CN101985656B

CN101985656B - Method for detecting single nucleotide polymorphisms of AQP9 genes in native Chinese cattle

Info

Publication number: CN101985656B
Application number: CN201010531532XA
Authority: CN
Inventors: 陈宏�; 张婧敏; 房兴堂; 张春雷
Original assignee: Xuzhou Normal University
Current assignee: Xuzhou Normal University
Priority date: 2010-11-04
Filing date: 2010-11-04
Publication date: 2012-05-30
Anticipated expiration: 2030-11-04
Also published as: CN101985656A

Abstract

The invention discloses a method for detecting single nucleotide polymorphisms of AQP9 genes in native Chinese cattle, which is characterized by taking genomic DNA of the cattle to be detected containing the AQP9 genes as the template and the specific primer pair P6 as the primers and amplifying the exons 6 of the AQP9 genes in the cattle by PCR; and carrying out SSCP detection on the segments of the amplified products and sequencing the products. As the functions of the AQP9 genes relate to such growth traits as weight, height, length, chest circumference and the like, the method screens and detects the molecular genetic markers closely related to the growth traits of the cattle on the DNA level so as to be used for marker-assisted selection (MAS) of the growth traits for the Chinese cattle meat and rapidly build the cattle population with excellent genetic resources.

Description

A kind of method that detects place of china ox AQP9 gene mononucleotide polymorphism

Technical field

The invention belongs to the molecular genetics field, relate to SNP (SNP) with the functional gene of ox as molecular genetic marker, particularly a kind of method that detects the SNP of ox AQP9 gene.

Background technology

Beef cattle industries is super-speed development situation in recent years, has become the important industry of various countries' national economy and agricultural, and the development of China beef cattle also has great potential.In order to obtain competitive power in the world market, meat production person need be the basis with human consumer's food, under the prerequisite that guarantees the meat safe mass, improves the production performance of livestock raised for meat as possible.This is exactly how to produce the livestock product that the human consumer welcomes for the breeding work person, obtains greatest benefit simultaneously, that is to say and will cultivate the efficiency of feed utilization height, the livestock and poultry species of superior product quality (being).The height of efficiency of feed utilization is the height that energy utilizes after all.With regard to ox, fat is the important factor that influences its taste between flesh simultaneously, and it is to lean on the deposition of fat and properly distributed to form.Along with the raising day by day that develops living standards of the people rapidly of market economy, it is imperative with raising meat matter to accelerate the ox speed of growth.

Growth Traits is a quantitative character typical, that have important economic worth, and its heritability is low, is generally 0.2～0.3, carries out breeding with traditional breeding way and makes slow progress.And molecular breeding is technological; Then be progressively to carry out the transition to the genotype of handling quantitative character from the phenotype of handling quantitative character; (marker-assisted selection MAS), finally can realize molecular breeding (molecular breeding) to transfer marker assisted selection to from the selection by phenotype.Along with the development of molecular biology and molecular genetics, molecule marker is also constantly advancing.In the development course of molecule marker, it is broadly divided into three types: the I class is to be the molecule marker on basis with the molecular hybridization, like RFLP and dna fingerprint; The II class is to be the molecule marker on basis with PCR, like RAPD, microsatellite marker, PCR-RFLP, PCR-SSCP and PCR-RMAPD (the polymorphic DNA of random amplification micro-satellite primers) mark; The III class is to be the molecule marker of core with the dna sequencing, like SNP and EST.

SNP (SNP) typically refers in the genomic dna sequence polymorphum that the variation owing to single Nucleotide (A/T/C/G) causes, comprises the variation of replacement, insertion, disappearance and the Tumor-necrosis factor glycoproteins copy number of base.Can be divided between gene coding region SNPs (cSNPs), gene periphery SNPs (pSNPs) and gene three types of SNPs (iSNPs) according to its distribution position in genome.Wherein for the nonsynonymous mutation of coding region SNPs, they possibly have direct material impact to gene function.Moreover, in population genetic research, these SNPs are also significant in the research of population genetic and organic evolution as genetic marker.At present the SNP detection technique has a lot, and wherein the determined dna sequence method is a SNP detection method the most accurately, still; Its testing cost is extremely expensive; And need large-scale instruments such as dna sequencing appearance, simultaneously, in the order-checking process, need very those skilled in the art and experience; So the determined dna sequence method is not a kind of actual desirable SNP detection method that is applied to produce.And some other SNP detection method operation is more loaded down with trivial details, and limitation is bigger, or requires experiment condition high, is not suitable for general clinical labororatory and uses.PCR-SSCP is as a kind of method that detects transgenation, and through constantly improving, it is easy, quick, sensitive, does not need special instrument, is more suitable for the needs of clinical experiment.Therefore, utilize PCR-SSCP and dna sequencing combined techniques to detect SNP and can suitably reduce testing cost, not only accurately but also practical.

(aquaporin AQP) is the specificity duct that extensively is present in transhipment water on protokaryon and the eukaryotic cell membrane to aquaporin.Wherein AQP3,7,9 and AQP10 between gene structure and protein sequence close; Except that penetrating to water molecules, neutral small molecules such as glycerine and urea are also had permeability, become second subfamily in the AQP family; Water-glycerol channel (aquaglyceroporin, AQP).AQP9 is that Kuriyame in 1997 etc. find in carrying out human adipose tissue gene order systems analysis process; At organs such as white corpuscle, liver, testis, spleen and brains discovery is arranged all also afterwards; At human multiple tissue expression is arranged all, its molecular structure and similar to the permeability and other the aquaporin of water, and observe AQP9 and strengthened the penetrating ability of adipocyte glycerine; So think that AQP9 begins to transport glycerine when steatolysis forms the glycerol concentration gradient between cytolemma.Other has research to show that liver plasma membrane surfaces A QP9 can accomplish the absorption process of glycerine.Adipocyte AQP7 is transported to blood with the glycerine that lipid mobilization produces; Glycerine arrives liver then needs AQP9 to accomplish the capture process from blood; Finally take by glycerine as the additional blood sugar of raw material glyconeogenesis, therefore accomplished glycerine by the transportation of adipocyte to liver by AQP7 and AQP9 coordination at liver.The dysfunction of AQP9 will influence glyconeogenesis and cause hypoglycemic appearance, and also closely related with insulin resistant.Big quantity research shows that AQP9 is keeping energy i (in vivo) metabolism and balance, and lipogenesis and individual aspect such as grow all has crucial effects.Therefore, research Mammals AQP9 gene genetic variation and molecular genetic characteristic have most important theories and practice significance.

The AQPs wide expression of each organ-tissue in vivo has considerable biological action.Not only the normal physiological function of a plurality of tracts has vital role to water-glycerol channel albumen in the body to keeping, and the transportation of the glycerine of its adjusting also has the important physical meaning for the body substance metabolism.Along with developing rapidly of molecular biology and genetic engineering technique; The distribution of specific situation of AQPs gene histoorgan in body reaches in the research of protein molecular biological heredity, structure aspects also day by day to be goed deep into; Become the focus that numerous scholars pay close attention to, clinical also had more widely use.AQP9 participates in the transhipment of glycerine in the body, in tissue, deposits much relations with metabolism of fat, lipid, so AQP9 produces material impact to lipid content in the body and individual growth development character.Existing data shows that there are relation in the polymorphum of AQP7 gene and label of pig fat deposition description proterties, finds that growing of this gene pairs pig has remarkable influence, is a comparatively ideal marker site.At present, do not see the research that makes a variation about animal AQP9 gene genetic both at home and abroad.The research in Chinese Cattle AQP9 gene genetic variation field does not appear in the newspapers, and the functional study of this gene locus and heritable variation thereof are still blank with the related research of growth traits (as: proterties such as body weight, height, chest measurement).Because the AQP9 gene function relates to the growth traits of animal; Detection method provided by the invention is that the SNPs of AQP9 gene and the foundation of growth traits relation are laid a good foundation; For use in the marker assisted selection (MAS) of Chinese Cattle growth traits, set up the good ox population of genetic resources fast.

Summary of the invention

The problem that the present invention solves is to utilize PCR-SSCP and dna sequencing combined techniques to detect the SNP of ox AQP9 gene; And itself and growth traits carried out association analysis; Verify whether it can be used as the molecule marker of assisted Selection in the ox molecular breeding; For Chinese Cattle genetic breeding field marker assisted selection (MAS) provides valuable data in growth seed selection in earlier stage, thereby accelerate fine-variety breeding speed and improve the population quality.

The present invention realizes through following technical scheme:

The SNP sequence of ox AQP9 gene, its gene mononucleotide polymorphism sequence comprises:

The 47575th of ox AQP9 gene is the SNP of C or T, and the 47615th is the SNP of C or T, and the 47690th is the SNP of A or G.

The method of the SNP (SNP) of above-mentioned detection place of china ox water-glycerol channel albumen 9 (AQP9) gene is:

Comprising the AQP9 gene order with place of china ox genomic dna is template; Utilize Auele Specific Primer pcr amplification ox AQP9 gene; Utilize native polyacrylamide gel electrophoresis detection product single stranded conformational then and carry out sequencing analysis, can identify accurately that the SNP of place of china ox kind AQP9 gene is:

The corresponding SNPs of AA genotype is NC_007308:g.47575C, 47615C, 47690A; The corresponding SNPs of AB genotype is NC_007308:g.47575C/T, 47615C/T, 47690A/G; The corresponding SNPs of BB genotype is NC_007308:g.47575T, 47615T, 47690G;

Described primer is to being:

The P6 upstream primer: 5 '-AAGGAAGACCAAGCGATGT-3 ' 19nt;

The P6 downstream primer: 5 '-GTGTGTGAAGATGCTTTGTGA-3 ' 21nt.

The condition of described pcr amplification is:

25 μ L reaction systems comprise 1.0 μ L (1U/ μ L) Taq archaeal dna polymerase, and 10 * Buffer, 2.5 μ L (contain Mg ²⁺), 2.0 μ L (2.5mmol/L) dNTPs, 1.0 μ L (50.0ng/ μ L) ox genomic dna or comprise the DNA sample of AQP9 gene order, each 0.5 μ L of 10 μ mol/L upstream and downstream primers and sterilization ultrapure water 17.5 μ L.

Described pcr amplification reaction program is:

94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s of 30～35 circulations, 61 ℃ of annealing 45s, 72 ℃ are extended 45s; 72 ℃ are extended 10min.

Described polyacrylamide gel electrophoresis is 10% polyacrylamide gel electrophoresis.

The present invention utilizes PCR-SSCP and dna sequencing combined techniques that the SNP that the generation of the missense mutation on ox AQP9 gene the 47575th and the 47615th site proteins encoded conformation changes is detected; When the 47575th site sports T by C; Coding triplet CUC changes into UUC; Protein coding amino acid in the transcription corresponding position changes; The leucine that peptide chain is 260 becomes phenylalanine(Phe) (Leu260Phe), and when the 47615th site sported T by C, coding triplet CCG changed into CUG; Protein coding amino acid in the transcription corresponding position changes; The proline(Pro) that peptide chain is 273 becomes leucine (Pro273Leu), and this makes that being distributed widely in the water-proteic sterie configuration of glycerol channel albumen 9 coded by said gene that has important physiological function on the cytolemma possibly change, so that the proteic biological function of influence.

The invention discloses the nucleotide polymorphisms of the functional gene AQP9 relevant, the SNPs of AQP9 gene carried out gene type and gene frequency analysis with the ox growth traits, and and the red ox growth traits in Jiaxian County between carried out association analysis; The result shows that the Nucleotide polymorphic site of AQP9 gene can become the mark of molecular genetic assistant breeding.

To above-mentioned ox AQP9 gene nucleotide polymorphum, the invention also discloses its examination and detection method, through PCR-SSCP and dna sequencing combined techniques, can be simply, fast, cost is low, detect the polymorphum of its mononucleotide accurately.Because the AQP9 gene function relates generally to Growth Traits such as body weight, height, chest measurement; Detection method provided by the invention is that the SNPs of AQP9 gene and the foundation of growth traits relation are laid a good foundation; So that be applied to the marker assisted selection of place of china ox kind growth traits, set up the good ox population of genetic resources fast.

Description of drawings

Fig. 1 is the PCR product electrophoretogram in ox AQP9 gene P6 site.

Fig. 2 is the PCR-SSCP electrophoretogram in ox AQP9 gene P6 site.

Fig. 3 is the different genotype sequencer map of ox AQP9 gene SNP s.

Fig. 4 is ox AQP9 gene P6 site wild-type and the Nucleotide of mutant and the compare of analysis of aminoacid sequence.

Embodiment

The present invention is with the 72bp fragment of AQP9 gene conserved sequence design primer amplification AQP9 gene extron 6 zones and 3 ' UTR; Genome with the red ox in Jiaxian County, western Shandong ox, 3 kinds of Qin Chuan ox is a template respectively; Carry out pcr amplification; Utilize native polyacrylamide gel electrophoresis to detect the product single strand conformation polymorphism then, polymorphic product is sought the mononucleotide polymorphic of this amplified fragments after checking order; Mononucleotide polymorphic to finding carries out the proterties correlation analysis; And its detection method is provided; Make the nucleotide polymorphisms of AQP9 gene become a kind of can be fast, the convenient molecular genetic marker that detects, for accelerating to set up ox population foundation is provided with high-quality economic characters.

Below in conjunction with the present invention being done further detailed description, said is to explanation of the present invention rather than qualification.

A, PCR-SSCP and dna sequencing combined techniques are to the detection of ox AQP9 gene SNP s

1, the collection of ox sample

The present invention specifically with the population of 3 place of china ox kinds as detected object, specifically gather sample and see table 1.

The collection of table 1 ox sample

2, the separation of blood sample genomic dna, extraction, purifying

1) freezing blood sample (being mainly hemocyte) room temperature is thawed, and transferase 45 00 μ L to 1.5mL Eppendorf centrifuge tube adds equal-volume PBS liquid; Abundant mixing; The centrifugal 10min of 12000r/min (4 ℃), abandoning supernatant, repetition above-mentioned steps to supernatant is transparent, deposition is faint yellow;

2) in centrifuge tube, add DNA extraction buffer 500 μ L, shake, make the hemocyte deposition break away from centrifuge tube tube wall, 37 ℃ of water-bath 1h;

3) add Proteinase K to 3 μ L (20mg/mL) and mixing, 55 ℃ are spent the night to clarification, and defecator not can add 1 μ L Proteinase K mixing and continue digestion until clarification as yet;

4) reaction solution is cooled to room temperature, adds the saturated phenol 500 μ L of Tris-, gentleness is shaken centrifuge tube 20min, makes its abundant mixing; 4 ℃, the centrifugal 10min of 12000r/min changes supernatant in another 1.5mL centrifuge tube over to, repeats once;

5) add chloroform 500 μ L, abundant mixing 20min, 4 ℃, the centrifugal 10min of 12000r/min changes supernatant in another 1.5mL centrifuge tube over to;

6) add chloroform, primary isoamyl alcohol mixed solution (24: 1) 500 μ L, abundant mixing 20min, 4 ℃, the centrifugal 10min of 12000r/min changes supernatant in another 1.5mL centrifuge tube over to;

7) add the NaAc damping fluid of 0.1 times of volume and the ice-cold absolute ethyl alcohol of 2 times of volumes, mix and rotate centrifuge tube, separate out, preserve 30～60min for-20 ℃ until the flocks of white;

8) 4 ℃, the centrifugal 10min of 12000r/min, abandoning supernatant precipitates 2 times with 70% ice-cold ethanol rinsing DNA;

9) 4 ℃, the centrifugal 10min of 12000r/min makes the ethanol volatilization clean under the abandoning supernatant, room temperature;

10) dried DNA is dissolved in the TE liquid of 80～100 μ L, and 4 ℃ of preservations are dissolved until DNA fully, and 0.8% agarose gel electrophoresis detects its quality ,-80 ℃ of preservations.

11) adding 10%SDS in the dna solution of 500 μ L, to make its final concentration be 0.1%, adds Proteinase K to final concentration and reach 50 μ g/mL;

12) 5 ℃ are incubated about 10h;

13) equal-volume phenol, chloroform, primary isoamyl alcohol (25: 24: 1) and the extracting of chloroform difference are once;

14) the centrifugal 5min phase-splitting of 12000r/min is drawn the upper strata water to another centrifuge tube;

15) add 1/10 volume 3mol/L sodium-acetate and the 2 times of ice-cold absolute ethyl alcohol deposit D of volume NA;

16) outwell liquid, dry after 70% washing with alcohol, add the dissolving of 60 μ L sterilization ultrapure water, 4 ℃ to be detected.

3, pcr amplification and product detect

(1) amplimer design

The primer of the present invention all is to design voluntarily with Primer premier5.0 software, and is synthetic by Shanghai biotechnology Services Co., Ltd.According to AQP9 gene order (GenBankacc.No.:NC_007308) the design primer of ox among the GenBank, with amplification AQP9 gene coding region and part non-coding area sequence.Amplification the primer sequence, annealing temperature and amplified fragments expectation size are specifically seen table 2.

Table 2AQP9 gene PCR primer sequence

(2) PCR reaction system

The PCR reaction system adopts mixes the application of sample method; Promptly, calculate the total amount of various reactive components, join in 1 1.5mL centrifuge tube according to the number of the required PCR reaction of the quantity of the required various components of each reaction system and 1 secondary response; Fully instantaneous centrifugal behind the mixing; Divide again to install in each 0.2mLEppendorfPCR pipe, add template DNA then, instantaneous more centrifugal laggard performing PCR amplification.

The total system of reaction in 6 sites of amplification ox AQP9 gene is 25 μ L, and the add-on and the concentration of various moitys are seen table 3.

Table 3PCR reaction system

(3) PCR response procedures

94 ℃ of preparatory sex change 4min;

72 ℃ are extended 10min;

Genomic dna to 555 samples of 3 ox kinds carries out pcr amplification, obtains the ox AQP9 gene amplification fragment of 555 individuals.

(4) electrophoresis detection of pcr amplification product

In 5 μ L pcr amplification products, add 2 μ L sample-loading buffers, contain electrophoresis on the sepharose of EB, 80V, 40min 1.2%.On the gel imaging appearance, take a picture and observe amplification, see Fig. 1.1.2% agarose gel electrophoresis analytical results shows that the amplified production band in AQP9 gene P6 site is clear, and specificity is good, can satisfy the needs that carry out next step operation.

4, pcr amplification product being carried out SSCP detects

The PCR product in ox AQP9 gene P6 site carries out sscp analysis after detecting.Specific operation process is following:

(1) preparation of electrophoresis apparatus: after abundant washed glass plate dried, each was sealed with a plastic glue strip with supporting two sheet glass intermediary both sides and bottom, and respectively presss from both sides the iron clamp that two electrophoresis chambers carry at the plastic glue strip on both sides, with leakproof glue.

(2) making of glue: see table 4.

The composition of the acrylamide gel of table 4 different concns

(3) encapsulating: with behind the glue mixing fast to going in the sheet glass interlayer, insert comb, place on the offset plate frame or place on the desktop of level.

(4) dress glue: treat that (about 45～60min) pulled out comb, take out the adhesive tape of bottom, in the tank of electrophoresis chamber bottom, add an amount of 1 * TBE earlier after gelling admittedly; Offset plate is fixed on the DYCZ-24B type electrophoresis chamber with clip, notices that the bottom does not produce bubble, the back adds 1 * TBE in the tank on electrophoresis chamber top; Attention will cover the about 1cm in glue hole, and whether inspection leaks electrophoresis liquid, if leak; Again above step is washed well with small syringe at last until not leaking.

(5) 250V prerunning 10min prepares point sample simultaneously.

(6) sex change: get 4 μ L PCR products and mix with 6 μ L sample-loading buffers, 98 ℃ of sex change 10min place ready trash ice rapidly after the taking-up, guarantee to begin point sample behind the 5min at least.

(7) electrophoresis: beginning 10min 300～400V high voltage electrophoresis, then about 140～200V electrophoresis 14h.

(8) flushing: after electrophoresis finishes, take out offset plate, carefully lever up sheet glass with blunt knife, cut the upper left corner to make marks, cut the well burr then, appropriate amount of deionized water is washed once, continues about 15s.

(9) dyeing: flushing finishes the back and adds staining fluid, on shaking table, rocks 8～10min.

(10) flushing: dyeing is outwelled staining fluid after finishing, and adds appropriate amount of deionized water gently about hand rolling 15s.

(11) colour developing: at first with a small amount of colour developing liquid evenly flushing once, about 15s of time all pours remaining staining fluid into pallet after outwelling again, the shaking table jog is till band occurring clearly.

(12) flushing: outwell staining fluid, with deionized water rinsing 2 times.

(13) imaging: gel imaging system is taken pictures or scanner scanning.

Through experiment repeatedly, set up 10% SEPIGEL 305 (29: 1) gel, 1 * tbe buffer liquid, 4 ℃, the top condition combination of 130V electrophoresis 14h, the result sees Fig. 2.According to pictorial display, ox AQP9 gene P6 site has polymorphum, and judges that there are 3 kinds of genotype (AA, AB, BB) in this site.

5, polymorphic product is carried out sequential analysis

The individual amplified production of every kind of genotype of 3 ox kinds checks order; The AQP9 gene order of ox (GenBank acc.No.:NC_007308) is compared among sequencing result and the GenBank; Find that this fragment is respectively at 47575 places; There are 1 C＞T respectively in 47615 places and 47690 places, the sudden change of C＞T and A＞G, and sequencing result is seen Fig. 3.Have 3 kinds of genotype (AA, AB, BB) according to this site of sequencing result final decision, and with base sequence be C-C-A be defined as allelotrope A, T-T-G is defined as allelotrope B.Find through the aminoacid sequence comparison; The missense mutation of Nucleotide causes its coded amino acid to change on ox AQP9 gene the 47575th and the 47615th site; Make the leucine of 260 of peptide chains become phenylalanine(Phe) (Leu260Phe); The proline(Pro) that peptide chain is 273 becomes leucine (Pro273Leu), sees Fig. 4.This will influence the sterie configuration of its proteins encoded to a great extent, so that this proteic biological function of influence.

The frequency statistics analysis in b, place of china ox AQP9 gene SNP s site

Genotype frequency is meant that certain genotype number of individuals of a certain proterties in the colony accounts for the ratio of total individual number.P _AA=N _AA/ N, wherein P _AARepresent the AA genotype frequency in a certain site; N _AAHas the genotypic number of individuals of AA in the expression colony; N is for detecting the total quantity of colony.

Gene frequency is meant that a certain gene number is to the relative ratios of its allelotrope sum in the colony.The formula that calculates can be write as: P _A=(2N _AA+ N _Aa1+ N _Aa2+ ...+N _Aan)/2N.In the formula, P _AExpression allelotrope A frequency, N _AAHas the genotypic individual amount of AA, N in the expression colony _AaiHave Aai genotype individual amount in the expression colony, a1-an is n the different multiple allelomorphos of allelotrope A; Statistics is seen table 5.

Genotype and the gene frequency distribution table of table 53 an ox kind AQP9 gene SNP s

Can find out that from table 5 in 3 kinds being studied, the AB individuality accounts for the overwhelming majority, the AB genotype is the advantage allelotype.And allelotrope A and B frequency are almost equal, all near 0.5000.

The association analysis of c, ox AQP9 gene SNP s locus gene effect

The genotype (AA, AB and BB) of genotype data: PCR-SSCP and the identification of dna sequencing combined techniques.

Association analysis sample: 362 of the red oxen in Jiaxian County that complete growth traits record is arranged.

Production data: the growth traits data of the red ox in Jiaxian County (body weight, height, body length, chest measurement etc.).

The association analysis model:

Utilize the dependency of SPSS (17.0) software analysis gene locus, male animal, age and parity factor and growth traits.Earlier data are carried out descriptive analysis, determine whether to exist outlier, utilize least-square analysis that data are proofreaied and correct again; According to data characteristics, utilize multivariate linear model analyzing gene type effect.Model is following:

Y _ijk＝μ+S _i+A _j+G _k+e _ijk

Wherein: Y _IjkThe observed value of-Di ijk individuals body chi proterties; μ-be the kind average; Si-is an i individuals sex effect value; A _j-be j individuals age effect value; G _kThe genotype effect value of-Di k individuals; e _Ijk-random residual effect.

To body weight, height, body length, chest measurement, the hip width of AQP9 gene P6 site SNP and the red ox in Jiaxian County, point of the buttocks is wide, buttocks is long and high 8 growth traitss of hip cross are carried out the least square analysis, the result sees table 6.

Table 6AQP9 gene P6 site is to the variance analysis of the red ox growth traits in Jiaxian County

Annotate: the colleague has same letter and representes difference not remarkable (P＞0.05), the significant difference with different subscripts letters, and level of signification P＜0.05 represented in small letter, and level of signification P＜0.01 is represented in capitalization.

Can know that by table body weight, height, chest measurement and the buttocks of the red ox of this site different genotype and Jiaxian County lived forever in dependency (P＜0.05), not have dependency (P＞0.05) with other indexs.The result of multiple comparisons shows that the genotypic individuality of AB is all significant higher on body weight, chest measurement and buttocks are long than the genotypic individuality of BB, and the genotypic individuality of AA is also significant high on height than the genotypic individuality of BB simultaneously.Explain that the BB genotype is an inferior position genotype, this possibly be that the mononucleotide of AQP9 gene coding region is prominent

Change causes its synthetic protein to change, and finally causes the variation of growth traitss such as body weight.Thus, in breeding work from now on, the seed selection AA that can try one's best, AB genotype kind eliminate the genotypic individuality of a part of ox AQP9 gene BB, accelerate to have the foundation of high-quality economic characters ox population.The SNP that has also confirmed simultaneously ox AQP9 gene can be used as the hereditary and selection that a candidate molecules genetic marker that improves the ox growth traits is applied to the ox fine quality.

Claims

1. method that detects the SNP of place of china ox AQP9 gene; It is characterized in that, be template with place of china ox genomic dna sequence, utilizes Auele Specific Primer to pcr amplification ox AQP9 gene; Utilize native polyacrylamide gel electrophoresis to detect the product single strand conformation polymorphism then; Polymorphic product can accurately be identified place of china ox kind AQP9 gene after order-checking mononucleotide polymorphism site is for this gene: g.47575C＞T, 47615C＞T, 47690A＞G variant sites; The SNP of ox AQP9 gene is: compare with GenBank acc.No.:NC_007308 sequence; The corresponding SNPs of AA genotype is: g.47575C, and 47615C, 47690A; The corresponding SNPs of AB genotype is: g.47575C/T, and 47615C/T, 47690A/G; The corresponding SNPs of BB genotype is: g.47575T, and 47615T, 47690G; Described primer is to being:

Upstream primer: 5 '-AAGGAAGACCAAGCGATGT-3 ' 19nt;

Downstream primer: 5 '-GTGTGTGAAGATGCTTTGTGA-3 ' 21nt.

2. the method for claim 1; It is characterized in that the condition of described pcr amplification is: 25 μ L reaction systems comprise 1.0 μ L Taq archaeal dna polymerases; 10 * Buffer, 2.5 μ L; 2.5mmol/LdNTPs2.0 μ L, the DNA sample 1.0 μ L of 50.0ng/ μ L ox genomic dna sequence, each 0.5 μ L of 10pmol/ μ L upstream and downstream primer and sterilization ultrapure water 17.5 μ L;

Described pcr amplification reaction program is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s of 30～35 circulations, 61 ℃ of annealing 45s, 72 ℃ are extended 45s; 72 ℃ are extended 10min.

3. the method for claim 1 is characterized in that, described polyacrylamide gel electrophoresis is 10% polyacrylamide gel electrophoresis.