CN104109664A - GSK3alpha gene promoter SNP as genetic label of pork carcass character and application thereof - Google Patents

Abstract

The invention belongs to the technical field of pig genetic label preparation, and specifically relates to the preparation of a molecular label related with pig carcass characters and an application of the molecular label in marker assisted selection. The molecular label is prepared by cloning the GSK3alpha gene promoter sequence, and the nucleotide sequence of the molecular label is represented by the SEQ ID No.1 in the sequence table. C154-A154 base mutation happens in the 154th base of a sequence represented by the SEQ ID No. 2 in the sequence table, and causes the Hin1IPCR-RFLP polymorphism. The invention further discloses a preparation method of the molecular label and an application of a polymorphism detection method. A novel genetic label is provided for the pig marker assisted selection.

Description

GSK3 α gene promoter SNP is as genetic marker and the application of hog on hook proterties

Technical field

The invention belongs to animal molecular marker preparing technical field, be specifically related to a kind of and clone hog on hook trait related gene GSK3 α promoter gene fragment, the preparation of molecule marker and the application in marker assisted selection.

Background technology

Inherited genetic factors exists difference on the impact of pig important economical trait in different kinds or same kind Different Individual, and these difference parts are owing to there being polygenic minor effect effect.But these economic characters also exist many single-gene effects, these genes are exactly the major gene that affects pig economic characters, these genes can carry out for marker assisted selection the improvement (Rosenvold of pig economic characters, K and Andersen, H.J. Factors of significance, for pork quality-a review. Meat Science 2003,64:219-237.).The selection breeding of pig has been carried out several thousand, so strong selection has produced many new variations with good phenotypic effect, these variations provide the method (Andersson of new breeding improvement to us, L. Genetic dissection of phenotypic diversity in farm animals. Nat Rev Genet, 2001,2:130-138.).Because carcass trait belongs to low-heritability traits, by traditional selection breeding, can not select fast and effectively, and carcass trait is also more difficult aspect mensuration, cost is very expensive (Gao also, Y et.al. Application of genomic technologies to the improvement of meat quality of farm animals. Meat Science, 2007,77:36-45.).Therefore, the major gene of utilize identifying carries out molecular marker assisted selection, and the control by genetic aspect improves hog on hook proterties and is one and well selects.

The major gene that current isolation identification affects pig economic characters also only only limits to several important genes that affect meat quality, such as: halothane, sour meat gene rNand iGF2gene, and the major gene that affects carcass trait also not studies have reported that, so we also need to find the more major gene that affects hog on hook proterties, and molecule mechanism and regulatory mechanism that it is affected to hog on hook proterties carry out deep research.An important factor that affects hog on hook proterties is protein anabolism process, therefore the important candidate gene that affects hog on hook proterties is found emphasis in this research at pig muscle protein synthesis path, disclose it and grow the mechanism of action and the regulatory mechanism in metabolic process at pig muscle, and detect the transgenation that affects hog on hook proterties.

(glycogen synthase kinase 3 GSK3) is a kind of serine/threonine proteinoid kinases to glycogen synthase kinase 3.This kinases in 1980 in muscle tissue, be first found and purifying out, because find that at first it is one of the kinases (Woodgett of phosphorylation glycogen synthetase, J.R. and Cohen, P. Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5). Biochim Biophys Acta, 1984, 788:339-347), cause that glycogen synthetase phosphorylation makes its inactivation, so be named as glycogen synthase kinase 3.In Mammals, GSK3 has hypotype GSK3 α and the GSK3 β of two height homologies, by two different genes encodings, wide expression in different tissues and cell, especially the highest (the Woodgett of expression amount in cerebral tissue, J.R. Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J, 1990,9:2431-2438.).Two kinds of hypotypes are in the homology in their kinase catalytic region nearly 97%, but hold at its C-end and N-, homology is lower, and holds and have a sequence that is rich in glycine at the N of GSK3 α.

GSK3 α gene has participated in many signal paths, important signal path (the Frame such as Wnt, Hedgehog and IGF-1 have wherein just been comprised, S and Cohen, P. GSK3 takes centre stage more than 20 years after its discovery. Biochemical Journal, 2001,359:1-16).Regular Insulin or IGF-1 act on target cell, activate phosphinositides 3 kinases (PI3-kinase) in downstream, then PI3K activates AKT, make the latter's phosphorylation (Ser-21, GSK3 α and GSK3 β, Ser-9), thereby the activity that suppresses GSK3, then glycogen synthetase (GS) and eukaryotic translation initiation factor (eIFB) dephosphorylation under the effect of phosphoesterase, thereby make the synthetic (Rommel that normally carries out of glycogen and protein, C et.al. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI (3) K/Akt/mTOR and PI (3) K/Akt/GSK3 pathways. Nature Cell Biology, 2001, 3:1009-1013).GSK3 α plays an important role in protein synthesis and Glycogen Metabolism process, and muscle tissue is the position that needs a large amount of protein synthesis.Therefore, we infer that pig GSK3 α gene may be one and affect the important gene that pig muscle grows.In addition, up to the present still do not see the relevant report of research pig GSK3 α gene promoter.So applicant is using pig GSK3 α gene as the important candidate gene that affects hog on hook proterties, study this polymorphism of gene promoter region mutational site in colony, and the relation of these polymorphic sites and pig production character has been carried out to further analysis, expectation can find to affect the important polymorphic site of hog on hook proterties.

Summary of the invention

The promoter region that the object of the invention is to the gene GSK3 α gene that clone is relevant to hog on hook proterties, find gene mutation site, and set up corresponding pleiomorphism detecting method and carry out proterties association analysis, for the marker assisted selection of pig provides a kind of useful molecule marker.

the present invention realizes by following technology:

As a GSK3 α gene promoter region sequence relevant to hog on hook proterties for molecule marker application, total length is 1387bp, and its nucleotide sequence is as described in sequence table SEQ ID NO:1.According to the design of DNA sequence dna as described in the sequence table SEQ ID NO:1 primer obtaining, by increasing and checking order, obtained GSK3 α gene promoter partial nucleotide sequence, this sequence length is 337bp, wherein there is the base mutation of a C154-A154 at 154bp place, causes Hin1I PCR-RFLP polymorphism.Applicant has designed a kind of primer pair that detects gene fragment sudden change, and the forward primer of described primer pair is: 5'TAGGAAGCGTGAGGATGCA 3'; Reverse primer is: 5'TCGACTGGGTGTAACTGAA 3'.

A method of preparing the described GSK3 α gene promoter sequence relevant to hog on hook proterties, according to following steps:

(1) take the cDNA of pig GSK3 α gene is information probes, at pig genome database, do homologous sequence screening, obtain a BAC clone that comprises pig GSK3 α gene, with BAC clone sequence, make template design primer, the genomic dna of place of china pig variety plum mountain pig of take is template, carries out pcr amplification, PCR product purification and cloning and sequencing, obtain pig GSK3 α gene promoter sequence, the nucleotide sequence as described in sequence table SEQ ID NO:1.

(2) selecting 3 Large Whites and 3 plum mountain pigs is test materials, from blood, extract pig genomic dna, with the nucleotides sequence described in SEQ ID NO:1, classify template design primer as, pcr amplification, PCR product purification, cloning and sequencing, obtain pig GSK3 α gene promoter partial nucleotide sequence, obtain the nucleotide sequence as described in sequence table SEQ ID NO:2.

Applicant is applied in the molecule marker relevant to hog on hook proterties of preparation in the association analysis of pig molecule mark assistant breeding in an embodiment of the present invention.

effect of the present invention is:found a molecule marker relevant to hog on hook proterties, for the molecular marker assisted selection of pig provides a new molecule marker.

More detailed technical scheme refers to the embodiment in < < embodiment > >.

Accompanying drawing explanation

Sequence table SEQ ID NO:1 is the promotor nucleotide sequence of the present invention's pig growth traits genes involved GSK3 α gene of cloning.

Sequence table SEQ ID NO:2 is the promoter region partial nucleotide sequence of the present invention's pig growth traits genes involved GSK3 α gene of cloning.

Fig. 1: techniqueflow chart of the present invention.

Fig. 2: be in the present invention pig GSK3 α gene for the DNA sequence dna amplification electrophoresis result of proterties association analysis, swimming lane M:DNA molecular weight standard (DL2000 ladder) in figure.

Fig. 3: the tri-kinds of genotype electrophoretograms of Hin1I-RFLP that are pig GSK3 α gene promoter sequence in the present invention.In figure: M:DNA molecular weight standard (DL2000 ladder).

Fig. 4: be the pig GSK3 α gene promoter partial nucleotide sequence that the present invention increases and obtains, clip size is 337bp, and in figure, underscore is respectively forward and reverse primer sequence.Shown in bracket is base mutation.

Embodiment

embodiment 1: acquisition and the polymorphic detection of pig GSK3 α promoter gene fragment

(1) pig GSK3 α gene promoter region DNA sequence dna amplification

(1) design of primers:

CDNA(GenBank accession number with pig GSK3 α gene: HM214803) be information probes, utilize the BLAST instrument of American National biotechnology information center (NCBI) to do homologous sequence screening in Genomic Biology pig genome database.Obtain a BAC clone (GenBank accession number: CH242-504I8) that comprises pig GSK3 α gene, with BAC clone sequence, make template design primer, the genomic dna of place of china pig variety plum mountain pig of take is template, obtains pig GSK3 α gene promoter sequence.With Primer Premier 5.0 software design primer amplification SEQ ID NO:1, described sequence, their sequence size is respectively 1387bp.The DNA sequence dna of described primer is as follows:

Forward primer (F1): 5 '-TGAGGGTAGTTTCATTCCAG-3 '

Reverse primer (R1): 5 '-TCTCTCGGCCCTCACAGAGT-3 '

(2) pcr amplification reaction

PCR reaction system is 25 μ L, and wherein template is that pig DNA is 100ng, and dNTPs concentration is 10mmol/L, and every primer concentration is 10mmol/ L, and the Taq archaeal dna polymerase of 2U adds deionized water to cumulative volume 25 μ L.PCR response procedures: 94 ℃ of denaturation 4min; Then 94 ℃ of sex change 40s, 59 ℃ of annealing 40s, 72 ℃ of extensions 1min20s, totally 35 circulations; Last 72 ℃ are extended 7min.PCR product detects through 1% agarose gel electrophoresis.

(3) recovery of PCR product, purifying, Cloning and sequencing

PCR product purified (reclaiming the operation of test kit specification sheets according to the UNIQ-10 pillar DNA glue of Shanghai Sheng Gong biotechnology company limited).Carrier linked system forms: 10 * buffer, 1 μ L; PMD18-T Vector(50ng), 1 μ L; The pcr amplified fragment reclaiming, 8 μ L; Cumulative volume is 10 μ L and is mixed evenly, and in 16 ℃, connects the connection of spending the night in 4 hours or 4 ℃.The connection product of 10 μ L and 100 μ L competent cells are mixed, on ice, place 30min, then thermal shock 90s in the circulator bath of 42 ℃.Rapidly pipe is transferred to 5min in ice bath.Add 400 μ L LB liquid nutrient mediums, 1h is cultivated in 37 ℃ of recoveries.4000r/min abandons part upper strata liquid after centrifugal 6 minutes, get 100 μ L and transfer on LB culture medium flat plate.After liquid is absorbed, be inverted plate, in 37 ℃, cultivate 12-16h.More than the single bacterium colony of picking adds and cultivates 6h in the LB substratum that contains penbritin, then carry out pcr amplification, electrophoresis detection, picking positive colony.Identify that correct bacterium liquid send Beijing AudioCodes biotechnology limited liability company to carry out sequencing.

(2) PCR-RFLP diagnostic method is set up

(1) primer sequence and pcr amplification condition

Select 3 Large Whites and 3 plum mountain pigs, the GSK3 α gene promoter sequence obtaining by embodiment 1 is test materials, has designed following primer, and primer sequence is as follows:

Forward primer F1:TAGGAAGCGTGAGGATGCA;

Reverse primer R1:TCGACTGGGTGTAACTGAA.

With this primer, in 3 " Large Whites " and 3 " plum mountain pig " genomic dnas, carry out pcr amplification respectively.PCR reaction system is 25 μ L, and wherein template DNA is 50ng, and dNTPs concentration is 10mmol/L, and every primer concentration is 10mmol/ L, 1U's taqarchaeal dna polymerase, adds deionized water to cumulative volume 25 μ l.PCR response procedures: 94 ℃ of denaturation 4min; Then 94 ℃ of sex change 40s, 61 ℃ of annealing 40s, 72 ℃ of extensions 20s, totally 35 circulations; Last 72 ℃ are extended 7min.PCR product detects through 1.5% agarose gel electrophoresis.

(2) RFLP detects

PCR product endonuclease reaction volume is 10 μ l, PCR product 6 μ L wherein, distilled water 2.7 μ L, 1 * buffer Tango, 1 μ L, restriction enzyme Hin1I is 0.3 μ L (10U/ μ L), by centrifugal after sample blending, 37 ℃ of incubators are placed 6h, with 2% agarose gel electrophoresis, detect enzyme and cut result, record genotype, under ultraviolet lamp, take pictures.

With primer amplification pig genomic dna, obtained 337bp specific amplification fragment, the 154th of this fragment is 1 A-C base transition, and this mutational site can cause the variation of Hin1I restriction enzyme site.Enzyme is cut and is produced three kinds of genotype, and AA genotype only has 337bp mono-band, and CC genotype has 184bp and 153bp two bands, and heterozygote AC genotype has 337bp, three bands of 184bp and 153bp.

embodiment 2: the polymorphism of molecule marker in different swinerys distributes

Utilize the method for PCR-Hin1I-RFLP, in 3 external western pig breeds (great Bai, long white and Du Luoke) and 3 Chinese native pig breeds (Mei Shan, Tongcheng and peaceful) swinery, detect pig GSK3 α gene pleiomorphism, detected result is as shown in table 1.Each genotype of GSK3 α gene, at great Bai, is grown A allelotrope in white and duroc and is preponderated, and in Mei Shan, Tongcheng and peaceful pig, C allelotrope is preponderated.

Table 1 not genotype and the gene frequency of GSK3 α gene Hin1I polymorphism in consanguinity pig variety

embodiment 3: the application of molecule marker in the production traits

Test swinery for statistical study is great Bai * Mei Shan F2Dai resource colony that pig genetics and breeding key lab of the Ministry of Agriculture is set up, and the main carcass trait dividing for association comprises skin rate, bone rate, fat meat rate, lean ratio, thin fat meat ratio, carcass weight, dressing percentage, leaf fat weight, caul-fat weight, lactones rate, long to atlas trunk, long to first rib chest trunk, the thickness of backfat between 6-7 lumbar vertebrae, the shoulder thickness of backfat, the thickness of backfat between chest lumbar vertebrae, the buttocks thickness of backfat, the average thickness of backfat, leg stern ratio, eye muscle is high, and eye muscle is wide, eye muscle area.

Applicant adopts SAS statistical software (SAS Institute Inc, Version 8.0) glm program to carry out single mark variance analysis, and carries out test of significance, and the model that adopts is:

Yijkl=μ+Gi +Sk+Yl(+bijklXijkl)+eijkl

Yij is proterties phenotypic number, and μ is mean value, and Gi is genotype effect; Sk, Yl are fixed effect, are respectively sex, annual effect, and bijkl is the regression coefficient of slaughter weight or slaughter age, and carcass trait be take slaughter age as concomitant variable; Eijkl is residual error effect.

The association analysis that pig GSK3 α gene Hin1I enzyme is cut genotype and carcass trait

^*expression significant difference ( p< 0.05), ^*represent difference extremely significantly ( p< 0.01)

Statistic analysis result between different genotype and the production traits is in Table 2.The thickness of backfat the and on average thickness of backfat and three proterties of buttocks fat thickness exist relevant (P<0.05) of conspicuous level between Hin1I site and eye muscle area, chest lumbar vertebrae in the carcass trait of statistics.And there is relevant (P<0.01) of utmost point conspicuous level in Hin1I site and buttocks thickness of backfat proterties.The genotypic eye muscle area of CC is far away higher than AA and AC genotype (P<0.05), and between the genotypic average thickness of backfat of while CC, the buttocks thickness of backfat and chest lumbar vertebrae, the thickness of backfat is significantly lower than AA and AC genotype (P<0.05).

SEQUENCE LISTING

<110> Sichuan Agricultural University

<120> GSK3 α gene promoter SNP is as genetic marker and the application of hog on hook proterties

<130> 2013

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1387

<212> DNA

<213> pig (Sus scrofa)

<400> 1

tgagggtagt ttcattccag ttccaggtat ttccgggaca cacacacaca cacacacaca 60

cacacacaca cacacacaca tcatatgaac tgtttcccag cgcggtttgg gactactgcc 120

cttccgggta taaactgata agtggggcga cataatgagt gcccaaaacc cgaggctact 180

cctagcagcc ggcttctttt gaggcttccg ggatcctgta atttaggaag cgtgaggatg 240

caaaaacctg aatggggcgg gggaggttcc aaacaaatta tattttcacc tctttcaaca 300

ttccgctcag tctcctggtg taagtcacat tgctagaaaa agctagacaa tatccgaaga 360

ctgtattgtc ctgagacgcc ggcagaaact ttgtttccta aatcgtttgc ggagagggtt 420

tccttctgga tttgtggaaa cttggatttt ggagtgttag gactccattt tccaccaccg 480

cctggtcacc tacggctttt ttgagggacc tgggggcggg gcaggatgcg ctgaaacttt 540

cttcagttac acccagtcga ctgccgttct ttcacctttc gctcgcgctc tctcaaagtt 600

accaattaga ctacacctcc cggcaagcct ctgcactttc gcctcataaa gggtcaggat 660

gcggtctggc ctttccctca gcccgcttct ggatcatgat agatcccttc ccctcagtca 720

atgggaaggc tgccttgaac tacatttccc agaagccaca aaggctcgct gtggcaacta 780

cgctgaagcg ttggtgttga ctacatttcc cagcaggggc tgcggcctag cgtatgaggg 840

aggaggaatg ctgtacttac tccatttcct atcctatctc cggaactgca gtattcgctt 900

gaggcttagg ggaaatcaca gaggactcca tttcccggcg ttctttggaa gtagctttac 960

acctgcaatg cggtgagggc gtggcgacaa ctccatttcc cagctggcct tgacgctgga 1020

tagtttcggg tccctggtct ccgactcagt ttcccagagt gcacctggcg cctgtgggca 1080

tacagccaat agactaaggc agcgactgaa ctccgtttcc cggagtgcat caggcctata 1140

cggctcactg atggaggcac tgcgactatg ttcctaaggc acacacaggt tgttgcggtg 1200

gagtgagtgt gagactctga cccagcttcc gaaagtgctt cagagtcagc tgtcagcaca 1260

gtcggccaac ttactgccca gaactccaat tcccgacgtg cagctcatca tccccgcggt 1320

ctactctgag gtctttgcag actccatttc ccggcgtgcc acgcggcact ctgtgagggc 1380

cgagaga 1387

<210> 2

<211> 337

<212> DNA

<213> pig (Sus scrofa)

<220>

<221> mutation

<222> (154)..(154)

<400> 2

taggaagcgt gaggatgcaa aaacctgaat ggggcggggg aggttccaaa caaattatat 60

tttcacctct ttcaacattc cgctcagtct cctggtgtaa gtcacattgc tagaaaaagc 120

tagacaatat ccgaagactg tattgtcctg agacgccggc agaaactttg tttcctaaat 180

cgtttgcgga gagggtttcc ttctggattt gtggaaactt ggattttgga gtgttaggac 240

tccattttcc accaccgcct ggtcacctac ggcttttttg agggacctgg gggcggggca 300

ggatgcgctg aaactttctt cagttacacc cagtcga 337

Claims (5)

1. as a GSK3 α promoter gene fragment relevant to hog on hook proterties for molecule marker application, its nucleotide sequence is as described in sequence table SEQ ID NO:2.

2. gene fragment according to claim 1, is characterized in that: the base mutation that has a C154-A154 at the 154bp place described in sequence table SEQ ID NO:2, causes Hin1I PCR-RFLP polymorphism.

3. it is as follows that test right requires the DNA sequence dna of the forward and reverse primer of genetic marker described in 1:

Forward primer: 5'TAGGAAGCGTGAGGATGCA 3'

Reverse primer: 5'TCGACTGGGTGTAACTGAA 3'.

4. the method for preparation gene fragment as claimed in claim 1 or 2, according to following steps:

(1) take the cDNA of pig GSK3 α gene is information probes, at pig genome database, do homologous sequence screening, obtain a BAC clone that comprises pig GSK3 α gene, with BAC clone sequence, make template design primer, the genomic dna of place of china pig variety plum mountain pig of take is template, carry out pcr amplification, PCR product purification and cloning and sequencing, obtain pig GSK3 α gene promoter sequence;

(2) extract pig genomic dna, with the nucleotides sequence described in SEQ ID NO:1, classify template design primer as, pcr amplification, PCR product purification and order-checking, obtain pig GSK3 α gene promoter partial nucleotide sequence, obtains the nucleotide sequence as described in sequence table SEQ ID NO:2.

5. genetic marker claimed in claim 2 application in the thickness of backfat, the buttocks thickness of backfat and average thickness of backfat proterties association analysis between pig eye muscle area, chest lumbar vertebrae.