polymerase chain reaction (PCR) product, namely in the purification of the PCR product, cutting gel containing a target fragment off from agarose gel with a low-melting point under an ultraviolet lamp, and putting the gel into a tube to purify the PCR product; C, performing a connection reaction, namely connecting the purified PCR product with a pMD18-T vector, and sterilizing; D, preparing a competent cell; and E, converting, namely putting the competent cell into the tube under the aseptic condition, adding a connection product into the tube, uniformly mixing, putting the mixture on ice, adding a liquid culture medium without antibiotics, and performing shake culture, wherein in the cloning and sequencing of the PCR product, the base mutation of C and T exists on the fragment. The preparation method is simple and efficient, has low false positive rate and high detectable rate, and can perform early species selection and in-vivo species selection when used in porcine breeding, shorten generation intervals, save time and cost and increase the speed of the porcine breeding greatly.

Description

Pork quality trait related gene BTG3 and preparation method and application

Technical field

The invention belongs to animal gene engineering technology field, be specifically related to a kind of pork quality trait related gene BTG3, also relate to the preparation method of a kind of pork quality trait related gene BTG3, also relate to the purposes of a kind of pork quality trait related gene BTG3 simultaneously.

Background technology

In year surplus the history of raising pigs of China reaches 5000, pork is China people's main meat product.For many years, the Swine Production of China is based on self-sufficient, and make progress seldom the breeding aspect.Modern times, American-European countries utilized the modern breeding means, was major objective with the raising speed of growth, minimizing feed intake, increase lean ratio, and the speed of growth of pig and lean ratio are increased substantially.But be accompanied by the raising of production level, meat quality but greatly reduces.Along with the Chinese society expanding economy, the variation from " having enough " to " eating " takes place in people's diet idea, and the human consumer is more and more higher to the requirement of meat quality.Therefore, when improving the lean ratio of pig, how to take into account the emphasis that meat quality becomes current pig breeding work.

Since the eighties in 20th century, along with molecular biology and genetic development, the directive breeding working efficiency of pig greatly improves.At present, marker assisted selection (MAS), influence pig important quantity proterties genome area location (QTL), (Economic traits loci ETLs) successfully has been applied in the middle of pig breeding both domestic and external puts into practice influence the major gene of pig important economical trait with genome scanning (genome scanning) method and candidate gene method (Candidate gene approach) research.Wherein, by fluothane (Hal) gene genotype directly being selected to reduce pig stress sensitive, raising meat quality only, there is the simplification DNA detection method of the halothane genotype of commercial value to become patent and (the Peng Zhong town etc. that are used widely, pig quantitative character gene and mark research evolution thereof. external livestock technology, 1999,26 (1): 28-32).(LeRoy etc. such as Le Roy, Evidence for a new major gene influencing meat quality in pigs.Genet Res, 1990,55 (1): 33-40) find that in hampshire and hybrid thereof unfavorable allelotrope RN-can make muscle glycogen content rising in the muscle, terminal pH value reduces, cause sour meat and cooking loss, pork technology quality descends, and influences the output of ham.(Casas-Carrillo etc. such as Casas-Carrillo, Relationship of growth hormone and insulin-like growth factor-1 genotypes with growth and carcass traits in swine.Anim Genet, 1997,28 (2): 88-93.) hybridizing in the colony with 18 no relevant sows with 6 boars, discovery is positioned at para-insulin like growth factor 1 on No. 5 karyomit(e)s of pig in a boar family (Insulin like growth factor 1, IGF-1) there be chain (LOD=2.3) in genotype and daily postweaning gain.(Milan etc. such as Milan, A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle.Science, 2000,288 (5469): 1248-51) by large-scale sequencing analysis is carried out in the candidate region, find single adenosine phosphate activating enzymes γ 3 subunit genes (AMP-activated protein kinase (AMPK), γ-subunit 3, PRKAG3) missense mutation of the codon of coding the 200th amino acids is directly related with the sour meat phenotype of hampshire, and this site of RN pig is disadvantageous allelotrope Q (being glutamine).(Te Pas 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 myogemn 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 MyoD is than L-system (selection lean ratio) height, in F-system, the thickness of backfat and myoblastic negative correlation (the Te Pas etc. that are expressed as, Messenger ribonucleic acid expression of the myoD gene family in muscle tissue at slaughter in relation toselection for porcine growth rate.J Anim Sci, 2000,7:69-77).

This research department is devoted to the isolation identification work of pig critical function gene for a long time, once utilized representative differential display technique, biochip technology, expressed sequence analytical technology (SAGE) etc. to separate some genes relevant, and the significant gene of some of them effect had been carried out functional analysis with economic characters.What the BTG3 gene screened from the skeletal muscle SAGE library of in vain long and Tongcheng pig fetal development different times just grows the gene with remarkably influenced to skeletal muscle, it is one of antiproliferative gene family BTG/TOB family member, 110 amino acid of this family member's protein N terminal have high homology, this homologous region comprises homologous region A-box two weak points, high conservative and B-box, A-box has antiproliferative effect, and B-box has with many target molecule bonded functions.More and more studies show that, this gene family member suppresses the propagation of various kinds of cell and stimulates their differentiation (Matsuda etc., In search of a function for theTIS21/PC3/BTG2/TOB family.FEBS Lett, 2001,497:67-72; Winkler GS. The Mammalian anti-proliferative BTG/Tob protein family.J Cell Physiol.2010.222 (1): 66-7).The BTG1 gene is had strong impulse effect (Matsuda etc., In search of a function for the TIS21/PC3/BTG1/TOB family.FEBS Lett, 2001,497:67-72 by external a plurality of studies have shown that to myoblastic differentiation; Busson etc., Coactivation of nuclear receptors and myogenic factorsinduces the major BTG1 influence on muscle differentiation.Oncogene, 2005,24 (10): 1698-710), the BTG3 gene mainly relies on the effect of p53 performance inhibition of cell proliferation, be considered to vascularization, neural formation and bone forming process have material impact (Yoshida etc., ANA, a novel member ofTob/BTG1 family, is expressed in the ventricular zone of the developing central nervous system.Oncogene, 1998.16:2687-2693; Rahamani, APRO4 negatively regulates Src tyrosine kinase activity in PC12 cells.J Cell Sci, 2006.119 (4): 646-658; Ou etc., The candidate tumor suppressor BTG3 is a transcriptional target of p53 thatinhibits E2F1.EMBO J, 2007.26 (17): 3968-3980).The applicant studies show that, the BTG3 gene is to the influence of BTG1 gene pairs sarcoplast early development is similar but effect is bigger than BTG1 gene, it may promote that earlier the propagation of Skeletal Muscle Cell is increasing of myofiber number pig embryonic stage, promote myocyte's differentiation then, this two aspect all has remarkably influenced (Feng etc. for the output and the quality of muscle Molecularc Haracterization of the BTG2 and BTG3 Genes in fetal muscle development of lean And fatty pig breeds.Gene, 2007.403:170-177).Rehfeld thinks the heritable variation and the very high (Rehfeld etc. of heritability of myofibrillar quantity and volume, Myogenesis and postnatal skeletal muscle cell growth as influenced by selection.Livestock Production Science, 2000,66:177-188), thereby utilize the gene influence myofiber early development to carry out meat and growth traits that energy directional transformation pig is selected in breeding.

Up to the present, except that the applicant, still do not see the report of research pig BTG3 gene function.The polymorphism of research mutational site in colony, and carry out the powerful measure that the proterties association analysis is the research gene function.The applicant has studied pig BTG3 gene pleiomorphism and genotype and proterties association analysis, has found the molecule marker of remarkably influenced pig flesh characters.

Summary of the invention

The objective of the invention is to be to provide a kind of pork quality trait related gene BTG3, this gene is a kind of antiproliferative gene, can suppress myoblastic growth, short its is divided into myofiber, and the low expression of this gene can make animal obtain muscle growth speed faster.

Another object of the present invention is the preparation method who has been to provide a kind of pork quality trait related gene BTG3, and is easy to implement the method, easy and simple to handle, utilizes the acquisition goal gene that this method can be simply rapidly and efficiently.

A further object of the present invention is to be to provide a kind of pork quality trait related gene BTG3 application in the pig marker assisted selection, is that the genotypic individuality of CT has the tender degree of muscle preferably in detection site.

In order to realize above-mentioned purpose, the present invention adopts following technical measures:

A kind of pork quality trait related gene BTG3, its cDNA sequence is the nucleotide sequence shown in the SEQ ID NO:1.

The purpose fragment total length of the BTG3 gene of pcr amplification is 135bp, and its sequence as shown in Figure 2.

The base mutation that C47-T47 is as shown in Figure 2 arranged in the segmental dna sequence dna of BTG3 gene purpose that is obtained causes Mvu I-RFLP (Restriction Fragment Length Polymorphism) polymorphism.

The preparation method of a kind of pork quality trait related gene BTG3 the steps include:

1, design of primers:

MRNA sequence (the GenBank accession number: NM_006806) be the information probe of personnel selection BTG3 gene, 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 pig ESTs (fragment length is greater than the 100bp) sequence more than 90%, use DNAstar (online download address: http://www.dnastar.com/forms.aspx then? forms=reg) the SeqMan program construction pig EST-contig in, and obtaining the concensus sequence (consensus) that is spliced to form by contig, nucleotide sequence is shown in sequence table SEQ ID NO:1.The concensus sequence of acquisition and people's mRNA are done the homology comparison to confirm the exactness of sequence.(online download address: http://www.premierbiosoft.com/primerdesign/index.html) length is the fragment of 135bp between design primer amplification BTG3 gene cDNA 782～916 sites to use Primer Premier 5.0 softwares then.Primer sequence is as follows:

B3POLF:5 '-GATATTCCCACCTCTTCCA-3 ' (forward),

B3POLR:5 '-GGTTTATTCTTCCTTCCCT-3 ' (oppositely);

2, the purifying of PCR product, clone and order-checking:

The purifying of PCR product: under ultraviolet lamp, contain the segmental gel of purpose from the sepharose cutting-out, put into 1.5mL Ependorff pipe, use PCR product purification test kit (Tiangen then, Beijing) purified pcr product, according to test kit specification sheets operation, concrete steps are the S1 liquid of 300 μ L in the gel of every 100mg, melt fully in 65 ℃ of incubation 10min to gel, the S1 liquid that will contain DNA changes the recovery post over to, and the centrifugal 30s of 9200g makes slurries extrude by Minicolumn.Waste liquid in the following pipe is outwelled, and the W1 liquid that adds 500 μ L again is to pipe, and the centrifugal 15s of 9200g outwells the waste liquid in the pipe.Add the W1 liquid of 500 μ L again, leave standstill 1min, the centrifugal 30s of 9200g takes off Minicolumn and packs in the 1.5ml Ependoff pipe, adds aqua sterilisa or the TE liquid of 25 μ L, leaves standstill after the 1min, and the centrifugal 1min of 9200g is stored in the Ependorff pipe with eluted dna.

3, ligation: the PCR product and the pMD18-T carrier (available from TAKARA company) of purifying are connected, the ligation cumulative volume is 5 μ l, comprising 2.5 μ l, 2 * ligation buffer, 0.5 μ l carrier, 1 μ l purified pcr product adds 1 μ l aqua sterilisa at last and puts 4 ℃ of water-baths and spend the night.

4, the preparation of competent cell: the single bacterium colony (promega of DH5 α of picking from 37 ℃ of fresh flat boards of having cultivated 16～20h, USA) be inoculated among the 2ml LB, in 37 ℃ of shaking culture 3h, switching 1ml bacterium liquid is in the saline bottle that contains 30ml LB, continuation is treated OD at 37 ℃ of about 4h of shaking culture ₆₀₀Reach 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, with the resuspended precipitation of CaCl2 that 10ml ices the 0.1mol/L of precooling, ice bath 30min, repeat 4 ℃ 4, the centrifugal 10min of 000g once, with the resuspended precipitation of CaCl2 of the 0.1mol/L of 4ml ice precooling, it is standby to put 4 ℃ of preservations.

5, 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 42 ℃ of heat shock 90s behind the 30min on ice, do not shake the Ependorff pipe therebetween, take out back ice bath 3～4min, the LB liquid that adds 400 μ l antibiotic-frees is supported base, 37 ℃ of shaking culture 45min.Getting 100 μ l coats in advance 4h and has been coated with isopropylthio-(37 ℃ keep flat to be inverted behind the 1h and cultivate for IPTG, Isopropylthio-β-D-galactoside) and on the agar plate that contains Amp of X-gal.

Clone son is accredited as male by PCR and is used to serve the order-checking of extra large Ying Jun Bioisystech Co., Ltd.Each one clone's of Da Bai, Du Luoke and three kinds of Mei Shan is checked order respectively or mixes order-checking, sequencing result Seqman ^TMSoftware (online download address: http://www.dnastar.com/forms.aspx? forms=reg) SNPs is sought in comparison, sequencing result is presented at this fragment 47bp place and has C, T base mutation, and the change of the C-T of this place causes amino acid to become Serine by proline(Pro).A kind of isolating pig flesh characters gene BTG3, its sequence is the nucleotide sequence shown in the SEQUENCE NO.1.

The application of a kind of pork quality trait related gene BTG3 in the pig marker assisted selection, its application process is:

1, the clone of pig BTG3 Gene Partial dna sequence dna:

Synthetic primer B3POLF:5 '-GATATTCCCACCTCTTCCA-3 ' (forward), length is the fragment of 135bp between B3POLR:5 '-GGTTTATTCTTCCTTCCCT-3 ' (oppositely) amplification BTG3 gene cDNA 782～916 sites.The pcr amplification reaction system is 20 μ l, dna profiling 1 μ l wherein, 1 * PCR buffer (Promega, USA), 0.3 μ M of each primer, 1.5mM MgCl2,75 μ M of each dNTPs, 1UTaq DNA polymerase (Promega, USA).The pcr amplification program: 95 ℃ of 5min, circulate 35 times, 94 ℃ of 30s, 55 ℃ of 20s, 72 ℃ of 20s, last 72 ℃ are extended 5min.Pcr amplification product detects through 3% (mass ratio) agarose gel electrophoresis, and the result that takes pictures under ultraviolet lamp demonstration is 135bp specific amplified fragment (seeing Fig. 2 for details).

2, the PCR-RFLP diagnostic method is set up:

Amplification PCR products is carried out enzyme with MvaI and is cut, and PCR product endonuclease reaction volume is 5 μ l, 1 * buffer0.5 μ l wherein, and PCR product 3 μ l, restriction enzyme 0.3 μ l (10U) supplies 5 μ l with H2O, and with centrifugal behind the sample mixing, 37 ℃ of enzymes are cut 4-8h.Detect enzyme with 3% (mass ratio) agarose gel electrophoresis and cut the result, gel imaging system imaging, record genotype.Enzyme is cut the back common property and is given birth to three kinds of genotype, wherein the CC genotype has 48bp and 87bp two bands, and heterozygote CT type has 135bp, 48bp and 87bp three bands, the TT type has only 135bp one band, can significantly differentiate banding pattern (Fig. 4) with the agarose gel electrophoresis detection of 3% (mass ratio).

3, mark property association analysis:

After utilizing the PCR-RFLP method that sample is carried out gene type, calculate its each SNPs site χ between kind by PopGen 32 softwares (the online download) ²The value and the P value of testing significance of difference, the generalized linear model program in the application SPSS software are come the related of analyzing gene type and the production traits.Analytical model is as follows:

Y _ijk1＝μ+G _i+B _j+S _k+ε _ijk1

Y _Ijk1The expression observed value, μ represents average, G _iExpression genotype effect, B _jThe expression combined effect, S _kExpression sex effect, ε _Ijk1The expression residual error, suppose obey N (0, I σ ²) distribute.

Use this model and each proterties analyzed and obtained a calibrated phenotypic number, then with calibrated phenotypic number as new character value, set up following analytical model again:

Yij＝μ+GENOTYPEi+ε _ij

Yij is new character value, and μ is the population mean of new character value, GENOTYPE _iBe genotype effect, ε _IjBe random error, suppose that obeying N (0, σ 2) distributes.

Use this model and can analyze the genotype effect, and carry out the multiple comparisons between the genotype effect.

The present invention provides a new genetic marker for the molecular breeding of pig.According to the present invention, pig production character gene BTG3 can be used for the pig marker assisted selection.

The present invention compared with prior art has the following advantages and effect: simple efficient, false positive rate is low, the recall rate height; Be used for that pig breeding can be chosen seeds in early days, the live body seed selection, shorten the generation interval, save time and cost, accelerate pig breeding speed greatly.

Description of drawings

Fig. 1 is a kind of pork quality trait related gene BTG3 preparation method's a schema;

Fig. 2 is a kind of electrophoretogram of pork quality trait related gene BTG3 the 5th exon district extension increasing sequence;

Clip size is 135bp (agarose gel concentration is 2%).M:DNA molecular weight standard (DL2000ladder);

There are two allelotrope of C, T in the 47bp place that Fig. 3 finds for a kind of pork quality trait related gene BTG3 order-checking;

Fig. 4 is two kinds of genotype electrophoretograms of MvaI-RFLP in a kind of pork quality trait related gene BTG3 the 5th exon district; M:DNA molecular weight standard (DL2000ladder)

Fig. 5 is a kind of pork quality trait related gene BTG3 nucleotides sequence tabulation.

Embodiment

Embodiment 1:

The preparation method of a kind of pork quality trait related gene BTG3 the steps include:

A, design of primers:

MRNA sequence (the GenBank accession number: NM_006806) be the information probe of personnel selection BTG3 gene, 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 pig ESTs (fragment length is greater than the 100bp) sequence more than 90%, use the SeqMan program (the online download) among the DNAstar to make up pig EST-contig then, and obtaining the concensus sequence (consensus) that is spliced to form by contig, nucleotide sequence is shown in sequence table SEQ ID NO:1.The concensus sequence of acquisition and people's mRNA are done the homology comparison to confirm the exactness of sequence.Use then that length is the fragment of 135bp between Primer Premier5.0 software (online download) design primer amplification BTG3 gene cDNA 782～916 sites.Primer sequence is as follows:

B3POLF:5 '-GATATTCCCACCTCTTCCA-3 ' (forward),

B3POLR:5 '-GGTTTATTCTTCCTTCCCT-3 ' (oppositely);

The purifying of B, 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 1.5mL Ependorff pipe, use PCR product purification test kit (Tiangen then, Beijing) purified pcr product, according to test kit specification sheets operation, concrete steps are the S1 liquid of 300 μ L in the gel of every 100mg, melt fully in 65 ℃ of incubation 10min to gel, the S1 liquid that will contain DNA changes the recovery post over to, and the centrifugal 30s of 9200g makes slurries extrude by Minicolumn.Waste liquid in the following pipe is outwelled, and the W1 liquid that adds 500 μ L again is to pipe, and the centrifugal 15s of 9200g outwells the waste liquid in the pipe.Add the W1 liquid of 500 μ L again, leave standstill 1min, the centrifugal 30s of 9200g, take off Minicolumn and pack in the 1.5ml Ependorff pipe, add aqua sterilisa or the TE liquid of 25 μ L, leave standstill after the 1min, the centrifugal 1min of 9200g is stored in the Ependorff pipe with eluted dna.

C, ligation: the PCR product and the pMD18-T carrier (available from TAKARA company) of purifying are connected, the ligation cumulative volume is 5 μ l, comprising 2.5 μ l, 2 * ligation buffer, 0.5 μ l carrier, 1 μ l purified pcr product adds 1 μ l aqua sterilisa at last and puts 4 ℃ of water-baths and spend the night.

The preparation of D, competent cell: the single bacterium colony of DH5 α of picking (laboratory deposit from 37 ℃ of fresh flat boards of having cultivated 16～20h, definitely can not realisticly test the chamber deposit, must write source or document source exactly) be inoculated among the 2ml LB, in 37 ℃ of shaking culture 3h, switching 1ml bacterium liquid is in the saline bottle that contains 30ml LB, continuation is treated OD at 37 ℃ of about 4h of shaking culture ₆₀₀Reach 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, with the resuspended precipitation of CaCl2 that 10ml ices the 0.1mol/L of precooling, ice bath 30min, repeat 4 ℃ 4, the centrifugal 10min of 000g once, with the resuspended precipitation of CaCl2 of the 0.1mol/L of 4ml ice precooling, it is standby to put 4 ℃ of preservations.

E, conversion: 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 42 ℃ of heat shock 90s behind the 30min on ice, do not shake the Ependorff pipe therebetween, take out back ice bath 3～4min, the LB liquid that adds 400 μ l antibiotic-frees is supported base, 37 ℃ of shaking culture 45min.Getting 100 μ l coats in advance 4h and has been coated with IPTG (front is write middle literal exactly, the English bracket of using) (Isopropylthio-β-D-galactoside, isopropylthio-) and on the agar plate that contains Amp of X-gal, 37 ℃ keep flat to be inverted behind the 1h and cultivate.

Clone son is accredited as male by PCR and is used to serve the order-checking of extra large Ying Jun Bioisystech Co., Ltd.Each one clone's of Da Bai, Du Luoke and three kinds of Mei Shan is checked order respectively or mixes order-checking, sequencing result Seqman ^TMSNPs is sought in software (the online download) comparison, and sequencing result is presented at this fragment 47bp place and has C, T base mutation, and the change of the C-T of this place causes amino acid to become Serine by proline(Pro).A kind of isolating pig flesh characters gene BTG3, its sequence is the nucleotide sequence shown in the SEQUENCE NO.1.

The distribution situation of PCR-RFLP-MvaI polymorphism in each pig variety is:

1, the clone of pig BTG3 Gene Partial dna sequence dna:

Synthetic primer B3POLF:5 '-GATATTCCCACCTCTTCCA-3 ' (forward), length is the fragment of 135bp between B3POLR:5 '-GGTTTATTCTTCCTTCCCT-3 ' (oppositely) amplification BTG3 gene cDNA 782～916 sites.The pcr amplification reaction system is 20 μ l, dna profiling (detected swinery blood extract DNA) 1 μ l wherein, 1 * PCR buffer (Promega, USA), 0.3 μ M of each primer, 1.5mMMgCl2,75 μ M of each dNTPs, 1U Taq DNA polymerase (Promega, USA).The pcr amplification program: 95 ℃ of 5min, circulate 35 times, 94 ℃ of 30s, 55 ℃ of 20s, 72 ℃ of 20s, last 72 ℃ are extended 5min.Pcr amplification product detects through 2% agarose gel electrophoresis, and the result that takes pictures under ultraviolet lamp demonstration is 135bp specific amplified fragment (seeing Fig. 2 for details).

2, the PCR-RFLP detection method is set up:

Amplification PCR products is cut with the MvaI enzyme, and PCR product endonuclease reaction volume is 5 μ l, 1 * buffer0.5 μ l wherein, and PCR product 3 μ l, restriction enzyme 0.3 μ l (10U) supplies 5 μ l with H2O, and with centrifugal behind the sample mixing, 37 ℃ of enzymes are cut 4-8h.Detect enzyme with 3% (mass ratio) agarose gel electrophoresis and cut the result, gel imaging system imaging, record genotype.Enzyme is cut the back common property and is given birth to three kinds of genotype, wherein the CC genotype has 48bp and 87bp two bands, and heterozygote CT type has 135bp, 48bp and 87bp three bands, the TT type has only 135bp one band, can significantly differentiate the banding pattern (see figure 4) with the agarose gel electrophoresis detection of 3% (mass ratio).

3, the distribution frequency analysis of genotype in each kind:

Utilize the PCR-MvaI-RFLP method to detect seven pig kinds: comprising Da Bai, long white, Du Luoke, Tongcheng, painted face in Beijing opera, peaceful and Yushan, Jiangxi, these pig kinds contain the genotype of each pig kind substantially.By PopGen32 software (online download) calculate its each SNPs site between kind χ 2 values and the P value of testing significance of difference, the gene frequency of the pig kind that test is used is as shown in table 1, in seven pig varieties, all only detect CC and CT genotype, and all be that C allelotrope is dominant in each pig variety, wherein two of Large White gene frequencies are the most approaching, in vain long and Du Luoke takes second place, and four domestic variety pigs all are that C allelotrope has comparative advantage.

Genotype frequency and the gene frequency of table 1 pig BTG3 gene in seven pig varieties

The chi square test result such as the table 2 of genotype frequency between several kinds.χ ²Check shows, has utmost point significant difference between Da Bai and landrace and other kind, has significant difference between long white, the Da Bai, and Du Luoke and peaceful pig significant difference are not remarkable with domestic other breed difference.

Table 2 pig BTG3 gene PCR-RFLP-MvaI genotype frequency distribution χ ²Assay

Annotate: shoulder motes ^*Expression P＜0.05; Shoulder motes ^*Expression P＜0.01; Df=2, χ ² _0.05(2)=5.99, χ ² _0.01(2)=9.21

Embodiment 2:

The application of a kind of pork quality trait related gene BTG3 in the pig marker assisted selection, its application process is:

1, the clone of pig BTG3 Gene Partial dna sequence dna:

Synthetic primer B3POLF:5 '-GATATTCCCACCTCTTCCA-3 ' (forward), length is the fragment of 135bp between B3POLR:5 '-GGTTTATTCTTCCTTCCCT-3 ' (oppositely) amplification BTG3 gene cDNA 782～916 sites.The pcr amplification reaction system is 20 μ l, dna profiling 1 μ l wherein, 1 * PCR buffer (Promega, USA), 0.3 μ M of each primer, 1.5mM MgCl2,75 μ M of each dNTPs, 1UTaq DNApolymerase (Promega, USA).The pcr amplification program: 95 ℃ of 5min, circulate 35 times, 94 ℃ of 30s, 55 ℃ of 20s, 72 ℃ of 20s, last 72 ℃ are extended 5min.Pcr amplification product detects through 3% (mass ratio) agarose gel electrophoresis, and the result that takes pictures under ultraviolet lamp demonstration is 135bp specific amplified fragment (seeing Fig. 2 for details).

2, the PCR-RFLP diagnostic method is set up:

Amplification PCR products is cut with the MvaI enzyme, and PCR product endonuclease reaction volume is 5 μ l, 1 * buffer0.5 μ l wherein, and PCR product 3 μ l, restriction enzyme 0.3 μ l (10U) uses H ₂O supplies 5 μ l, and with centrifugal behind the sample mixing, 37 ℃ of enzymes are cut 4-8h.Detect enzyme with 3% (mass ratio) agarose gel electrophoresis and cut the result, gel imaging system imaging, record genotype.Enzyme is cut the back common property and is given birth to three kinds of genotype, wherein the CC genotype has 48bp and 87bp two bands, and heterozygote CT type has 135bp, 48bp and 87bp three bands, the TT type has only 135bp one band, can significantly differentiate banding pattern (Fig. 4) with the agarose gel electrophoresis detection of 3% (mass ratio).

3, mark property association analysis:

Pig BTG3 gene 3 ' UTR sequence PsuI-RFLP pleomorphism site is used generalized linear model program in the SPSS software come the related of analyzing gene type and the production traits, genotype detection result shows that the CC genotype has 107 in 88 individualities, and the CT genotype has 81.The result of the proterties significant difference between the different genotype (least square mean and standard error analysis) sees Table 3, and other proterties does not have significant difference between different genotype.

Analytical results shows that the genotype in this site is significantly related with the existence of muscle shearing force, and the genotypic muscle shearing force of CC is significantly higher than CT genotype (P＜0.05), and other proterties does not have significant difference between different genotype.The CT genotype is the selective marker that helps reducing the muscle shearing force, and C allelotrope is the unfavorable mark that improves the tender degree of muscle, and T allelotrope is the favourable mark that improves the tender degree of muscle.

Table 3 pig BTG3 gene M vaI-enzyme is cut the association analysis of genotype and several meat proterties

^*Expression significant difference (p＜0.05), * * are represented difference extremely significantly (p＜0.01).

SEQUENCELISTING

 

＜110〉Animal Husbandry and Veterinary Inst., Hubei Academy of Agricultural Sciences (C

＜120〉pork quality trait related gene BTG3 and preparation method and application

＜130〉pork quality trait related gene BTG3 and preparation method and application

<160>?3

<170>?PatentInversion3.1

 

<210>?1

<211>?1466

<212>?DNA

＜213〉pig

 

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ttccgggccgcgagccccccgcgcgccgcttcagaaccgcgctcgttcgctcgcgcgcgc 60

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gctcgctgcgctccctgtcgccctgcgcctgccggctaaggcggcgtcctcgattctcgg 180

tccggagacggtcaggcagccaaatctgtagtccgagagcggcggcggcggcggcagcag 240

cattgagaaatatgaagaacgaaattgctgctgtagtcttctttttcacaaggctagttc 300

gaaaacatgataagttgaaaaaagaagcagttgaaaggtttgctgagaaattgactctaa 360

tacttcaagaaaaatataaaaatcactggtatccggaaaaaccatcaaaaggacaggcct 420

acagatgcattcgggtcaataagtttcagagagttgatcctgatgtcctgaaagcctgtg 480

agaacagctgcatcttatacagtgatctgggcttgccaaaggaacttactctgtgggtgg 540

acccctgtgaggtgtgctgtcggtatggagagaaaaacaatgcattcatcgttgccagct 600

ttgaaaatgaggaggagaataaggatgaaatttccaagaaagttaccagggcccttgata 660

aggttacctctgattatcattcaggatcctcttcttcagatgaagaaacaagtaaggaag 720

tagaagtgaaacccaattcggtgactgcgacccctagccctgtgtaccagatttcagaac 780

tgatattcccacctcttccaatgtggcaccctttgcccagaaaaaagccaggaatgtacc 840

gaggaaatggtcatcaaaatcactaccctcctcctgttccatttggttatccaaatcagg 900

gaaggaagaataaaccatatcgcccaattccagtaacatgggtacctcctcctggaatgc 960

attgtgaccggaatcactggattaatcctcacatgttagcaccccactagctgcttttga 1020

ttctgttggtgtcatgttgagagaaggtagaataagcctgacatattaaaaagttcttac 1080

tcaaagtagtaaagttagatgggccaaaccatcaaacttatttttatagagaagttattg 1140

aaaataatctttcctaaaagatatatatgcactttagatatattgatatagtttgagaaa 1200

ctttattaaagttagtcaagtgcctgagtttttaatattggacttgagtatttatatatt 1260

gtgcatcaactctgttggatatgagaacactgtaggagtgggcaatatgttctagcacct 1320

ttgagcatttactctatggagagtatgtaagttatttatacacaaggaaatctattttat 1380

gtcattgtttagaattgcgtgaaatcatgtagttgcaaataaaaagtagtttgaggcata 1440

aaaaaaaaaaaaaaaaaaaaaaaaaa 1466

  

<210>?2

<211>?19

<212>?DNA

＜213〉synthetic

 

<400>?2

gatattcccacctcttcca 19

 

 

<210>?3

<211>?19

<212>?DNA

＜213〉synthetic

 

<400>?3

ggtttattcttccttccct 19

 

 

Claims (3)

1. isolating pig flesh characters gene BTG3, its sequence is the nucleotide sequence shown in the SEQUENCE NO.1.

2. the described a kind of pork quality trait related gene of claim 1 BTG3The preparation method, the steps include:

A,Design of primers:

The mRNA sequence of personnel selection BTG3 gene is 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 pig ESTs sequence more than 90%, with the SeqMan program construction pig EST-contig among the DNAstar, the concensus sequence that acquisition is spliced to form by contig, use then that length is the fragment of 135 bp between Primer Premier 5.0 software design primer amplification BTG3 gene cDNAs 782 ~ 916 sites, primer sequence is as follows:

B3POLF：5’-?GATATTCCCACCTCTTCCA?-3’?,

B3POLR：5’-?GGTTTATTCTTCCTTCCCT?-3’?；

B,The purifying of PCR product, clone and order-checking:

The purifying of PCR product: under ultraviolet lamp, contain the segmental gel of purpose from the sepharose cutting-out, put into 1.5 mL Ependorff pipes, with PCR product purification test kit purified pcr product, step is the S1 liquid of 300 μ L in the gel of every 100mg, melt fully in 65 ℃ of incubation 10min to gel, change the S1/DNA mixture over to the recovery post, the centrifugal 30s of 9200g makes slurries extrude by Minicolumn, waste liquid in the following pipe is outwelled, the W1 liquid that adds 500 μ L again is to pipe, and the centrifugal 15s of 9200g outwells the waste liquid in the pipe, the W1 liquid that adds 500 μ L again, leave standstill 1min, the centrifugal 30s of 9200g takes off Minicolumn and packs in the 1.5 ml Ependorff pipes, the aqua sterilisa or the TE liquid that add 25 μ L, leave standstill after the 1min, centrifugal 1 min of 9200g is stored in the Ependorff pipe with eluted dna;

C, ligation: the PCR product of purifying is connected with the pMD18-T carrier, the ligation cumulative volume is 5 μ l, comprising 2.5 μ l, 2 * ligation buffer, 0.5 μ l carrier, 1 μ l purified pcr product adds 1 μ l aqua sterilisa at last and puts 4 ℃ of water-baths and spend the night;

The preparation of D, competent cell: the single colony inoculation of DH5 α of picking is in 2ml LB from 37 ℃ of fresh flat boards of cultivating 16 ~ 20 h, in 37 ℃ of shaking culture 3 h, transfer 1 ml bacterium liquid in the saline bottle that contains 30 ml LB, continue to treat OD at 37 ℃ of shaking culture 4h ₆₀₀Reach at 0.3 ~ 0.4 o'clock saline bottle is put ice bath cooling 10 ~ 15 min from the shaking table taking-up, then bacterium liquid is changed in the centrifuge tube in 4 ℃ 4, centrifugal 10 min of 000g are with collecting cell, centrifuge tube is inverted to abandon clean nutrient solution, with the resuspended precipitation of CaCl2 that 10 ml ice 0.1 mol/L of precooling, ice bath 30 min, repeat 4 ℃ 4, centrifugal 10 min of 000g once, with the resuspended precipitation of CaCl2 of 0.1 mol/L of 4 ml ice precooling, it is standby to put 4 ℃ of preservations;

E, transform: get 100 ~ 120 μ l competent cells under the sterile state in 1.5 ml Ependorff pipes, the connection product of 5 μ l is added mixing, place 42 ℃ of heat shock 90 s behind 30 min on ice, take out back ice bath 3 ~ 4 min, the LB liquid that adds 400 μ l antibiotic-frees is supported base, 37 ℃ of shaking culture 45 min, get 100 μ l and coat in advance that 4 h have been coated with on the agar plate that contains Amp of IPTG and X-gal, 37 ℃ keep flat to be inverted behind 1 h and cultivate, sequencing result is presented at this fragment 47bp place and has C, the T base mutation, the change of the C-T of this place causes amino acid to become Serine by proline(Pro).

3. the application of the described a kind of pork quality trait related gene BTG3 of claim 1 in the pig marker assisted selection.

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