CN103333899B - Cloning and application of CDC16 gene molecular marker related to pork quality character - Google Patents
Cloning and application of CDC16 gene molecular marker related to pork quality character Download PDFInfo
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Abstract
The invention belongs to the technical field of livestock molecular marker preparation, and in particular relates to cloning and application of a porcine CDC16 gene molecular marker. The molecular marker provided by the invention is cloned from a CDC16 gene; and the sequence of the CDC16 molecular marker is shown as a SEQ ID NO:1. An A / G basic group mutation at the No. 570bp in the SEQ ID NO:1 leads to PCR-RFLP-Csp6I polymorphisms, which can be applied to association analysis of pork quality character. The invention provides a novel molecular marker for pork quality character marker assisted 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 SEQ ID NO:1.The invention still further relates to mononucleotide polymorphism site in the polynucleotide sequence as shown in SEQ ID NO:1 and the method for mononucleotide polymorphism site as described in detecting.
Background technology
Pork is the main source of urban and rural residents of China animal protein, along with people are to the increase of meat requirement, also relatively improves the requirement of meat quality.The development of molecular biotechnology, make people can find at DNA level the key-gene or molecule marker closely linked with it that control Meat Quality, marker assisted selection is applied in breeding process, to improve selection process, improve pig flesh characters better, meet the needs of people, to obtain larger economic benefit.
What meat definition was accepted extensively the most is the definition of Hoffmarm, and namely meat should consider sensory attribute, nutritive value, technical quality and food safety.Sensory attribute refers to outward appearance (as color and luster), moisture holding capacity, hardness, eating quality refers to tenderness, succulence, local flavor and flavour, stink, marble grain, nutritive value refers to lipid content, lipid acid composition, protein content, other nutrient contg, technical factor refers to Coefficient shrinkage, fatty hardness, separate tissue, oxidative stability, society's quality refers to animal welfare, environment, and food safety refers to microorganism health (without Salmonellas, Campylobacter), noresidue (microbiotic, heavy metal, sterilant).Affect pork qualitative factor a lot, there are inherited genetic factors and non-genetic factor, from heredity, pig flesh characters to be evaluated and seed selection is the key improving meat quality, Meat Quality belongs to quantitative character mostly, controlled by minor-polygene, there is major gene effect in some quantitative character, great majority belong to QTL.Many Meat Qualities could can only measure after animal slaughtering, so conventional herd breeding can only express compatriot or half-sibs test, this just certainly will strengthen seed selection cost, and genetic progress is slow.Can only could measure when animal slaughtering by Meat Quality, need expensive sib test, therefore, utilizing molecule marker to carry out assisted Selection (MAS), to control Meat Quality be significantly.
The development of molecular biotechnology and the structure of pig high-density gene mapping, make breeder can find the key-gene affecting Meat Quality or the molecule marker being close to lock with it on DNA level, these key-genes or molecule marker are once confirmation in theory, just can carry out marker assisted selection breeding.
Anaphase stimulate complex body (The Anaphase-Promoting Complex, APC) be a kind of part of ubiquitin, be one of moiety of the Cycle Regulation in cell mitogen in cyclin degeneration system, and cyclin control whole mitotic process.Vertebrates APC is made up of 8 subunits, and they are BimE(APC1 respectively), CDC27(APC3), CDC16(APC6) and CDC23(APC8).It is estimated that, human cell disintegrate cyclin 16(Cell divisioncyclin16, CDC16) be made up of 619 amino acid, be approximately 30% with the homology of yeast CDC16.By immunofluorescence technique, find that CDC16 and CDC27 is in whole mitotic division process, comprises Spindle when occurring, is gathered on centrosome.Kittler etc. build short RNA interfering (shor interfering RNAs, esiRNAs) library with endoribonuclease, find that CDC16 gene is relevant with cell fission.
People CDC16 gene DNA total length 37777bp, comprises 18 exons, 17 introns, and 5'-UTR is long is 186bp, 3'-UTR232bp.Mouse CDC16 gene DNA total length 12576bp, comprises 7 aobvious sons, 6 introns, and 5'-UTR is long is 110bp, 3'-UTR299bp.Old friend is obviously different from mouse CDC16 gene structure.People CDC16 gene is positioned at No. 13 karyomit(e) q34, and this gene is positioned at mouse (Mus musculus) No. 8 karyomit(e) A1.1-1.2.
The pig CDC16 assignment of genes gene mapping is in No. 11 karyomit(e)s (SSC11), pig CDC16 protein comprises 617 amino acid, is respectively 97.6%, 95.3% with the homology of people, mouse and yeast, 38.5, it is also closely similar that the hydrophobicity of the CDC16 protein of people and pig distributes.
But the domestic and international correlative study about pig CDC16 gene is little at present.
Summary of the invention
The object of the invention is to clone pig Meat Quality gene C DC16 fragment, find the mutational site of CDC16 gene and the detection method as pig flesh characters gene pleiomorphism, for 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, provides a kind of pork quality trait related gene CDC16 and the application in pig marker assisted selection thereof, is the molecule marker that pig marker assisted selection provides.
In order to solve the problem, the technical solution adopted in the present invention is: the application of a kind of pork quality trait related gene CDC16 in pig molecule mark assisted Selection, there is the base mutation of an A/G at the 570bp place of DNA sequence dna as shown in SEQ ID NO:1 of this pork quality trait related gene CDC16 gene, causes PCR-RFLP-Csp6I polymorphism.
Above-mentioned CDC16 gene polynorphisms utilizes comparative genomics method according to the conserved regions design primer of the CDC16 gene of people, with the genomic dna of pig for template amplification, amplified fragments utilizes SSCP technology and sequencing technologies to find SNP, and utilize PCR-RFLP to carry out gene type, and recycling SAS software GLM(general linear model) analyze associating of SNP and Meat Quality.
Applicant obtains a kind of gene fragment relevant to pig flesh characters by clone, and its nucleotide sequence is as described in sequence table SEQ ID NO:1.There is the base mutation of 1 A/G at the 570bp place of sequence table SEQ ID NO:1, causes PCR-RFLP-Csp6I polymorphism.
Prepare the primer pair of detection above-mentioned sequence table SEQ ID NO:1 gene fragment sudden change, described primer pair,
Forward primer is 5 '-GACACGGCAACATGCTAG-3 '.
Reverse primer is 5 '-TTGGGATGGA A G A AG ACC-3 '.
Utilize the molecule marker relevant to pig flesh characters of above-mentioned preparation to carry out the application of association analysis to external pig kind and place of china kind, thus complete the present invention.
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 Csp6I to carry out enzyme and cut detection polymorphism by analysis.In the segment of the 696bp of amplification, there is the restriction enzyme site of 1 Csp6I, there are 3 kinds of genotype in the A/G site that electrophoresis detection result is presented at CDC16 gene, GG type (570bp, 126bp), AG type (126bp, 570bp, 696bp) and AA genotype (696bp).Restriction enzyme digestion and electrophoresis result confirms the sudden change existed at CDC16 gene the 7th intron base positions 58478bp place.
Associate between genotype-Meat Quality: utilize the general linear model in SAS software (General Linear Model, GLM) program to carry out association analysis to genotype and proterties, model the following is Y
ijkn=μ+h
i+ l
j+ g
k+ ε
ijkn, Y
ijknfor trait phenotypes value, μ is population mean, h
ifor pasture effect, l
jfor ageing, g
kfor the effect of POSTN genotype different loci, ε
ijknfor random error effect, Normal Distribution (0, σ
2).Analysis shows that CDC16 gene SNP site has remarkably influenced (P<0.01) to the thickness of backfat.
The present invention is by the molecule marker for pig flesh characters, for marker assisted selection (MAS) establishes solid basis, illustrating the molecular mechanism of Meat Quality further, theoretical foundation will be provided for improving meat quality, improve Swine Production economic benefit, and guide the breeding practice of pig.
Accompanying drawing explanation
Fig. 1 is the electrophoresis result figure of the pcr amplification product of CDC16 gene fragment of the present invention.
In figure: M:100bp DNA Ladder Marker, 1-7 represents different swimming lane.
Fig. 2 is the Csp6I restriction enzyme digestion and electrophoresis result in CDC16 of the present invention Gene A/G site.
In figure: swimming lane 2,4,6,7:AG genotype; Swimming lane 3,5:GG genotype: swimming lane 1,8:AA genotype;
M:100bp DNA Ladder Marker
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.
The Csp6I polymorphism of PCR-RFLP technology for detection CDC16 gene.
Design of primers: utilize comparative genomics method according to the 6th intron of the CDC16 gene (the GenBank number of including is NW_003541041) of people to the 8th intron, BLAST is made at GenBank with this gene, CDC16 gene candidate SNPs site is filtered out after sequence alignment, choose the 58478bp candidate SNP s site of CDC16 gene, with NW_003541041.seq sequence for standard, design primer, uses the existence in this site of PCR-RFLP technical identification.
Primer is: F:5 '-GACACGGCAACATGCTAG-3 ',
R：5′-TTGGGATGGAAGAAGACC-3′
DNA sample from 8 kinds totally 867, wherein pig farm duroc 62, Large White 118 are planted by Yiyang institute of agricultural sciences; Pig farm Large White 106 is planted by Xiang Tan agricultural university; Zheng Hongzhong pig farm, Yueyang Large White 136, new five rich pig farm landrace 45; Local variety Ning Xiang Swine 68, the black pig in the Land of Peach Blossoms 70, Daweizi pig 62, Shaziling pig 62 and Wuzhi Mountain pig 54.Get fritter and extract DNA for examination pig ear tissue.
PCR condition: PCR reaction system (cumulative volume 20 μ L): 10 × Buffer2 μ L, 2mmol/L dNTPs1.6 μ L, 20mmol/LMgCl
21.6 μ L, Taq archaeal 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, ddH
2o12.6 μ L.
Response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 50.5 DEG C of annealing 30s, 72 DEG C extend 1min, totally 35 circulations; 10min is extended, last 4 DEG C of preservations after 72 DEG C.
Get 10 μ L pcr amplification products, after adding 2 μ L tetrabromophenol sulfonphthalein sample-loading buffer mixings, point sample, on the sepharose (containing 0.05%EB) of 1%, then puts 6 μ L100bp DNA Markers 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 Csp6I enzyme of PCR primer is cut
In 10 μ L PCR primer, add 0.8 μ L10 × restriction endonuclease damping fluid, 0.2 μ L restriction enzyme and 1 μ L distilled water, cumulative volume is 12 μ L, 37 DEG C of digestion 4-10h.The 2% agarose gel electrophoresis analysis of A/G site, 5V/cm electrophoresis 0.5h, observations under ultraviolet lamp is also taken pictures, and enzyme cuts result as Fig. 2, amplified production checks order, sequence is as shown in SEQ ID NO:1, and am-plified fragments is between 34275bp to the 34972p of pig CDC16 gene, is the segment of the 6th intron to the 8th intron, 696bp altogether, for PCR-RFLP-Csp6I molecule marker, Fig. 2 is GG, AG and AA electrophoresis result of CDC16 gene in the present invention, 3 kinds of genes of PCR-RFLP.M:DNA molecular weight standard (DL1500ladder) in figure.
The present invention is using pig CDC16 gene as the candidate gene of pig flesh characters, mix as test materials with 5 local pig breeds (Ning Xiang Swine, the black pig in the Land of Peach Blossoms, Daweizi pig, Shaziling pig and Wuzhi Mountain pig), 3 external pig kinds (duroc, Large White, landrace) and Du × length × Dasanyuan, PCR-RFLP method is adopted to detect the CDC16 Gene A/gene frequency in G site, genotype frequency, and analyze its genetic construction, analyze the dependency of this gene and Meat Quality.
The distribution situation of PCR-RFLP-Csp6I polymorphism in each kind is as shown in table 1 below, as can be seen from Table 1, in these three native breeds of Daweizi pig, Ning Xiang Swine and Wuzhi Mountain pig, G gene is advantage allelotrope, wherein Daweizi pig G gene frequency the highest (0.6667), and A gene is advantage allelotrope in Shaziling pig and Land of Peach Blossoms pig, in 3 adventive durocs, landrace, Large White, A gene is advantage allelotrope, wherein duroc A gene frequency the highest (0.7302); From genotype distribution frequency, GG type distributes and preponderates in native breed, and AA type is preponderant genotype in adventive; Hardy-Weinberg balance check result shows the P<0.05 of Duroc, Wuzhi Mountain pig, Daweizi pig, and remaining 5 kind is all in Hardy-Weinberg equilibrium state.
The Hardy-Weinberg balance check in table 1 different varieties CDC16 Gene A/G site
The application of molecule marker of the present invention in the association analysis of pig flesh characters mark property
Using SAS8.02(Statistical Analysis System) statistical software carries out association analysis to CDC16 genotype and pig flesh characters.Model is as follows:
Y
ijkn=μ+h
i+l
j+g
k+ε
ijkn
Y
ijknfor trait phenotypes value, μ is population mean, h
ifor pasture effect, l
jfor ageing, g
kfor CDC16 genotype different loci is to the effect of proterties, ε
ijknfor random error effect, Normal Distribution (0, σ
2).
Table 2 pig CDC16 Gene A/G site associates with Meat Quality
In table, each row mark same letter represents difference significantly, the expression significant difference (lower same) of the different letter of mark
As shown in Table 2, in duroc, between AG genotype percentage of water loss and AA, GG genotype, difference is extremely remarkable, 4.72% (p<0.05) is reduced than AA genotype, GG genotype storage loss reduce 0.34% (p<0.05) than AA genotype storage loss, and AA, AG, GG genotype surveys mean ph value, cold cuts rate, between yellowish pink grade and marble grain grade, difference is all not remarkable.In Large White, GG genotype pH value improves 0.42(p<0.05 than AG genotype), cold cuts rate is also high than AG genotype, difference 2.74%(p<0.05), and difference is not remarkable between AA genotype cold cuts rate and GG genotype cold cuts rate, AA genotype storage loss reduce 0.64%(p<0.05 than GG genotype storage loss), but and difference is remarkable between AG genotype, the yellowish pink grade of GG, AG, AA genotype, between marble grain and percentage of water loss, difference is not remarkable.In landrace, AA genotype and the yellowish pink grade of AG genotype are all than GG genotype yellowish pink grade height 1.0(p<0.05), AG genotype marble grain grade is higher than GG genotype marble grain 0.88(p<0.05), GG genotype storage loss reduce 0.73%(p<0.05 than AA genotype storage loss), the difference of other proterties does not all reach conspicuous level.In Du × length × Dasanyuan is assorted, between GG, AG and AA genotype, in 6 proterties, difference is all not remarkable.
Table 3 Large White CDC16 gene C/T site and growth traits association analysis table
As shown in Table 3, in Large White, the GG genotype thickness of backfat reduces 2.56mm than the AG genotype thickness of backfat, and between AG genotype and AA genotype, difference is not remarkable; AG genotype 100kg age in days reduces by 3.64 days than GG genotype 100kg age in days, and between GG genotype and AA genotype, difference is not remarkable.
Claims (4)
1. a CDC16 gene fragment relevant to pig flesh characters, its sequence is as described in sequence table SEQ ID NO:1; There is the base mutation of an A/G at the 570bp place of sequence table SEQ ID NO:1, cause PCR-RFLP-Csp 6I polymorphism.
2. test right requires a primer pair for a kind of relevant to the pig flesh characters CDC16 gene fragment described in 1, and it is characterized in that: forward primer is 5 '-GACACGGCAACATGCTAG-3 ', reverse primer is 5 '-TTGGGATGGAAGAAGACC-3 '.
3. detect the method for as claimed in claim 1 relevant to pig flesh characters CDC16 gene fragment, described CDC16 gene fragment has an A/G base mutation at 570bp place, carries out this molecular genetic marker detection according to following steps:
The primer described in claim 2 is utilized to carry out pcr amplification, PCR primer 1% agarose gel electrophoresis detects, utilize restriction enzyme Csp 6I to carry out enzyme to PCR primer and cut qualification, finally detect the genotypic distribution of GG, AG and AA with 2% agarose gel electrophoresis.
4. the application of CDC16 gene fragment in pig molecule mark is assisted relevant to pig flesh characters described in claim 1, gene fragment, as described in sequence table SEQ ID NO:1, has the base mutation of 1 A/G at 570bp place, cause PCR-RFLP-Csp 6I polymorphism.
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| Kittler R.等.An endoribonuclease-prepared siRNA screen in human cells identifies genes essential.《Nature》.2004,第432卷第1036-1040页. * |
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