CN103667279B - The molecule marker of pig average daily gain genes involved Resistin and application thereof - Google Patents
The molecule marker of pig average daily gain genes involved Resistin and application thereof Download PDFInfo
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Abstract
The present invention relates to field of pig molecular marker preparation, refer to molecule marker and the application thereof of a boar average daily gain genes involved Resistin particularly.The present invention utilizes in Resistin gene order and comprises a SNP site, positioned at nucleotide sequence SEQ? ID? the 861bp place of NO.1, this site is the base mutation of T/C, causes Alw26I-RFLP polymorphism.This base mutation is arranged in Resistin gene the 3rd intron, does not change the aminoacid sequence of its translation.Therefore primer is designed near the SNP site of Resistin gene order, obtain molecule labelled series SEQ? ID? NO.1, thus qualification pig average daily gain proterties.The present invention utilizes existing PCR-RFLP technology, in Resistin gene 861bp place's discovery T-C sudden change, and finds this polymorphic site and average daily gain proterties significant correlation, has certain help to the research of pig growth traits.
Description
Technical field
The present invention relates to field of pig molecular marker preparation, refer to molecule marker and the application thereof of a boar average daily gain genes involved Resistin particularly.
Background technology
Livestock industry is in occupation of very important status in modern agricultural development, and its ratio occupied in agriculture production is commonly used to the important indicator of a measurement countries and regions development degree.Pork as the topmost meat sources of China, pig-breeding industry in China's livestock industry in occupation of very important status.At 20th century the mid-80, China's production of meat about 2,000 ten thousand tons, pork accounts for 85%, and within 2012, pork output reaches 5,335 ten thousand tons, accounts for meat total amount 63.63%.Although pork proportion declines gradually, it is dominate all the time.
In pig industry, feed cost accounts for the 65-75% of total cost of raising pigs.Therefore, reduce the feed intake in pig-breeding, improve food utilization efficiency, reduce feed cost and be related to raise pigs enterprise development and even existence.Research finds, the economic characters significant correlation such as the same food consumption of feed efficiency, average daily gain, intramuscular fat of pig (Zhao Kebin improves the strategy that growing-finishing pig feed conversion rate reduces feed cost. pig industry science, 2008,60-63).Therefore, the gene affecting the proterties such as pig feed intake, average daily gain, intramuscular fat can be used as the candidate gene of research pig feed transformation efficiency.
Phylaxin Resistin is by (SteppanCM such as Univ Pennsylvania USA Steppan, BaileyST, BhatS, BrownEJ, BanerjeeRR, WrightCM, PatelHR, AhimaRS, LazarMA. " Thehormoneresistinlinksobesitytodiabetes " [J] .Nature, 2001,409 (6818): 307 – 312) find in the adipocyte of maturation.The same year, Kim etc. utilize gene chip isolation technique to be separated the Resistin gene (Sung-EunKim obtaining mouse, HeHuang, MingZhao, XinjunZhang, AiliZhang.WntStabilizationof β-CateninRevealsPrinciplesforMorphogenReceptor-ScaffoldAss emblies [J] .Science, PublishedOnlineApril112013).Subsequently, Rajala etc. use subtractive screen library separating out fat cell when the gene of different differential period expression, also Resistin gene (RajalaMW has been isolated, LinY, RanallettaM, YanqXM, QianH, GinqerichR.Celltype-specificexpressionandcoregulationofm urineresistinandresistin-likemolecule-alphainadiposetiss ue [J] .MolEndocrinol, 2002,16:1920 – 30).Pan Yingbin etc. adopt in the promotor of PCR-SSCP method discovery Resistin gene, exon, intron and all find polymorphic site (Pan Yingbin, Liu Yanfen, Liu You, Gao Zhijie, Wang Qun, He Wanjuan, Tan Minjing. six kind pork insulin resistin gene single nucleotide polymorphism analysis. Journal of Agricultural Biotechnology, 2008,16 (3): 402-406).
Model red flag by the research of insulin sensitivity target tissue-Skeletal Muscle Cell Sugar intake function is shown Resistin can significantly suppress myocyte on glucose uptake (model red flag .Resistin is on the impact of Skeletal Muscle Cell Sugar intake function and mechanism analysis thereof. [master thesis], Nanjing: Nanjing Medical University, 2007).Solana etc. research show mouse Resistin gene can with receptor tyrosine kinase sample orphan receptor 1(ROR1) be combined in specific region, ROR1 extracellular, mediation 3T3-L1 preadipocyte differentiation and glucose uptake (BeatrizS á nchez-Solana, JorgeLaborda, VictorianoBaladr ó n.MouseResistinModulatesAdipogenesisandGlucoseUptakein3T 3-L1PreadipocytesThroughtheROR1Receptor [J] .MolecularEndocrinology, 2012,26 (1): 110-127).By studying further, Liu Feng etc. find that process LAN Resistin slows down beta Cell of islet RINm5F and breeds, show that Resistin can produce restraining effect (Liu Feng to insulin secretion, Qiu Jie, Zhang Chunmei, Ji Chenbo, Guo's tin melts the impact that .Resistin gene pairs beta Cell of islet RINm5F breeds. Chinese Journal of Contemporary Pediatrics, 2010,43-46).
Resistin can be used as signaling molecule adjustment fat growth growth and distributes and deposits simultaneously.The parameter that Chen little Dong is relevant to some steatolysis in food restriction and the pork fat of being satiated with food detects, find that in the fatty tissue of pig, Resistin significantly expresses, and Resistin expression level with fatty deposits be proportionate (Chen little Dong. the expression of Adipocyte Factor TNF-α and resistin and the regulating and controlling effect to pork fat metabolism thereof. [Ph.D. Dissertation], Wuhan: Hua Zhong Agriculture University, 2004).Cieslak finds in Polish Large White colony, Resistin gene is with thickness of backfat significant correlation (CieslakJ, Nowacka-WoszukJ, BartzM, Fijak-NowakH, GrzesM, SzydlowskiM, SwitonskiM.AssociationstudiesontheporcineRETN, UCP1, UCP3andADRB3genespolymorphismwithfatnesstraits [J] .MeatScience, 2009,83 (3): 551-554).
More than research shows, Resistin is by insulin action, and blood-sugar content in control agent, has close ties with the propagation of body fat cell, differentiation and the deposition level with fat simultaneously, by regulating Resistin expression amount, can control volume self-energy metabolic balance.
Because carbohydrate metabolism and fatty deposits have substantial connection in the growth traits of pig and body, therefore can study Resistin gene and whether pig growth traits be had an impact.Because Resistin is at important role (Yang Qing such as adjustment lipid metabolism, insulin resistance, energy balance, food consumption, inflammation, Zhou Lei, Gan Li, thunderclap, the expression of old little winter .Resistin gene and study on regulation. biochemical tenth the seminar abstract of a thesis compilation of national animal physiological, 2008), study the relation between the energy metabolism of Resistin and pig, food consumption, the economic characters for improvement pig are significant.
Summary of the invention
Technical problem to be solved by this invention is just to provide the molecule marker of a kind of pig average daily gain genes involved Resistin, and its nucleotide sequence is as shown in SEQIDNO.2.
Present invention also offers a kind of primer pair for obtaining described molecule marker, described primer pair is:
Forward primer: 5 '-CCCTCAGGTGAGTACAGAACT-3 ';
Reverse primer: 5 '-AAGCCTGCAAGTGGGAATGAG-3 '.
Present invention also offers a kind of preparation method of molecule marker, there is the pig genomic dna of Resistin gene for template, adopt following primer pair:
Forward primer: 5 '-CCCTCAGGTGAGTACAGAACT-3 ';
Reverse primer: 5 '-AAGCCTGCAAGTGGGAATGAG-3 '.
Carry out pcr amplification, its molecule marker obtained is SEQIDNO.2.
Present invention also offers the method for the high average daily gain proterties of a kind of molecular markers for identification pig, with the genomic dna of pig to be measured for template, adopt following primer pair:
Forward primer: 5 '-CCCTCAGGTGAGTACAGAACT-3 ';
Reverse primer: 5 '-AAGCCTGCAAGTGGGAATGAG-3 ';
Carry out pcr amplification, wherein, have the pig of 349bp band, it has Resistin gene, as SEQIDNO.1.
Present invention also offers the application of a kind of molecule marker in pig some growth proterties and feed efficiency proterties related molecular marker assistant breeding.
Present invention also offers a kind of application of primer pair in pig some growth proterties and feed efficiency proterties related molecular marker assistant breeding of molecule marker.
Qualification contains a method for the pig of Resistin gene, comprises the following steps to be:
1) primer pair:
Forward primer: 5 '-CCCTCAGGTGAGTACAGAACT-3 ';
Reverse primer: 5 '-AAGCCTGCAAGTGGGAATGAG-3 ';
2) pcr amplification condition
PCR reacts cumulative volume 10 μ l, and wherein pig genomic DNA template 1 μ l, forward and reverse primer each 0.2nmol/ μ l, PCRmix5 μ l to be measured, finally add deionized water to cumulative volume 10 μ l;
3) PCR reaction conditions is: after 95 DEG C of denaturation 5min, and circulate 30 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 40s; Last 72 DEG C extend 5min; PCR primer detects through 2%W/V agarose gel electrophoresis;
4) RFLP detects
PCR primer endonuclease reaction volume is 10 μ l, wherein PCR primer 5 μ l, deionized water 3.5 μ l, and 10 × buffer1 μ l, restriction enzyme A lw26I are 0.5 μ l (10U/ μ l),
By centrifugal after sample blending, 12h placed by 37 DEG C of incubators, detects enzyme cut result with 3.5%W/V agarose gel electrophoresis, and record genotype, takes pictures under ultraviolet lamp;
Enzyme cuts generation three kinds of genotype, and TT genotype only has 349bp mono-band, and CC genotype has 183bp and 166bp two band, and heterozygote TC genotype has 349bp, 183bp and 166bp tri-band.
The principle of the invention
Comprise a SNP site in Resistin gene order, be positioned at the 861bp place of nucleotide sequence SEQIDNO.1, this site is the base mutation of T/C, causes Alw26I-RFLP polymorphism.This base mutation is arranged in Resistin gene the 3rd intron, does not change the aminoacid sequence of its translation.Therefore primer is designed near the SNP site of Resistin gene order, obtain molecule labelled series SEQIDNO.1, thus qualification pig average daily gain proterties.
In order to realize above-mentioned object, the present invention adopts following technical measures:
A kind of acquisition of the molecule marker relevant to pig some growth proterties and feed efficiency proterties:
1) design of primers of pig Resistin gene and partial dna sequence increase
With the template sequence of pig Resistin gene sequence information (the GenBank number of including: ENSSSCG00000013575) as design of primers, utilize biology primer-design software Oligo7.0 to design primer, primer sequence is as follows:
Forward primer: 5 '-CCCTCAGGTGAGTACAGAACT-3 ';
Reverse primer: 5 '-AAGCCTGCAAGTGGGAATGAG-3 ';
2) pcr amplification reaction:
TIANampGenomicDNAKit test kit (purchased from American invitrogen company) is utilized to be that (this colony derives from Hubei gold woods breeding station to great Bai castrating pig experimental population from purebred U.S.A,) (slaughter determining and sampling are divided into 21 batches and carry out, measure individual amount and amount to 236, to be all U.S.A be, and purebred great Bai castrates boar) extract genomic dna in ear tissue, concrete operation method carries out with reference to TIANampGenomicDNAKit test kit specification sheets.
PCR reacts: reaction cumulative volume is 30 μ l, and wherein pig DNA template 3.0 μ l, PCRmix15 μ l, forward primer and reverse primer each 0.6nmol/ μ l, finally add deionized water to cumulative volume 30 μ l.PCR reaction conditions is: after 95 DEG C of denaturation 5min, and circulate 30 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 40s, and last 72 DEG C extend 5min.PCR primer detects through 1% (W/V) agarose gel electrophoresis, and obtain part Resistin fragment, length is 349bp, and its sequence is for shown in SEQIDNO.2.
3) purifying of PCR primer, order-checking
The purifying of PCR primer: cut the gel containing object fragment from low melting-point agarose gel under ultraviolet lamp, puts into 1.5ml centrifuge tube, then uses PCR primer purification kit (purchased from Beijing hundred Imtech) purified pcr product.DNA solution after purifying is delivered to the forward and reverse order-checking of Shanghai Ying Jun company.
349bp specific amplification fragment is obtained with primer amplification pig genomic dna, as shown in Figure 2, forward and reverse sequencing result finds that the 861st of the Resistin gene (shown in SEQIDNO.1) at this fragment place finds T-C conversion (as shown in Figure 3), causes an Alw26I restriction enzyme site
.
Based on a detection method for the molecule marker relevant to pig average daily gain, its step is as follows:
1) primer sequence
Forward primer: 5 '-CCCTCAGGTGAGTACAGAACT-3 ';
Reverse primer: 5 '-AAGCCTGCAAGTGGGAATGAG-3 ';
This primer amplification fragment length 349bp, its sequence is for shown in SEQIDNO.2.
2) pcr amplification condition
PCR reacts cumulative volume 10 μ l, and wherein pig genomic DNA template 1 μ l, forward and reverse primer each 0.2nmol/ μ l, PCRmix5 μ l, finally add deionized water to cumulative volume 10 μ l.PCR reaction conditions is: after 95 DEG C of denaturation 5min, and circulate 30 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 40s; Last 72 DEG C extend 5min.PCR primer is through 2%(W/V) agarose gel electrophoresis detection.
3) RFLP detects
PCR primer endonuclease reaction volume is 10 μ l, wherein PCR primer 5 μ l, deionized water 3.5 μ l, 10 × buffer1 μ l, restriction enzyme A lw26I are 0.5 μ l (10U/ μ l), by centrifugal after sample blending, 12h placed by 37 DEG C of incubators, with 3.5%(W/V) agarose gel electrophoresis detect enzyme cut result, record genotype, take pictures under ultraviolet lamp.
Enzyme cuts generation three kinds of genotype, and TT genotype only has 349bp mono-band, and CC genotype has 183bp and 166bp two band, and heterozygote TC genotype has 349bp, 183bp and 166bp tri-band.
To the molecule marker of pig average daily gain proterties with the application in pig some growth proterties and the relevant molecular mark of feed efficiency proterties, step is as follows:
The average daily gain of collected specimens, food consumption, prediction food consumption, residue food consumption, feed conversion rate and intramuscular fat 6 indexs.According to the group structure of collected specimens, mixture model is used to come the genotype effects of statistical study Resistin gene SNP site and the relation with feed efficiency proterties thereof:
Y
ij=μ+genotypei+ε
ij+G
Wherein, Y
ijfor processing rear character value, μ is population mean, and ε ij is random error, and G is a batch effect, assuming that obey N(0, σ
2) distribution.Can analyze genotype effects, the multiplicity completing genotype effects compares.SPSS16.0 software (AsiaAnalyticsChina) is adopted to carry out data processing and statistical study.
Statistical study finds the T>C mutated-genotype of this gene, and wherein TT type and the different of CC type and average daily gain significant correlation (P=0.038), with food consumption, predict food consumption and remain food consumption not significant correlation.Compared between two the individuality that genotype is TT, CC, TC by least squares means, result shows that TT type genotypic mean day weight gain is higher than CC type genotype individuals, without significant difference between TC type genotype and other two kinds of genotype.
Compared with prior art, the present invention has the following advantages:
The present invention utilizes existing PCR-RFLP technology, in Resistin gene 861bp place's discovery T-C sudden change, and finds this polymorphic site and average daily gain proterties significant correlation, has certain help to the research of pig growth traits.
Accompanying drawing explanation
Fig. 1 is the technology of the present invention schema.
Fig. 2 is pig Resistin gene group amplification electrophoresis result schematic diagram.
M:DL2000 molecular weight marker;
Fig. 3 is the T>C sudden change that in the present invention, pig Resistin gene sequencing finds.
Fig. 4 is three kinds of genotype (TTCCTC) electrophoresis result schematic diagram of the Alw26I-RFLP of pig Resistin gene extron.
M:50bpDNA molecular weight marker; P:PCR product.
Embodiment
In order to explain the present invention better, illustrate main contents of the present invention further below in conjunction with specific embodiment, but content of the present invention is not only confined to following examples.
Embodiment 1:
A kind of acquisition of the molecule marker relevant to pig average daily gain proterties:
(1) design of primers of pig Resistin gene and partial dna sequence increase
With the template sequence of pig Resistin gene sequence information (the GenBank number of including: ENSSSCG00000013575) as design of primers, utilize biology primer-design software Oligo7.0 to design primer, primer sequence is as follows:
Forward primer: 5 '-CCCTCAGGTGAGTACAGAACT-3 ';
Reverse primer: 5 '-AAGCCTGCAAGTGGGAATGAG-3 ';
(2) pcr amplification reaction:
TIANampGenomicDNAKit test kit (purchased from American invitrogen company) is utilized to be that (slaughter determining and sampling are divided into 21 batches and carry out great Bai castrating pig experimental population (this colony derives from Hubei gold woods breeding station) from purebred U.S.A, measure individual amount and amount to 236, to be all U.S.A be, and purebred great Bai castrates boar) extract genomic dna in ear tissue, concrete operation method carries out with reference to TIANampGenomicDNAKit test kit specification sheets.
PCR reacts: reaction cumulative volume is 30 μ l, and wherein pig DNA template 3.0 μ l, PCRmix15 μ l, forward primer and reverse primer each 0.6nmol/ μ l, add deionized water to cumulative volume 30 μ l.PCR reaction conditions is: after 95 DEG C of denaturation 5min, and circulate 30 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 40s, and last 72 DEG C extend 5min.PCR primer is through 1(W/V) detection of % agarose gel electrophoresis, obtain part Resistin fragment, length is 349bp, and its sequence is for shown in SEQIDNO.2.
(3) purifying of PCR primer, order-checking
The purifying of PCR primer: cut the gel containing object fragment from low melting-point agarose gel under ultraviolet lamp, put into 1.5ml centrifuge tube, then PCR primer purification kit (purchased from Beijing hundred Imtech) purified pcr product is used, operate according to test kit specification sheets, concrete steps are that the ratio adding 400 μ lGSBuffer according to 100mg blob of viscose adds GSBuffer, put 50 DEG C of incubation 10min, agarose blob of viscose is dissolved completely, within every two minutes, put upside down mixing once; The glue dissolved is transferred to centrifugal adsorbing column, and centrifugal adsorbing column is placed in waste collection pipe, the centrifugal 30s of 10000rpm, discards waste liquid; Put back by centrifugal adsorbing column in waste collection pipe, add 500 μ lWashBuffer in centrifugal adsorbing column, the centrifugal 30s of 10000rpm, abandons filtrate.Use 500 μ lWashBuffer solution washings in the same way more once; Centrifugal adsorbing column is put in meeting waste collection pipe, the centrifugal 1min of top speed; Careful taking-up centrifugal adsorbing column, is inserted in an aseptic 1.5ml centrifuge tube, is added 30 μ l distilled waters in adsorption film central authorities, after room temperature leaves standstill 2-10min, and the centrifugal 1min of top speed; Take out centrifugal adsorbing column, 1.5ml centrifuge tube (DNA solution) is placed in-20 DEG C and saves backup.DNA solution after purifying is delivered to the forward and reverse order-checking of Shanghai Ying Jun company.
349bp specific amplification fragment is obtained with primer amplification pig genomic dna, as shown in Figure 2, forward and reverse sequencing result finds that the 861st of the Resistin gene (shown in SEQIDNO.1) at this fragment place finds T-C conversion (as shown in Figure 3), causes an Alw26I restriction enzyme site
Embodiment 2:
Based on a detection method for the molecule marker relevant to pig average daily gain proterties, its step is as follows:
PCR-RFLP diagnostic method is set up
(1) primer sequence
Forward primer: 5 '-CCCTCAGGTGAGTACAGAACT-3 ';
Reverse primer: 5 '-AAGCCTGCAAGTGGGAATGAG-3 ';
This primer amplification fragment length 349bp, its sequence is for shown in SEQIDNO.2.
(2) pcr amplification condition
PCR reacts cumulative volume 10 μ l, and wherein pig genomic DNA template 1 μ l, forward and reverse primer each 0.2nmol/ μ l, PCRmix5 μ l, finally add deionized water to cumulative volume 10 μ l.PCR reaction conditions is: after 95 DEG C of denaturation 5min, and circulate 30 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 40s; Last 72 DEG C extend 5min.PCR primer is through 2%(W/V) agarose gel electrophoresis detection.
(3) RFLP detects
PCR primer endonuclease reaction volume is 10 μ l, wherein PCR primer 5 μ l, deionized water 3.5 μ l, 10 × buffer1 μ l, restriction enzyme A lw26I are 0.5 μ l (10U/ μ l), by centrifugal after sample blending, 12h placed by 37 DEG C of incubators, with 3.5%(W/V) agarose gel electrophoresis detect enzyme cut result, record genotype, take pictures under ultraviolet lamp.
Enzyme cuts generation three kinds of genotype, and TT genotype only has 349bp mono-band, and CC genotype has 183bp and 166bp two band, and heterozygote TC genotype has 349bp, 183bp and 166bp tri-band, as shown in Figure 4.
Embodiment 3:
From the application of the molecule marker that pig average daily gain proterties is correlated with in different swinery body polymorphic detection, the steps include:
Utilizing PCR-Alw26I-RFLP to have detected purebred U.S.A is that great Bai castrates pig experimental population (this colony derives from Hubei gold woods breeding station) (236 U.S.As are that purebred great Bai castrates boar colony).The genotype of this mutational site in experimental population and gene frequency as shown in table 1, detected result shows, there are three kinds of genotype in Resistin gene in experimental population, wherein TT type individuality has 104, TC type individuality has 112, CC type individuality has 20, and the detected result of enzyme cutting type conforms to sequencing result, and detection method of the present invention is reliable.This result shows that Resistin gene is that great Bai castrating pig experimental population allelic T preponderates in purebred U.S.A.
Embodiment 4:
The association analysis of a kind of molecule marker relevant to pig average daily gain proterties and some growth proterties and feed efficiency proterties
The swinery of the present embodiment is that great Bai castrates pig from this Hubei gold purebred U.S.A of woods breeding station, and slaughter determining and sampling are divided into 21 batches and carry out, and measure individual amount and amount to 236, be all U.S.A is purebred great Bai castrating boar.
The proterties analyzed is some growth proterties and the proterties relevant to feed efficiency proterties mainly, comprising: average daily gain, food consumption, prediction food consumption, residue food consumption, feed conversion rate and intramuscular fat 6 indexs.According to the group structure of collected specimens, applicant uses mixture model to come the genotype effects of statistical study Resistin gene SNP site and the relation with some growth and feed efficiency proterties thereof:
Yij=μ+genotypei+ε
ij+G
Wherein, Yij is character value after process, and μ is population mean, and ε ij is random error, and G is a batch effect, assuming that obey N(0, σ
2) distribution.Can analyze genotype effects, the multiplicity completing genotype effects compares.SPSS16.0 software (software comes from AsiaAnalyticsChina) is adopted to carry out data processing and statistical study.
Carry out association analysis to pig Resistin Gene A lw26I-RFLP pleomorphism site and some growth and feed efficiency proterties, known by table 1: in 236 individualities, TT type individuality has 104, TC type individuality has 112, and CC type individuality has 20.
The present embodiment adopts the generalized linear model in SPSS16.0 software to be that great Bai castrates in swinery body and carried out preliminary association analysis to some growth and feed efficiency proterties to the T>C mutational site of pig Resistin gene polymorphic in U.S.A.Preliminary Analysis Results is in table 1.Statistical study finds the T>C mutated-genotype of this gene, and wherein TT type and the different of CC type and average daily gain significant correlation (P=0.038), with food consumption, predict food consumption and remain food consumption not significant correlation.Compared between two the individuality that genotype is TT, CC, TC by least squares means, result shows that TT type genotypic mean day weight gain is higher than CC type genotype individuals, without significant difference between TC type genotype and other two kinds of genotype.
The association analysis of table 1Resistin gene polymorphism sites genotype and some growth proterties and feed efficiency proterties detects
Note: the proterties average in table is mean number ± standard deviation.
ADG: average daily gain; FI: daily ingestion amount; PFI: prediction daily ingestion amount; RFI: residue food consumption; FCR: feed conversion rate; IMF: intramuscular fat.
Other unspecified part is prior art.Although above-described embodiment is to invention has been detailed description; but it is only the present invention's part embodiment; instead of whole embodiment, people can also obtain other embodiments according to the present embodiment under without creative prerequisite, and these embodiments all belong to scope.
Claims (3)
1. the application of molecule marker in pig average daily gain associated genotype detects of pig average daily gain genes involved Resistin, the nucleotide sequence of described molecule marker is as shown in SEQIDNO.2, and described pig is purebred U.S.A is that great Bai castrates pig.
2. the application of primer pair in pig average daily gain associated genotype detects of molecule marker described in amplification claim 1, described primer pair comprises:
Forward primer: 5'-CCCTCAGGTGAGTACAGAACT-3',
Reverse primer: 5'-AAGCCTGCAAGTGGGAATGAG-3';
Described pig is purebred U.S.A is that great Bai castrates pig.
3. carry out a method for pig average daily gain associated genotype detection based on the molecule marker described in claim 1, it is characterized in that: comprise PCR-RFLP step, wherein:
1) PCR primer sequence
Forward primer: 5'-CCCTCAGGTGAGTACAGAACT-3';
Reverse primer: 5'-AAGCCTGCAAGTGGGAATGAG-3';
This primer amplification fragment length 349bp, its sequence is for shown in SEQIDNO.2;
2) pcr amplification condition
PCR reacts cumulative volume 10 μ l, and wherein pig genomic DNA template 1 μ l, forward and reverse primer each 0.2nmol/ μ l, PCRmix5 μ l, finally add deionized water to cumulative volume 10 μ l; PCR reaction conditions is: after 95 DEG C of denaturation 5min, and circulate 30 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 40s; Last 72 DEG C extend 5min; PCR primer detects through the agarose gel electrophoresis of 2%;
3) RFLP detects
PCR primer endonuclease reaction volume is 10 μ l, wherein PCR primer 5 μ l, deionized water 3.5 μ l, 10 × buffer1 μ l, restriction enzyme A lw26I are 0.5 μ l, by centrifugal after sample blending, 12h placed by 37 DEG C of incubators, agarose gel electrophoresis with 3.5% detects enzyme and cuts result, and record genotype, takes pictures under ultraviolet lamp;
Enzyme cuts generation three kinds of genotype, and TT genotype only has 349bp mono-band, and CC genotype has 183bp and 166bp two band, and heterozygote TC genotype has 349bp, 183bp and 166bp tri-band; Described pig is purebred U.S.A is that great Bai castrates pig.
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| CN103451180A (en) * | 2013-08-23 | 2013-12-18 | 中国农业大学 | Molecular marker related to sedimentary character of pork fat, and application thereof |
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| Resistin基因在肥胖易感大鼠与肥胖抗性大鼠脂肪组织中表达差异的研究;郭锡熔;《南京医科大学学报（自然科学版）》;20040930;第24卷(第5期);摘要、474页右栏、475页表1-2 * |
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