CN1974769A - Identification of MUC4 mononucleotide polymorphic site obviously affecting diarrhea resistance of weaned pig and application thereof - Google Patents
Identification of MUC4 mononucleotide polymorphic site obviously affecting diarrhea resistance of weaned pig and application thereof Download PDFInfo
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Abstract
The invention discloses a MUC4 mononucleotide polymorphism site for identifying the diarrhea resistance of weaned pig and genetic improvement of breeding pig and its application, which is characterized by that firstly, the mononucleotide polymorphism (SNP) A243G is identified, then the PCR-Hha I-RFLP method is used to make SNPA243G genotype judgment. The invention detects the polymorphic site by modern molecular biology technology, selects favorable allelic type individual seed reservation by Marker Assisted Selection (MAS), can obviously improve the diarrhea resistance of weaned piglets in population, and greatly reduces the death rate of the piglets.
Description
Technical field
The present invention relates to the MUC4 mononucleotide polymorphic site differentiating remarkably influenced diarrhea of weaned piglets resistance and can be used for kind of swine improvement.
Background technology
The annual whole world Production of Livestock and Poultry of giving of livestock and poultry transmissible disease causes huge financial loss, and the loss that developed country causes thus accounts for 17% (1,700,000,000 pound) of annual volume, in developing country up to 35%-50%.Piglet diarrhoea is the most general and very important disease, and the piglet mortality ratio that causes because of dysentery reaches 11.5%-29.5%, and wherein the M ﹠ M that causes because of intestinal bacteria accounts for 56.2% and 24.7% of whole diarrhea of pigs incidence and mortality.Colibacillosis has a strong impact on piglet growth and grows, cause feeding cost to increase, bring the tremendous economic loss to pig industry, adopt vaccine inoculation or adopt the anti-microbial type medicine that this disease is prevented and treated, on the one hand long-time use causes resistance easily, the abuse of medicine will cause the livestock product drug residue and become medicine food in addition, finally influence human beings'health.What deserves to be mentioned is pathogenic colon bacillus (Eschoerchia coli, E.coli) antigenic characteristic is variable, the E.coli thalline O antigen serotype of having found just has 164 kinds, give and to develop vaccine and pharmacological agent brings very big difficulty, therefore fundamentally carry out the genetic mechanism research of pig, will open up grice diarrhoea breeding for disease resistance new way the E.coli resistance.
Soderlind O etc. studies show that, mainly be again to cause in the colibacillosis by enterotoxigenic escherichia coli (Enterotoxigenic Eschoerchia coli ETEC) F4, ETEC F4 through stick be settled in host's intestinal epithelial cell after, the effect of performance steric restriction is not got rid of by the cleaning of intestinal peristalsis or secretory product, and a large amount of breeding, the secretion enterotoxin, cause cell malabsorption, cause diarrhoea, even cause death.The F4 adhesin can be divided into F4ab, F4ac, F4ad by discovery and names such as Rskov according to its amynologic characteristic.
Reported first such as Sellwood 1 pig that the diarrhoea that E.coli F4ac adhesin is caused has resistance, its resistance be since mucous membrane of small intestine lack corresponding acceptor.E.coil F4 ac adherent acceptor is followed Mendelian inheritance, and dominant gene S expresses has receptor protein, pig only to show as the sensitivity to E.coli F4ac, and recessive gene s is expressed receptor albumen not, shows as resistance.Bijlsma finds encode the simultaneously acceptor of F4ab and two kinds of adhesins of F4ac of a site, and the heredity of two acceptors is than close linkage (θ=0.02), reports the chain of Transferrins,iron complexes site (Tf) and F4ac acceptor site simultaneously.Usefulness Sweden Yorkshires such as Edfors-Lilja * European wild boar resource family is a material, through the linkage analysis of three generations pedigree, F4ab and F4ac receptor mapping in No. 13 karyomit(e)s, and has been determined F4ab and F4ac acceptor site close linkage (θ=0.01).Grange etc. have done a series of detailed researchs to the character of F4 acceptor, be separated to two kinds of mucus type sialoglycoprotein (Intestinemucus glycoprotein) at the chitterlings epithelial cell, molecular weight is 210kDa (IMTGP-1) and 240kDa (IMTGP-2) respectively, and these two kinds of albumen are considered to pig to F4ab and F4ac susceptibility and the important factor of determination of resistance.The cooperation of the R﹠D institution of 5 countries such as Denmark is positioned the QTL site of enterotoxic Escherichia coli F4ab and F4ac acceptor for a segment length 7cM zone on No. 13 karyomit(e) q41 of pig by further Fine Mapping.The MUC4 gene is positioned at this zone, analyzes in conjunction with its biochemical functions, thinks that the MUC4 gene very likely is an important candidate gene of enterotoxic Escherichia coli F4ab and F4ac acceptor.Given this, the applicant carried out in a deep going way pig MUC4 gene SNPs (single nucleotide polymorphism) search, evaluation and influence the research of piglet diarrhoea proterties, carry out the breeding for disease resistance work of diarrhea of weaned piglets disease in the hope of setting up the marker assisted selection technology.
Summary of the invention
The purpose of this invention is to provide a MUC4 mononucleotide polymorphic site of differentiating remarkably influenced diarrhea of weaned piglets resistance, detect this polymorphic site by modern molecular biology technique, utilize marker assisted selection (MAS) to select favourable aflelotype unity to reserve seed for planting, can significantly improve the sick ability of diarrhea of population weanling pig, significantly reduce the piglet mortality ratio.
The object of the present invention is achieved like this:
(1) discriminating of single nucleotide polymorphism (SNP) A243G: according to one section 560 bp EST of pig MUC4 gene (expressed sequence tag) sequence (sequence number BF075199), with public Primer 5.0 or Oligo 6.0 software design primers F 1 and R1 (F1:5 '-CAG GAT GCC CAA TGG CTC TAC-3 ' and R1:5 '-CCC CGA AGTTGT GAA AGG AAG-3 ') amplification pig genomic dna (thymus nucleic acid).In the reaction system of the polymerase chain reaction of 25 μ L (PCR), comprise 25ng pig genomic dna, 1.0mM MgCl, 100mM dNTP, each 10pmol of forward and reverse primer, 2 unit archaeal dna polymerases (Taq enzyme) and 1 * PCRbuffer (damping fluid) (Chinese Shanghai is given birth to the worker).Amplification condition is: 95 ℃ of 3min; 94 ℃ of 30s, 65.5 ℃ of 30s, 72 ℃ of 45s, totally 35 circulations; Extend 10min at 72 ℃ at last.The amplified fragments of 538bp adopts QIAquick PCR Purification Kit (QIAGEN, Germany) purifying, by the order-checking of Beijing China big-and-middle living development in science and technology company limited.Sequencing result is adopted public SEQMAN software analysis, disclose this fragment and contain an intron, there is the part exon its both sides, and the homology of the exon of both sides and pig est sequence is respectively 98% and 97%, illustrate that this section sequence is the part of the MUC4 gene of pig.By finding relatively that with people's MUC4 genome sequence this intron is the 18th intron.During with the SeqMan software analysis sequencing result of public DNAStar, find in introne 18, to exist a mononucleotide polymorphic site (SNPs), i.e. SNP A243G site.
(2): the 243rd nucleotide site that is positioned at pig MUC4 sequence is:
1 caggatgccc aatggctcta ctctttcccc acggagctcc gaggagaccc tttttcagta
61 tggaatgacc tgtgagtctg gtcctcagag tcctcgggta gatgggcagg gctgctgcat
121 ggtctccctc aggcctgtgg gaaccagacc tctgtccttt gtgagagagg ggtagaaagg
181 aagagatcta aagatgctgg tgctaccccc agatttgtcc cctctgtccc caacccacct
241 gcacctctct acctgggaga ggggacaagg gaggtgggtg gccctcagtc actagagtga
301 ggatttccag actcagaagc tggaagtcac tctgtctctc cacatcccct gccccaaccc
361 caaaccatat aagggtaagg ggtggacaca acagaaatac agccagaggc ccaccctggt
421 aacaccacca tcatgccttg tgtgttttag gggagatcaa cggaaccagc ctccttggca
481 agaggaatga ccatgtgtct tataacttca cccctgtctt cctttcacaa cttcgggg
(3) SNP A243G declare type:
This polymorphic site has changed the restriction enzyme site of HhaI restriction enzyme, can adopt PCR-HhaI-RFLP (Restriction fragment length polymorphism, RFLP restriction fragment length polymorphism) method to judge the genotype in this site.HhaI digestion with restriction enzyme F1/R1 primer amplification genes of individuals group DNA gained purpose fragment, 20 μ L enzymes are cut and are contained 15 μ L PCR products in the system, 0.2 μ L (10U/ μ L) HhaI restriction enzyme, 2 μ L, 10 * buffer (100mM Tris-HCLpH 7.5,100mM MgC12,10mM DTT, 500mM NaCL), 2.5 μ L dd-H
2O (distilled water).Behind the system mixing, place 37 ℃ of temperature to bathe 4～6 hours or spend the night.In every pipe endonuclease reaction system, add the incidental 10 * loading buffer of 2 μ L restriction endonuclease test kits, mixing, with 5V/cm voltage electrophoresis 1～2hr, ultraviolet transillumination platform observations is also taken pictures on 2.5% sepharose.
Specifically declare the type method as shown in Figure 2.
Another object of the present invention is achieved in that the present invention utilizes this polymorphic site and flanking DNA sequence information, adopts molecular biotechnology to differentiate idiotype, at diarrhea characteristic of disease shape seed selection boar.
The present invention has differentiated MUC4 single nucleotide polymorphism (SNP) site that influences weanling pig diarrhea characteristic of disease shape, detect this polymorphic site by modern molecular biology technique, utilize marker assisted selection (MAS) to select favourable aflelotype unity to reserve seed for planting, can significantly improve the sick ability of diarrhea of population weanling pig, significantly reduce the piglet mortality ratio.
Description of drawings
Fig. 1 is the sequence peak figure in the pig MUC4 SNP A243G mutational site of the present invention's discriminating;
Fig. 2 declares type figure for the PCR-HhaI-RFLP in SNPA243G site, and wherein 1 and 3 is the AG type; 2,4 and 6 is the GG type; 5 and 7 is the AA type; 8 is molecular weight marker;
Fig. 3 is the detected result of sticking of ECF18ab and brush border, (a) is adhesion type not (b) to be adhesion type.
Embodiment
Below in conjunction with embodiment and contrast accompanying drawing the present invention is described in further detail.
Select boar core group individuality, utilize above-mentioned PCR-HhaI-RFLP method to differentiate the genotype in MUC4 SNPA243G site, select favourable aflelotype unity to reserve seed for planting, the anti-piglet diarrhoea performance of improvement population after colony's subculture seed selection.
Embodiment:
Utilize self make up, with white Du Luoke and Chinese painted face in Beijing opera pig for parent for generations, to be used for the localized pig resource of functional gene family be laboratory animal.795 resource family s-generations (F2 generation) boar individual feeding is butchered to 240 ages in days, get small intestine jejunum part, extract the intestinal epithelial cell brush border, utilize E.coli F4ab, F4ac and F4ad bacterial strain to stick chitterlings epithelial cell brush border, by the result of sticking of phase microscope bacterial detection.The brush border of each sample detection more than 20, if have on the brush border more than 10% and have 2 bacterial adhesions at least, declaring this sample is adhesion type, if have the brush border of 2 bacterial adhesions at least less than 10%, but the brush border that 1-2 bacterial adhesion arranged is more than 10%, declaring this sample is weak adhesion type, otherwise is judged to not adhesion type.Stick design sketch as shown in Figure 3.
Adopting above-mentioned PCR-Hha I-RFLP method that 795 F2 are carried out SNP A243G genotype for individuality judges, with the Freq process of SAS software package (SAS9.0.2002.SAS institute Ine.Cary.NC.USA.), the genotype and the external phenotype of sticking the chitterlings brush border of ETECF4ab/ac in each SNP site are carried out the dependency statistical study.Wherein SNP A243G polymorphism is relevant extremely significantly with the susceptibility that infects of ETEC F4ab/ac, and A allelotrope is tumor susceptibility gene, and G allelotrope is resistant gene; Frequency of genotypes AA and AG individuality are to the susceptible that infects of ETEC F4ab/ac, and GG type individuality has resistivity (p＜0.0001) to infecting of ETECF4ab/ac.GG type individuality is the required genotype individuality of seed selection improvement of the sick type pig of diarrhea.In commercial boar core group, subculture seed selection GG type individuality can further be improved the diarrhea characteristic of disease shape of population, reduces the mortality ratio of piglet.
Table 1SNP A243G polymorphism and ETEC F4 stick the dependency of phenotype
| Genotype | Stick phenotype | x 2 | df | P | RR | 95％L | 95％U | ||
| AA AG GG AA AG GG AA AG GG | F4ab+ 26 141 70 F4ac+ 47 276 81 F4ad+ 17 109 115 | F4ab- 20 154 376 F4ac- 2 27 362 F4ad- 30 180 322 | AA-AG AA-GG AG-GG AA-AG AA-GG AG-GG AA-AG AA-GG AG-GG | 1.2123 44.257 89.84 1.3012 138.15 382.2 0.0412 2.0776 10.598 | 1 1 1 1 1 1 1 1 1 | 0.2709 ＜.0001 ＜.0001 0.254 ＜.0001 ＜.0001 0.8391 0.1495 0.0011 | 1.1825 3.6012 3.0453 1.053 5.2459 4.9818 0.959 1.3745 1.4332 | 0.8937 2.5828 2.3813 0.9841 4.2729 4.0788 0.6379 0.9113 1.155 | 1.5648 5.0214 3.8944 1.1267 6.4405 6.0847 1.4417 2.073 1.7784 |
Annotate: x
2The expression chi-square value; Df represents degree of freedom; RR represents the relative risk rate; 95%L represents the fiducial interval lower limit; 95%U represents the fiducial interval upper limit; P＜0.01 expression difference is extremely remarkable; P＞0.05 expression difference is not remarkable; The brush border of+expression bacterial adhesion chitterlings, the brush border that-expression bacterium is not sticked chitterlings.
Claims (2)
1, MUC4 mononucleotide polymorphic site of differentiating remarkably influenced diarrhea of weaned piglets resistance, it is characterized in that: this polymorphic site is as follows:
1 caggatgccc aatggctcta ctctttcccc acggagctcc gaggagaccc tttttcagta
61 tggaatgacc tgtgagtctg gtcctcagag tcctcgggta gatgggcagg gctgctgcat
121 ggtctccctc aggcctgtgg gaaccagacc tctgtccttt gtgagagagg ggtagaaagg
181 aagagatcta aagatgctgg tgctaccccc agatttgtcc cctctgtccc caacccacct
241 gcacctctct acctgggaga ggggacaagg gaggtgggtg gccctcagtc actagagtga
301 ggatttccag actcagaagc tggaagtcac tctgtctctc cacatcccct gccccaaccc
361 caaaccatat aagggtaagg ggtggacaca acagaaatac agccagaggc ccaccctggt
421 aacaccacca tcatgccttg tgtgttttag gggagatcaa cggaaccagc ctccttggca
481 agaggaatga ccatgtgtct tataacttca cccctgtctt cctttcacaa cttcgggg
2, the application of MUC4 mononucleotide polymorphic site in seed selection improvement boar diarrhea characteristic of disease shape.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010066118A1 (en) * | 2008-12-11 | 2010-06-17 | 江西农业大学 | Muc13 molecular marker for identifying the f4ac adhesin-caused diarrhea resistance of the weanling pig and the use thereof |
| CN101603089B (en) * | 2009-07-20 | 2011-08-10 | 江西农业大学 | Molecular marker for identifying local pig anti-F4ac piglet diarrhea in China and breeding application |
| CN104878113A (en) * | 2015-06-12 | 2015-09-02 | 江西农业大学 | Main-effect SNP (Single Nucleotide Polymorphism) marker affecting enterotoxigenic escherichia coli F41 diarrheal disease resistance of weaned piglet and application |
-
2006
- 2006-09-13 CN CNA200610124511XA patent/CN1974769A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010066118A1 (en) * | 2008-12-11 | 2010-06-17 | 江西农业大学 | Muc13 molecular marker for identifying the f4ac adhesin-caused diarrhea resistance of the weanling pig and the use thereof |
| CN101532052B (en) * | 2008-12-11 | 2010-11-24 | 江西农业大学 | Susceptible/resistant MUC13 molecular marker capable of identifying F4 weaning brash of piglets and application |
| CN101603089B (en) * | 2009-07-20 | 2011-08-10 | 江西农业大学 | Molecular marker for identifying local pig anti-F4ac piglet diarrhea in China and breeding application |
| CN104878113A (en) * | 2015-06-12 | 2015-09-02 | 江西农业大学 | Main-effect SNP (Single Nucleotide Polymorphism) marker affecting enterotoxigenic escherichia coli F41 diarrheal disease resistance of weaned piglet and application |
| CN104878113B (en) * | 2015-06-12 | 2018-04-27 | 江西农业大学 | Influence main effect SNP marker and the application of the enterotoxigenic escherichia coli F41 diarrhoeal diseases resistances of weanling pig |
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