CN102485892A - Traceablility SNP marker in pig GIGYF2 gene and detection method thereof - Google Patents

Abstract

The invention relates to a traceablility SNP marker in a pig GIGYF2 gene and a detection method thereof. The SNP marker which is positioned in a genomic DNA fragment in the pig GIGYF2 gene is obtained through DNA pool sequencing. The genomic DNA fragment in the pig GIGYF2 gene, which is represented by SEQ ID NO.1 and comprises bps with the number of 452, comprises parts of twenty-three introns, and there is a base mutation from 187A to 187C at the 187th bit. The distribution case of the SNP marker of the invention in allelic genes in a test colony comprising ten pig kinds or lines is analyzed, and results show that frequencies of the marker in the allelic genes of different kinds or lines are close, so the polymorphism is abundant, there is a small difference among the frequencies of the allelic genes of different kinds or lines, and heterozygosities are greater than 0.3, so a case that the SNP marker of the invention can be used for pig product DNA traceablility is preliminarily determined.

Description

A SNP mark and detection method thereof that can be used for tracing to the source in the pig GIGYF2 gene

Technical field

The invention belongs to the food safety field, be specifically related to a SNP mark and detection method thereof that can be used for tracing to the source in the boar GIGYF2 gene.

Background technology

Along with the raising of standard of living and health care consciousness, People more and more is paid attention to food-safety problem.How pork product guarantees its safety and sanitation as important protein matter source in people's meals, ensures that human consumer's healthy and life security 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, in the hope of reducing the generation of food safety incident to the full extent.The traceability system of meat product has also been set up in each big city of China one after another; The management of tracing to the source through meat product; The information of accurate and detailed relevant product can be provided for the human consumer; Help the operator and in time find the hidden danger that exists in each link, for the human consumer provides an approach that obtains effective authentic communication.What is more important has been strengthened the supervision of government department to meat quality amount safety greatly, and the coping mechanism of setting up food safety risk for country rapidly provides effective information, will help the stable of society.

But the label technique that China's meat product traceability system is adopted exist label lose, write down go on business, indicia patterns is smudgy and the easy shortcoming such as artificial change of label; And the DNA technology of tracing to the source that developed countries adopted is based on the biology techniques of individual dna fingerprinting, and absolute unalterable feature and uniqueness are arranged, and not influenced by human factor.And because the DNA technology of tracing to the source easily somatotype, good reproducibility, detection means simple and fast, the technology of tracing to the source fast that is acknowledged as tool development potentiality and using value at present in the world that becomes such as with low cost.

The generation of the DNA technology of tracing to the source comes from the heredity and variation of DNA, and the molecule marker that is used to make up individual dna fingerprinting has AFLP mark (AFLP), SSR mark (microsatellite marker) and SNP mark (SNP).

Height reliability that AFLP has and ease-to-operate, but that AFLP exists is blunt to template reaction performance, mispairing possibly take place bands of a spectrum and disappearance, the relatively high shortcoming of cost.Compare the SNP mark, the allelotrope of microsatellite marker is numerous, and is polymorphic abundant, but also just because of this, make that the banding pattern of SSR mark is complicated, brings difficulty to dna fingerprint identification robotization and mass-producing.The SNP mark is a third generation molecular genetic marker, is meant the variation of single Nucleotide on the same site of genome, and one shows as two equipotential genes, and very suitable high throughput automated analysis is so become animal identification identification molecule marker of greatest concern day by day.

But be used for SNP mark that meat product traces to the source and be different from one SNP mark, for one SNP mark, it must have following characteristic at least: (1) degree of variation is high, and gene frequency is approaching in kind or strain; (2) The genotypes distribution and allele frequencies difference is little between kind; (3) heterozygosity is more than or equal to 0.3.

In secular breeding work, the investigator has accumulated a large amount of SNP marks, is distributed on some karyomit(e) but these SNP marks are often concentrated; Like No. 1 karyomit(e), No. 12 karyomit(e)s etc., and other chromosome dyad; As No. 5, No. 8,14, No. 15 karyomit(e)s etc. then lack the SNP mark; Therefore in order to satisfy pork product is carried out the requirement that DNA traces to the source, we carry out the research work of New SNP marker.

The GIGYF2 gene of pig is positioned at No. 15 karyomit(e)s of pig, and human GIGYF2 gene is positioned on No. second karyomit(e), is made up of 31 exons and 30 introns, comprises 4 spliced bodies.Research shows GIGYF2 albumen participation tyrosine receptor signal pathway.

Summary of the invention

Technical problem to be solved by this invention provides a SNP mark and detection method thereof that can be used for tracing to the source in the boar GIGYF2 gene.SNP of the present invention is marked in the market pig cultivation, can be widely used in DNA source tracing of pork products and detection thereof, traces to the source especially for the security of pork product.

For realizing above-mentioned purpose, the present invention adopts following technical scheme:

Described SNP mark; Be arranged in pig GIGYF2 gene one fragment gene group dna fragmentation; Be to obtain, and in the test colony that comprises 10 pig varieties or strain (amounting to 213 individuals), analyze the distribution situation that this SNP mark SNP is marked at test colony medium position gene through DNA pond (pool) order-checking; Find that this molecule marker is approaching in different kinds and the gene frequency in the strain; Rich polymorphism, the gene frequency distributional difference is little between kind or strain, and this SNP mark of preliminary judgement can be used as DNA source tracing of pork products.

Described pig GIGYF2 gene one fragment gene group dna fragmentation, shown in SEQ ID NO 1,452bp comprise part 23 introns, and there is a base mutation 187A → 187C at its 187th bit base place altogether.In this dna fragmentation, its A allelotrope has only site of 452bp, and C allelotrope has 187bp and two sites of 265bp, and these two allelotrope can be formed three kinds of frequency of genotypes AA, AC, CC.

The detection method of described SNP mark adopts the PCR-RFLP method to carry out, and specifically may further comprise the steps:

(1) the DNA pond (pool) of structure pig;

(2) the forward and reverse primer of design separates the dna fragmentation of GIGYF2 gene, and order-checking is also analyzed;

(3) foundation of mutational site detection method;

(4) the ear tissue appearance of 10 kinds of collection and strain amounts to 213 individuals;

(5) detect the distribution that SNP is marked at test colony, statistical study judges whether that the DNA that is applicable to pork product traces to the source.

Wherein, in the above-mentioned detection method, the forward and reverse primer sequence of the dna fragmentation of separation GIGYF2 gene is respectively shown in SEQ ID No 2 and SEQ ID No 3.

The present invention adopts the DNA pond to detect the SNP mark that exists in the pig GIGYF2 gene; In the test colony that comprises 10 pig varieties or strain; Analyze the distribution situation that this SNP is marked at test colony medium position gene; Find this be marked at different kinds or the gene frequency in the strain approaching, rich polymorphism, the gene frequency distributional difference is little between kind or strain; And heterozygosity is all greater than 0.3, during the security that this SNP mark of preliminary judgement can be used as DNA source tracing of pork products and pork product is traced to the source.

Embodiment

Below in conjunction with specific embodiment technical scheme of the present invention is described in further detail.

Embodiment 1

Searching of SNP mark

(1) structure of DNA pond (pool)

The ear tissue of difference random acquisition duroc, Shen farming pig individual each 2 (regardless of sex), behind the extraction DNA, the DNA that equivalent is drawn 4 individuals puts into same centrifuge tube, and mixing is for use.

(2) design of primers

CDNA sequence (Genbank:NW 001885690) with pig GIGYF2 gene is a template, and the design primer separates pig GIGYF2 gene (DNA with a Shen farming pig is the template of pcr amplification), and primer is following:

Forward primer: 5 '-TTTCTCCCTAAGTCGTCG-3 ' (referring to sequence SEQ ID No 2)

Reverse primer: 5 '-CGTGCTGCTAAGTTTCTA-3 ' (referring to sequence SEQ ID No 3)

PCR reaction TV is 20 μ l; The about 100ng of pig GIGYF2 genomic dna wherein; Contain 1 * buffer (Promega company), 1.5mmol/L MgCl2, dNTP (worker biotech firm is given birth in Shanghai) final concentration is 150 μ mol/L; The primer final concentration is 0.2 μ mol/L, 2U Taq archaeal dna polymerase (Promega company).

The pcr amplification program is: 94 ℃ of 4min, and the 94 ℃ of 30s that circulate then 30 times, 56 ℃ of annealing 30s, 72 ℃ of 30s, last 72 ℃ are extended 10min.

The PCR reaction product detects with 1% agarose gel electrophoresis.

(3) sequencing analysis

The pig GIGYF2 gene PCR product that obtains is carried out purifying: under uv lamp, contain the segmental gel of purpose from the sepharose cutting-out; Put into 1.5ml Ependorff pipe; With PCR product purification test kit (day root biochemical technology ltd) purifying, deliver to Shanghai and give birth to the order-checking of the biological ltd of worker.

Through test, this dna fragmentation through spliced pig GIGYF2 gene is 452bp, and its sequence is shown in SEQ ID No 1.There is a base mutation (187A-187C) through analyzing the 187th base place that finds this dna fragmentation; Through the molecular biology software analysis, find that the base mutation at the 187th base place causes Eco31I-RFLP (Restriction Fragment Length Polymorphism) polymorphum.

(4) foundation of RFLP detection method

Endonuclease reaction TV 10 μ l, 1 * buffer, 10 μ l wherein, PCR product 3～5 μ l, restriction enzyme Eco31I are 0.5 μ l (5U), use H ₂O supplies 10 μ l, and 37 ℃ of water-bath 6h take by weighing the 0.6g agarose and are dissolved in 15.75ml DEPC (worker biotech firm is given birth in the Shanghai) treating water; Cooling (60 ℃) slightly; Add the 5 * formaldehyde gel electrophoretic buffer of 5ml and 37% formaldehyde solution of 4.25ml, the mixing glue detects enzyme and cuts the result behind the electrophoresis.The result finds: in this dna fragmentation, A allelotrope has only site of 452bp, and C allelotrope has 187bp and two sites of 265bp, and these two allelotrope can be formed three kinds of frequency of genotypes AA, AC, CC.

SNP mark of the present invention specifically is the 187th bit base that is arranged in sequence SEQ ID NO 1, and substituting of A → C taken place for it, can detect with the PCR-Eco31I-RFLP method.

Embodiment 2

Allelic distribution situation

(1) design of test colony

Test group: gather Pietrain (24), Shen Nong (32), Da Shen (31), Pi Shen (10), long Shen (26), Pi Dashen (19), the Shen of growing up (25), Du Dashen (7), Du Pishen (8) and the individual ear tissue of Du Pi Dashen (31); Extract DNA, amount to 213 dna samples.

The purpose of test colony is to detect SNP and is marked at the distribution situation in the different varieties.

(2) genotype detection

Utilize forward primer: 5 '-TTTCTCCCTAAGTCGTCG-3 ' (referring to sequence SEQ ID No 2)

Reverse primer: 5 '-CGTGCTGCTAAGTTTCTA-3 ' (referring to sequence SEQ ID No 3)

Increase, detect all idiotypes of test colony with identical RFLP method.

(3) statistical study

Write down all individual genotype, and calculate gene frequency and heterozygosity, the result is as shown in table 1 below.

Table 1

Analyze the allelic distribution situation of this SNP marker site; Find that this molecule marker is except A gene frequency in cultivating kind skin Shen is identical with the C gene frequency; All be that the A gene frequency accounts for outside the clear superiority in other kind, especially in the Pietrain kind; Heterozygosity is lower than 0.3 in Pietrain kind, Du Dashen and Du Pishen strain, and Pietrain is an adventive, possibly have hereditary difference, and Du Dashen and Du Pishen strain possibly be because the too small heterozygosity that causes of sample content is low; In addition, the heterozygosity H of all the other kinds or strain is all greater than 0.3, so we think that tentatively the DNA that this SNP mark can be used for pork product traces to the source.

Should be noted that at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although with reference to preferred embodiment the present invention is specified, those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement the technical scheme of invention; And not breaking away from the spirit and the scope of technical scheme of the present invention, it all should be encompassed in the claim scope of the present invention.

Claims (7)

1. a fragment gene group dna fragmentation of pig GIGYF2 gene, its sequence are shown in SEQ ID NO 1, and 452bp comprises part the 23rd intron altogether.

2. a fragment gene group dna fragmentation of pig GIGYF2 gene according to claim 1 is characterized in that, there is a base mutation 187A → 187C at described dna fragmentation the 187th bit base place.

3. a fragment gene group dna fragmentation of pig GIGYF2 gene according to claim 1 is characterized in that there is an A allelotrope at the 452bp place of described dna fragmentation, and respectively there is a C allelotrope at its 187bp and 265bp place.

4. a SNP mark is characterized in that, is arranged in a fragment gene group dna fragmentation of claim 1 or 2 or 3 described pig GIGYF2 genes.

5. the detection method of the described SNP mark of claim 4 is characterized in that, adopts the PCR-Eco31I-RFLP method to carry out, and specifically may further comprise the steps:

(1) the DNA pond of structure pig;

(2) the positive and negative primer of design separates the dna fragmentation of pig GIGYF2 gene, and order-checking is also analyzed;

(3) foundation of mutational site detection method;

(4) gather the ear tissue appearance of 10 kinds or the strain of pig, amount to 213 individuals;

(5) detect the distribution that SNP is marked at test colony, whether this mark of analysis and judgement is applicable to that the DNA of pork product traces to the source.

6. the detection method of SNP mark according to claim 5 is characterized in that, the sequence of the forward and reverse primer of the dna fragmentation of separation pig GIGYF2 gene is respectively shown in SEQ ID NO 2 and SEQ ID NO 3.

7. the described SNP of claim 4 is marked at the application in the DNA source tracing of pork products.