CN101824416A - Pork quality trait related gene DGAT1 and application thereof in porcine marker-assisted selection - Google Patents
Pork quality trait related gene DGAT1 and application thereof in porcine marker-assisted selection Download PDFInfo
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- CN101824416A CN101824416A CN 201010164689 CN201010164689A CN101824416A CN 101824416 A CN101824416 A CN 101824416A CN 201010164689 CN201010164689 CN 201010164689 CN 201010164689 A CN201010164689 A CN 201010164689A CN 101824416 A CN101824416 A CN 101824416A
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Abstract
The invention relates to a pork quality trait related gene DGAT1 and an application thereof in porcine marker-assisted selection; a molecular marker which is applied to the porcine marker-assisted selection and relates to pork quality traits is measured; the molecular marker is obtained by cloning a DGAT1 gene, and has the nucleotide sequence shown in a sequence table SEQ ID No: 1; and the overall length of the sequence is 327bp, and an A-G basic group mutation exists at the position of 265bp of the sequence, so that the PCR-RFLP-Ava II polymorphism is caused. The invention provides a new molecular marker for the pork quality trait marker-assisted selection.
Description
Technical field:
The invention belongs to technical field of molecular biology, relate to a kind of existence of the molecule marker relevant with pig flesh characters, specifically, relate to a kind of DGAT1 gene as SEQ ID NO:1 shown in relevant with pig flesh characters, further say, relate to detect the existence of the mononucleotide polymorphism site in the polynucleotide sequence of this gene.
Background technology:
Pork is the main source of China urban and rural residents animal protein, along with the increase of people to the meat requirement, to the also raising relatively of requirement of meat quality.And this mainly is by Gene Handling, and has the key-gene effect.The development of Protocols in Molecular Biology, make people can seek the key-gene or the molecule marker closely linked of control meat proterties at dna level with it, in breeding process, be used for marker assisted selection, to improve the selection process, improve pig flesh characters better, satisfy people's needs, to obtain bigger economic benefit.
DGAT is a kind of glyceroyl transferring enzyme (DiacylgycerolAcyltransferase; DGAT); this enzyme and metabolism of fat, lipid deposit much relations in tissue; its main mechanism is to make diacylglycerol (diac ylgycerol; DAG) add fatty acid acyl form triacylglycerol (triacylgycerol, TAG).Wherein DAG is monoacylglycerol (monoacylgycerol; MAG) at acyl-CoA monoacylglycerol acyltransferase (Acyl CoA:monoacylgycerola cyltransferases; MGAT) add under the effect that fatty acid acyl generates, TAG is that DAG adds that under the effect of DGAT enzyme fatty acid acyl generates.
DGAT is a kind of glyceroyl transferring enzyme; this enzyme and metabolism of fat, lipid deposit much relations in tissue; its main mechanism is to make diacylglycerol add that fatty acid acyl forms triacylglycerol; so the DGAT genes involved may produce material impact to intramuscular fat content; thereby influence meat, this gene is most probably for influencing the candidate gene of meat proterties intramuscular fat content.
But, at present both at home and abroad seldom about the correlative study of pig DGAT1 gene.
Summary of the invention:
Technical problem to be solved by this invention is: at above-mentioned the deficiencies in the prior art, provide a kind of pork quality trait related gene DGAT 1 and the application in the pig marker assisted selection thereof, provide the molecule marker of usefulness for the pig marker assisted selection.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of pork quality trait related gene DGAT 1, its dna sequence dna is shown in sequence table SEQ ID NO:1.There is the base mutation of 1 A → G at the 265bp place of described SEQ ID NO:1, causes the PCR-RFLP-AvaII polymorphism.
The polymorphism of above-mentioned DGAT1 gene is to utilize the comparative genomics method, the 2854bp that selects people DGAT1 gene is between the 3180bp, it is the segment of the 1st intron to the 2 introns, the consequence devised primer in the DGAT1 gene candidate SNP s site that filters out according to sequence alignment, genomic dna with pig is that template is carried out pcr amplification, utilize restriction enzyme A vaII that the PCR product is carried out enzyme and cut evaluation, whether the 265bp place of the DGAT1 gene fragment that obtains the clone exists the sudden change of A → G.
Described clone DGAT1 gene the primer is:
Forward primer: 5 '-ATGAGGTTCTCCGCTGCCCGCT-3 '
Reverse primer: 5 '-TTGTACCCCTCTGCAGGCATC-3 '.
Described detection DGAT1 transgenation the primer is:
Forward primer is 5 '-CCTCACTITCCCGCATCTTC-3 ',
Reverse primer is 5 '-GCTCCCTATTCTTCCTTTCCAG-3 '.
The application of above-mentioned pork quality trait related gene DGAT 1 in the pig molecule mark assisted Selection.
Utilize the molecule marker relevant of above-mentioned preparation can carry out the application of association analysis to external pig kind and place of china kind with pig flesh characters.
The pig DGAT1 assignment of genes gene mapping is in No. 4 karyomit(e)s (SSC4), and with the QTL close linkage that influences the thickness of backfat and intramuscular fat content, the homology of the amino acid of this genes encoding and mouse, people, ox is respectively 85%, 86%, 92%.The present invention selects the candidate gene of pig DGAT1 gene as pig flesh characters.Utilize the DGAT1 gene conservative district design primer of comparative genomics method according to the people, genomic dna with pig is a template amplification, amplified fragments utilizes SSCP technology and sequencing technologies to find SNP, and utilize PCR-RFLP to carry out gene type, utilize SAS software GLM (general linear model) to analyze the relational degree of SNP and meat proterties again.
SNP finds to set up with detection method: the applicant has designed amplification and has comprised this SNP primer, can adopt AvaII to carry out enzyme by analysis and cut the detection polymorphism.On the segment of the 327bp that increases, the restriction enzyme site that has 1 AvaII, the A265G site that electrophoresis detection result is presented at the DGAT1 gene fragment exists 3 kinds of genotype, AA type (64bp, 263bp), AG type (64bp, 263bp, 327bp) and GG genotype (327bp).Wherein, can't detect clearly with agarose gel electrophoresis because the 64bp segment is too little.The restriction enzyme digestion and electrophoresis result has confirmed the sudden change in the existence of 265bp place.
Association results between genotype-meat proterties: utilize general linear model in the SAS software (GeneralLinear Model, GLM) program is carried out association analysis to genotype and proterties, model the following is Y
Ijkn=μ+h
i+ l
j+ g
k+ ε
Ijkn, Y
IjknBe the proterties phenotypic number, μ is a population mean, h
iBe pasture effect, 1
jBe age effect, g
kBe the effect of DGAT1 genotype different loci to proterties, ε
IjknBe the random error effect, and Normal Distribution (0, σ
2).Analysis revealed DGAT1 gene A 265G site SNP has remarkably influenced (P＜0.05) to storage loss etc., and this site has no significant effect other meat proterties such as the thickness of backfat.
The present invention will be the molecule marker of pig flesh characters, (MAS) establishes solid basis for marker assisted selection, further illustrates the molecular mechanism of meat proterties, will provide theoretical foundation for improving meat quality, can improve the Swine Production economic benefit, and guide the breeding practice of pig.
Description of drawings:
Fig. 1 is the sequence of DGAT1 gene fragment of the present invention, and the variant sites at 265bp place marks expression with ().
Fig. 2 is the electrophoresis result figure of the pcr amplification product of DGAT1 gene fragment of the present invention.
Among the figure: M:100bp DNA Ladder Marker.
Fig. 3 is AvaII (Eco47I) the restriction enzyme digestion and electrophoresis figure as a result in DGAT1 gene A 265G of the present invention site.
Among the figure: swimming lane 2:AG genotype; Swimming lane 1,3:GG genotype; Swimming lane 4,5:AA genotype; M:100bp DNA Ladder Marker.
Embodiment:
Below in conjunction with specific embodiment the present invention is done to explain further, but concrete enforcement do not done any qualification to the present invention.
The AvaII polymorphism of PCR-RFLP technology for detection DGAT1 gene:
Design of primers
Utilize the comparative genomics method according to the 1st intron to the 2 introns of people's DGAT1 gene (the GenBank number of including is AY116586), make BLAST with this gene at GenBank, filter out DGAT1 gene candidate SNP s site behind the sequence alignment, choose the 3122bp candidate SNP s site of DGAT1 gene, with the AY116586.seq sequence is standard, the design Auele Specific Primer increases from the genomic dna of pig ear tissue extraction with these primers.
Primer is: M-F:5 '-ATGAGGTTCTCCGCTGCCCGCT-3 '
M-R：5′-TTGTACCCCTCTGCAGGCATC-3′
The PCR condition:
PCR reaction system (cumulative volume 20 μ L): 10 * buffer, 2 μ L, 2mmol/L dNTPs 1.6 μ L, 20mmol/L MgCl
21.6 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.4 μ L, dna profiling (100ng/ μ L) 1 μ L, each 0.4 μ L of upstream and downstream primer (10pmol/ μ L), ddH2O 12.6 μ L.
Response procedures: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 40s, 60 ℃ of annealing 40s, 72 ℃ are extended 40s, totally 35 circulations; Extend 8min after 72 ℃, last 4 ℃ of preservations.
Get 10 μ L pcr amplification products, add 2 μ L tetrabromophenol sulfonphthalein sample-loading buffer mixings after, point sample is put 6 μ L 50bp or 100bp DNA Markers then as reference on 2% sepharose (containing 0.05%EB).5V/cm electrophoresis 0.5～1.0h.Electrophoresis end back is observed amplification and is taken pictures in gel imaging system, the result as shown in Figure 2.
Add 0.8ul 10 * restriction endonuclease damping fluid, 0.2ul restriction enzyme and 1ul distilled water in 10ul PCR product, cumulative volume is 12ul, 37 ℃ of digestion 16h.With 1% agarose gel electrophoresis analysis, 5V/cm voltage electrophoresis 0.5h, observations and taking pictures under the ultraviolet lamp, result exist AA, AG and GG genotype after showing the AvaII process as shown in Figure 3.
The result: the order-checking of the purified rear clone of pcr amplification product, sequencing result show the amplification segment be the 2854bp of pig DGAT1 gene between the 3180bp, be the segment of the 1st intron to the 2 introns, 327bp altogether, sequence is seen Fig. 1 shown in SEQ ID NO:1.Sequencing result shows and has AA, AG and GG genotype, has the sudden change of A → G at the 265bp place of DGAT1 gene, has further confirmed to have polymorphism in the PCR product.
For further this molecular genetic marker of proof is stable molecule marker, but not the transgenation that exists in the colony, the present invention has analyzed the distribution situation of PCR-RFLP-AvaII molecule marker polymorphism in each kind.
The present invention is a test materials with 5 local pig breeds (Ningxiang pig, the black pig in the Land of Peach Blossoms, big defensive wall pig, sand mountain range pig and Wuzhi Mountain pig) and 3 external pig kinds (duroc, Large White, landrace), adopt the PCR-RFLP method that gene frequency, the genotype frequency in DGAT1 gene A 265G site are detected, and analyze its genetic construction, tentatively inquire into the dependency of this gene and meat proterties.
The distribution situation of PCR-RFLP-AvaII polymorphism in each kind is as shown in table 1 below, as can be seen from Table 1, the G gene is an advantage allelotrope in Ningxiang pig, big defensive wall pig, sand mountain range pig, Land of Peach Blossoms pig, these 5 local variety of Wuzhi Mountain pig, sand mountain range pig G gene frequency the highest (0.8188) wherein, and the A gene is an advantage allelotrope in 3 adventive durocs, landrace, Large White, wherein duroc A gene frequency the highest (0.7911); From the genotype distribution frequency, the GG type distributes in native breed and preponderates, and the AA type is a preponderant genotype in adventive.
The Hardy-Weinberg balance check is the result show, this molecule marker all is in equilibrium state 8 kinds, shows that this molecule marker is stable molecular genetic marker.
The Hardy-Weinberg balance check result in table 1 different varieties DGAT1 gene 265 sites
The application of molecule marker of the present invention in the association analysis of pig flesh characters mark property
Utilization SAS8.02 (Statistical Analysis System) statistical software carries out association analysis to DGAT1 genotype and pig flesh characters.Model is as follows:
Y
ijkn＝μ+h
i+l
j+g
k+ε
ijkn
Y
IjknBe the proterties phenotypic number, μ is a population mean, h
iBe pasture effect, l
jBe age effect, g
kBe the effect of DGAT1 genotype different loci to proterties, ε
IjknBe the random error effect, and Normal Distribution (0, σ
2).The result is as shown in table 2 below:
Table 2 pig DGAT1 gene A 265G site and meat proterties association analysis table
Annotate: go up in the table each row mark same letter and represent that difference is not remarkable, the expression significant differences of the different letters of mark (down with)
By last table 2 as can be known, in duroc, AA genotype lean ratio has improved 4.66% (p＜0.05) than GG genotype lean ratio, and fat ratio, marble grain then significantly are lower than the GG genotype, have reduced by 29.55% and 14.69% (p＜0.05) respectively; It is all not remarkable that AA, AG and GG genotype are surveyed between the average thickness of backfat, percentage of water loss and the storage loss difference.In Large White, it is remarkable that AA and AG genotype are surveyed the average thickness of backfat and lean ratio and GG genotypic difference, the AA type is surveyed the average thickness of backfat and has been reduced by 19.18% (p＜0.05) than GG type, AA type lean ratio is higher than GG type 3.28% (p＜0.05), but difference is not remarkable between AA and the AG genotype, and AA type storage loss are lower than GG type 32.55% (p＜0.05); Difference is all not remarkable between AA, AG and GG genotype fat ratio, marble grain and the percentage of water loss.In landrace, AA genotype fat ratio is lower than GG genotype 26.77% (p＜0.05), and the difference on other proterties does not all reach conspicuous level.
Sequence table
＜110〉Agricultural University Of Hunan
＜120〉pork quality trait related gene DGAT 1 and the application in the pig marker assisted selection thereof
<130>PHW10366
<160>1
<170>PatentIn?version?3.3
<210>1
<211>327
<212>DNA
＜213〉pig
<400>1
atgaggttct?ccgctgcccg?ctgcccaggg?ctccgctggt?gcctgctcac?cagggtgcct 60
gtctcctgct?ttccaggtgc?caccgcctgc?aggattcttt?gttcagttca?gacagtggtt 120
tcagcaacta?ccgtggcatc?ctgaattggt?gtgtggtcat?gctggtgagt?agggtggcat 180
cttggggcag?gtggggccca?ggctggtggg?gagagaggtc?acggagacag?tccctgcagt 240
ggccagacac?cggagctccc?gaggaccaca?cgcttaaccc?ttgtccctgt?tgagttgcaa 300
gagatgcctg?cagaggggta?caactag 327
Claims (6)
1. pork quality trait related gene DGAT 1, its dna sequence dna is shown in sequence table SEQ ID NO:1.
2. pork quality trait related gene DGAT 1 as claimed in claim 1 is characterized in that: there is the base mutation of 1 A → G at the 265bp place of described SEQID NO:1, causes the PCR-RFLP-AvaII polymorphism.
3. pork quality trait related gene DGAT 1 as claimed in claim 2, it is characterized in that: the polymorphism of described DGAT1 gene is to utilize the comparative genomics method, the 2854bp that selects people DGAT1 gene is between the 3180bp, it is the segment of the 1st intron to the 2 introns, the consequence devised primer in the DGAT1 gene candidate SNP s site that filters out according to sequence alignment, genomic dna with pig is that template is carried out pcr amplification, utilize restriction enzyme A vaII that the PCR product is carried out enzyme and cut evaluation, whether the 265bp place of detecting the DGAT1 gene fragment that obtains the clone exists the sudden change of A → G.
4. pork quality trait related gene DGAT 1 as claimed in claim 3 is characterized in that: described clone DGAT1 gene the primer is:
Forward primer: 5 '-ATGAGGTTCTCCGCTGCCCGCT-3 '
Reverse primer: 5 '-TTGTACCCCTCTGCAGGCATC-3 '.
5. pork quality trait related gene DGAT 1 as claimed in claim 3 is characterized in that: described detection DGAT1 transgenation the primer is:
Forward primer is 5 '-CCTCACTITCCCGCATCTTC-3 ',
Reverse primer is 5 '-GCTCCCTATTCTTCCTTTCCAG-3 '.
6. each described pork quality trait related gene DGAT 1 application in the pig molecule mark assisted Selection in the claim 1 to 5.
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| CN103224941A (en) * | 2013-05-27 | 2013-07-31 | 湖南农业大学 | Related molecular marker for detecting meat quality traits of pigs and application thereof |
| CN103333899A (en) * | 2013-07-03 | 2013-10-02 | 湖南农业大学 | Cloning and application of CDC16 gene molecular marker related to pork quality character |
| CN105063021A (en) * | 2015-06-18 | 2015-11-18 | 中国农业大学 | SNP molecular marker associated with pig fat deposition, and applications thereof |
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| CN105755131A (en) * | 2016-04-09 | 2016-07-13 | 华中农业大学 | Genetic marker associated with pig meat quality characters and carcass characters |
| CN107058557A (en) * | 2017-05-12 | 2017-08-18 | 安徽省农业科学院畜牧兽医研究所 | Influence the function candidate gene and its screening technique of pig flesh characters |
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Non-Patent Citations (2)
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| 《EMBL-Bank Sequence Database》 20071128 Yuan Z.R.等 登录号：EU258765 1 * |
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| CN103224941A (en) * | 2013-05-27 | 2013-07-31 | 湖南农业大学 | Related molecular marker for detecting meat quality traits of pigs and application thereof |
| CN103224941B (en) * | 2013-05-27 | 2016-06-22 | 湖南农业大学 | A kind of detect the relevant molecular marker of pig flesh characters and application |
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| CN105063021A (en) * | 2015-06-18 | 2015-11-18 | 中国农业大学 | SNP molecular marker associated with pig fat deposition, and applications thereof |
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| CN105755131A (en) * | 2016-04-09 | 2016-07-13 | 华中农业大学 | Genetic marker associated with pig meat quality characters and carcass characters |
| CN105755131B (en) * | 2016-04-09 | 2020-07-14 | 华中农业大学 | Genetic marker associated with pork quality traits and carcass traits |
| CN107058557A (en) * | 2017-05-12 | 2017-08-18 | 安徽省农业科学院畜牧兽医研究所 | Influence the function candidate gene and its screening technique of pig flesh characters |
| CN107937557A (en) * | 2017-11-14 | 2018-04-20 | 中国农业大学 | One and the relevant SNP site of pig economic characters and its application |
| CN107937557B (en) * | 2017-11-14 | 2020-05-05 | 中国农业大学 | SNP (single nucleotide polymorphism) site related to economic traits of pigs and application thereof |
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