CN104480216A - Molecular marker related to pH (Potential of Hydrogen) value character of pig muscle - Google Patents
Molecular marker related to pH (Potential of Hydrogen) value character of pig muscle Download PDFInfo
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- CN104480216A CN104480216A CN201410853566.9A CN201410853566A CN104480216A CN 104480216 A CN104480216 A CN 104480216A CN 201410853566 A CN201410853566 A CN 201410853566A CN 104480216 A CN104480216 A CN 104480216A
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Abstract
The invention belongs to the technical field of preparation of livestock molecular markers, and particularly relates to an SNP (Single Nucleotide Polymorphism) molecular marker related to a pH (Potential of Hydrogen) value character of pig muscle. The molecular marker is obtained by LDHA (Lactate DehydrogenaseHydroxyacyl) gene cloning and has a nucleotide sequence as shown in Figure 4.In the sequence as shown in Figure 4, the 495th position has allele G or A base substitution, and the 635th position of the sequence has allele C or T base substitution. The new molecular marker is provided for assistant selection of pig markers.
Description
Technical field
The invention belongs to technical field of livestock molecular marker preparation, be specifically related to the application of a molecule marker relevant to pork pH value proterties.
Background technology
The main source of China's meat product is pork, and along with the development of pig-breeding industry, its output can meet the demand of people, so become the hot issue of pig breeding research to the improvement of meat quality.The improvement of meat can from heredity, and the many-side such as nutrition and diseases prevention and treatment is considered, wherein, inherited genetic factors is immanent cause.Carry out breed breeding by molecular genetic means, from source, effectively can improve meat quality.
The evaluation index of meat comprise yellowish pink, pH value, Coefficient shrinkage, drip loss, marble grain etc. (Cui Min. brief talk the factor [J] affecting meat quality. Chinese Pigs industry, 2014,9 (1): 57-60.), and pH value and yellowish pink and Coefficient shrinkage and ATP, glycogen and lactic acid content have close relationship, meanwhile, according to the pH value of butchering when rear glycogenolysis stops, PSE meat can be distinguished, normal meat and DFD meat, so pH value is the very important index judging meat.(Chen Song, Feng Yuerong, Cao refined duckweed .pH value is on the impact [J] of slaughter meat quality. meat industry, 2009 (6): 21-23.) but, the mensuration of pH value after butchering, can only significantly limit the development of seed selection work.So, use Molecular tools, select the auxiliary seed selection of candidate gene affecting pH value, become the important approach improving meat.
LDHA gene is the member in lactic dehydrogenase enzyme family, and it mainly expresses in skeletal muscle.Serum lactic dehydrogenase is the important enzyme of anaerobic glycolysis final step in organism, is that coenzyme catalysis Lactic acid and Pyruvic acid transforms mutually with NADH.Pyruvic acid changes into lactic acid under the catalysis of serum lactic dehydrogenase, and reaction discharges a small amount of energy simultaneously and utilizes for body.Therefore, the activity of serum lactic dehydrogenase will directly affect glucolytic speed, thus cause the difference of pH value.So the present invention selects LDHA to be candidate gene, by the amplification to population sample, utilize sequenator to check order to DNA fragmentation, from order-checking peak figure, directly read the genotype in single nucleotide polymorphism (SNP) site, thus filter out the molecule marker relevant to pH value proterties.
In the present invention, increased in the intron region of LDHA gene, find two new SNP site, the perfect further SNP information storage of pig, simultaneously significant to the improvement of meat.
Summary of the invention
The object of the present invention is to provide single nucleotide polymorphism (SNP) molecule marker relevant to pig muscle pH value proterties.Another object of the present invention is to provide the application of the single nucleotide polymorphism molecule marker relevant to pig muscle pH value proterties.
The present invention is achieved through the following technical solutions:
Applicant clone from the pig LDHA gene of report obtains a SNP marker relevant to pig muscle pH value proterties, and the nucleotide sequence of this molecule marker is as follows:
TAATGACTCATCTAGTATCGCTGCCTAAATATTTTCTTCATAGTGGATATCTTGACCTATGTGGCTTGGAAGATAAGTGGCTTTCCCAAAAACCGTGTTATTGGAAGTGGTTGCAATTTGGATTCAGCCCGGTTCCGTTACCTAATGGGGGAAAGGCTGGGAGTTCACCCCCTAAGCTGTCATGGGTGGATCCTTGGGGAGCATGGAGACTCTAGTGGTAAGTATAACTTATTTTCTCTGTGAAAGAAAGATGGTGTAAAAATTGATAGGAGTATCATTACTAAATCAGAGCCTAAATCAGATGTCCATTCTTTAGGGTAGAATGATTTCCAGGAATCTTTTATTTTGGTATAATTATATCCTAAGAATTGTAAAGATTTTTAGCATTCTGTATGACATTTTCTGGTATAAGGGATATGGGATGAAATTTCTTTCTTACAGTAGACAGACATATAATAAGATGATAGCTCTTAGGCTACTTCTTTTAATATCACRGCTGTTTATACTCAAACTGTTTAGCCCTGAAAGAGTCAGCCAAGCCAAATGGAGGGTATGAGAACAAATATTATTATCACAGTTGAAAGATGTTATCATTTGTGCAGAGTCCTCAGTTCCTTATGCCAAGAAAATTCCTRTTATGCTCTCATCCTGTTTCATCTCAATAGGTTAGGAAAATAGGTTAAAATAAGAGTATAACTGATAGTATAAAACTGATAGTTTTACATAATCTAATCATTGAAAAGTTAAAAGTTGGGGTATTGGTAGTCTTGACCATCTCTACCAAAAGGGGGTAATCTTCCACAGATAAGACAATTTTAGAAGAATAGGTCAGGATGACTAGTTTGCTGGCTGGCGCATGAAAATAAATATATTCTATACTATTTCTTTTAGTGCCTGTGTGGAGCGGAGTAAATGTTGCTGGTGTCTCCCTGAAGAATCTGCACCCTGAATTAGGCACTGATGCAGATAAGGAACACTGGAAAGCAGTTCACAAACAGGTGGTGGACAGGTAATAGATCTCA
Above-mentioned sequence is 1022bp altogether, and the R in the 495th is G or A; R in 635th is C or T.
Applicant devises the primer pair (this primer pair is also the primer pair of molecule marker of the present invention of increasing) of the above-mentioned LDHA gene fragment that increases, and its DNA sequence dna is as follows:
Forward primer: 5'-TAATGACTCATCTAGTATCGCTGCC-3',
Reverse primer: 5'-TGAGATCTATTACCTGTCCACCACC-3'.
Implementing concrete grammar of the present invention is: extract pig genomic dna, designs primer according to the pig LDHA gene sequence information (sequence of its nucleotide sequence as shown in sequence table SEQ ID NO:1 or accompanying drawing 4) that NCBI announces.Carry out pcr amplification with these primer pairs, obtain the fragment of 1022bp, its electrophorogram as shown in Figure 3.PCR primer is delivered to the order-checking of order-checking company of Wuhan Qing Ke biotech firm, analyze and obtain SNP, and carry out the application of the association analysis between genotype and pig muscle pH value proterties, for pig marker assisted selection provides new molecule marker.
More detailed technical scheme is see " embodiment ".
Accompanying drawing explanation
Sequence table SEQ ID NO:1 is the partial nucleotide sequence in the present invention's pig LDHA gene complete sequence of cloning, and this sequence is the sequence after allelic mutation, and its base of 495 sports A by G; 635th bit base sports T by C.
Fig. 1: be main technical flows schematic diagram of the present invention.
Fig. 2: be LDHA Gene Partial sequence PCR primer electrophorogram.Description of reference numerals: in Fig. 2: M swimming lane is DNA molecular amount mark (DL2000); Amplified production is 1022bp.
Fig. 3: be LDHA Gene Partial sequence result figure.
Fig. 4: the nucleotide sequence being molecule marker prepared by the present invention, SNP site is the 495th and the 635th, and the R in the 495th in above-mentioned sequence is G or A; R in 635th is C or T.
Embodiment
Embodiment 1
(1) pig extracting genome DNA
The pig variety extracting pig genomic dna in the present invention is Large White (Hubei gold woods original seed herding company limited).The genomic DNA kit (TIANamp Genomic DNA Kit) that swinery body DNA sample all adopts day root biochemistry (Beijing) Technology Co., Ltd. to produce extracts, and concrete operation step is as follows:
1, the ear sample of Large White is put into the EP pipe of 2mL, after being cut into pasty state with the ophthalmologic operation of alcohol swab wiped clean, add 200 μ L damping fluid GA (this test kit carries).
2, add 20 μ L Proteinase K Solution (this test kit carries), mixing is placed in 56 DEG C of water-baths to digest spends the night.
3, add 200 μ L damping fluid GB, fully put upside down mixing, place 10min for 70 DEG C, solution strain is limpid, of short duration centrifugal, removes the globule of inside pipe wall.
4, add 200 μ L dehydrated alcohols, fully, now may there is flocks in vibration mixing 15s, of short duration centrifugal after, remove the globule of inside pipe wall.
5, all join in an adsorption column CB3 (adsorption column puts into collection tube) by previous step gained solution and flocks, the centrifugal 30s of 12,000rpm, outwells waste liquid, is put back in collection tube by adsorption column CB3.
6, in adsorption column CB3, add 500 μ L damping fluid GD (this test kit carries), the centrifugal 30s of 12,000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
7, in adsorption column CB3, add 600 μ L rinsing liquid PW (this test kit carries), the centrifugal 30s of 12,000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
8, repetitive operation step 7.
9, adsorption column CB3 is proceeded in a clean centrifuge tube, the unsettled dropping in middle part to adsorption column 50-200 μ L elution buffer TE (this test kit carries), room temperature places 2-5min, and 12, the centrifugal 2min of 000rpm, by solution collection in centrifuge tube.
10, after integrity qualification and Concentration Testing being carried out to the DNA extracted, be placed in-80 DEG C and save backup.
(2) pcr amplification and order-checking
Download the sequence of LDHA gene (number of including of GenBank: NC_010444.3) from NCBI, design primer amplification, the DNA sequence dna of primer is as follows:
Forward primer: 5'-TAATGACTCATCTAGTATCGCTGCC-3',
Reverse primer: 5'-TGAGATCTATTACCTGTCCACCACC-3'.
PCR reaction system: reaction is totally 10 μ L, comprises DNA profiling 40ng, 2 × PCR Mix 5 μ L, forward primer 3pmoL, reverse primer 3pmoL, ddH
2o 3.4 μ L.
PCR response procedures: 95 DEG C of denaturation 5min, 94 DEG C of 30s, 59.9 DEG C of 30s, 72 DEG C of 50s, circulate 35 times, and 72 DEG C extend 5min, 15 DEG C of stopped reaction.
PCR primer detects through 2.0% agarose gel electrophoresis, and product is 1002bp.
PCR primer through qualification delivers to the order-checking of Wuhan Qing Ke biotech firm, and sequencing analysis result as shown in Figure 4.
(3) data relation analysis
The present invention measures 100kg body weight age in days, 100kg body weight live body Large White (sample amounts to 235 Large Whites) thickness of backfat and eye muscle area, and in conjunction with Full-automatic feed transformation efficiency Analytical system, calculate average daily gain (Average daily gain, ADG), feed conversion rate (Feed conversion rate, and residue food consumption (Residual feed intake FCR), RFI) these three indexs, and the group structure of center Large White group is measured according to Ministry of Agriculture boar, adopt general linear model (GLM) to single SNP site and swinery sports school just after each trait data carry out association analysis, illustrate the genotype affecting these proterties, statistical model is as follows:
Y
ij＝μ+genotype
i+ε
ij
Wherein Y
ijfor phenotypic number; μ is colony's average; Genotype
ifor genotype effects; ε
ijfor random error; Separate and submit to N (0, σ
2).
Table 1 LDHA gene SNP site the 495th genotype and the association analysis of butchering latter 24 hours pH
Table 2LDHA gene SNP site the 635th genotype and the association analysis of butchering latter 24 hours pH
The genotypic results of 2 SNP site in LDHA gene and phenotypic character are carried out association analysis, and the GG genotype individuals pH average of SNP site the 495th R=G or A is significantly lower than other genotype individuals (P < 0.05); The TC genotype individuals pH average of the 635th R=C or T is higher than other genotype individuals (P < 0.05).
Claims (4)
1. the SNP marker relevant to Large White muscle pH value proterties, it is characterized in that, the nucleotide sequence of described molecule marker is as follows:
TAATGACTCATCTAGTATCGCTGCCTAAATATTTTCTTCATAGTGGATATCTTGACCTATGTGGCTTGGAAGATAAGTGGCTTTCCCAAAAACCGTGTTATTGGAAGTGGTTGCAATTTGGATTCAGCCCGGTTCCGTTACCTAATGGGGGAAAGGCTGGGAGTTCACCCCCTAAGCTGTCATGGGTGGATCCTTGGGGAGCATGGAGACTCTAGTGGTAAGTATAACTTATTTTCTCTGTGAAAGAAAGATGGTGTAAAAATTGATAGGAGTATCATTACTAAATCAGAGCCTAAATCAGATGTCCATTCTTTAGGGTAGAATGATTTCCAGGAATCTTTTATTTTGGTATAATTATATCCTAAGAATTGTAAAGATTTTTAGCATTCTGTATGACATTTTCTGGTATAAGGGATATGGGATGAAATTTCTTTCTTACAGTAGACAGACATATAATAAGATGATAGCTCTTAGGCTACTTCTTTTAATATCACRGCTGTTTATACTCAAACTGTTTAGCCCTGAAAGAGTCAGCCAAGCCAAATGGAGGGTATGAGAACAAATATTATTATCACAGTTGAAAGATGTTATCATTTGTGCAGAGTCCTCAGTTCCTTATGCCAAGAAAATTCCTRTTATGCTCTCATCCTGTTTCATCTCAATAGGTTAGGAAAATAGGTTAAAATAAGAGTATAACTGATAGTATAAAACTGATAGTTTTACATAATCTAATCATTGAAAAGTTAAAAGTTGGGGTATTGGTAGTCTTGACCATCTCTACCAAAAGGGGGTAATCTTCCACAGATAAGACAATTTTAGAAGAATAGGTCAGGATGACTAGTTTGCTGGCTGGCGCATGAAAATAAATATATTCTATACTATTTCTTTTAGTGCCTGTGTGGAGCGGAGTAAATGTTGCTGGTGTCTCCCTGAAGAATCTGCACCCTGAATTAGGCACTGATGCAGATAAGGAACACTGGAAAGCAGTTCACAAACAGGTGGTGGACAGGTAATAGATCTCA
Above-mentioned sequence is 1022bp altogether, and the R in the 495th is G or A; R in 635th is C or T.
2. the primer pair of amplification molecule marker as claimed in claim 1, its DNA sequence dna is as follows:
Forward primer: 5'-TAATGACTCATCTAGTATCGCTGCC-3',
Reverse primer: 5'-TGAGATCTATTACCTGTCCACCACC-3'.
3. the application of molecule marker according to claim 1 in Large White muscle pH value proterties detects.
4. the application of primer pair according to claim 2 in Large White muscle pH value proterties detects.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105256045A (en) * | 2015-11-04 | 2016-01-20 | 中国农业科学院北京畜牧兽医研究所 | Method for identifying pH value of longissimus muscle of back of pig butchered for 24 hours and specialized primer pair thereof |
| CN110468217A (en) * | 2019-09-11 | 2019-11-19 | 湖南省畜牧兽医研究所 | SNP marker relevant to pig muscle pH and drip loss character and its application |
-
2014
- 2014-12-31 CN CN201410853566.9A patent/CN104480216A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 邱海芳: "猪SSC2背膘厚日增重QTL区基因的连锁定位和TEF-1靶基因的筛选", 《万方数据》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105256045A (en) * | 2015-11-04 | 2016-01-20 | 中国农业科学院北京畜牧兽医研究所 | Method for identifying pH value of longissimus muscle of back of pig butchered for 24 hours and specialized primer pair thereof |
| CN105256045B (en) * | 2015-11-04 | 2018-05-25 | 中国农业科学院北京畜牧兽医研究所 | It is a kind of identify pig kill after 24 it is small when longissimus dorsi muscle pH value size method and its special primer pair |
| CN110468217A (en) * | 2019-09-11 | 2019-11-19 | 湖南省畜牧兽医研究所 | SNP marker relevant to pig muscle pH and drip loss character and its application |
| CN110468217B (en) * | 2019-09-11 | 2021-03-23 | 湖南省畜牧兽医研究所 | SNP molecular marker related to pH and drip loss traits of pig muscle and application thereof |
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