CN104450729B - Pig flesh characters is correlated with the clone of WNT10B gene molecule marker and application - Google Patents
Pig flesh characters is correlated with the clone of WNT10B gene molecule marker and application Download PDFInfo
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Abstract
The invention belongs to domestic animal technical field of molecular biology, be specifically related to pig flesh characters and be correlated with the clone of WNT10B gene molecule marker and application.Molecule marker of the present invention is obtained by pig WNT10B gene clone, is the sequence of its WNT10B molecule marker as SEQ? ID? shown in NO:1.WNT10B gene polynorphisms utilizes comparative genomics method according to the WNT10B gene order design primer of people, with the genomic dna of pig for template amplification, amplified fragments utilizes order-checking screening SNP, and utilize PCR-RFLP to carry out gene type, find that there is the base mutation of an A/G at 552bp place, do you cause PCR-RFLP-Sal? I polymorphism, for pig flesh characters marker assisted selection provides new molecule marker, can utilize the situation of this marker detection China and foreign countries pig kind.
Description
Technical field
The invention belongs to domestic animal technical field of molecular biology, relate to a kind of nucleic acid molecule comprising pork quality trait related gene Nucleotide as shown in SEQ ID NO:1.The invention still further relates to mononucleotide polymorphism site in the molecular cloning method of the WNT0B gene as shown in SEQ ID NO:1 and polynucleotide sequence and its application in pig molecule mark assisted Selection.
Background technology
Pork is topmost meat on China 1,300,000,000 population dining table, and within 2009, China delivers live pig number 6.45 hundred million for sale, occupies the whole world first.The speed of growth of external product boar reaches high level, but meat is bad is also undisputable fact, Meat problem is subject to extensive concern day by day, in many influence factors, intramuscular fat content (Intramuscular Fat Content, IMF) be important influence factor, IMF is the leading indicator evaluating Meat quality, the tenderness of pork can be affected, local flavor, marble grain, succulence etc., the heritability of pig intramuscular fat content is about 0.6, within the specific limits, IMF is higher, meat is better, local variety pig intramuscular fat content will far above external pig kind, thus meat is excellent.
Wnt10b gene is the member of classical Wnt signal transduction path, and Wnt10b is the signal protein in classical Wnt signal transduction path.Wnt albumen is combined with the acceptor be positioned on cytolemma by autocrine or paracrine, activates the signal transducers at different levels in born of the same parents, regulates fatty deposits.But, in livestock and poultry particularly in pig about the research of the Wnt signal transduction pathway of Wnt10b gene mediated is few, extremely unbecoming with the develop rapidly of current gene function genome era bio-science.
A critical function of Wnt signal transduction pathway is regulation and control fatty depositss, therefore biological function and the application thereof of Wnt10b gene in pig is furtherd investigate, from molecule, the research of biological cells and tissues level to intramuscular fat deposition Regulation Mechanism, excavate and develop new genetic marker, for improving meat, accelerate discipline development significant.The research of the signal pathway of Wnt10b gene mediated is one of the discipline development of pig functional genomics and meat improvement problem demanding prompt solution.
Wnt gene is being collectively referred to as of homologous gene wingless and int-1, Sharma etc. have found aptery gene wingless (wg) for 1973 in the research of growing drosophila embryos, Nusser etc. are when Study Mouse breast tumor, from mammary gland of mouse cancer, clone a kind of proto-oncogene, be called int-l at that time.Utilize conserved sequence design primer in these Wnt genes to carry out pcr amplification in recent years, in the many species from vertebrates to nematode, there is Wnt gene.
Wnt signal transduction is the key signaling pathways in a too many levels, multiaction site and animal development, the fetal development of different plant species, the process such as cytodifferentiation and adult eubolism such as to take part in from the mankind to nematode, the research of Wnt and signal transduction thereof mainly concentrates on the aspects such as cytodifferentiation, fetal development and various diseases.
Wnt signal transduction is a very conservative signal transduction pathway, this approach is all found in hydra and the mankind, according to the mode of Wnt transduction signal, Wnt signal transduction pathway is divided into canonical Wnt signal pathway (Canonical Wnt signal Pathway) and non-classical Wnt signal by way of (Noncanonical WntSignal Pathway).
Canonical Wnt signal pathway is also known as Wnt/ β catenin (β-catenin) signal pathway, the protein complexes that one can be responded to receptor signal is dynamically there is in tenuigenin, this complex body is also called β-catenin degraded complex body, by axle albumen (Axin), adenoma shape polyposis (Adenomatous polyposis coli, APC), glycogen synthase kinase (Glycogen synthetase kinase-3 β, GSK-3 β), the composition such as casein kinase (Casein kinase 1 α, CK1 α).When born of the same parents outer without Wnt protein signal time, the GSK-3 β phosphorylation in this protein complexes is dissociated the specific site of β-catenin albumen, causes β-catenin to degrade, makes the β-catenin in tenuigenin be in lower concentration, when there is Wnt albumen outside born of the same parents, Wnt albumen (as Wntl0b) and its specific receptors FZ (frizzled, and accessory receptor LDH receptor related protein 5/6 (low-density lipoprotein receptor-related protein 5/6 Frz), LR P5/6) combine after, activate albumen (dishevlled at random, Dv1), the Dv1 albumen activated inhibits GSK-3 'beta ' activity, thus β-catenin free in prevention kytoplasm degrades, endogenous β-catenin is caused to gather in tenuigenin and enter nucleus, with its downstream signaling molecule T cell factor/lymphocyte enhancement factor (Tcf/Lef) or Tsh, Xsox17 and histone acetyltransferase carbohydrate-binding protein (CBP) interact, activate the relevant target gene of canonical Wnt signal pathway, regulation and control adipocyte.
The approach of the Wnt albumen (as Wnt4, Wnt5a, Wnt11) that in non-classical Wnt signal pathway, β-catenin does not gather transducer cell signal is by other means called non-classical Wnt signal pathway, in this approach, Wnt only with Frz effect, do not need LRP5/6.Wnt/ cell planar polar approach (planar cell polaritypathway) is by small G-protein Rho etc., activate Jun kinases (c-Jun N-terminal kinase, JNK), regulate the mal-distribution of cytoskeleton and epithelial collaborative polarization, regulate the planar polar of cell, the stage regulation and control etc. of fetal development, finally cause core target gene to be expressed.Wnt/Ca
2+signal pathway is activated, by the Ca mediated in cell by Wnt5a and Wntll
2+calmodulin dependent protein kinase II and protein kinase C (protein kinase C, PKC) and calcineurin (calcineurin, CAN) etc. work, and cause Ca in cell
2+release, thus affect cellular adhesion and genetic expression.
At present, both at home and abroad to pig WNT10B gene and associated proteins thereof and this gene very few in the report research of different tissues and different varieties pig differential expression, the research carrying out the bioinformatic analysis of this gene and gene expression profile will for WNT10B gene molecule hereditary property, provide basic basis in gene functional research such as intramuscular fat depositions.The regulation and control that the present invention is intended to for disclosing the Meat Qualities such as WNT10B gene pairs pig intramuscular fat content lay the foundation.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, the clone providing a kind of pig flesh characters to be correlated with WNT10B gene molecule marker and application, by clone pig WNT10B gene cDNA molecule, finding the mutational site of WNT10B gene and detect this gene polynorphisms, is the molecule marker that pig flesh characters marker-assisted breeding provides.
In order to solve the problem, the technical solution adopted in the present invention is: a kind of pig flesh characters is correlated with WNT10B gene, its DNA sequence dna, as shown in SEQ ID NO:1, at the base mutation that there is 1 A/G at 552 bp places of SEQ ID NO:1 sequence, causes PCR-RFLP-Sal I polymorphism.
The present invention with the WNT10B gene order of people (the GenBank number of including is for NM_003394.3) for Seed Sequences, BLASTN (Basic Local Alignment Search ToolNucleotide) is utilized in GenBank, special primer is designed with reference to the pig EST of its homology more than 80% (Expression Sequence Tags), utilize RACE (Rapid Amplification of cDNA End), clone pig WNT10B gene cDNA total length, the DNA sequence dna of this pig WNT10B gene is as shown in SEQ ID NO:1.WNT10B gene polynorphisms utilizes comparative genomics method according to the WNT10B gene order design primer of people, with the genomic dna of pig for template amplification, amplified fragments utilizes order-checking screening SNP, and utilize PCR-RFLP to carry out genotyping technique, find that there is the base mutation of an A/G at the 552nd bp place, cause PCR-RFLP-Sal I polymorphism, and utilize the situation of this marker detection China and foreign countries pig kind.
Test materials: 25 age in days Shaziling pig are provided by Shaziling pig resource field, xiangtan, hunan province city, get longissimus dorsi muscle-80 DEG C preservation, 5 '-RACE Version 2.0 test kit is purchased from Invitrogen company, 3 '-RACESMARTer
tMrACE cDNA Amplification Kit test kit is purchased from Clontech company.
The synthesis of Total RNAs extraction and cDNA the 1st chain: carry out the synthesis of gene first chain cDNA to total serum IgE with SUPERSCRIPT II RT enzyme and Auele Specific Primer GSP-1, uses the cDNA of RNase Mix to synthesis to go RNA process, finally carries out purifying to cDNA.
Design of primers: according to GenBank people Wnt10b gene (accession number NM_003394.3) sequence conservation design primer, the software of design of primers is Primer Premier 5.0, the principle of design of primers is that Auele Specific Primer length is at 23-28 Nucleotide, GC content is at 50%-70%, annealing temperature is 65 DEG C-70 DEG C, uses nest-type PRC to increase.
Encoding sequence pcr amplification: with the pig cDNA synthesized for template, at conserved regions design primer through cloning and sequencing, obtains this Gene Partial CDS (coding domain sequence) sequence.
5 '-RACE amplification: use TdT enzyme and dCTP to add poly C to the cDNA end after purifying, uses bridging rivet primer AUAP (table 1) in primer GSP5 (Gene-Specific Primers) and test kit to carry out PCR first round amplification to the cDNA adding dC tail; Use the bridging universal amplification primer AUAP in primer GSP-3 and test kit to carry out nest-type PRC second and take turns amplification, take turns PCR primer to carry out electrophoresis and cut glue to object band reclaiming purifying by second, PCR primer after purifying is connected with pMD18-T, after conversion, positive colony is checked order, obtain the aim sequence of 268 bp.
3 '-RACE amplification: use the cDNA of synthesis to carry out first round pcr amplification for template.First round pcr amplification product is diluted 50 times, then uses primer GSP3 and UPM (Universal Primer A Mix) (table 1) to carry out second and take turns pcr amplification.Take turns PCR primer to carry out electrophoresis and cut glue to object band reclaiming purifying by second.PCR primer after purifying is connected with pMD18-T, picking positive colony order-checking after transforming.
Length 910 bp that the 5 '-RACE length that obtains is 268 bp by increasing, 3 '-RACE increases to be obtained and open reading frame length are the full length cDNA sequence that the sequence assembly of the encoding sequence of 1058 bp obtains WNT10B gene, thus complete the content of the clone of WNT10B gene of the present invention.
The primer sequence used in table 1 the present invention
It is below the screening of WNT10B gene molecule marker.
DNA sample: get fritter pig ear tissue and extract DNA, totally 5 kinds, wherein Large White 325, the black pig in the Land of Peach Blossoms 107, Shaziling pig 57, Ning Xiang Swine 67 and Daweizi pig 70, amount to 626.
Design of primers: utilize comparative genomics method according to the 1st exon of the WNT10B gene (the GenBank number of including is KF 569219) of pig, BLAST is made at GenBank with this gene, candidate SNP s site is filtered out after sequence alignment, with reference to GenBank primers, use this site of PCR-RFLP technical identification.
Prepared the primer pair of detection above-mentioned sequence table SEQ ID NO:1 gene fragment sudden change, described primer pair sequence is as follows: forward primer is 5 '-AATCTGAAGCGGAAATGC-3 ' (SEQ ID NO:12); Reverse primer is 5 '-TAAAGGGACTCCAGGGTT-3 ' (SEQ ID NO:13).
PCR condition: PCR reaction system (cumulative volume 20 μ L): 10 × Buffer 2 μ L, 2mmol/L dNTPs1.6 μ L, 20mmol/L MgCl
21.6 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.4 μ L, DNA profiling (100ng/ μ L) 1 μ L, upstream and downstream primer (10pmol/ μ L) each 0.4 μ L, dd H
2o12.6 μ L.
Response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 50s, totally 35 circulations; 8min is extended, last 4 DEG C of preservations after 72 DEG C.
Get 10 μ L pcr amplification products, after adding 2 μ L tetrabromophenol sulfonphthalein sample-loading buffer mixings, point sample is on the sepharose (containing 0.05%EB) of 1%, and 6 μ L 100bp DNA Markers are as reference.5V/cm electrophoresis 0.5-1.0h, by the PCR primer Hou Songboshang biotech company order-checking after purifying.
The Sal I enzyme of PCR primer is cut.
In 10 μ L PCR primer, add 0.8 μ L 10 × restriction endonuclease damping fluid, 0.2 μ L restriction enzyme and 1 μ L distilled water, cumulative volume is 12 μ L, 37 DEG C of digestion 4-10h.The 2% agarose gel electrophoresis analysis of A/G site, 5V/cm electrophoresis 0.5h, observations under ultraviolet lamp is also taken pictures, enzyme cuts result as Fig. 1, amplified production checks order, and sequence is as shown in SEQ ID NO:1, and am-plified fragments is between the 1st exon to exon 2,732bp, is PCR-RFLP-Sal I molecule marker altogether.
SNP finds to set up with detection method: applicant devises amplification and comprises this SNP primer, and the sudden change in A/G site can adopt Sal I to carry out enzyme and cut detection polymorphism by analysis.In the segment of the 732bp of amplification, there is the restriction enzyme site of 1 Sal I, there are 3 kinds of genotype in the A/G site that electrophoresis detection result is presented at WNT10B gene, AA genotype (732bp), AG type (180bp, 552bp and 732bp) and GG type (180bp and 552bp).
Utilize the pig molecule mark of above-mentioned preparation to be applied to external pig kind and place of china kind, thus complete the present invention.
The present invention will establish solid basis for Meat Quality marker assisted selection (MAS) such as pig intramuscular fat contents, for improving Swine Production economic benefit, and guide the breeding practice of pig to provide theoretical foundation.
Accompanying drawing explanation
Fig. 1 is the Sal I restriction enzyme digestion and electrophoresis result in WNT10B of the present invention Gene A/G site.
In figure: swimming lane 1,5,9,10:AA genotype; Swimming lane 3,4,7,8:AG genotype: swimming lane 2,6:GG fundamental mode; M:100 bp DNA Ladder Marker.
Embodiment
Below in conjunction with specific embodiment, the present invention is explained further, but concrete enforcement does not do any restriction to the present invention.
With the WNT10B gene order of people (the GenBank number of including is for NM_003394.3) for Seed Sequences, BLASTN is utilized in GenBank, special primer is designed with reference to the pig EST of its homology more than 80% (ExpressedSequence Tags), RACE technology is utilized to clone pig WNT10B gene cDNA total length, WNT10B gene polynorphisms utilizes comparative genomics method according to the WNT10B gene order design primer of people, with the genomic dna of pig for template amplification, amplified fragments utilizes order-checking screening SNP, find that there is the base mutation of an A/G at 552bp place, cause PCR-RFLP-Sal I polymorphism, and utilize this have detected distribution situation that this is marked at China and foreign countries' pig kind.
Get 25 age in days Shaziling pig longissimus dorsi muscles, the synthesis of Total RNAs extraction and cDNA the 1st chain uses SUPERSCRIPT II RT enzyme and Auele Specific Primer GSP-1 to carry out the synthesis of gene first chain cDNA to total serum IgE, uses the cDNA of RNase Mix to synthesis to go RNA process.
Utilize RACE test kit to carry out 5 '-RACE and 3 '-RACE to increase, 5 '-RACE and 3 '-RACE is spliced to obtain the full length cDNA sequence of WNT10B gene, thus complete the clone of WNT10B gene of the present invention.
Design of primers: utilize comparative genomics method according to No. 12 karyomit(e) of the WNT10B gene (the GenBank number of including is NM_003394.3) of people, BLAST is made at GenBank with this gene, WNT10B gene candidate SNPs site is filtered out after sequence alignment, choose WNT10B gene candidate SNPs site, with reference to GenBank sequence NM_003394.3 primers, use this site of PCR-RFLP technical identification.Forward primer is 5 '-AATCTGAAGCGGAAATGC-3 ', and reverse primer is 5 '-TAAAGGGACTCCAGGGTT-3 '.
PCR reaction conditions: PCR reaction system (cumulative volume 20 μ L), comprises 10 × Buffer 2 μ L, 2mmol/L dNTPs 1.6 μ L, 20mmol/L MgCl
21.6 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.4 μ L, DNA profiling (100ng/ μ L) 1 μ L, upstream and downstream primer (10pmol/ μ L) each 0.4 μ L, dd H
2o 12.6 μ L.
PCR response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 50s, totally 35 circulations; 8min is extended, last 4 DEG C of preservations after 72 DEG C.
Get 10 μ L pcr amplification products, after adding 2 μ L tetrabromophenol sulfonphthalein sample-loading buffer mixings, point sample, on the sepharose (containing 0.05%EB) of 2%, puts 6 μ L 100bp DNA Markers as reference.5V/cm electrophoresis 0.5-1.0h.
The Sal I enzyme of PCR primer is cut: in 10 μ L PCR primer, add 0.8 μ L 10 × restriction endonuclease damping fluid, 0.2 μ L restriction enzyme and 1 μ L distilled water, cumulative volume is 12 μ L, 37 DEG C of digestion 4-10h.The 2% agarose gel electrophoresis analysis of A/G site, 5V/cm electrophoresis 0.5h, observations under ultraviolet lamp is also taken pictures.
The Sal I polymorphism of PCR-RFLP technology for detection WNT10B gene.
The present invention with pig WNT10B gene for research object, using 4 local pig breeds (Daweizi pig, Shaziling pig, Ning Xiang Swine, the black pig in the Land of Peach Blossoms), 1 external pig kind (Large White) as experimental subjects, wherein, Large White 325, Daweizi pig 70, Shaziling pig 57, the black pig of Ning Xiang Swine 67 and the Land of Peach Blossoms 107, amount to 626.
Applicant devises amplification and comprises this SNP primer, and the sudden change in A/G site adopts Sal I to carry out enzyme and cuts detection polymorphism by analysis.In the fragment of the 552bp of amplification, there is the restriction enzyme site of 1 Sal I, there are 3 kinds of genotype in the A/G site that electrophoresis detection result is presented at WNT10B gene, AA genotype (732bp), AG type (180bp, 552bp, 732bp) and GG type (180bp, 552bp).
WNT10B Gene A/the gene frequency in G site, genotype frequency are detected, and analyzes its genetic polymorphism.
The distribution situation of PCR-RFLP-Sal I polymorphism in each kind is as shown in table 2 below, as shown in Table 2, in the black pig in the Land of Peach Blossoms, Daweizi pig, these 3 Hunan local variety of Ning Xiang Swine, A gene is advantage allelotrope, its gene frequency is respectively 0.9019,0.8000,0.6866, and sand ridge A and G gene frequency very nearly the same.In adventive Large White, A gene is advantage allelotrope, and gene frequency is 0.6677; According to suitability chi square test, meet Mendelian inheritance law of segregation genotype frequency and compare 1:2:1.
The gene frequency in table 2 different varieties WNT10B Gene A/G site and genotype frequency distribution
Table 3 WNT10B-552 A → position, Building G different genotype is to the effect of pig growth traits
As shown in Table 3, AA, AG and GG tri-kinds of genotype are in correction age in days and correction thickness of backfat difference remarkable (p>0.05).
Claims (4)
1. pig flesh characters is correlated with a WNT10B gene, and its DNA sequence dna, as shown in SEQ ID NO:1, at the base mutation that there is 1 A/G at 552 bp places of SEQ ID NO:1 sequence, causes PCR-RFLP-SalI polymorphism.
2. test right requires pig flesh characters described in 1 to be correlated with the primer pair of base mutation of WNT10B gene, it is characterized in that, described primer pair sequence is: forward primer is 5 '-AATCTGAAGCGGAAATGC-3 ', and reverse primer is 5 '-TAAAGGGACTCCAGGGTT-3 '.
3. the application in the pig WNT10B gene according to claim 1 molecular marker assisted selection of being correlated with at pig flesh characters.
4. the primer pair according to claim 2 application of being correlated with in the molecular marker assisted selection of WNT10B gene at pig flesh characters.
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| CN105255878B (en) * | 2015-11-16 | 2018-01-26 | 上海市农业科学院 | It is used for SNP marker and its application traced to the source on No. 6 chromosomes of pig |
| CN105838795B (en) * | 2016-04-27 | 2019-08-06 | 华中农业大学 | The molecular labeling and its application of fat thickness at back of pig and intramuscular fat trait related gene SVEP1 |
| CN106544412B (en) * | 2016-08-30 | 2020-12-04 | 华中农业大学 | Molecular marker related to boar sperm motility character and application thereof |
| CN110079614B (en) * | 2019-05-23 | 2021-03-16 | 华中农业大学 | Molecular marker related to pig muscle fiber area and intramuscular fat content, detection method and application thereof |
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