CN104087595B - Hog on hook and Meat Quality SNP genetic marker and application - Google Patents
Hog on hook and Meat Quality SNP genetic marker and application Download PDFInfo
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Abstract
The invention belongs to domestic animal genetic marker preparing technical field, it is specifically related to the preparation and application of the SNP genetic marker of a kind of hog on hook and Meat Quality, from pig OLR1 gene, clone obtains a kind of hog on hook as genetic marker application and the genetic fragment of Meat Quality, its nucleotide sequence is as shown in sequence table SEQ ID NO:1 and Fig. 4, the base having a C/T at the 408bp of sequence shown in Fig. 4 is replaced, and this sudden change causes Pst I RFLP polymorphism.The invention also discloses the genetic marker preparing hog on hook and the method for Meat Quality genetic marker and preparation application in hog on hook and Meat Quality polymorphic detection, the marker assisted selection for pig provides a new labelling.
Description
Technical field
The invention belongs to the genetic marker preparing technical field of pig, be specifically related to a boar OLR1 genetic fragment as trunk
With genetic marker and the application of Meat Quality, described genetic marker is cloned from pig OLR1 gene.
Background technology
Along with the development of Protocols in Molecular Biology, progressively occur in that the molecular marker assisted selection with molecular marker as core
Penetrating into equimolecular breeding technique with molecular marker, these technology combine with traditional breeding method and have greatly accelerated entering of pig breeding
Journey.Molecular marker assisted selection can analyze the genetic constitution of individuality from molecular level rapidly and accurately, thus realizes base
Because of directly selecting of type, carry out molecular breeding, be a genetic engineering important application in modern cattle breeding, pass through molecule
The method of labelling, can not only shorten the breeding time limit, greatly reduces the manpower and materials consumption of breeding；It addition, molecular marker is various
Property more make molecular marker application potential in animal breeding be greatly improved (Lu Shaoxiong, Wu Changxin., 2000).For assisting
The molecular marker selected includes protein labeling, microsatellite marker, single nucleotide polymorphism (single nucleotide
Polymorphism, is called for short SNP) labelling etc..
SNP marker refers to the DNA sequence polymorphism caused by genome single nucleotide variations, including base transition, transversion, list
Base insertion or disappearance etc., be acknowledged as up-to-date third generation DNA molecular marker.Its quantity is many, rich polymorphism.Its detection
Method includes PCR-SSCP, PCR-RFLP, order-checking and SNP chip etc..Restriction fragment length polymorphism
(Restriction Fragment Length Polymorphism, RFLP) is the limit according to different cultivars (individual) genome
The restriction enzyme site base of property restriction endonuclease processed is undergone mutation, or there occurs the insertion of base, disappearance between restriction enzyme site, causes enzyme action
Clip size there occurs that change, this change can be detected by sepharose electrophoresis, thus comparable different cultivars is (individual
Body) the difference (i.e. polymorphism) (BeuzenN.D., et al.2000) of DNA level.
Oxidized LDL receptor 1 (oxidized low-density lipoprotein receptor1,
OLR1) it is that a class can be in conjunction with the receptor of OxLDL ELISA (oxLDL).Sawamura etc. (1997) are first at cattle endothelium
Be found that OLR1 gene on cell, and successful clone human endothelial cell OLR1 gene, the most in succession cloned rat, mice and
The cDNA sequence of the OLR1 genes such as rabbit (Miki et al., 1998；Kume et al.,1998).OLR1 in these species
The encoding amino acid sequence of gene is more similar.Pig OLR1 gene has 5 introns and 6 exons, encodes 274 amino
Acid, and the similarity that people's OLR1 gene has 79%, the highest with the similarity of cattle, reach 84% (Yamanaka et al., 1998；
Ming et al.,2001).OLR1 gene is widely believed that closely related with the formation of cardiovascular disease, this external lipid metabolism
During play an important role.Patricia etc. (2005) study the effect of OLR1 first in adipose cell, it was demonstrated that
OLR1 is in the presence of PPAR γ and its relevant anti-diabetic part, and at regulation and control lipid metabolism, thus regulation insulin supports potentially
Anti-aspect plays an important role.Juwon A etc. (2007) research proves extracellular signal-regulated kinase (ERK) and c-Jun amino
Terminal Kinase (JNK) path is the important function path that OLR1 plays a role in adipose cell.Grandson superfine (2009) studies table
Bright OLR1 gene is played a role by P38MAPK path during mice preadipocyte differentiation, to suppression adipose cell
Propagation has certain effect.Li Tuo (2013) analyzes the phase of OLR1 gene genetic polymorphism and Meat Quality in Qinchuan Cattle colony
Guan Xing, finds to there is C in g.l0497 site > A sudden change, CC homozygous genotype has aobvious with Qinchuan Cattle eye muscle area and loin depth
Write dependency.At present, the research of OLR1 gene is concentrated mainly on the species such as people, mice, rat and cattle, grinds pig OLR1 gene
Studying carefully report less, applicant has carried out polymorphism research and association analysis to this gene in pig, and the genetic improvement for pig provides
Important theoretical foundation.
Summary of the invention
It is an object of the invention to obtain the SNP genetic marker relevant to hog on hook and Meat Quality and application.From pig
OLR1 gene clone obtains a special DNA fragmentation, by finding SNP site, and sets up corresponding SNP detection method, point
Analysing itself and hog on hook character and the relation of Meat Quality, the marker assisted selection for pig provides a kind of new genetic marker.
The present invention is realized by following technical proposal:
Applicant is by the separating clone to pig OLR1 gene, it was found that a kind of relevant to hog on hook and Meat Quality
SNP marker, its nucleotide sequence is as follows:
CGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCCTGGCCACTGATTACATGCATG
TCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCAGCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAG
ATCCCTTTGGTTCTGTGGCTTCATGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCC
TCCCTACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATCTCTGAGGAAACC
CAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTTGTAAGTTTCCTATTCTTTTCCTAATCTGC
TGGTGAAGTTACTGCCGCATTCGCAGAGTGTCCTCTTCCTGCTGGAAAGAACCCTGGGAAATTTCTGAAT
CTCTCTCCTCAGCCCTCTCATGCAGAGGCTTGCCCCTTGACTCATTCAGACCTTCTCTTGAGTTTTCCTTCTTACAT
TTATTACTGATTTGAAGCTTTAAGACAAGGCCAAGTGCCTGACCCAAGGT
The R at 408bp in above-mentioned sequence is C or T, and this sudden change causes Pst I-RFLP polymorphism.
Applicant devises the primer expanding above-mentioned pig OLR1 genetic fragment to (this primer something lost to being also the detection present invention
Pass labelling primer to), its DNA sequence is as follows:
Forward primer: 5 ' CGGTGGAAGAGCATTTGT 3 ',
Reverse primer: 5 ' ACCTTGGGTCAGGCACTT 3 '.
The present invention establishes the method for a kind of screening molecular marker relevant to hog on hook and Meat Quality, its step bag
Include:
Extract pig genomic DNA, according to the pig OLR1 gene sequence information announced in NCBI, design pcr amplification primer thing pair
(its nucleotide sequence is as shown in sequence table SEQ ID NO:4 and SEQ ID NO:5), with this primer to carrying out PCR amplification, obtains
The amplified fragments of a length of 583bp, its nucleotide sequence such as sequence table SEQ ID NO:1 (R at its 408bp is C or T, or ginseng
See Fig. 4, wherein: the R at R in this sequence display base mutation position, i.e. 408bp is C or T), SEQ ID NO:2 (Mei Shan
Pig: at its 408bp, the base of sudden change is T) and SEQ ID NO:3 (Large White: the base of the sudden change at its 408bp is C)
Shown in, this sudden change causes Pst I-RFLP polymorphism, and then to the PCR primer enzyme action typing obtained, carries out genotype and pig
Association analysis between carcass trait and Meat Quality, the marker assisted selection for pig provides a new genetic marker.
The genetic marker of the present invention can apply in hog on hook and Meat Quality detection.The primer of present invention design is to also
Can be applicable in hog on hook and Meat Quality detection.
More detailed technical scheme is as described in " detailed description of the invention ".
Accompanying drawing explanation
Sequence table SEQ ID NO:1 is the nucleotide sequence of genetic marker prepared by the present invention, and sequence length is 583bp,
Wherein (i.e. show at R) at the 408bp of this sequence and there is an allelic mutation (T/C replacement).
Sequence table SEQ ID NO:2 is the nucleotide sequence of the place of china pig variety " prunus mume (sieb.) sieb.et zucc. mountain pig " of clone.Sequence length
For 583bp, at the 408bp of this sequence, wherein there is a T/C replace (base of the sudden change having shown that is T).
Sequence table SEQ ID NO:3 is the nucleotide sequence of the external pig kind " Large White " of clone.Sequence length is
, at the 408bp of this sequence, wherein there is a C/T replace (base of the sudden change having shown that is C) in 583bp.
Sequence table SEQ ID NO:4 and SEQ ID NO:5 is the nucleotide sequence of the primer pair that the present invention designs.
Fig. 1: be Large White, Landrace, prunus mume (sieb.) sieb.et zucc. mountain pig and peaceful pig OLR1 sequence alignment result, the English of overstriking in figure respectively
Letter represents SNP site.
Fig. 2: be the amplification of pig OLR1 gene the 5th intron.
Agarose concentration is 1.5%；Description of symbols in figure: swimming lane M:DL200 0Marker；Swimming lane 1-4 is respectively DABAI
Amplified fragments in pig, Landrace, prunus mume (sieb.) sieb.et zucc. mountain pig and peaceful pig, clip size is 583bp.
Fig. 3: pig OLR1 gene Pst I-RFLP testing result.
Agarose concentration is 1.5%；Description of symbols in figure: swimming lane M is DL2000Marker；CC genotype, 583bp；CT
Genotype, 583bp, 172bp, 411bp；TT genotype, 172bp, 411bp.
Fig. 4: being the nucleotide sequence of pig OLR1 gene the 5th intron that the present invention clones, wherein the r at 408bp is C
Or T sudden change, this sudden change causes Pst I-RFLP polymorphism.
Detailed description of the invention
The acquisition of embodiment 1 pig OLR1 genetic fragment and the foundation of pleiomorphism detecting method
The test pig kind of the present invention is external blood relationship pig variety Large White and Landrace；Place of china pig variety prunus mume (sieb.) sieb.et zucc. mountain pig
With peaceful pig, sample is real with molecular breeding Hubei Province emphasis by Animal Husbandry and Veterinary Inst., Hubei Academy of Agricultural Sciences's animal embryo
Test room to provide, for general types.
The extraction of pig genomic DNA:
Use the genomic DNA kit that Beijing hundred Tyke Bioisystech Co., Ltd produces (by this test kit description
Operate) extract, comprise the following steps that described:
(1) take the ear tissue of 20-50mg pig, put in the centrifuge tube of 2ml after being cut into pasty state with ophthalmologic operation, add
200 μ l lysate TL, with the piping and druming of rifle head uniformly.
(2) 20 μ l E.C. 3.4.21.64 (20mg/ml) are added, the most reverse fully mixing, 55 DEG C of water-baths digest overnight.
(3) add 200 μ l and combine liquid CB (this test kit carries), the most reverse mixing, place 10min for 70 DEG C.
(4) 100 μ l isopropanols are added after cooling, the most reverse fully mixing.
(5) drawing said mixture with the rifle head of 1mL, add in adsorption column AC, 10000rpm is centrifuged 30s, outwells collection
Waste liquid in pipe.
(6) adding 500uL mortifier and remove liquid IR (this test kit carries), 12000rpm is centrifuged 30s, abandons waste liquid.
(7) adding 700 μ l rinsing liquid WB (this test kit carries), 12000rpm is centrifuged 30s, outwells waste liquid.
(8) repetitive operation step 7.
(9) putting back in collecting pipe by adsorption column AC, 12000rpm is centrifuged 2min, removes rinsing liquid as far as possible, in order to avoid residual
Ethanol suppression downstream reaction.
(10) take out adsorption column AC, put in a clean centrifuge tube, add 50-100 μ l to the middle part of adsorbed film
Elution buffer EB, room temperature is placed 3-5min, 12000rpm and is centrifuged 1min, collected in centrifuge tube by solution.
(11) concentration and quality to the DNA extracted carry out saving backup at detection is placed on-20 DEG C.
Genome sequence according to OLR1 gene (the GenBank number of logging in: NC_010447.4, http: //
Www.ncbi.nlm.nih.gov/nuccore/NC_010447.4) designing following primer pair, its DNA sequence is as follows:
Forward primer: 5 ' CGGTGGAAGAGCATTTGT 3 ', corresponding sequence is the sequence shown in SEQ ID NO:4.
Reverse primer: 5 ' ACCTTGGGTCAGGCACTT 3 ', corresponding sequence is the sequence shown in SEQ ID NO:5.
Above-mentioned primer is utilized to carry out PCR amplification in Large White, Landrace, prunus mume (sieb.) sieb.et zucc. mountain pig and peaceful pig genomic DNA,
PCR reaction system 25 μ L, in system the concentration of each component be 100ng template DNA, 1 × Taq buffer 2.5mmol/L,
MgCl21.5mmol/L, dNTPs 2.5mmol/L, above-mentioned forward and reverse primer each 0.5mmol/L, 1U Taq DNA polymerase, PCR
Operation program as follows: 94 DEG C of 4min of denaturation；Then 94 DEG C of 30s, 53 DEG C of 40s, 72 DEG C of 50s, 35 circulations；Last 72 DEG C are continued
Renew and stretch 10min.
(biological purchased from the raw work in Shanghai through Gel Extraction Kit test kit to the PCR primer of aforementioned four pig variety
Engineering company limited, operates according to test kit description) purification, (the raw work biological engineering in Shanghai is entrusted in order-checking to cloning and sequencing
Technology Co., Ltd. is carried out), the size obtaining fragment is 583bp, its nucleotide sequence such as sequence table SEQ ID NO:2 (Mei Shan
Pig) and SEQ ID NO:3 (Large White) shown in.Carry out sequence alignment through ClusterW software, find wherein to be positioned at this fragment
At 408bp, (at the 5th intron 52bp), C/T variation causes the change of Pst I restriction enzyme site (CTGCA ↓ G) polymorphism.
Take 5.5 μ L PCR primer and add restricted enzyme 0.5 μ L (10U/ μ L), 10 × buffer 1 μ L, ddH2O 3μ
L, puts 37 DEG C of isothermal reactions overnight, by the agarose gel gel electrophoresis analysis of 1.5%, observes at gel imaging system and remembers
Record enzyme action genotyping result.This site of Pst I nonrecognition when base at 408bp is all C, is designated as CC type (583bp), works as sudden change
This site of Pst I enzyme identification when site base is all T, is designated as TT type (172bp+411bp), when C and T all in the presence of be designated as CT type
(583bp+172bp+411bp).Enzyme action genotyping result such as Fig. 3 of the PCR-Pst I-RFLP of pig OLR1 gene.
The polymorphism distribution detection checking in different swinerys of the molecular marker of embodiment 2 present invention
In two China's Native pig breeds (prunus mume (sieb.) sieb.et zucc. mountain pig, peaceful pig) and two external pig kind (Large White, Landrace) detections
Pig OLR1 gene the 5th intron district PCR-Pst I-RFLP polymorphism distribution frequency, testing result is as shown in table 1.Large White,
In Landrace, C gene frequency is higher, and in prunus mume (sieb.) sieb.et zucc. mountain pig, peaceful pig, TT gene frequency is preponderated, the allelic frequency of T
Higher, there is significant difference in the pig of external blood relationship and place of china pig variety.
Table 1 OLR1 gene the 5th intron district polymorphic distribution results in different cultivars of Pst I enzyme action
The molecular marker of embodiment 3 present invention clone and the association analysis of pig production character and application
The most relevant to hog on hook and Meat Quality in order to determine pig OLR1 gene pleiomorphism, select 259 DABAI × prunus mume (sieb.) sieb.et zucc.s
Mountain F2In generation, (hybridizing method was common method.Blood sample entrusts key lab of the pig genetics and breeding Ministry of Agriculture of Hua Zhong Agriculture University to gather)
Sources group is test material, and the Pst I-RFLP method using embodiment 1 to be set up carries out polymorphic detection, uses SAS system
Meter software (SAS Institute Inc, Version 8.0) GLM program carries out single labelling variance analysis, analyzes pig OLR1 gene
Pst I-RFLP different genotype and hog on hook and the dependency relation of Meat Quality, used model is:
Model Yijk=μ+Gi+Sj+Yk(+bijkXijk)+eijk
Yij is trait phenotypes value, and μ is meansigma methods, GiFor genotype effects, Sj、YkFor fixed effect, respectively sex, year
Degree effect, bijkFor slaughter weight or the regression coefficient of slaughter age, carcass trait with slaughter weight as covariant, Meat Quality
With slaughter age as covariant, eijkFor residual error effect.
As can be seen from Table 2: during genotype difference, fat thickness between skin rate, lean meat percentage, 6-7 lumbar vertebra, buttocks fat thickness, the back of the body are the longest
There is significant difference (P < 0.05) in the character such as flesh water content, fat thickness, average backfat thickness, longissimus dorsi muscle between shoulder fat thickness, Thoracolumbar disk
There is pole significant difference (P < 0.01) in the character such as intramuscular fat content.
Table 2 pig OLR1 gene PCR-Pst I-RFLP genotype and trunk and the statistical analysis table of Meat Quality
Table 1 illustrates: (1), above numerical value are least square mean value ± standard error；Containing same letter, colleague represents that difference is not
Significantly, different lowercase alphabets show that significant difference (P < 0.05), different capitalizations represent that difference is extremely notable (P < 0.01)；Gene
Type effect * represents that P ＜ 0.05, * * represent P ＜ 0.01.
(2), fat thickness, breast between skin rate, lean meat percentage, fat meat rate, thin fat meat ratio, shoulder fat thickness, 6-7 lumbar vertebra in These parameters
Between lumbar vertebra, fat thickness, buttocks fat thickness, average backfat thickness are carcass trait；Longissimus dorsi muscle water content and longissimus dorsi muscle intramuscular fat content
For Meat Quality.
Leading reference
Juwon A,Matthew K,Franklin,et al.Linoleic acid regulates the
expression of oxidized low density lipoprotein receptor(OLR1)in 3T3-
L1adipocytes via ERK and JNK pathways[J].FASEB Journal,2007,21(5):A455APR
Patricia C,Hong-Ping Guan,Michael L.PPARy regulates adipocyte
cholesterol metabolism via oxidized LDL receptorl.Clin Invest,2005,115(8):
2244-2256.
Li Tuo. the association analysis of Qinchuan Cattle OLR1 gene pleiomorphism and Meat Quality and interacting with bta-miR-370
Research [D]. Xibei Univ. of Agricultural & Forest Science & Technology, 2013.
Grandson is superfine. the fatty acid effect [J] to mice Preadipocyte In Vitro proliferation and differentiation and OLR1 gene transcript expression. and west
North agriculture and forestry science and technology college journal, 2009,37 (3): 1-6.
Claims (3)
1. hog on hook and a Meat Quality SNP genetic marker, its nucleotide sequence is as follows:
CGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCCTGGCCACTGATTACATG
CATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCAGCAGGCATGTCTATAAAACCATCCAGAAGATGGC
AAAGATCCCTTTGGTTCTGTGGCTTCATGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATG
TTCCTCCCTACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATCTCTGAGGA
AACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTTGTAAGTTTCCTATTCTTTTCCTAAT
CTGCTGGTGAAGTTACTGCCGCATTCRGCAGAGTGTCCTCTTCCTGCTGGAAAGAACCCTGGGAAATTTCTGAATCT
CTCTCCTCAGCCCTCTCATGCAGAGGCTTGCCCCTTGACTCATTCAGACCTTCTCTTGAGTTTTCCTTCTTACATTT
ATTACTGATTTGAAGCTTTAAGACAAGGCCAAGTGCCTGACCCAAGGT
The R at 408bp in above-mentioned sequence is C or T, causes Pst I-RFLP polymorphism.
2. the method screening hog on hook and Meat Quality genetic marker, it is characterised in that comprise the following steps:
From pig ear tissue, extract genomic DNA, design primer, its DNA sequence such as sequence table SEQ according to pig OLR1 gene order
Shown in ID NO:4 and 5, with forward primer CGGTGGAAGAGCATTTGT and reverse primer ACCTTGGGTCAGGCACTT at pig base
Because group DNA carries out PCR amplification, PCR primer purification and cloning and sequencing, it is thus achieved that the nucleoside as shown in sequence table SEQ ID NO:1
Acid sequence.
3. the application in hog on hook and Meat Quality detect of the genetic marker described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410344261.5A CN104087595B (en) | 2014-07-18 | Hog on hook and Meat Quality SNP genetic marker and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410344261.5A CN104087595B (en) | 2014-07-18 | Hog on hook and Meat Quality SNP genetic marker and application |
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| Publication Number | Publication Date |
|---|---|
| CN104087595A CN104087595A (en) | 2014-10-08 |
| CN104087595B true CN104087595B (en) | 2017-01-04 |
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