CN103333897B - Clone and application of pork quality character correlation POSTN gene molecular marker - Google Patents
Clone and application of pork quality character correlation POSTN gene molecular marker Download PDFInfo
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- CN103333897B CN103333897B CN201310220869.2A CN201310220869A CN103333897B CN 103333897 B CN103333897 B CN 103333897B CN 201310220869 A CN201310220869 A CN 201310220869A CN 103333897 B CN103333897 B CN 103333897B
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Abstract
The invention belongs to the technical field of the preparation of the livestock molecular marker, and particularly relates to clone and an application of a pig POSTN gene molecular marker. The molecular marker is obtained by cloning the POSTN gene, and the sequence of the POSNT molecular marker is shown as SEQ ID NO.1. A C/T base mutation occurs on the 709th bp position of the SEQ ID NO.1, the polymorphism of PCR-RFLP-HaeIII is caused, and the molecular marker can be used for the correlation analysis of the pork quality characters. A novel molecular marker can be provided for the auxiliary selection of the pork quality character marker.
Description
Technical field
The invention belongs to domestic animal technical field of molecular biology, relate to a kind of nucleic acid molecule comprising pig gene nucleotide as shown in SEQ ID NO:1.The invention still further relates to mononucleotide polymorphism site in the polynucleotide sequence as shown in SEQ ID NO:1 and the method for mononucleotide polymorphism site as described in detecting.
Background technology
Pork is the main source of urban and rural residents of China animal protein, along with people are to the increase of meat requirement, also relatively improves the requirement of meat quality.And this is mainly by Gene Handling, and there is major gene effect.The development of molecular biotechnology, make people can find at DNA level the key-gene or molecule marker closely linked with it that control Meat Quality, marker assisted selection is applied in breeding process, to improve selection process, improve pig flesh characters better, meet the needs of people, to obtain larger economic benefit.
What meat definition was accepted extensively the most is the definition of Hoffmarm, and namely meat should consider sensory attribute, nutritive value, technical quality and food safety.Sensory attribute refers to outward appearance (as color and luster), moisture holding capacity, hardness, eating quality refers to tenderness, succulence, local flavor and flavour, stink, marble grain, nutritive value refers to lipid content, lipid acid composition, protein content, other nutrient contg, technical factor refers to Coefficient shrinkage, fatty hardness, separate tissue, oxidative stability, society's quality refers to animal welfare, environment, and food safety refers to microorganism health (without Salmonellas, Campylobacter), noresidue (microbiotic, heavy metal, sterilant).Affect pork qualitative factor a lot, there are inherited genetic factors and non-genetic factor, from heredity, pig flesh characters to be evaluated and seed selection is the key improving meat quality, Meat Quality belongs to quantitative character mostly, controlled by minor-polygene, there is major gene effect in some quantitative character, great majority belong to QTL.Many Meat Qualities could can only measure after animal slaughtering, so conventional herd breeding can only express compatriot or half-sibs test, this just certainly will strengthen seed selection cost, and genetic progress is slow.Can only could measure when animal slaughtering by Meat Quality, need expensive sib test, therefore, utilizing molecule marker to carry out assisted Selection (MAS), to control Meat Quality be significantly.
The development of molecular biotechnology and the structure of pig high-density gene mapping, make breeder can find the key-gene affecting Meat Quality or the molecule marker being close to lock with it on DNA level, these key-genes or molecule marker are once confirmation in theory, just can carry out marker assisted selection breeding.
Mouse bone-forming cell specific factor (periostin, osteoblast specific fctor, POSTN) gene POSTN is named as Osf2, with the POSTN gene of mouse for probe, examination people blastodisc and osteosarcoma cDNA library, the POSTN gene of clone 2 kinds of people is multi-form, according to inferring, a kind of coding 779 amino acid, molecular weight is 87.0KD, another 836 amino acid of encoding, molecular size range is 93.3KD, POSTN albumen contains 1 typical signal peptide, 1 cysteine rich territory and 4 150 amino acid whose folding repeating structures, 1 C-terminal domains, the homology of mouse and people POST N protein is 89.2%, the homology of mature form is 90.10%.The Northern blot hybridization of mouse bone-forming cell system detects the transcript of the POSTN gene of a 3.4kb, RNA Dot hybridization analysis, and POSTN gene is expressed in the scleroblast and lung of mouse, in other organ, do not measure expression.
Gillan etc. have found that an EST containing POSTN gene clones, POSTN genes encoding 782 amino acid according to inferring, RNA dot blot assay shows, this gene is wide expression in the aorta of grownup, stomach, gi tract, placenta, uterus, mammary tissue.This gene is expressed in foetal calf serum, but does not express in the calf serum of new life.The discoveries such as Gillan, the hysteroma of cultivating secrete POSTN albumen, but do not secrete in normal uterine epithelial cell, suffer from the ascites of 20 people uterus carcinoma patient find POSTN albumen from 21.POSTN recombinant protein contributes to the adhesion of uterine epithelial cell, and the antibody capable anti-adhesion of integrin ITGB3 and ITGB5, and he thinks that OSTN is part as integrin IT-GB3 and ITGB5 and promotes the migration of cell adhesion and uterine epithelial cell accordingly.The discoveries such as Shao, POSTN overexpression in the breast tumor of most people, the tumor cell line energy accelerating growth of the transfection of overexpression POSTN albumen, and after the xenotransplantation of non-responsiveness animal, accelerate vascularization.It is up-regulated expression due to vascular endothelial growth factor receptor KDR in a way that the blood of POSTN mediation occurs in.
People POSTN gene DNA total length 36096bp, cDNA total length is 3213bp, and containing 23 exons, 22 introns, 5'-UTR is long is 691bp, 3'-UTR11bp.Mouse (Mus musculus) DNA total length 29908bp, cDNA total length is 3187bp, and containing 22 exons, 21 introns, 5'-UTR is long is 18bp, 3'-UTR133bp.This gene of people is positioned at HSA13q13.3, and mouse POSTN gene is positioned at No. 3 karyomit(e) 3C.
But the domestic and international correlative study about pig POSTN gene is little at present.
Summary of the invention
The object of the invention is to clone pig Meat Quality gene POSTN fragment, find the mutational site of POSTN gene and the detection method as pig flesh characters gene pleiomorphism, for marker-assisted breeding provides a kind of useful molecule marker.
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, provides a kind of pork quality trait related gene POSTN and the application in pig marker assisted selection thereof, is the molecule marker that pig marker assisted selection provides.
In order to solve the problem, the technical solution adopted in the present invention is: the application of a kind of pork quality trait related gene POSTN in pig molecule mark assisted Selection, there is the base mutation of a C/T at the 709bp place of DNA sequence dna as shown in SEQ ID NO:1 of this pork quality trait related gene POSTN gene, causes PCR-RFLP-Hae Ш polymorphism.
Above-mentioned POSTN gene polynorphisms utilizes comparative genomics method according to the conserved regions design primer of the POSTN gene of people, with the genomic dna of pig for template amplification, amplified fragments utilizes SSCP technology and sequencing technologies to find SNP, and utilize PCR-RFLP to carry out gene type, and recycling SAS software GLM(general linear model) analyze associating of SNP and Meat Quality.
Applicant obtains a kind of gene fragment relevant to pig flesh characters by clone, and its nucleotide sequence is as described in sequence table SEQ ID NO:1.There is the base mutation of 1 C/T at the 709bp place of sequence table SEQ ID NO:1, causes PCR-RFLP-Hae Ш polymorphism.
Prepare the primer pair of detection above-mentioned sequence table SEQ ID NO:1 gene fragment sudden change, described primer pair,
Forward primer is 5 '-AGGAGCACTTATTCTTGT-3 '.
Reverse primer is 5 '-AAATGCGTTATTCACAGG-3 '.
Utilize the molecule marker relevant to pig flesh characters of above-mentioned preparation to carry out the application of association analysis to external pig kind and place of china kind, thus complete the present invention.
SNP finds to set up with detection method: inventor has devised amplification and comprise this SNP primer, the sudden change in C/T site can adopt Hae Ш to carry out enzyme and cut detection polymorphism by analysis.In the fragment of the 1249bp of amplification, there is the restriction enzyme site of 1 Hae Ш, there are 3 kinds of genotype in the C/T site that 2% agarose gel electrophoresis detected result is presented at POSTN gene fragment, TT type (1249bp), CT type (541bp, 708bp, 1249bp) and CC genotype (541bp, 708bp).Restriction enzyme digestion and electrophoresis result confirms the sudden change existed at POSTN gene the 9th intron base positions 12835bp place.
Associate between genotype-Meat Quality: utilize the general linear model in SAS software (General Linear Model, GLM) program to carry out association analysis to genotype and proterties, model the following is Y
ijkn=μ+h
i+ l
j+ g
k+ ε
ijkn, Y
ijknfor trait phenotypes value, μ is population mean, h
ifor pasture effect, l
jfor ageing, g
kfor the effect of POSTN genotype different loci, ε
ijknfor random error effect, Normal Distribution (0, σ
2).Analysis shows that POSTN gene SNP site has remarkably influenced (P<0.01) to the thickness of backfat, and also there is remarkably influenced in this site to other Meat Quality.
The present invention is by the molecule marker for pig flesh characters, for marker assisted selection (MAS) establishes solid basis, illustrating the molecular mechanism of Meat Quality further, theoretical foundation will be provided for improving meat quality, improve Swine Production economic benefit, and guide the breeding practice of pig.
Accompanying drawing explanation
Fig. 1 is the electrophoresis result figure of the pcr amplification product of POSTN gene fragment of the present invention.
In figure: M:100bp DNA Ladder Marker, 1-7 represents different swimming lane.
Fig. 2 is the Hae Ш restriction enzyme digestion and electrophoresis result in POSTN of the present invention gene C/T site.
Swimming lane 2:TT genotype; Swimming lane 1,3,4,7:CT genotype: swimming lane 5,6:CC genotype; M:100bp 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.
The Hae Ш polymorphism of PCR-RFLP technology for detection POSTN gene.
Design of primers: utilize comparative genomics method according to the 8th intron of the POSTN gene (the GenBank number of including is NM_001206351) of people to the 10th intron design primer, after GenBank makes BLAST sequence alignment, POSTN gene candidate SNPs site is filtered out with this gene, choose the 12835bp candidate SNP s site of POSTN gene complete sequence, design Auele Specific Primer, increases from the genomic dna of pig ear tissue extraction with these primers.
Primer is: F:5 '-GAGTGCCAAGGAGCAGAAAT-3 ',
R：5′-CCCGCTAAGGTGTGTTTGTT-3′
DNA sample from 8 kinds totally 772, wherein pig farm duroc 57, Large White 118 are planted by Yiyang institute of agricultural sciences; Pig farm Large White 106 is planted by north, Xiang Tan agricultural university; Zheng Hongzhong pig farm, Yueyang Large White 136, new five rich pig farm landrace 38; Local variety Ning Xiang Swine 67, the black pig in the Land of Peach Blossoms 70, Daweizi pig 62, Shaziling pig 63 and Wuzhi Mountain pig 55.Get fritter and extract DNA for examination pig ear tissue.
PCR condition: PCR reaction system (cumulative volume 20 μ L): 10 × Buffer2 μ L, 2mmol/L dNTPs1.6 μ L, 20mmol/LMgCl
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, ddH
2o12.6 μ L.
Response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 50.5 DEG C of annealing 30s, 72 DEG C extend 1min, totally 35 circulations; 10min 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%, then puts 6 μ L100bp DNA Markers as reference.5V/cm electrophoresis 0.5 ~ 1.0h.In gel imaging system, observe amplification after electrophoresis terminates and take pictures, result as shown in Figure 1.By the PCR primer Hou Songboshang biotech company order-checking after purifying.
The Hae Ш enzyme of PCR primer is cut: in 10 μ L PCR primer, add 8 μ L10 × restriction endonuclease damping fluids, 0.2 μ L restriction enzyme and 2 μ L distilled waters, cumulative volume is 20.2 μ L, 37 DEG C of digestion 10h.The 1% agarose gel electrophoresis analysis of C/T site, 5V/cm electrophoresis 0.5h, observations under ultraviolet lamp is also taken pictures, and enzyme cuts result as Fig. 2, and amplified production checks order, sequence is as shown in SEQ ID NO:1, am-plified fragments is between pig POSTN gene 12127bp to 13375bp, is the segment of the 8th intron to the 10th intron, altogether 1249bp, for PCR-RFLP-Hae III molecule marker, Fig. 2 is TT, CT and CC electrophoresis result of 3 kinds of genes of POSTN gene PCR-RFLP in the present invention.M:DNA molecular weight standard (DL100bp ladder) in figure.
The present invention is using pig POSTN gene as the candidate gene of pig flesh characters, mix as test materials with 5 local pig breeds (Ning Xiang Swine, the black pig in the Land of Peach Blossoms, Daweizi pig, Shaziling pig and Wuzhi Mountain pig), 3 external pig kinds (duroc, Large White, landrace) and Du × length × Dasanyuan, PCR-RFLP method is adopted to detect the POSTN gene C/gene frequency in T site, genotype frequency, and analyze its genetic construction, analyze the dependency of this gene and Meat Quality.
The distribution situation of PCR-RFLP-Hae Ш polymorphism in each kind is as shown in table 1 below, as can be seen from Table 1, in Ning Xiang Swine, Daweizi pig, Shaziling pig, Land of Peach Blossoms pig, these 5 local variety of Wuzhi Mountain pig, C gene is advantage allelotrope, wherein there is not T gene in Land of Peach Blossoms pig and Wuzhi Mountain pig, Shaziling pig C gene frequency the highest (0.9921) in other 3 local pigs; In 3 adventive durocs, landrace, Large White, C gene is also advantage allelotrope, duroc C gene frequency the highest (0.7368); From genotype distribution frequency, CC type is preponderated.
The gene frequency in table 1 different varieties POSTN gene C/T site and genotype frequency
The application of molecule marker of the present invention in the association analysis of pig flesh characters mark property.
Using SAS8.02(Statistical Analysis System) statistical software carries out association analysis to POSTN genotype and pig flesh characters.Model is as follows:
Y
ijkn=μ+h
i+l
j+g
k+ε
ijkn
Y
ijknfor trait phenotypes value, μ is population mean, h
ifor pasture effect, l
jfor ageing, g
kfor POSTN genotype different loci is to the effect of proterties, ε
ijknfor random error effect, Normal Distribution (0, σ
2).
Table 2 pig POSTN gene C/T site and Meat Quality association analysis table
In table, each row mark same letter represents difference significantly, the expression significant difference (lower same) of the different letter of mark
As shown in Table 2, in duroc, CC genotype percentage of water loss reduces 8.98% (p<0.05) than CT genotype percentage of water loss, TT genotype storage loss reduce 0.89%(p<0.05 than CC genotype storage loss), CC, CT, TT genotype surveys mean ph value, cold cuts rate, between yellowish pink grade and marble grain grade, difference is all not remarkable.
In Large White, CT genotype percentage of water loss reduces 4.48%(p<0.05 than TT genotype), marble grain grade is then lower than TT genotype, difference 0.80(p<0.05), the genotypic yellowish pink grade of CC yellowish pink grade more genotypic than TT is high 0.50(p<0.05), but difference is not remarkable between CC and CT genotype, CC genotype storage loss reduce 0.57%(p<0.05 than CT genotype storage loss), CC, CT, between TT genotype pH value and cold cuts rate, difference is not remarkable.In landrace, the yellowish pink grade of CT genotype is than TT genotype yellowish pink grade height 0.75(p<0.05), not remarkable with CC genotypic difference, CC genotype storage loss are higher than TT genotype storage loss 0.44%(p<0.05), the difference in other proterties does not all reach conspicuous level.In DLY ternary is assorted, CC genotype pH value is higher by 0.47 than TT genotype pH value, CC genotype percentage of water loss reduces 5.27%(p<0.05 than TT genotype percentage of water loss), but difference is not remarkable between CC and CT genotype, the yellowish pink grade of CT genotype grade height more yellowish pink than CC genotype 1.47(p<0.05), and difference is not remarkable between CC and TT genotype, in other proterties, difference is not remarkable.
Table 3 Large White POSTN gene C/T site and growth traits association analysis table
As shown in Table 3, in Large White, the CC genotype thickness of backfat is than TT genotype thickness of backfat 3.18mm(p<0.01), and CC, CT and TT genotype difference remarkable (p>0.05) on 100kg age in days.
Claims (4)
1. a POSTN gene fragment relevant to pig flesh characters, its sequence is as described in sequence table SEQ ID NO:1; The base mutation of 1 C/T is had at the 709bp place of sequence table SEQ ID NO:1.
2. test right requires the primer pair of a kind of relevant to the pig flesh characters POSTN gene fragment described in 1, and described primer pair, forward primer is 5 '-AGGAGCACTTATTCTTGT-3 ', and reverse primer is 5 '-AAATGCGTTATTCACAGG-3 '.
3. detect the method for as claimed in claim 1 relevant to pig flesh characters POSTN gene fragment, according to following steps:
The primer described in claim 2 is utilized to carry out pcr amplification, PCR primer 2% agarose gel electrophoresis detects, utilize restriction enzyme Hae Ш to carry out enzyme to PCR primer and cut qualification, finally detect the genotypic distribution of CC, CT and TT with 2% agarose gel electrophoresis.
4. the application of POSTN gene fragment in pig molecule mark assisted Selection relevant to pig flesh characters described in claim 1.
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| CN104032004B (en) * | 2014-06-12 | 2016-06-29 | 北方民族大学 | A kind of method for identifying molecules of Islamic meat product |
| CN107267644A (en) * | 2017-07-31 | 2017-10-20 | 湖南农业大学 | Application of the pig NDEL1 gene molecule markers in meat detection |
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| CN101701262B (en) * | 2009-11-17 | 2012-05-30 | 华中农业大学 | Molecular marker relative to pig meat quality traits and application |
| CN102719523B (en) * | 2011-12-28 | 2014-06-25 | 中山大学 | Molecule labeling method for maker-assisted selection of pig backfat thickness |
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Effective date of registration: 20160111 Address after: 516626, Guangdong province Shanwei city red grass Town Fen Village Patentee after: SHANWEI BAOSHAN PIG FARM CO., LTD. Address before: 410128 Hunan province Changsha Furong District Agricultural Road No. 1 Patentee before: Hunan Agricultural University |
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