CN103589716B - One of pig SNCG gene can be used for the SNP marker of tracing to the source and detection method thereof - Google Patents
One of pig SNCG gene can be used for the SNP marker of tracing to the source and detection method thereof Download PDFInfo
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Abstract
The invention discloses SNP marker and detection method thereof that of a boar SNCG gene can be used for tracing to the source.Described SNP marker is obtained by DNA pond (pool) order-checking, altogether 1599bp, and it comprises part the 3rd intron sequences, and there is a base mutation 986C-986T at the 986th bit base place, is its sequence as SEQ? ID? shown in NO.1.In the test colony comprising 11 pig varieties or strain, analyze the distribution situation of this SNP marker at test colony allelic, find that the gene frequency of this molecule marker in different kinds or strain is close, rich polymorphism, between kind or strain, gene frequency distributional difference is little, and heterozygosity is all greater than 0.3, this SNP marker of preliminary judgement can be used as DNA source tracing of pork products.
Description
Technical field
The invention belongs to field of food safety, relate to SNP marker and detection method thereof that of a boar SNCG gene can be used for tracing to the source.
Background technology
Along with the raising of standard of living and health care consciousness, people more and more focus on food-safety problem.How pork product, as protein source important in people's meals, guarantees its safety and sanitation, ensures that the healthy and life security of human consumer has become global hot issue.Countries in the world government takes a series of technical measures to strengthen the security control to meat product one after another, to reducing the generation of food safety affair to the full extent.The each big city of China also establishes the traceability system of meat product one after another, by the management of tracing to the source of meat product, the information of accurate and detailed related products can be provided for human consumer, be conducive to the hidden danger existed in each link of operator's Timeliness coverage, for human consumer provides an approach obtaining effective authentic communication.What is more important, greatly strengthen the supervision of government department to meat quality amount safety, the coping mechanism setting up rapidly food safety risk for country provides effective information, will contribute to the stable of society.
But the label technique that China's meat product traceability system adopts exists, and label is lost, record is gone on business, indicia patterns is smudgy and label such as easily artificially to change at the shortcoming; And the DNA tracing technology that developed countries adopts is the biology techniques based on individual DNA fingerprinting, there is absolute unalterable feature and uniqueness, is not affected by human factors.And because the easy somatotype of DNA tracing technology, reproducible, detection means simple and fast, with low cost etc. become the quick tracing technology being acknowledged as most development potentiality and using value at present in the world.
The generation of DNA tracing technology comes from the heredity and variation of DNA, and the molecule marker for building individual DNA fingerprinting has AFLP to mark (amplified fragment length polymorphism), SSR marker (microsatellite marker) and SNP marker (single nucleotide polymorphism).
AFLP mark has height reliability and ease-to-operate, but also exists simultaneously, to template reaction performance blunt, bands of a spectrum, the shortcomings such as mispairing and disappearance, cost are relatively high may occur.Compare SNP marker, the allelotrope of microsatellite marker is numerous, polymorphic abundant, but also just because of this, makes the banding pattern of SSR marker complicated, bring difficulty to DNA fingerprint identification automatization and mass-producing.SNP marker is third generation molecular genetic marker, refers to the variation of single core thuja acid on the same site of genome, generally shows as two allelotrope, and very suitable high throughput automated analysis, so day by day become animal identification identification molecule marker of greatest concern.
But be different from general SNP marker for the SNP marker that meat product is traced to the source, for single SNP marker, it at least must have following characteristics: (1) degree of variation is high, kind or strain allelic frequency close; (2) between kind, the genotypes distribution and allele frequencies difference is little; (3) heterozygosity is more than or equal to 0.3.
In long-term breeding work, investigator have accumulated a large amount of SNP marker, but these SNP marker often integrated distribution on some karyomit(e), as No. 1 karyomit(e), No. 12 karyomit(e)s etc., and other chromosome dyad, as No. 5, No. 8, No. 14, No. 18 karyomit(e)s etc. then lack SNP marker, and therefore carry out to pork product the requirement that DNA traces to the source in order to meet, we carry out the research work of New SNP marker.
Pig SNCG gene is positioned on No. 18 karyomit(e)s of pig, and this gene is studied very few in pig.The SNCG gene of people is called γ synapse nucleoprotein gene (γ-synuclein, SNCG), the Breast Cancer-Specific gene 1 (BreastCancerSpecificGene1 that Recent study finds, BCSGl) being proved with γ synapse nucleoprotein is same albumen, itself and mammary cancer and ovarian cancer closely related, specific high expression level in the mammary cancer and ovarian cancer of most progressive stage, and report relative less with the relation of other tumours.SNCG gene is new mankind's kinds cancer broad spectrum biomarker, and this paper is in the famous magazine of world's cancer research " american cancer research magazine " and " international oncology magazine " upper publication.
Summary of the invention
Technical problem to be solved by this invention is the SNP marker and detection method thereof that provide one of a boar SNCG gene to can be used for tracing to the source, this SNP marker is in market pig is cultivated, can be widely used in DNA source tracing of pork products and detection thereof, the security especially for pork product is traced to the source.
For achieving the above object, the present invention realizes by the following technical solutions.
One of described pig SNCG gene can be used for the SNP marker of tracing to the source, altogether 1599bp, and comprise part the 3rd intron sequences, and there is a base mutation 986C → 986T at the 986th bit base place, its DNA sequence dna is as shown in SEQIDNO.1.
Described SNP marker is obtained by DNA pond (pool) order-checking, and in the test colony (amounting to 235 individualities) comprising 10 pig varieties or strain, analyze the distribution situation of this SNP marker at test colony allelic, find that the gene frequency of this molecule marker in different kinds and strain is close, rich polymorphism, between kind or strain, gene frequency distributional difference is little, and this SNP marker of preliminary judgement can be used as DNA source tracing of pork products.
In described pig SNCG gene, one can be used for the detection method of the SNP marker of tracing to the source, and adopts PCR-Bpu1102I-RFLP method to carry out, comprises the following steps:
(1) the DNA pond (pool) of pig is built;
(2) design the DNA fragmentation that forward and reverse primer is separated SNCG gene, check order and analyze;
(3) foundation of PCR-Bpu1102I-RFLP detection method and the detection in mutational site;
(4) gather the ear tissue sample of 10 kinds and strain, amount to 235 individualities;
(5) detect the distribution of SNP marker in test colony, statistical study, and further whether verification experimental verification is applicable to during pork product traces to the source.
Wherein, in above-mentioned detection method, the forward and reverse primer sequence of the DNA fragmentation of described separation SNCG gene is as follows:
Forward primer: 5'-TTTGTGATGGTGCTCTGCGTTT-3'(SEQIDNO.2)
Reverse primer: 5'-CCCTGCCTGAAGGTGGTTGT-3'(SEQIDNO.3)
The present invention detects the SNP marker existed in pig SNCG gene by DNA pond, in the test colony comprising 10 pig varieties or strain, analyze the distribution situation of this SNP marker at test colony allelic, find that the gene frequency of this molecule marker in different kinds or strain is close, rich polymorphism, between kind or strain, gene frequency distributional difference is little, and heterozygosity is all greater than 0.3, during the security that this SNP marker of preliminary judgement can be used as DNA source tracing of pork products and pork product is traced to the source.
Trace to the source because single SNP traces to the source to mark to complete, therefore tracing to the source in simulation test, combined in the Novel SNP molecular marker site of the SNCG gene of the present invention's acquisition and other existing published SNP marker site (amounting to 12 SNP site) carries out tracing to the source and tests.At slaughterhouse random choose 100 individualities, detect the genotype of each individual 12 SNP site, add up the genotype results of each individuality, find that 100 individualities all have genotype obstructed separately, individual differentiation can be realized; Simultaneously, picking 20 parts of muscle samples in these 100 individualities at random, the genotype in same detection statistics 12 SNP marker sites, then compare with 100 individual genotype, find all can find and 20 parts of on all four individualities of muscle samples genotype, namely the source finding 20 parts of pig muscles by tracing to the source in 100 individualities is individual, therefore judges that this SNP marker of the present invention may be used for pork product and traces to the source.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in further detail.
Searching of embodiment 1 molecule marker
(1) structure of DNA pond (pool)
The respectively ear tissue of random acquisition duroc, plum mountain pig individual each 2 (regardless of sex), after extracting DNA, equivalent is drawn 4 individual DNA and is put into same centrifuge tube, mixes stand-by.
(2) design of primers
With the DNA sequence dna of pig SNCG gene (Genbank:EF104639) for template, design primer is separated pig SNCG gene (template being pcr amplification with the DNA of a duroc), and primer is as follows:
Forward primer: 5'-TTTGTGATGGTGCTCTGCGTTT-3'(SEQIDNO.2)
Reverse primer: 5'-CCCTGCCTGAAGGTGGTTGT-3'(SEQIDNO.3)
It is 20 μ l that PCR reacts cumulative volume, and wherein pig SNCG genomic dna is about 100ng, containing 1 × buffer (Promega company), and 1.5mmol/LMgCl
2, dNTP (Shanghai Sheng Gong biotech firm) final concentration is 150 μm of ol/L, and primer final concentration is 0.2 μm of ol/L, 2UTaqDNA polysaccharase (Promega company).
Pcr amplification: 94 DEG C of 4min, (94 DEG C of 30s, 62 DEG C of annealing 40s, 72 DEG C of 40s) circulate 30 times, and last 72 DEG C extend 10min.
PCR reaction product 1% agarose gel electrophoresis detects.
(3) cloning and sequencing analysis
The pig SNCG gene PCR product obtained is cloned as follows.
The purifying of PCR primer: cut the gel containing object fragment from sepharose under ultraviolet lamp, puts into 1.5mlEpendorff pipe, with PCR primer purification kit (Tian Gen biochemical technology company limited) purifying.
Ligation: the PCR primer of purifying is connected with pMD18-T carrier (the precious biotech firm in Dalian), ligation cumulative volume is 10 μ l, comprising 5 μ lsolutionI (the precious biotech firm in Dalian), the carrier T (the precious biotech firm in Dalian) of 0.5 μ l, the purified pcr product of 2.5 μ l, finally adds 2 μ l aqua sterilisas and puts 4 DEG C of water-baths and spend the night.
Transform: get 100-120 μ l competent cell (Tian Gen biochemical technology company limited) under sterile state in 1.5mlEpendorff pipe, the connection product of 5 μ l is added mixing, 30min is placed on ice, 42 DEG C of heat shock 90s, do not shake Ependorff pipe therebetween, ice bath 3-4min after taking out, adds the LB liquid nutrient medium of 400 μ l antibiotic-frees, and 37 DEG C keep flat to be inverted after 1h and cultivate.
Bacterium colony PCR identifies: the bacterial strain after bacterium colony PCR identifies is in LB substratum 37 DEG C of overnight incubation, and the multiple clone of picking delivers to the order-checking of Shanghai raw work biological company limited immediately.
After tested, this DNA sequence dna through spliced pig SNCG gene is 1599bp altogether, and comprise part the 3rd intron sequences, sequence is as shown in SEQIDNO.1.Find that its 986th base place exists a base mutation 986C → 986T by analysis.Analyzed by molecular biology software, find that the base mutation at the 986th base place causes Bpu1102I-RFLP (RestrictionFragmentLengthPolymorphism, i.e. Bpu1102I-RFLP site) polymorphism.
(4) foundation of PCR-Bpu1102I-RFLP detection method and the detection in mutational site
Endonuclease reaction cumulative volume 10 μ l, wherein 1 × buffer10 μ l, PCR primer 3-5 μ l, restriction enzyme Bpu1102I are 0.5 μ l (5U), use H
2o supplies 10 μ l, 37 DEG C of water-bath 4h, take 0.6g agarose to be dissolved in 15.75mlDEPC (Shanghai Sheng Gong biotech firm) and to process in water, slightly cool (60 DEG C), add 5 × formaldehyde gel damping fluid of 5ml and 37% formaldehyde solution of 4.25ml, mixing glue, detects enzyme and cuts result after electrophoresis.Found that: in SEQIDNO.1 sequence, C allelotrope only has 1599bp fragment, T allelotrope has 985bp and 6146bp two fragments, and these two allelotrope can form three kinds of genotype, CC, CT, TT.And undergo mutation at the 986th bit base of this SEQIDNO.1 sequence, C → T.
The allelic distribution situation of embodiment 2
(1) design of colony is tested
Test group: gather the ear tissue that Pietrain (21), Shen Nong (17), great Bai (43), great Shen (52), long Shen (16), Du Shen (23), Pi great Shen (11), Shen of growing up (25), Du great Shen (7) and Du Pi great Shen (20) are individual, extract DNA, amount to 235 DNA samples.
The object of test colony is to detect the distribution situation of SNP marker in different varieties.
(2) gene test
Forward primer: 5'-TTTGTGATGGTGCTCTGCGTTT-3'(SEQIDNO.2)
Reverse primer: 5'-CCCTGCCTGAAGGTGGTTGT-3'(SEQIDNO.3)
Carry out increase (condition is as embodiment 1), by the idiotype that identical PCR-Bpu1102I-RFLP method detection experiment colony is all.
(3) statistical study
Record the genotype of all individualities, and calculate gene frequency and heterozygosity, result is as following table 1.
Table 1
As known from Table 1: the gene frequency of SNP marker of the present invention in each kind is close, rich polymorphism; Between kind or strain, the distributional difference of gene frequency C and T is little; The heterozygosity H of all kinds or strain is all greater than 0.3, in therefore tentatively thinking that DNA that this SNP marker may be used for pork product traces to the source and security traces to the source.
Embodiment 3SNP molecule marker operability is traced to the source verification experimental verification
Above-mentioned SNP marker be can be used in traceability mark by tentative confirmation, and in order to ensure in the operability in mark of tracing to the source, the present invention resamples in different location again, carries out operability and to trace to the source verification experimental verification.
Reviving ear tissue and each 100 parts of the muscle sample (each pig individuality gathers ear tissue sample and muscle sample all simultaneously) of slaughterhouse random acquisition pig individuality, ear tissue sample numbering E1-E100, muscle sample numbering M1-M100, the DNA extracting muscle sample and ear tissue sample is respectively for subsequent use.
In 100 individual ear tissue samples and detect the genotype (amounting to 12 SNP marker sites) of existing published 11 SNP site in the Bpu1102ISNP site of pig SNCG gene of the present invention and following table 2 in 20 muscle samples of random choose, add up the genotype results of each individuality.Cut banding pattern according to enzyme, a band is designated as 1, and occur that two bands are designated as 2, three bands are designated as 3.Record order is according to the order arrangement of table 2 site, SNP marker site as the Pvu II enzyme identification of ADAMTS-1 gene is 1, its genotype record result just makes number one, the SNP marker site of the Eam1104 I enzyme identification of ADD1 gene is 2, its genotype record result just comes second, the like, the Bpu1102ISNP molecule marker site of the SNCG gene of the application comes finally, its genotype results is just expressed as the 12nd numeral from left to right, and individual genotype can be expressed as: 212323233112.
Existing published 11 SNP site of table 2
Add up the genotype in 12 SNP marker sites of each individuality and meat sample, result shows 100 individualities, 20 parts of muscle samples have different genotype, can realize individual differentiation.Simultaneously, the genotype results in 12 of 20 parts of muscle samples SNP marker sites is compared with these 100 individual genotype, find all can find and these 20 parts of on all four individualities of muscle samples genotype, namely the source finding 20 parts of pig muscles by tracing to the source in 100 individualities is individual, realizes meat sample to individual trace to the source (comparison result is referring to table 4).The genotype of 100 individualities and 20 parts of muscle samples, respectively as following table 3 and table 4, wherein comes numeral the 12nd and is the genotype results in the SNP marker site of SNCG gene of the present invention by runic mark.
A table 3100 individual genotype
The genotype of table 420 part muscle sample and with 100 parts of individual comparison results
Claims (2)
1. one of a boar SNCG gene can be used for the SNP marker of tracing to the source, and obtained by the order-checking of DNA pond, altogether 1599bp, comprise part the 3rd intron sequences, there is a base mutation 986C → 986T at the 986th bit base place; The DNA sequence dna of described SNP marker is as shown in SEQIDNO.1.
2. one of pig SNCG gene according to claim 1 can be used for the application of SNP marker in DNA source tracing of pork products of tracing to the source.
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| CN104152447B (en) * | 2014-08-08 | 2017-03-29 | 南京农业大学 | A kind of SNP marker and HRM methods carry out Carnis Sus domestica DNA source tracing methods |
| CN105296640B (en) * | 2015-11-16 | 2018-10-02 | 上海市农业科学院 | SNP marker for tracing to the source and its application on No. 6 chromosomes of one pig |
| CN105296639B (en) * | 2015-11-16 | 2018-10-02 | 上海市农业科学院 | SNP marker for tracing to the source and its application on No. 6 chromosomes of one pig |
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