CN101875977B - Method for detecting mononucleotide polymorphism of scalper SREBP1c gene - Google Patents

Abstract

The invention discloses a method for detecting mononucleotide polymorphism of a scalper SREBP1c gene, which comprises the following steps of: using a scalper whole genome DNA to be detected containing the SREBP1c gene as a template, using a primer pair P as a primer, and performing PCR amplification on the scalper SREBP1c gene; using a restriction endonuclease Bg1 I to digest a PCR amplification product, and then performing sepharose gel electrophoresis on an amplified fragment after enzyme digestion; and identifying the mononucleotide polymorphism of the 10914th bit of the scalper SREBP1c gene according to the result of the sepharose gel electrophoresis. Because the functions of the SREBP1c gene relate to four important growth traits of body weight, daily gain, body length and chest circumference, the detection method provided by the invention lays a good foundation for establishing a relation between SNP and the growth traits of the SREBP1c gene, and is convenient to be used in a marker assistant selection (MAS) of the growth traits of Chinese scalper beef to quickly establish a scalper population with an excellent genetic resource.

Description

A kind of method that detects mononucleotide polymorphism of scalper SREBP 1 c gene

Technical field

The invention belongs to biological technical field, relate to the detection of gene mononucleotide polymorphism (SNP), the method for particularly a kind of detection scalper SREBP 1 c (the conjugated protein 1c of sterol regulatory element) gene mononucleotide polymorphism.

Background technology

Gene pleiomorphism is meant the difference of genome sequence between the Different Individual in different plant species or the same species; These differences be since in the karyomit(e) in the DNA allelotrope Nucleotide change and cause, mainly be the variation of the replacement, insertion, disappearance and the Tumor-necrosis factor glycoproteins copy number that comprise base.

SNP (Single Nucleotide Polymorphism; SNP) be one type of genetic marker system that the scholar Lander (1996) by the human genome research centre of Massachusetts Institute Technology proposes, just be meant in the genomic dna sequence polymorphum that the replacement owing to single Nucleotide (A/T/C/G) causes.Its variant form has: transversion, conversion, insertion and disappearance etc., mainly conversion or the transversion by single base causes.SNPs with nucleotide variation of conversion hysteria accounts for 2/3.

Position according to SNP generation in the genome; Can be divided into following 3 types: gene coding region SNP (Coding-region SNPs; CSNPs), gene periphery SNP (Perigenic SNPs; SNP pSNPs) and between gene (Intergenic SNPs, iSNPs).

Research shows that the cSNP that is positioned at the coding region is fewer, because it is significant in heredopathia research, therefore, the research of the cSNP in the coding region is more paid close attention to.CSNP in the gene coding region can be divided into 2 kinds again: a kind of is synonym cSNP (Synonymous cSNP) in the coding region, and promptly the change of encoding sequence can't influence the change of aminoacid sequence in its protein of translating due to the SNP; Another kind is the non-synonym cSNP (Non-Synonymous cSNP) in the coding region, i.e. the change of base sequence will cause the change of coded amino acid, thereby cause the change of aminoacid sequence in the protein, possibly finally have influence on proteinic function.

Molecular breeding; Be molecular marker assisted selection breeding (Molecular Mark-Assist Selection; MAS), this technology is by dna molecular marker genetic resources or breeding material to be selected, and the comprehensive proterties of livestock and poultry is carried out breed improvement; It is a method of utilizing modern molecular biology and traditional genetic breeding to combine, carries out breeding of new variety.In the beef cattle breeding, people expectation, through closely related to growth traits, and with the selection of the closely linked dna marker of quantitative character, reach early stage seed selection and improve the purpose of breeding value accuracy, thereby in the livestock and poultry breeding, obtain bigger genetic progress.

The molecular genetic marker assisted Selection combines modern biotechnology exactly with conventional system of selection; Through the selection of genetic marker being selected indirectly the quantitative trait locus (QTL) of certain proterties of control; Enable to utilize simultaneously the phenotype information of marker site information and quantitative character; More accurately estimate the breeding value of animal individual, improve efficiency of selection, accelerate the breeding progress.Marker assisted selection has mainly experienced three phases: the fs is the genetic analysis between each proterties of domestic animal; Subordinate phase is the marking phase of protein (enzyme) mark to quantitative character; Three phases is the molecular genetic marker stage.Along with molecular marking technique is day by day ripe and abundant, make the mark that covers whole genome become possibility, through and QTL between linkage analysis, realize the target of molecular marker assisted selection.(single nucleotidepolymorphism SNP) has been widely used in the assignment of genes gene mapping, clone, genetic breeding and multifarious research as new genetic marker to SNP.

Conjugated protein (the Sterol Regulatory Element Binding Proteins of sterol regulatory element; SREBPs) gene family is because the montage mode of mRNA is different, but and produces different 3 kinds of protein: SREBP-1a, SREBP-2 (SREBP-1b) and the SREBP-1c of the identical structure of function.SREBP-1a and SREBP-1c are produced by same gene, but because because its first exon is different, cause separately promotor different and produce transcribe (the Exon 1a and the Exon 1c) of different positions.

In SREBPs family, SREBP-1a and SREBP-1c are main existence forms, and SREBP-1c is called adipocyte decision and differentiation factor again.SREBP-1a and SREBP-1c mainly regulate the fatty acid metabolism involved enzyme, and the effect of SREBP-1c mainly concentrates on the synthetic and glucose metabolism of lipid.The SREBP1c gene (GenBank:NC_007317) of ox is positioned on No. 19 karyomit(e), and its full length gene is 17482bp, and 21 exons are arranged.

At present, focus mostly on the mankind and rat, mainly comprise the correlative study of this gene and adipocyte differentiation factor, Regular Insulin and leptin etc. for the research of SREBP1c gene.About the research of SREBP1c gene genetic variation be more common in both at home and abroad with the pig be laboratory animal that do with research fat and meat proterties aspect, do not see the scalper SREBP 1 c gene genetic polymorphism and to the report of ox growth traits correlation effect research.The research in scalper SREBP 1 c gene heritable variation field at present is deficient, and the related research of the functional study of this gene locus and heritable variation thereof and Growth Traits (as: weight, body weight, day weight gain, height, the body proterties such as tiltedly length, chest measurement, point of the buttocks is wide of being born) is still blank.

Summary of the invention

The technical problem that the present invention solves is to provide a kind of method of mononucleotide polymorphism of scalper SREBP 1 c gene of detection, seeks the SNP related with economic characters as molecule marker, and quickening has the foundation of high-quality economic characters ox population.

The present invention realizes through following technical scheme:

A kind of method that detects mononucleotide polymorphism of scalper SREBP 1 c gene is a template with the ox complete genome DNA to be measured that comprises the SREBP1c gene, is primer with primer to P, the pcr amplification scalper SREBP 1 c gene; After restriction enzyme Bgl I digestion pcr amplification product, the amplified fragments after again enzyme being cut carries out agarose gel electrophoresis; Identify the SNP of the 10914th of scalper SREBP 1 c gene according to the agarose gel electrophoresis result;

Described primer to P is:

Upstream primer: agcccgacag cccggtctgc caggac 26;

Downstream primer: cctcagccct gtccttcttc 20.

Described pcr amplification reaction program is:

94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 59 ℃ of annealing 30s, 72 ℃ are extended 15s, 30～35 circulations; 72 ℃ are extended 10min.

Described agarose gel electrophoresis is that mass concentration is 4% agarose gel electrophoresis.

Saidly identify that according to the agarose gel electrophoresis result SNP of the 10914th of scalper SREBP 1 c gene is: the GG genotype shows as 130bp and 25bp band; The GA genotype shows as 155bp, 130bp and 25bp band; The AA genotype shows as the 155bp band; Wherein, single nucleotide polymorphism A A genotype is the sudden change homozygous genotype.

Compared with prior art, the present invention has following beneficial technical effects:

The present invention possibly produce the SNP that the proteins encoded conformation changes to the missense mutation on scalper SREBP 1 c gene the 10914th site and detect; When the 10914th site sports A by G; Coding triplet GGC changes into AGC; Protein coding amino acid in the transcription corresponding position changes, and (the sweet basic acid mutation that peptide chain is 502 is the acid of silk base; Gly 502Ser), two, the three grades of configurations in the proteic space of the protein-bonded transcriptional regulator SREBP1c of sterol regulatory element coded by said gene with important physiological function are changed, so that the proteic biological function of influence.

Mononucleotide polymorphism of scalper SREBP 1 c gene detection method provided by the invention; Sport the transition mutations of A by G to the 10914th site; 3 ' end through at newly-designed upstream primer is introduced base mispairing, constructs the cleavage site of being discerned as restriction enzyme Bgl I.When becoming A, near the mispairing position, can't form restriction enzyme Bgl I recognition site behind the pcr amplification SREBP1c gene, and suddenly change when not taking place, can form Bgl I recognition site behind the pcr amplification SREBP1c gene by G; Can detect the SREBP1c gene mononucleotide polymorphism accurately, fast and easily through the electrophoresis detection somatotype: the GG genotype shows as 130bp and 25bp band; The GA genotype shows as 155bp, 130bp, 25bp band; The AA genotype shows as the 155bp band; And then to the allelotrope of the SREBP1c gene SNP of 3 ox colonies and the change monitoring of genotype frequency.

Because the SREBP1c gene function relates to growth traitss such as birth weight, body weight, day weight gain, height, body weight, body are tiltedly long, point of the buttocks is wide; Detection method provided by the invention is that the SNP of SREBP1c gene and the foundation of growth traits relation are laid a good foundation; For use in the marker assisted selection (MAS) of the meat growth traits of Chinese Cattle, set up the good ox population of genetic resources fast.

Description of drawings

Fig. 1 is the 534bp PCR product electrophoresis result of scalper SREBP 1 c gene 10509bp～11042bp;

Fig. 2 is the 155bp PCR product electrophoresis result of scalper SREBP 1 c gene 10888bp～11042bp;

Fig. 3 introduces BglI restriction enzyme site PCR product enzyme for mispairing and cuts the electrophoresis detection result that the SNP of the 10914th of scalper SREBP 1 c gene is identified in the back;

Fig. 4 is different genotype (GG, GA, the AA) sequencer map of scalper SREBP 1 c gene SNP.

Embodiment

The present invention is through the 10914th site missense mutation possibly produce the SNP that the proteins encoded conformation changes and detects to scalper SREBP 1 c gene; For use in the marker assisted selection of the meat growth traits of Chinese Cattle, set up the good ox population of genetic resources fast.Below in conjunction with the detection of concrete sample is related with proterties the present invention is done further detailed description, said is to explanation of the present invention rather than qualification.

A, scalper SREBP 1 c gene contain the PCR primer design of nt10914 sudden change

Hereford cow (NC_NC_007317.3) sequence so that NCBI was announced is reference, utilizes the Primer5.0 design to increase and comprises the PCR primer in scalper SREBP 1 c gene exon 9 zones, and its primer sequence is following:

Upstream primer F1:cgact acatc cgctt cctt 19;

Downstream primer R1:cctca gccct gtctt tcttc 20;

With above-mentioned primer to the ox genome amplification; Can the increase gene fragment of the 534bp that comprises scalper SREBP 1 c gene exon 9 regional 10509bp～11042bp, the segmental electrophoresis detection in amplification back is as shown in Figure 1, wherein; Swimming lane 1～6 is for detecting fragment, and swimming lane M is Marker; To the fragment of amplification check order identify after, wherein, the sequence of the 10888bp～10919bp is as follows:

agccc?gacag?cccgg?tct?

agga?c?

gcc?ag；

When the G of 10914bp (being the 1504th in SREBP1c gene C DS district) sports A; The 502nd the codon of causing encoding sports AGC by GGC (sequence shown in the frame line

); Promptly sport 502Ser, make that like this change (glycocoll Gly changes into Serine Ser) has taken place coded amino acid in the translation process by 502Gly.

Because (SNP site) no nature restriction enzyme site at the 10914bp place; Can not be through the direct enzyme cutting check; And if the artificial design PCR primer mispairing at 10906bp～10908 places is GCC by TTG (sequence shown in the frame line

) mispairing; When not suddenling change in 10914 sites; The 10906bp of pcr amplification SREBP1c gene product～10915bp sequence is gcc agga^c gg; Formed the restriction enzyme site of restriction enzyme Bgl I (GCCNNNN^NGG); When 10914bp sported A by G, the 10906bp of pcr amplification SREBP1c gene product～10915bp sequence was gcc agga^c ag, and restriction enzyme Bgl I can not discern; So just can detect this site SNP polymorphum; Therefore designing pcr amplification primer P is:

Upstream primer: agcccgacag cccggtctgc cagga c 26;

Downstream primer: cctcagccct gtccttcttc 20;

Wherein, the base mismatch of introducing is the GCC of upstream primer the 10th～12bp; The fragment of the 155bp of 10888bp～11042bp that the corresponding gene fragment that increases of primer P is the SREBP1c gene, the segmental electrophoresis detection in amplification back is as shown in Figure 2, and wherein, swimming lane 1～4 is for detecting fragment, and swimming lane M is Marker.

B, carry out the SREBP1c gene fragment of pcr amplification ox to be measured with primer P

1, ox sample collection and extracting genome DNA

(1) 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: Henan Nanyang Cattle (265), Qin Chuan, Shaanxi ox (235), the red ox in Jiaxian County, Henan (441);

The collection of table 1 ox sample

Kind	Sample number	The sample title	Sample source	Sample mode
					Nanyang Cattle (NY cattle)	265	Blood sample	Pick up from Nanyang City, Henan Province nan yang yellow cattle seed stock breeding station (national ox protects kind of a field)	No. 16 syringe needles of jugular vein blood sampling
Qin Chuan ox (QC cattle)	235	Blood sample	Pick up from cattle farm, Qin Chuan, Shaanxi Province, ox stock breeding center, Qin Chuan, Shaanxi Province and Dali County, Shaanxi Province	No. 16 syringe needles of jugular vein blood sampling
					The red ox in Jiaxian County (JX cattle)	441	Blood sample	Pick up from red ox stock breeding center, Jiaxian County, Pingdingshan City, Henan Province (the red ox in national Jiaxian County protects kind of a field) and the red ox in Jiaxian County and protect kind of each villages and small towns, district	No. 16 syringe needles of jugular vein blood sampling

(2) 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 abandons supernatant, makes the ethanol volatilization clean under the 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

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 or the 2.0mL 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 0.2mL Eppendorf PCR pipe, add template DNA then, instantaneous more centrifugal laggard performing PCR amplification;

The PCR reaction system is seen table 2:

Table 2PCR reaction system

Sterilization ultrapure water (H 2O)	10.8μL
		2 * Buffer (includes Mg 2+, dNTPs etc.)	12.5μL
Primer P upstream primer (10pmol/L)	0.5μL
		Primer P downstream primer (10pmol/L)	0.5μL
Taq archaeal dna polymerase (2.5U/ μ L)	0.25μL
		Dna profiling (50ng/ μ L)	0.45μL
TV	25μL

The PCR response procedures:

(4) PCR product purification and order-checking

After accomplishing, pcr amplification carries out agarose gel electrophoresis; The glue of cutting that carries out the PCR product then reclaims and purifying: under uv lamp, contain the segmental gel of purpose from the sepharose cutting-out; Put into the 1.5mL centrifuge tube; Reclaim purification kit (sky, Beijing root biochemical technology ltd) purified pcr product with the PCR product then, according to the operation of test kit specification sheets, concrete steps are following:

1) at first in adsorption column, add 500 μ L balance liquid BL, the centrifugal 1min of 12000r/min outwells the waste liquid in the collection tube, adsorption column is relay reclaim in the collector.

2) single target DNA band is downcut from sepharose put into clean centrifuge tube, take by weighing weight.

3) in blob of viscose, add equal-volume solution PC, 60 ℃ of water-baths are placed about 10min, constantly leniently spin upside down centrifuge tube therebetween, fully dissolve to guarantee blob of viscose.

4) will go up a step gained solution and add in the adsorption column, the centrifugal 1min of 12000r/min outwells the waste liquid in the collection tube, and adsorption column is reentered in the collection tube.

5) in adsorption column, add 700 μ L rinsing liquids, the centrifugal 1min of 12000r/min outwells waste liquid, and adsorption column is reentered in the collection tube.

6) in adsorption column, add 500 μ L rinsing liquids, the centrifugal 1min of 12000r/min outwells waste liquid, and centrifugal adsorption column is put into collection tube, and the centrifugal 2min of 12000r/min removes rinsing liquid as far as possible.Adsorption column is placed room temperature or 50 ℃ of incubator numbers minute, thoroughly dry.

7) adsorption column is put in the clean centrifuge tube, to an amount of elution buffer of the unsettled dropping in adsorption film mid-way, room temperature is placed 2min.The centrifugal 1min of 12000r/min collects dna solution.

8) in order to improve the yield of DNA, can be with in the centrifugal solution that the obtains centrifugal adsorption column of add-back again, repeating step 7.

Genomic dna to 941 samples of 3 ox kinds carries out pcr amplification, obtains to comprise in the scalper SREBP 1 c gene of 941 individuals the dna fragmentation of the 155bp in this SNP site.

C, BglI enzyme are cut the SREBP1c gene fragment of digestion pcr amplification

1, BglI endonuclease reaction digestion system (25～30 μ L): 10～15 μ LPCR products, 10 * damping fluid (containing BSA), 2.5～3.0 μ L, BglI (10U/ μ L) is 1.0～1.5 μ L, 11.5～16.5 μ L sterilization pure water (H ₂O);

2, enzyme is cut digestion condition: digest 5～10h in 37 ℃ of constant incubators.

Agarose gel electrophoresis analysis behind d, the BglI digestion PCR product

1, agarose gel electrophoresis

Make 4% sepharose, 120V voltage electrophoresis 50min, EB dyeing;

2, treat that the different dna fragmentation of molecular weight separates when clear, forms images at BIO-RAD Gel Doc 2000 gel imaging systems;

3, according to agarose gel electrophoresis interpretation of result SNP polymorphum:

Analyze with the photograph of BIO-RAD Gel Doc 2000 gel imaging systems, judge the polymorphum of SNP:

When the 10914bp of SREBP1c gene sports A by G; Owing to introduced base mispairing, when not suddenling change in 10914 sites, the 10906bp of pcr amplification SREBP1c gene product～10915bp sequence is gcc agga^c gg; Formed the restriction enzyme site of restriction enzyme Bgl I (GCCNNNN^NGG); When 10914bp sported A by G, the 10906bp of pcr amplification SREBP1c gene product～10915bp sequence was gcc agga^c ag, and restriction enzyme Bgl I can not discern; So just can detect this site SNP polymorphum;

The agarose gel electrophoresis result of the 10914th polymorphum of scalper SREBP 1 c gene is:

The GG genotype shows as 130bp and 25bp band; The GA genotype shows as 155bp, 130bp, 25bp band; The AA genotype shows as the 155bp band; Because 25bp is less, thus in agarose electrophoretic analysis, detect less than, but still can differentiate GG genotype, GA genotype and AA genotype accurately through 130bp and 155bp band: not comprising the 155bp band is GG genotype individuality; Not comprising the 130bp band is that the AA genotype is individual; Comprising 155bp and 130bp band simultaneously is that the GA genotype is individual;

The electrophoresis detection result of SNP as shown in Figure 3, wherein, swimming lane 1～4 does not comprise the 155bp band, and it is that the GG genotype is individual, comprises 130bp and 25bp band (25bp is because gene fragment is too little, and electrophoretic effects shows not obvious).Swimming lane 5, swimming lane 6 and swimming lane 8 comprise 155bp, 130bp, 25bp band (25bp is because gene fragment is too little, and electrophoretic effects shows not obvious), are GA genotype individuality.Swimming lane 7 comprises the 155bp band simultaneously, is AA genotype individuality.Swimming lane M be Marker I (600bp, 500bp, 400bp, 300bp, 200bp, 100bp).

4, the sequence verification of the individual PCR product of different genotype

Utilize ABI 3730 sequenators that the individual PCR product of different genotype is carried out positive and negative two-way order-checking respectively; Simultaneously; Carry out the SNP position analysis; The result shows that individual its 10914 the sequencer map of the heterozygote GA genotype that comprises 155bp and 130bp band is expressed as G or A really, and shown in Fig. 4 b, the 9th peak is two peaks from left to right; And GG genotype, AA genotype are respectively G, A, shown in Fig. 4 a, 4c.

The frequency statistics analysis of e, place of china ox population SREBP1c gene SNP site

1, genotype frequency

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 _GG＝N _GG/N

P _AA＝N _AA/N

P wherein _GG, P _AARepresent GG, the AA genotype frequency in a certain site; N _GG, N _AAHave GG, the genotypic number of individuals of AA in the expression colony; N is for detecting the total quantity of colony.

2, gene frequency

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 _Aa3+ N _Aa4+ ...+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 mutually different multiple allelomorphos of allelotrope A.

The allelotrope that this institute relates to is G and A, so concrete gene frequency calculation formula is:

P _G＝(2N _GG+N _GC)/2N

P _A＝(2N _AA+N _GC)/2N

In the formula, P _G, P _ARepresent the allelic frequency of allelotrope G and A respectively, N _GG, N _GAAnd N _AARepresent the genotypic individual amount of GG, GA and AA respectively, N representes the total group number.

G gene frequency rangeability in different ox kind SREBP1c gene SNPs is 73.8%～93.2%, and A gene frequency rangeability is between 6.8%～26.2%, and is as shown in table 3.

The 10914th SNP gene frequency distribution table of table 3 scalper SREBP 1 c gene

The correlation analysis of f, Nanyang Cattle SREBP1c gene SNP site and growth traits

1, data processing method

Adopt SPSS16.0 (Statistical Product and Service Solutions; Version 16.0Edition) statistical software; Carried out test of significance to wherein writing down with body chi and weight data than the different genotype of more comprehensive Nanyang Cattle is individual; Because of the age all extremely remarkable to each item index influence, so each item index of statistical study same age bracket, to reject the influence (seeing table 4) at age.

1) the main proterties of measuring comprises: birth weight, and body weight, height, body is long, chest measurement, point of the buttocks is wide, average daily gain.

2) time of measuring: nascent, 6 monthly ages, 12 monthly ages, 18 monthly ages, 24 monthly ages.

3) colony that measures: 265 altogether of Nanyang Cattle colonies, sample and data information are all from Nanyang City, Henan Province nan yang yellow cattle seed stock breeding station (national ox protects kind of a field).

4) genotype and number

In this experiment, detect 3 kinds of genotype:

The GG type, N=157; The GA type, N=77; The AA type, N=31.

5) the general linear model of association analysis: call SPSS 16.0 software general linear model GLM (General Linear Models Procedure) each genotype is carried out test of significance to the influence of growth traits.Its body situation according to this experiment is set up following statistical model:

Y _ijk＝μ+A _i+G _j+E _ijk；

Wherein: Y _Ijk: individual phenotype record; μ: population mean; A _i: age effect; G _j: the genotype effect; E _Ijk: error immediately.

2, association analysis result

The result sees table 4; The body weight GG type of 6 monthly ages, 12 monthly ages, 18 monthly ages and 24 monthly ages individuality is significantly greater than GA type and AA type (P＜0.05 or P＜0.01); There were significant differences (P＜0.05 or P＜0.01) between oblique length of body that 6 monthly ages and 12 monthly ages are individual and chest measurement proterties aspect different genotype; The tiltedly long GG type of body of 24 monthly ages individuality is significantly greater than GA type and AA type (P＜0.05 or P＜0.01); The average daily gain GG type of 6 monthly ages and 24 monthly ages individuality is significantly greater than GA type and AA type (P＜0.01); The average daily gain GG type of 18 monthly ages individuality is significantly greater than CG type (P＜0.05).

Can find out from table 4, each item growth traits index of genotype GG Different Month all be higher than or the utmost point be higher than heterozygous genes type GA and mutant AA significantly.Therefore; The GG type of not undergoing mutation in Nanyang Cattle SREBP1c gene 10914G＞A polymorphic site is a preponderant genotype; This possibly be that SREBP1C gene 10914G＞A sudden change has caused the SREBP1c synthetic protein to change, finally caused the variation of body weight and body chi proterties.Thus, in breeding work from now on, should eliminate the individuality of the 10914G＞A sudden change in ox SREBP1C gene extron 9 zones in advance, accelerate to have the foundation of high-quality economic characters ox population.

The association analysis of table 4 Nanyang Cattle SREBP1c gene 10914G＞A loci polymorphism and growth traits

Annotate: numeric representation mean standard error in the table.Superscript has same letter and representes difference not remarkable (P＞0.05), alphabetical different table differential different significantly (P＜0.05)

The nucleotides sequence tabulation

< 110>Xibei Univ. of Agricultural & Forest Science & Technology

< 120>a kind of method that detects mononucleotide polymorphism of scalper SREBP 1 c gene

<160>3

<210>1

<211>26

<212>DNA

< 213>synthetic

<400>1

agcccgacag?cccggtctgc?caggac 26

<210>2

<211>19

<212>DNA

< 213>synthetic

<400>2

cctcagccct?gtccttcttc 20

<210>3

<211>32

<212>DNA

< 213>ox

<400>3

agcccgacag?cccggtcttt?gaggacggcc?ag 32

Claims (3)

1. a method that detects mononucleotide polymorphism of scalper SREBP 1 c gene is characterized in that, is template with the ox complete genome DNA to be measured that comprises the SREBP1c gene, is primer with primer to P, the pcr amplification scalper SREBP 1 c gene; After restriction enzyme Bgl I digestion pcr amplification product, the amplified fragments after again enzyme being cut carries out agarose gel electrophoresis; Identify the SNP of the 10914th of scalper SREBP 1 c gene according to the agarose gel electrophoresis result:

The GG genotype shows as 130bp and 25bp band; The GA genotype shows as 155bp, 130bp and 25bp band; The AA genotype shows as the 155bp band; Wherein, single nucleotide polymorphism A A genotype is the sudden change homozygous genotype;

Described primer to P is:

Upstream primer: agcccgacag cccggtctgc caggac;

Downstream primer: cctcagccct gtccttcttc.

2. the method for detection mononucleotide polymorphism of scalper SREBP 1 c gene as claimed in claim 1 is characterized in that, described pcr amplification reaction program is:

94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s, 59 ℃ of annealing 30s, 72 ℃ are extended 15s, 30～35 circulations; 72 ℃ are extended 10min.

3. the method for detection mononucleotide polymorphism of scalper SREBP 1 c gene as claimed in claim 1 is characterized in that, described agarose gel electrophoresis is that mass concentration is 4% agarose gel electrophoresis.

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