CN105063059A - Cloning and application of pork quality character related GADD45G gene molecule marker - Google Patents
Cloning and application of pork quality character related GADD45G gene molecule marker Download PDFInfo
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Abstract
The invention belongs to the technical field of livestock molecular marker biology and particularly relates to cloning and application of a pork quality character related GADD45G gene molecule marker. The molecule marker provided by the invention is obtained by cloning a GADD45G gene and the sequence of the GADD45G molecule marker is shown in SEQ ID No.1. For polymorphism of the GADD45G gene, a primer is designed by using a comparative genomic method according to the GADD45G gene sequence of a human body, the genome DNA of a pig is used as a template for amplification, in an amplified fragment, sequencing is used for screening SNP, PCR-RFLP is used for genetic typing, that A/G base mutation exists at 640th bp in the SEQ ID No.1 is found, PCR-RFLP-PstI polymorphism is caused and the molecule marker can be used for association analysis of pork quality characters. According to the invention, the novel molecular marker is provided for pork quality character marker assistant selection.
Description
Technical field
The invention belongs to domestic animal technical field of molecular biology, relate to a kind of nucleic acid molecule comprising pig gene nucleotide as shown in SEQIDNO:1.The invention still further relates to the mononucleotide polymorphism site in the molecular marker clone method of the GADD45G gene as shown in SEQIDNO:1 and polynucleotide sequence and its detection method.
Background technology
Pork is topmost meat on the nearly 1,400,000,000 population dining tables of China, and China's domestic pork output in 2014 is 5,671 ten thousand tons, and live pig is delivered for sale and reaches 73,510 ten thousand.State's Import stud pigs is very fast relative to the speed of growth place of china kind, but meat is bad is also undisputable fact, Meat problem is subject to extensive concern day by day, in many influence factors, intramuscular fat content (IntramuscularFatContent, IMF) be important influence factor, IMF is the leading indicator evaluating Meat quality, the tenderness of pork can be affected, local flavor, marble grain, succulence etc., the heritability of pig intramuscular fat content is about 0.6, within the specific limits, the higher meat of IMF is better, local variety pig intramuscular fat content will far above external pig kind, thus meat is excellent.
Retarded growth and DNA damage inducible protein 45 (growtharrestandDNAdamageinduciblegamma45, GADD45G) are the important signal transducers of a class, and participate in the several functions regulating cell.GADD45G gene family comprises Gadd45 α, and Gadd45 β, Gadd45 γ 3 genes, play biological action widely, comprises DNA reparation, programmed cell death and immunity moderation reaction.GADD45G genetically deficient can cause the propagation of human cell, causes tumour, and the propagation of this gene pairs cell plays restraining effect simultaneously, promotes apoptosis.And the overexpression of GADD45G gene can cause the division Forming ability of cell to decline, affect DNA and repair.Have report to show, GADD45G gene can regulate and control the speed of growth of mouse.The research in the demethylation of human cancerous disease and gene of GADD45G gene is a lot, but the research of this gene on pig is few.
GADD45G gene is positioned on No. 14 karyomit(e)s of pig, and the GADD45G assignment of genes gene mapping is in No. 2, No. 9 chromosome long arm No. 2 districts of people band No. 1 subzone to 2 subzone, and the GADD45G assignment of genes gene mapping is in No. 13 karyomit(e)s of mouse.GAdd45G gene is positioned at No. 4, No. 17 karyomit(e)s No. 1 district band of rat.
The present invention is by finding with the GADD45G gene cDNA comparison of people, the homology of this cDNA of pig and people reaches 82.9%, infer that this gene same loci of corresponding pig also identical mutation occurs by the SNP site of this gene of people found, the method of PCR – RFLP is used to find the new SNP site of pig GADD45G gene with this, detect in different pig kind further, and association analysis is carried out for finding new genetic marker to meat correlated character, for the qualification of GADD45G gene basic function and marker assisted selection are laid a good foundation.
At present, very few about the research of pig to pig GADD45G gene both at home and abroad, carrying out this gene molecule genetic marker research will for GADD45G gene molecule hereditary property, provides foundation in gene functional research such as intramuscular fat depositions.The regulation and control that the present invention is intended to for disclosing the Meat Qualities such as GADD45G gene pairs pig intramuscular fat content lay the foundation.
Summary of the invention
The object of the invention is to clone pig Meat Quality GADD45G gene cDNA molecule, find the mutational site of GADD45G gene and the detection method of gene pleiomorphism, for Meat marker-assisted breeding provides a kind of useful molecule marker.
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, the clone providing a kind of pig flesh characters to be correlated with GADD45G gene molecule marker and the application in pig marker assisted selection thereof, use molecule marker for pig flesh characters marker assisted selection provides.
In order to solve the problem, the technical solution adopted in the present invention is: a kind of GADD45G gene fragment relevant to pig flesh characters, and its sequence is as described in SEQIDNO:1; There is the base mutation of 1 A/G at the 640bp place of this SEQIDNO:1 sequence, causes PCR-RFLP-Pst I polymorphism.
The present invention with the GADD45G gene order of people (the GenBank number of including is for NM_003394.3) for Seed Sequences, BLASTN (BasicLocalAlignmentSearchToolNucleotide) is utilized in GenBank, special primer is designed with reference to the pig EST (ExpressionSequenceTags) of its homology more than 80%, clone pig GADD45G gene cDNA molecule, the DNA sequence dna of this GADD45G gene is as shown in SEQIDNO:1.GADD45G gene polynorphisms utilizes comparative genomics method according to the GADD45G gene order design primer of people, with the genomic dna of pig for template amplification, amplified fragments utilizes order-checking screening SNP, and utilize PCR-RFLP to carry out genotyping technique, find that there is the base mutation of an A/G at 640bp place, cause PCR-RFLP-PstI polymorphism, by analysis, there are 3 kinds of genotype in this A/G site, AA type (804bp), AG type (164bp+640bp+804bp) and GG type (164bp+640bp).
It is below the screening of GADD45G gene molecule marker.
DNA extraction: get fritter pig ear tissue and extract DNA, totally 5 kinds, wherein Large White 325, the black pig in the Land of Peach Blossoms 107, Shaziling pig 57, Ning Xiang Swine 67 and Daweizi pig 70, amount to 626.
Design of primers: utilize comparative genomics method to be Seed Sequences according to the GADD45G gene (the GenBank number of including is NM_003394.3) of people, the est sequence that BLAST screens the pig of homology more than 80% is made at GenBank, candidate SNP s site is filtered out after sequence alignment, with reference to GenBank primers, PCR-RFLP technology is used to carry out somatotype to this site.Forward primer is 5 '-AAACTTGCTGCTTGCG-3 ' (SEQIDNO:2); Reverse primer is 5 '-GGATGGAGGCGACACT-3 ' (SEQIDNO:3).
Pcr amplification condition: PCR reaction system (cumulative volume 20 μ L): 10 × Buffer2 μ L, 2mmol/LdNTPs1.6 μ L, 20mmol/LMgCl
21.6 μ L, Taq DNA polymerase (5U/ μ L) 0.4 μ L, DNA profiling (100ng/ μ L) 1 μ L, upstream and downstream primer (10pmol/ μ L) each 0.4 μ L and ddH
2o12.6 μ L.
PCR response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 50s, totally 35 circulations; 8min is extended, last 4 DEG C of preservations after 72 DEG C.
Get 10 μ LPCR amplified productions, after adding 2 μ L tetrabromophenol sulfonphthalein sample-loading buffer mixings, point sample, on the sepharose (containing 0.05%EB) of 2%, then puts 6 μ L100bpDNAMarkers as reference.5V/cm electrophoresis 0.5-1.0h.In gel imaging system, observe amplification after electrophoresis terminates and take pictures, result as shown in Figure 1.By the PCR primer Hou Songboshang biotech company order-checking after purifying.
The PstI enzyme of PCR primer is cut: in 10 μ LPCR products, add 0.8 μ L10 × restriction endonuclease damping fluid, 0.2 μ L restriction enzyme Pst I, 10 × Buffer2 μ L, then add the deionized water of 18 μ L, 37 DEG C of digestion 3-4h.The 2% agarose gel electrophoresis analysis of A/G site, 5V/cm electrophoresis 0.5h, observations under ultraviolet lamp is also taken pictures, enzyme cuts result as Fig. 1, amplified production checks order, and sequence is as shown in SEQIDNO:1, and am-plified fragments is between the 1st exon to exon 2,804bp, is PCR-RFLP-PstI molecule marker altogether.
SNP finds to set up with detection method: applicant devises amplification and comprises this SNP primer, and the sudden change in A/G site can adopt PstI to carry out enzyme and cut detection polymorphism by analysis.In the segment of the 804bp of amplification, there is the restriction enzyme site of 1 PstI, there are 3 kinds of genotype in the A/G site that electrophoresis detection result is presented at GADD45G gene, AA type (804bp), AG type (164bp+640bp+804bp) and GG type (164bp+640bp).
Hardy-Weinberg balances comptibility test: whether detect is equilibrium population χ2-test,chi-square test.Result show this SNP site except Daweizi pig group be not in Hardy-Weinberg equilibrium state (P<0.05), Ning Xiang Swine, Shaziling pig, Land of Peach Blossoms pig and Large White are all in Hardy-Weinberg equilibrium state (P>0.05).
The association analysis of genotype and production performance: to use in SAS9.0 software GLM (GeneralLinearModel) program by the association analysis carried out with drag between genotype and proterties:
Y
i＝μ+G
i+e
Y in formula
ifor proterties observed value, μ represents colony's average, G
irepresent genotype effects, e represents residual error effect.
Utilize the pig molecule mark of above-mentioned preparation to be applied to external pig kind and place of china kind, thus complete the present invention.
The present invention will establish solid basis for marker assisted selection (MAS), for improving Swine Production economic benefit, and guide the breeding practice of pig to provide theoretical foundation.
Accompanying drawing explanation
Fig. 1 is the Pst I restriction enzyme digestion and electrophoresis result in GADD45G of the present invention Gene A/G site.
In figure: swimming lane 1 and 6:AA genotype; Swimming lane 3 and 5:AG genotype; Swimming lane 4:GG fundamental mode; Swimming lane 2:DNAMarker (M).
Embodiment
Below in conjunction with specific embodiment, the present invention is explained further, but concrete enforcement does not do any restriction to the present invention.
Design of primers: utilize comparative genomics method to be Seed Sequences according to the GADD45G gene (the GenBank number of including is NM_003394.3) of people, the est sequence that BLAST screens the pig of homology more than 80% is made at GenBank, GADD45G gene candidate SNPs site is filtered out after sequence alignment, choose GADD45G gene candidate SNPs site design primer, the gene DNA extracted from pig ear tissue with this primer amplification.
Primer sequence: forward primer is 5 '-AAACTTGCTGCTTGCG-3 ' (SEQIDNO:2);
Reverse primer is 5 '-GGATGGAGGCGACACT-3 ' (SEQIDNO:3).
DNA extraction: the present invention adopts traditional phenol chloroform-primary isoamyl alcohol method, extract 5 different pig kinds (wherein Large White 325, the black pig in the Land of Peach Blossoms 107, Shaziling pig 57, Ning Xiang Swine 67 and Daweizi pig 70, amount to 626) DNA, be placed in-20 DEG C and save backup.Get fritter pig ear tissue and extract DNA.
Pcr amplification condition: PCR reaction system cumulative volume is 20 μ L:10 × Buffer2 μ L, 2mmol/LdNTPs1.6 μ L, 20mmol/LMgCl
21.6 μ L, Taq DNA polymerase (5U/ μ L) 0.4 μ L, DNA profiling (100ng/ μ L) 1 μ L, upstream and downstream primer (10pmol/ μ L) each 0.4 μ L and ddH
2o12.6 μ L.
PCR response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 50s, totally 35 circulations; 8min is extended, last 4 DEG C of preservations after 72 DEG C.
After reaction terminates, get 10 μ LPCR amplified productions, after adding 2 μ L tetrabromophenol sulfonphthalein sample-loading buffer mixings, point sample, on the sepharose (containing 0.05%EB) of 2%, then puts 6 μ L100bpDNAMarkers as reference.5V/cm electrophoresis 0.5-1.0h.In gel imaging system, observe amplification after electrophoresis terminates and take pictures, result as shown in Figure 1.By the PCR primer Hou Songboshang biotech company order-checking after purifying.
Polymorphic detection: the PCR primer through electrophoresis detection gets 10 μ L, add 0.8 μ L10 × restriction endonuclease damping fluid, 0.2 μ L restriction enzyme Pst I, 10 × Buffer2 μ L, add the deionized water of 18 μ L again, mixing, after centrifugal several minutes, under 37 DEG C of constant temperature, enzyme cuts 3-4h, carries out electrophoresis detection with 2% sepharose, 5V/cm electrophoresis 0.5h, takes pictures under ultraviolet lamp and judges genotype.Enzyme cuts result as Fig. 1, and amplified production checks order, and sequence is as shown in SEQIDNO:1, and am-plified fragments is between the 1st exon to exon 2, and 804bp, is PCR-RFLP-PstI molecule marker altogether.
Comprise this SNP primer by design amplification, the sudden change in A/G site adopts PstI to carry out enzyme and cuts detection polymorphism by analysis.In the fragment of the 804bp of amplification, there is the restriction enzyme site of 1 PstI, there are 3 kinds of genotype in the A/G site that electrophoresis detection result is presented at GADD45G gene, i.e. AA type (804bp), AG type (164bp+640bp+804bp) and GG type (164bp+640bp).
GADD45G Gene A/the gene frequency in G site, genotype frequency are detected, and analyzes its genetic polymorphism.
The distribution situation of PCR-RFLP-PstI polymorphism in each kind is as shown in table 1, as shown in Table 1, detected result finds, in 5 swinerys, all there is AA genotype, GG genotype and AG genotype, wherein in Land of Peach Blossoms swinery body, the allelic frequency of A is the highest, be 0.83, G gene frequency is 0.17, and the highest with AA type genotype frequency, is 0.68.In addition, except Shaziling pig and Large White colony, all the other colonies are all G allelotrope is advantage allelotrope.As known from Table 1, Ning Xiang Swine, Shaziling pig, Daweizi pig colony AG genotype frequency are higher, and Daweizi pig AG genotype frequency is up to 0.84.In a word, this gene is in the rich polymorphism of these 5 pig kinds.
The gene frequency in table 1 different varieties GADD45G Gene A/G site and genotype frequency distribution
Table 2GADD45G-552A → G site different genotype effect
This research application method of least squares corrects the thickness of backfat to the polymorphism of Pst I restriction enzyme site and carries out association analysis with reaching 100kg age in days and reach 100kg, in conjunction with see table 2, found that this site and reach 100kg age in days to there is significant correlation (P<0.05), its concrete influence shows as: A allelotrope has the effect of growth promoting effects, AA genotype reaches 100kg age in days (165.99 ± 10.32Day) significantly lower than AG genotype (172.48 ± 13.72Day), AG genotype reaches 100kg age in days (172.48 ± 13.72Day) significantly lower than GG genotype (183.03 ± 16.02Day), but with reach 100kg and correct the thickness of backfat and do not find significant correlation.
Claims (5)
1. a GADD45G gene fragment relevant to pig flesh characters, its sequence is as described in SEQIDNO:1; There is the base mutation of 1 A/G at the 640bp place of this SEQIDNO:1 sequence, causes PCR-RFLP-Pst I polymorphism.
2. test right requires the primer pair of the base mutation of the GADD45G gene fragment relevant to pig flesh characters described in 1, and it is characterized in that, the sequence of described primer pair is as follows:
Forward primer is 5 '-AAACTTGCTGCTTGCG-3 ',
Reverse primer is 5 '-GGATGGAGGCGACACT-3 '.
3. test right requires the method for the GADD45G gene fragment relevant to pig flesh characters described in 1, and it is characterized in that, the method carries out the detection in the A/G mutational site of GADD45G gene fragment according to following steps:
The primer pair described in claim 2 is utilized to carry out pcr amplification, PCR primer 2% agarose gel electrophoresis detects, utilize restriction enzyme PstI to carry out enzyme to PCR primer and cut qualification, finally detect the genotypic distribution of AA, AG and GG with 2% agarose gel electrophoresis.
4. the application of GADD45G gene fragment according to claim 1 in the molecular marker assisted selection relevant to pig flesh characters.
5. the application of primer pair according to claim 2 in the molecular marker assisted selection relevant to pig flesh characters.
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| CN106544412A (en) * | 2016-08-30 | 2017-03-29 | 华中农业大学 | A kind of molecular marker related to sperm motility of boars character and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106544412A (en) * | 2016-08-30 | 2017-03-29 | 华中农业大学 | A kind of molecular marker related to sperm motility of boars character and application |
| CN106544412B (en) * | 2016-08-30 | 2020-12-04 | 华中农业大学 | Molecular marker related to boar sperm motility character and application thereof |
| CN106544418A (en) * | 2016-10-14 | 2017-03-29 | 华中农业大学 | The clone of pig birth weight character correlation 1 gene molecule markers of HAI and its application |
| CN106544418B (en) * | 2016-10-14 | 2019-11-15 | 华中农业大学 | The clone of pig birth weight character correlation HAI-1 gene molecule marker and its application |
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Application publication date: 20151118 |