CN103451180B - Molecular marker related to sedimentary character of pork fat, and application thereof - Google Patents
Molecular marker related to sedimentary character of pork fat, and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker, and particularly relates to cloning of a pig ECI1 gene segment and application as the molecular marker. A nucleotide sequence of the molecular marker is shown in SEQ ID NO.1; a C157-T157 base mutation is formed at the 157th bit, resulting in Bsm I-RFLP (restricted fragment length polymorphisms).
Description
Technical field
The present invention relates to animal molecular marker field, specifically, relate to the application of a kind of ECI1 gene relevant to label of pig fat deposition description proterties as molecule marker.
Background technology
Pig industry is the main body of China's Animal husbandry production, and its output value is all first of the livestock industry gross output value for a long time all the time.Along with the raising of people's living standard, the quality and quantity demand of meat product is improved constantly, how to cultivate high lean ratio, focus that the pig kind of high intramuscular fat is current pig breeding work.
China's pig resources enriches, and for pig breeding provides good material, gene and molecule marker also for studying important economical trait provide good material.Zang Zhushi China distinctive plateau type pig kind, small volume and thin skin, meat are tender delicious, with rich flavor, meat is fine and smooth, unique flavor, have " treasure on plateau " good reputation (Liu Xuan, strong Ba Yang ancestor, Wang Qiang etc. hide the analysis of pig reproductive trait multigentic effect. heredity, 2010,32 (5): 480-485).The southern regions of the Yunnan Province microtia pig is the small-sized local variety of Xishuangbanna Prefecture, Yunnan Province, there is the ecotope of the district's hot humid adapting to tropical rain forest, advantage (the Ma Junsong such as precocious easily fertilizer, strong stress resistance, the thin bone of skin are thin, Fresh & Tender in Texture, nutritious, Ma Qiwen, Duan Yong, Lian Linsheng. original ecological breeding version receives microtia fattening of pig performance, trunk and meat quality determination. raises pigs, 2009, (2): 37-38).Huaihe River pig is the ancient local variety of original Soil Development in Huaibei Plain, have that reproductivity is high, fine and tender taste, at local pig breed lean ratio comparatively high (Zhang Donghong, Wang Like, Tao Li, Wang Chonglong, Li Qinggang, Chen Liangyun. Huaihe River pig and the change of filial generation diameter of muscle fiber and with the research of meat relation. herding and animal doctor, 2005,37(9): 9-11).Hide the local pig breed of pig, the southern regions of the Yunnan Province microtia pig and Huai Zhushi China characteristic, have that slow, the heavy fat ability of the speed of growth is strong, degeneration-resistant, disease-resistant, crude feed tolerance, especially meat is fine and smooth, meat is tender delicious, unique flavor.Large White (Large White Yorkshire) is famous, the widest leading bacon hogs kind of distribution in the world, fast growth, but meat is poor.Huai Pig new line utilizes Huaihe River pig to be the Shanxi Lean meat Pig New Line that basic material is cultivated, there is the advantage (Li Qinggang such as meat is excellent, lean ratio is high, the price of deed is high, litter size is many, disease resistance is strong, anti-stress, accommodative ability of environment are strong, Xu Jingen, Tao Li. Huai Pig new line I system's trunk and meat guality Breeding Progress from generation to generation. raise pigs, 2009, (4): 33-34).The pig of above different fatty character is the good material of research label of pig fat deposition description character gene.
Along with the develop rapidly of molecular quantitative, Protocols in Molecular Biology, about molecular genetic marker and mark
The research of note assisted Selection is extensively carried out, and applies in Animal Breeding, creates tremendous influence.Fat deposition is one of major objective selected in pig breeding, and the label of pig fat deposition description of different varieties or type has obvious difference.Affect the gene of fatty deposits or mark from molecular level qualification is one of the focus studied of current pig functional gene (Aslan O, Hamill R, Davey G, McBryan J, Mullen A, Gispert M, Sweeney T:Variation in theIGF2gene promoter region is associated with intramuscular fat content inporcine skeletal muscle.Mol Biol Rep, 2012,9 (4): 4101 – 4110).Investigator is once by measuring the association analysis of gene polynorphisms and phenotypic character and quantitative expression, think fatty acid-binding protein gene (FABP) (Chang W, Richers-HaunerlandJ, Haunerlang NH.Induction of cardiac fabp gene expression by longchain fatty acids in cultured rat muscle cells.Mol Cell Biochem2001, 221:127-132), fatty acid synthetase (FAS) (Clarke R, Lbutlton RF.Lark M.Thesuccessful translocation of an endangered species with a complex socialorganisation:The black-eared miner.Abstract Volume of the23rdInternational Ornithological Congress.2002, 191-192), peroxisome proliferation-activated receptors (PPAR) (Li Fangqiong, Liu Haifeng, Zhu sharpens. and rosiglitazone and serum are on impact (English) [J] .Agricultural Science & Technology of PPAR α and PPAR γ genetic expression in the differentiation-inducing process of pig PECTORAL LIMB SKELETON, 2011, 5 (06): 893-896), long-chain acyl CoA synthetic enzyme (ACSL) (Zhan T, Poppelreuther M, Ehehalt R, F ü llekrug J.Overexpressed FATP1, ACSVL4/FATP4and ACSL1increasethe cellular fatty acid uptake of3T3-L1adipocytes but are localized onintracellular membranes.PLoS ONE2012, 7 (9): e45087) etc. relevant with the fatty deposits of pig.But at present, in the practice of pig molecular breeding, still lack definite functions, the remarkable major gene of effect and molecule marker.
△ 3-△ 2-diene ester acyl-CoA isomerase (ECI1) is the enzyme of organism kernel coding, in plastosome, participate in the necessary coenzyme of odd positions double bond metabolism in beta-oxidation process, it carrys out the reaction of catalysis β-oxidation by trans or cis-3 alkene ester acyl coenzyme A of different positions and length being converted into trans-2 alkene ester acyl coenzyme As, is the key coenzymes of this approach.Research shows; the unsaturated fatty acids β-oxidation of enzyme to mouse and people of this genes encoding has regulating and controlling effect (Palosaari PM; Kilponen JM; Sormunen RT; et al. △ 3-△ 2-enoyl-CoAisomerases.Characterization of the mitochondrial isoenzyme in the rat.JBIOL CHEM1990,265 (6): 3347-3353; Hiltunen JK, QinY.Beta-oxidation-strategies for the metabolism of a wide variety ofacyl-CoA esters.Biochim.Biophys.Acta.2000,1484:117 – 128).The change in a lot of fatty acid metabolism process can be caused after mouse mitochondrial ECI1 gene is knocked, increase (the Janssen Uand Stoffel W.Disruption of mitochondrial beta-oxidation of unsaturatedfatty acids in the3 of intermediate product unsaturated ethylene diacid in the steatosis of such as liver and urine, 2-trans-enoyl-CoA inomerase-deficient mouse.J BiolChem, 2002,277 (22): 19576-19584).
Based on the vital role that this gene plays in growth and development process, applicant infers that pig ECI1 gene may play an important role affecting in label of pig fat deposition description, therefore using the candidate gene of pig ECI1 gene as fatty deposits, electronic cloning and PCR order-checking is adopted to obtain pig ECI1 gene order, and PCR sequencing technologies screens the SNP site of dissimilar between herds in swine difference, establish method for quick, obtain the SNP site and genotype effects that affect label of pig fat deposition description.
Summary of the invention
The object of the invention is to clone the ECI1 gene relevant to label of pig fat deposition description proterties, find the mutational site of ECI1 gene and the pleiomorphism detecting method as label of pig fat deposition description genes involved, for marker-assisted breeding provides a kind of useful molecule marker.
In order to realize the object of the invention, first the present invention provides a kind of molecule marker relevant to label of pig fat deposition description proterties, and its nucleotide sequence, as described in sequence table SEQ ID NO.1, has 1 C157-T157 base mutation at the 157th, causes Bsm I-RFLP polymorphism.
The site of described base mutation is positioned at pig ECI1 gene the 3rd intron 54bp place, called after Intron3-C54T.
The present invention also provides a kind of preparation method of described molecule marker, and it is with pig genomic dna for template, and design primer, carry out pcr amplification, object is to increase and comprises the DNA sequence dna in Intron3-C54T site.The nucleotide sequence of described primer is as follows:
Forward primer: 5 '-GCTGCCTCCTCTCCCTCA-3 ';
Reverse primer: 5 '-GGAACAGCCAGCCACTTG-3 '.
PCR reaction system is 25 μ L, wherein 10 × PCR Buffer2.5 μ L, 10mmol/LdNTP mix2.0 μ L, the each 1 μ L of primer of 5pmol/ μ L, Taq archaeal dna polymerase (5U/ μ L) 0.5 μ L, DNA profiling 1 μ L(DNA is about 100ng), add dd H2O to 25 μ L.
PCR reaction conditions is 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 65 DEG C of renaturation 30s, 72 DEG C extend 20s, 36 circulations; 72 DEG C extend 7min; Be cooled to 4 DEG C of maintenances.
Specifically, the acquisition of described molecule marker is realized by following steps:
1, the cloning and sequencing of pig ECI1 gene
(http://www.ncbi.nlm.nih.gov/database/indem html) homology comparison people ECI1 gene (NM_001919) on NCBI website, obtain pig ESTs sequence, through repeatedly BLAST, comparison and splicing, finally obtain the electronic cloning sequence of 1401bp, nucleotide sequence is as shown in sequence table SEQ ID NO.2.According to SEQ ID NO.2 primers ECI1-1 and ECI1-2(in table 1), pcr amplification pig ECI1 gene mRNA sequence, purifying reclaims amplified production and is connected to pEASY-T3 cloning vector, transfection Trans1-T1 competent cell, cultivate and identify recombinant plasmid, order-checking, connects ECI1-1 and ECI1-2 Product Sequence, to confirm the exactness of described electronic cloning sequence.
2, pig ECI1 gene SNP screening
The pig ECI1 gene mRNA sequence obtained by cloning and sequencing is BLAT in UCSC website (http://genome.ucsc.edu/) pig genome database, obtain location and the DNA sequence dna of this gene, design 3 couples of primers ECI1-3, ECI1-4 and ECI1-5(in table 1).To hide pig, the southern regions of the Yunnan Province microtia pig and Yorkshire Pigs genomic dna for template, utilize above-mentioned 3 pairs of primers, all exons of amplification ECI1 gene and part of intron sequence.To increasing, the PCR primer obtained checks order, comparison, and the SNPs site of mutation frequency difference between screening colony, result finds C/T base mutation at pig ECI1 gene the 3rd intron 54bp place, called after Intron3-C54T.
3, pig ECI1 gene molecule marker detection method, concrete steps are as follows:
For the Intron3-C54T mutational site of the pig ECI1 gene screened, design primer ECI1-6(is in table 1), object amplification comprises the DNA sequence dna in Intron3-C54T mutational site, and nucleotide sequence is as shown in sequence table SEQ ID NO.1.
Wherein: there is a C/T base mutation being positioned at ECI1 the 3rd intron 54bp place at the 157bp place described in sequence table SEQ ID NO.1, this sudden change can by the identification of Bsm I restriction endonuclease.
Table 1. pig ECI1 gene PCR primer
4, pig ECI1 gene fatty deposits correlation function qualification
From pig ear tissue, extract genomic dna, carry out pcr amplification with primer ECI1-6 (see table 1) to pig genes of individuals group DNA profiling, obtaining length is 349bp sequence (see SEQ IDNO.1).PCR primer Bsm I endonuclease digestion, can identify Intron3-C54T site, qualification pig idiotype.This loci gene type frequency distribution in more different fatty type kind swinery, and correlation analysis is carried out to genotype in Huai Pig new line colony and the thickness of backfat, identify the fatty deposits function of this gene molecule marker.
Invention further provides and utilize Bsm I-RFLP different genotype individuality to apply with label of pig fat deposition description correlation analysis.
The present invention also provides a kind of primer pair for the described molecule marker that increases, and its nucleotide sequence is as follows:
Forward primer: 5 '-GCTGCCTCCTCTCCCTCA-3 ';
Reverse primer: 5 '-GGAACAGCCAGCCACTTG-3 '.
The present invention also provides the application of described molecule marker in pig marker assisted selection.
Beneficial effect of the present invention is:
The present invention is that the molecular breeding of pig provides a new genetic molecule mark, and described molecule marker is not by the restriction such as age, sex of pig, may be used for the early stage seed selection of pig, even just can select and remain exactly when pig is just born, greatly can shorten the generation interval, accelerate the breeding process of pig.
Described pig ECI1 gene molecule marker detection method detection method fast, accurately.By the application in pig marker assisted selection, the thickness of backfat of pig can be reduced.
Accompanying drawing explanation
Fig. 1 is the product gel electrophorogram that in the embodiment of the present invention 1, primer pair ECI1-1 and primer pair ECI1-2 carries out pcr amplification.
Fig. 2 is pig ECI1 gene Intron3-C54T mutational site order-checking peak figure in the embodiment of the present invention 2.
Fig. 3 is the product gel electrophorogram of ECI1 gene Intron3-C54T sudden change PCR-RFLP in the embodiment of the present invention 3.
Fig. 4 is ECI1 genotype and thickness of backfat cognation in Huai Pig new line of the present invention.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
The cloning and sequencing of embodiment 1 pig ECI1 gene
Adopt electronic cloning method, in NCBI (http://www.ncbi.nlm.nih.gov/) website, homology comparison is carried out to the ECI1 gene (NM_001919 and NM_010023) of people and mouse, search pig expressed sequence tag (EST), then splices.After repeatedly comparison, splicing extend, obtain the sequence of 1401bp, as shown in SEQ ID NO.2.
In order to verify the sequence that electronic cloning obtains, we devise 2 pairs of primers: primer pair ECI1-1 and primer pair ECI1-2(is in table 1); Wherein 18th ~ the 1165bp of ECI1-1 primer pair amplifies sequence, expected product length 1148bp; 1037th ~ the 1397bp of ECI1-2 primer pair amplifies sequence, expected product length 361bp.Primer sterilizing distilled water is diluted to 10ppm/ μ L.
Gather in Fromlingzhi, tibet Tibet Agricultural and Animal Husbandry College teaching practice pasture and hide pig liver tissue sample, be immersed in non-liquid nitrogen type sample retention liquid (RNAfixer, purchased from Beijing hundred Tyke Bioisystech Co., Ltd), take back laboratory.Adopt RNeasy Tissue Mini Kit(QIAGEN, Germany) extract liver organization total serum IgE, synthesize total cDNA with Reverse Transcriptase Reagents kit (Promega, the U.S.).
To hide the total cDNA of pig liver tissue for template, carry out pcr amplification with primer ECI1-1 and ECI1-2, amplified production 1.5% agarose gel electrophoresis, the results are shown in Figure 1.In figure, swimming lane M is DL2000Plus Marker, and other swimming lanes are amplified production, can find out that primer ECI1-1 is consistent with expected product length with ECI1-2 amplified production, be respectively 1148bp and 361bp.
By above-mentioned ECI1-1 and ECI1-2 amplified production through Gel Extraction Kit test kit (Shanghai biotechnology company limited) purifying, be connected to pEASY-T3 cloning and sequencing carrier, transfection Trans1-T1 competent cell, cultivate and identify recombinant plasmid, then checking order (Beijing Hua Da scientific & technical corporation).Sequencing result splices after removing carrier sequence, obtain the sequence that length is 1380bp, through DNAMAN comparison, 18th ~ 1397bp number of base sequence of the 1401bp sequence that this sequence and electronic cloning obtain is consistent, prove that this sequence (SEQ ID NO.2) is the sequence really expressed in pig liver tissue, be submitted to Genbank, sequence number is: KC447361.
This sequence (accession number: KC447361) is retrieved in UCSC website (http://genome.ucsc.edu/) pig genome database, discovery is positioned at pig No. 3 karyomit(e)s, be made up of 5 exons and 4 introns, comprise a complete CDS district, length 909bp, to encode 302 amino acid, its 5 ' UTR head of district 12bp.
By the ECI1 protein sequence comparison of the protein sequence of this sequence encoding and people (NP_001910), mouse (NP_034153) and ox (NP_00103986), find that homology is respectively 76%, 73% and 79%.With people ECI1 albumen tertiary structure for template (website: http://folding.stanfold.edu/villin/), adopt Swiss-Model method (website: http://swissmodel.expaxy.org//SWISS-MODEL.html), the tertiary structure of prediction pig ECI1 sequence.Found that pig ECI1 albumen tertiary structure is identical with people, formed by 3 polymeric subunits, the catalytic site of functional domain is that L-glutamic acid gathers E136.Therefore, sequence (SEQ ID NO.2) the pig ECI1 gene really that cloning and sequencing obtains is proved.
Embodiment 2 pig ECI1 gene SNP s site is screened
By pig ECI1 gene mRNA sequence BLAT in UCSC website (http://genome.ucsc.edu/) pig genome database, obtain genomic dna sequence, then 3 couples of primers ECI1-3, ECI1-4 and ECI1-5(are designed in table 1), all exons of amplification pig ECI1 gene and part of intron sequence.
Gather place of china kind " Tibetan pig " (picking up from Tibet Agricultural and Animal Husbandry College's teaching practice pasture), " the southern regions of the Yunnan Province microtia pig " (picking up from microtia pig conservation of resources field, Xishuangbanna Prefecture, Yunnan Province the southern regions of the Yunnan Province) and introduced variety " Yorkshire Pigs " (pig farm, Shunyi, Beijing) ear tissue sample, adopt phenol/chloroform method to extract tissue gene group DNA.
To hide pig, the southern regions of the Yunnan Province microtia pig and Yorkshire Pigs genomic dna for template, by primer pair ECI1-3, ECI1-4 and ECI1-5 amplification pig ECI1 gene order, amplify the sequence band of 686bp, 768bp and 701bp of expectation respectively.Carry out purifying with Gel Extraction Kit test kit (Shanghai biotechnology company limited) to the PCR primer of above-mentioned 3 pig kinds, each pig kind selects 10 individual PCR primer equivalent to mix pond, order-checking (Beijing Nuo Sai biotechnology company) respectively.Sequencing result, through the comparison of Chromas and DNAMAN software, finds that the C base mutation at the 380bp place (being positioned at ECI1 the 3rd intron 54bp place) of ECI1-3 primer PCR amplified fragments is T base (see figure 2).
Embodiment 3 pig ECI1 gene Intron3-C54T site PCR-RFLP detection method is set up
In order to detect pig ECI1 gene Intron3-C54T mutational site genotype quickly and easily, adopting the restricted enzyme cutting analysis of DNAMAN software, finding that this mutational site can be identified by Bsm I restriction endonuclease (recognition sequence is AATG); When this site base is T, there is Bsm I restriction enzyme site; When this site mutation is C, Bsm I restriction enzyme site disappears.For the Intron3-C54T mutational site of pig ECI1 gene, design primer ECI1-6(is in table 1), object amplification comprises the DNA sequence dna in Intron3-C54T site, and nucleotide sequence is as shown in sequence table SEQ IDNO.1.157bp place wherein described in sequence table SEQ ID NO.1 is C/T mutational site.
Pcr amplification is carried out with ECI1-6 primer pair pig genomic dna, PCR reaction system is 25 μ L, wherein 10 × PCR Buffer2.5 μ L, 10mmol/L dNTP mix2.0 μ L, the each 1 μ L of primer of 5pmol/ μ L, Taq archaeal dna polymerase (5U/ μ L) 0.5 μ L, DNA profiling 1 μ L(DNA is about 100ng), add dd H
2o to 25 μ L.PCR reaction conditions is the 1st step 95 DEG C of sex change 5min; 2nd step 95 DEG C sex change 30s; 3rd step 65 DEG C renaturation 30s; 4th step 72 DEG C extends 20s; Repeat 36 circulations of the 2nd to the 4th step, afterwards again 72 DEG C extend 7min, be finally cooled to 4 DEG C of maintenances.
Pcr amplification product concentration is the agarose gel electrophoresis of 1.5%, and ethidium bromide staining observes pcr amplification effect in gel imaging system, can see the clear band (see figure 3) that clip size is 349bp.This PCR primer Bsm I endonuclease digestion, reaction system is: 10 × Buffer1 μ L, Bsm I restriction endonuclease 0.25 μ L(concentration is 10U/ μ L), PCR primer 4 μ L, adds dd H
2o to 10 μ L.In 37 DEG C of environment, enzyme cuts 4-8h.Then, by the digestion products electrophoresis in 3% sepharose obtained, ethidium bromide staining, observes (see figure 4) in gel imaging system, judges genotype.When the 157bp place base of amplified fragments is T, this site of Bsm I restriction endonuclease identifiable design, pcr amplified fragment can be cut into 2 fragments that length is 159bp and 190bp, this genotype is designated as TT type; When the base at 157bp place is all C, Bsm I restriction endonuclease can not identify, namely pcr amplified fragment can not be cut open, and only have 1 length to be the band of 349bp in gel, this genotype is designated as CC type; When being 3 band of 349bp, 159bp and 190bp containing length in gel electrophoresis, this genotype is designated as heterozygous TC.
Embodiment 4 pig ECI1 gene Intron3-C54T loci polymorphism detects
Gather and hide pig (Fromlingzhi, tibet), the southern regions of the Yunnan Province microtia pig (In Xishuangbanna of Yunnan), Laiwu Pigs (Shandong), large Pu Lianhei pig (Shandong), Huaihe River pig (Anhui), Huai Pig new line (Anhui Province Kexin Pig Farming and Breeding Co., Ltd.), landrace (Beijing) and Yorkshire Pigs (Beijing) ear tissue sample, adopt phenol/chloroform method to extract pig genes of individuals group DNA sample.
Adopt the PCR-RFLP technology set up in embodiment 3, measure the PCR-Bsm I-RFLP genotype of hiding pig individual ECI1 gene Intron3-C54T, detected result is as shown in table 2.
Table 2 pig ECI1 gene Intron3-C54T loci polymorphism distributes
Hide pig, the southern regions of the Yunnan Province microtia pig, great Pu face pig, Laiwu Pigs and Huaihe River pig and all belong to the slow place of china pig variety of the speed of growth, back fat and intramuscular fat are all higher than in introduction pig.In table 2, visible these Chinese native pig breeds ECI1 gene Intron3-C54T site preponderant genotype is CC, and allele C frequency and Large White, Yorkshire Pigs difference are very large.Huai Pig new line utilizes Chinese native pig breed Huaihe River pig (25% Huaihe River pig blood lineage) and western pig breeds selection cross to form, and its genotype frequency and gene frequency distribution are between place of china pig and western pig breeds, and C gene frequency is 33.9%.Result shows genotype and allele distributions obvious difference between different fat deposition swinery of this gene locus, may be relevant to fatty deposits.
The correlation analysis of embodiment 5 Huai Pig new line colony ECI1 genotype and the thickness of backfat
286 Huai Pig new line (picking up from prosperous breeding company limited of raising pigs of Anhui section) genotype are shown in embodiment 4.The 90kg body weight thickness of backfat of these 286 all individualities of pig is collected from the prosperous breeding company limited breeding record of raising pigs of Anhui section.Adopting linear model (formula 1), application SAS(9.0) software carries out correlation analysis to ECI1 gene Intron3-C54T loci gene type and the thickness of backfat in Huai Pig new line colony.
Y=μ+G+bW+e(formula 1)
Y is the thickness of backfat, and μ is average, and G is genotype effects, and W is body weight concomitant variable, and b is regression coefficient, and e is residual effect.
In Huai Pig new line colony, the CC genotype individuals thickness of backfat is significantly higher than TC and TT genotype individuals; T allelotrope can reduce fat thickness at back of pig, and shows dominant effect.As can be seen here, allelotrope T is the molecule marker affecting fatty deposits, and hereditary effect is large, may be used for pig molecule mark assistant breeding.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (8)
1. a molecule marker relevant to label of pig fat deposition description proterties, is characterized in that, its nucleotide sequence, as shown in sequence table SEQ ID NO.1, has 1 C157-T157 base mutation at the 157th, causes Bsm I-RFLP polymorphism.
2. molecule marker according to claim 1, is characterized in that, the site of described base mutation is Intron3-C54T site, is positioned at pig ECI1 gene the 3rd intron 54bp place.
3. the preparation method of molecule marker according to claim 1, is characterized in that, with pig genomic dna for template, and design primer, carry out pcr amplification, object is to increase and comprises the DNA sequence dna in Intron3-C54T site;
The nucleotide sequence of described primer is as follows:
Forward primer: 5 '-GCTGCCTCCTCTCCCTCA-3 ';
Reverse primer: 5 '-GGAACAGCCAGCCACTTG-3 '.
4. preparation method according to claim 3, is characterized in that, PCR reaction system is 25 μ L, the wherein each 1 μ L of primer of 10 × PCR Buffer 2.5 μ L, 10mmol/L dNTP mix2.0 μ L, 5pmol/ μ L, Taq archaeal dna polymerase 0.5 μ L, DNA profiling 1 μ L, adds ddH
2o to 25 μ L.
5. preparation method according to claim 4, is characterized in that, the concentration of described Taq archaeal dna polymerase is 5U/ μ L.
6. preparation method according to claim 3, is characterized in that, PCR reaction conditions is 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 65 DEG C of renaturation 30s, 72 DEG C extend 20s, 36 circulations; 72 DEG C extend 7min; Be cooled to 4 DEG C of maintenances.
7., for a primer pair for the molecule marker described in claim 1 that increases, its nucleotide sequence is as follows:
Forward primer: 5 '-GCTGCCTCCTCTCCCTCA-3 ';
Reverse primer: 5 '-GGAACAGCCAGCCACTTG-3 '.
8. the application of molecule marker according to claim 1 in label of pig fat deposition description proterties marker assisted selection.
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| CN105018608A (en) * | 2015-07-08 | 2015-11-04 | 广西大学 | Gene ELOVL6 method for detecting pig fat deposition |
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| CN107022603B (en) * | 2016-07-07 | 2020-08-18 | 华中农业大学 | Molecular marker for pig backfat thickness character and application thereof |
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