CN103333897A - 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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- CN103333897A CN103333897A CN2013102208692A CN201310220869A CN103333897A CN 103333897 A CN103333897 A CN 103333897A CN 2013102208692 A CN2013102208692 A CN 2013102208692A CN 201310220869 A CN201310220869 A CN 201310220869A CN 103333897 A CN103333897 A CN 103333897A
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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 the domestic animal technical field of molecular biology, relate to a kind of nucleic acid molecule that comprises pig gene nucleotide shown in SEQ ID NO:1.The invention still further relates to the mononucleotide polymorphism site in the polynucleotide sequence shown in SEQ ID NO:1 and detect as described in the method for mononucleotide polymorphism site.
Background technology
Pork is the main source of China urban and rural residents animal protein, along with the increase of people to the meat requirement, to the also raising relatively of requirement of meat quality.And this mainly is by Gene Handling, and has the key-gene effect.The development of molecular biotechnology, make people can seek key-gene or the molecule marker closely linked with it of control meat proterties at dna level, in breeding process, be applied to marker assisted selection, to improve the selection process, improve pig flesh characters better, satisfy people's needs, to obtain bigger economic benefit.
What the meat definition was accepted extensively the most is the definition of Hoffmarm, and namely meat should be considered 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 tender degree, 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 be waterpower, fatty hardness, separate tissue, oxidative stability, society's quality refers to animal welfare, environment, and food safety refers to microorganism health (no Salmonellas, Campylobacter), noresidue (microbiotic, heavy metal, sterilant).It is a lot of to influence the pork qualitative factor, inherited genetic factors and non-genetic factor are arranged, evaluating with seed selection to pig flesh characters from the heredity is the key of improving meat quality, the meat proterties belongs to quantitative character mostly, controlled by minor-polygene, there is the key-gene effect in some quantitative character, and great majority belong to QTL.Many meat proterties can only could be measured behind animal slaughtering, so conventional seed selection can only be expressed compatriot or half-sibs test, this just certainly will strengthen the seed selection cost, and genetic progress is slow.Can only the meat proterties could measure when animal slaughtering, need expensive sib test, therefore, it is very significant utilizing molecule marker to carry out assisted Selection (MAS) control meat proterties.
The structure of the development of molecular biotechnology and pig high-density gene mapping, the person can seek the molecule marker that influences the key-gene of meat proterties or be close to lock with it at dna level to make the breeding work, these key-genes or molecule marker just can carry out the marker assisted selection breeding once affirmation in theory.
Mouse bone-forming cell specific factor (periostin, osteoblast specific fctor, POSTN) gene POSTN is named as Osf2, POSTN gene with mouse is probe, examination people blastodisc and osteosarcoma cDNA library, 2 kinds of people's of clone POSTN gene is multi-form, according to inferring, 779 amino acid of a kind of coding, molecular weight is 87.0KD, another 836 amino acid of encoding, the molecular weight size is 93.3KD, POSTN albumen contains 1 typical signal peptide, 1 halfcystine enrichment territory and 4 150 amino acid whose folding repeating structures, 1 C-end structure territory, the homology of mouse and people POST N albumen is 89.2%, the homology of mature form is 90.10%.The Northern blot hybridization of mouse bone-forming cell system detect a 3.4kb the POSTN gene transcription this, the RNA dot hybridization is analyzed, the POSTN gene is expressed in the scleroblast of mouse and lung, does not measure expression in other organ.
Gillan etc. have found an EST clone who contains the POSTN gene, 782 amino acid of POSTN genes encoding according to inferring, RNA Dot blot analysis revealed, this gene is wide expression in adult aorta, stomach, gi tract, placenta, uterus, mammary tissue.This gene is expressed in foetal calf serum, but does not express in new life's calf serum.Discoveries such as Gillan at the hysteroma secretion POSTN albumen of cultivating, but are not being secreted in normal uterine epithelial cell, find POSTN albumen from 21 ascites of suffering from 20 people the uterus carcinoma patient.The POSTN recombinant protein helps the adhesion of uterine epithelial cell, and integrates the antibody capable inhibition adhesion of plain ITGB3 and ITGB5, and he thinks that OSTN is the migration that promotes cell adhesion and uterine epithelial cell as the part of integrating plain IT-GB3 and ITGB5 accordingly.Discoveries such as Shao, POSTN overexpression in most people's breast tumor, 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 because the up-regulated expression of vascular endothelial growth factor receptor KDR in a way that the blood of POSTN mediation occurs in.
People POSTN gene DNA total length 36096bp, the cDNA total length is 3213bp, contains 23 exons, 22 introns, 5'-UTR is long to be 691bp, 3'-UTR11bp.Mouse (Mus musculus) DNA total length 29908bp, the cDNA total length is 3187bp, contains 22 exons, 21 introns, 5'-UTR is long to be 18bp, 3'-UTR133bp.This gene of people is positioned at HSA13q13.3, and mouse POSTN gene is positioned at karyomit(e) 3C No. 3.
But, at present both at home and abroad about the correlative study of pig POSTN gene seldom.
Summary of the invention
The objective of the invention is to clone pig flesh characters gene POSTN fragment, seek the mutational site of POSTN gene and as the detection method of 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: at above-mentioned the deficiencies in the prior art, provide a kind of pork quality trait related gene POSTN and the application in the pig marker assisted selection thereof, provide the molecule marker of usefulness for the pig marker assisted selection.
In order to address the above problem, the technical solution adopted in the present invention is: the application of a kind of pork quality trait related gene POSTN in the pig molecule mark assisted Selection, there is the base mutation of a C/T at the 709bp place of dna sequence dna shown in SEQ ID NO:1 of this pork quality trait related gene POSTN gene, causes PCR-RFLP-Hae Ш polymorphism.
The polymorphism of above-mentioned POSTN gene is to utilize the comparative genomics method according to people's POSTN gene conservative district design primer, genomic dna with pig is 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 the related of SNP and meat proterties.
The applicant obtains a kind of gene fragment relevant with pig flesh characters by the clone, and its nucleotide sequence is as described in the 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.
It is right to have prepared the primer that detects above-mentioned sequence table SEQ ID NO:1 gene fragment sudden change, and described primer is right,
Forward primer is 5 '-AGGAGCACTTATTCTTGT-3 '.
Reverse primer is 5 '-AAATGCGTTATTCACAGG-3 '.
Utilize the molecule marker relevant with pig flesh characters of above-mentioned preparation that external pig kind and place of china kind have been carried out the application of association analysis, thereby finished the present invention.
SNP finds to set up with detection method: the contriver has designed amplification and has comprised this SNP primer, and the C/T site mutation can adopt Hae Ш to carry out enzyme and cut the detection polymorphism by analysis.On the fragment of the 1249bp that increases, the restriction enzyme site that has 1 Hae Ш, the C/T site that 2% agarose gel electrophoresis detected result is presented at the POSTN gene fragment exists 3 kinds of genotype, TT type (1249bp), CT type (541bp, 708bp, 1249bp) and CC genotype (541bp, 708bp).The restriction enzyme digestion and electrophoresis result has confirmed the sudden change in the existence of 12835bp place, the 9th intron base position of POSTN gene.
Related between genotype-meat proterties: utilize general linear model in the SAS software (General Linear Model, GLM) program is carried out association analysis to genotype and proterties, model the following is Y
Ijkn=μ+h
i+ l
j+ g
k+ ε
Ijkn, Y
IjknBe the proterties phenotypic number, μ is population mean, h
iBe pasture effect, l
jBe age effect, g
kBe the effect of POSTN genotype different loci, ε
IjknBe the random error effect, Normal Distribution (0, σ
2).Analysis revealed 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 proterties.
The present invention will be the molecule marker of pig flesh characters, (MAS) establishes solid basis for marker assisted selection, further illustrates the molecular mechanism of meat proterties, will provide theoretical foundation for improving meat quality, improve the Swine Production economic benefit, and guide the breeding practice of pig.
Description of drawings
Fig. 1 is the electrophoresis result figure of the pcr amplification product of POSTN gene fragment of the present invention.
Among the figure: M:100bp DNA Ladder Marker, 1-7 represents different swimming lanes.
Fig. 2 is the Hae Ш restriction enzyme digestion and electrophoresis result in POSTN gene C of the present invention/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 done to explain further, but concrete enforcement do not done any restriction to the present invention.
The Hae Ш polymorphism of PCR-RFLP technology for detection POSTN gene.
Design of primers: utilize the comparative genomics method according to the 8th intron to the 10 introns design primer of people's POSTN gene (the GenBank number of including is NM_001206351), after making the BLAST sequence alignment, GenBank filters out POSTN gene candidate SNP s site with this gene, choose the 12835bp candidate SNP s site of POSTN gene complete sequence, the 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′
The DNA sample from totally 772 of 8 kinds, wherein 57 of pig farm durocs, 118 of Large Whites are planted by Yiyang institute of agricultural sciences; 106 of pig farm Large Whites are planted by north, Xiang Tan agricultural university; 136 of positive rainbow kind pig farm, Yueyang Large Whites, 38 of new five rich pig farm landraces; 55 of 70 of the black pigs of 67 of local variety Ningxiang pigs, the Land of Peach Blossoms, 62 of big defensive wall pigs, 63 of sand mountain range pigs and Wuzhi Mountain pigs.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, each 0.4 μ L of upstream and downstream primer (10pmol/ μ L), ddH
2O12.6 μ L.
Response procedures: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 50.5 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 35 circulations; Extend 10min after 72 ℃, last 4 ℃ of preservations.
Get 10 μ L pcr amplification products, add 2 μ L tetrabromophenol sulfonphthalein sample-loading buffer mixings after, point sample is put 6 μ L100bp DNA Markers then as reference on 2% sepharose (containing 0.05%EB).5V/cm electrophoresis 0.5～1.0h.Electrophoresis end back is observed amplification and is taken pictures in gel imaging system, the result as shown in Figure 1.Platinum biotech company's order-checking still will be sent behind the PCR product behind the purifying.
The Hae Ш enzyme of PCR product is cut: add 8 μ L10 * restriction endonuclease damping fluid, 0.2 μ L restriction enzyme and 2 μ L distilled waters in 10 μ L PCR products, cumulative volume is 20.2 μ L, 37 ℃ of digestion 10h.The C/T site is with 1% agarose gel electrophoresis analysis, 5V/cm voltage electrophoresis 0.5h, observations and taking pictures under the ultraviolet lamp, enzyme is cut result such as Fig. 2, and amplified production checks order, sequence is shown in SEQ ID NO:1, the amplification segment be pig POSTN gene 12127bp between the 13375bp, be the segment of the 8th intron to the 10 introns, 1249bp altogether, be PCR-RFLP-Hae III molecule marker, Fig. 2 is TT, CT and the CC electrophoresis result of 3 kinds of genes of POSTN gene PCR-RFLP among the present invention.M:DNA molecular weight standard among the figure (DL100bp ladder).
The present invention is with the candidate gene of pig POSTN gene as pig flesh characters, assorted with 5 local pig breeds (Ningxiang pig, the black pig in the Land of Peach Blossoms, big defensive wall pig, sand mountain range pig and Wuzhi Mountain pig), 3 external pig kinds (duroc, Large White, landrace) and Du * length * Dasanyuan is test materials, adopt the PCR-RFLP method that the POSTN gene C/gene frequency in T site, genotype frequency are detected, and analyze its genetic construction, analyze the dependency of this gene and meat proterties.
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, the C gene is advantage allelotrope in Ningxiang pig, big defensive wall pig, sand mountain range pig, Land of Peach Blossoms pig, these 5 local variety of Wuzhi Mountain pig, wherein in Land of Peach Blossoms pig and the Wuzhi Mountain pig T gene does not appear, sand mountain range pig C gene frequency the highest (0.9921) in other 3 local pigs; The C gene also is advantage allelotrope in 3 adventive durocs, landrace, Large White, duroc C gene frequency the highest (0.7368); From the genotype distribution frequency, the CC type is preponderated.
Gene frequency and the genotype frequency in table 1 different varieties POSTN gene C/T site
The application of molecule marker of the present invention in the association analysis of pig flesh characters mark property.
Utilization 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
IjknBe the proterties phenotypic number, μ is population mean, h
iBe pasture effect, l
jBe age effect, g
kBe the effect of POSTN genotype different loci to proterties, ε
IjknBe the random error effect, Normal Distribution (0, σ
2).
Table 2 pig POSTN gene C/T site and meat proterties association analysis table
Each row marks same letter represents that difference is not remarkable in the table, the expression significant differences of the different letters of mark (down with)
As shown in Table 2, in duroc, CC genotype percentage of water loss has reduced by 8.98% (p＜0.05) than CT genotype percentage of water loss, TT genotype storage loss have reduced 0.89%(p＜0.05 than CC genotype storage loss), difference is all not remarkable between CC, CT, TT genotype actual measurement mean ph value, cold cuts rate, yellowish pink grade and the marble grain grade.
In Large White, CT genotype percentage of water loss has reduced 4.48%(p＜0.05 than TT genotype), the marble grain grade then is lower than the TT genotype, differ 0.80(p＜0.05), the genotypic yellowish pink grade of CC than the genotypic yellowish pink grade height of TT 0.50(p＜0.05), but difference is not remarkable between CC and the CT genotype, and CC genotype storage loss have reduced 0.57%(p＜0.05 than CT genotype storage loss), difference is not remarkable between CC, CT, TT genotype pH value and the cold cuts rate.The yellowish pink grade of CT genotype is than the high 0.75(p of the yellowish pink grade of TT genotype＜0.05 in the landrace), not remarkable with the CC genotypic difference, CC genotype storage loss than TT genotype storage loss height 0.44%(p＜0.05), the difference on other proterties does not all reach conspicuous level.In the DLY ternary is assorted, CC genotype pH value is higher by 0.47 than TT genotype pH value, CC genotype percentage of water loss has reduced 5.27%(p＜0.05 than TT genotype percentage of water loss), but difference is not remarkable between CC and the CT genotype, the yellowish pink grade of CT genotype than the yellowish pink grade height of CC genotype 1.47(p＜0.05), and difference is not remarkable between CC and the TT genotype, and difference is not remarkable on other proterties.
Table 3 Large White POSTN gene C/T site and growth traits association analysis table
As shown in Table 3, in the 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 not significantly (p〉0.05) on the 100kg age in days.
Claims (4)
1. POSTN gene fragment relevant with pig flesh characters, its sequence is as described in the sequence table SEQ ID NO:1; The base mutation of 1 C/T is arranged at the 709bp place of sequence table SEQ ID NO:1, cause PCR-RFLP-Hae Ш polymorphism.
2. test right requires 1 described a kind of POSTN gene fragment relevant with pig flesh characters, and the primer of its sudden change is right, and forward primer is 5 '-AGGAGCACTTATTCTTGT-3 ', reverse primer is 5 '-AAATGCGTTATTCACAGG-3 '.
3. the method for preparing the POSTN gene fragment relevant with pig flesh characters as claimed in claim 1, according to following steps:
Utilize the described primer of claim 2 to carry out pcr amplification, the PCR product detects with 2% agarose gel electrophoresis, utilize the PCR product of restriction enzyme Hae Ш to carry out enzyme and cut evaluation, detect CC, CT and the genotypic distribution of TT with 2% agarose gel electrophoresis at last.
4. described POSTN gene fragment the application in pig molecule mark assisted Selection relevant with pig flesh characters of claim 1.
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| CN103898120A (en) * | 2014-03-12 | 2014-07-02 | 湖南农业大学 | Molecular clone of porcine NR1H4 gene and application |
| CN104032004A (en) * | 2014-06-12 | 2014-09-10 | 北方民族大学 | Molecular identification method of halal meat food |
| 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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