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CN106480174B - It is a kind of for the genetic molecule labeling method of pig disease resistant breeding and application - Google Patents

It is a kind of for the genetic molecule labeling method of pig disease resistant breeding and application Download PDF

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CN106480174B
CN106480174B CN201610825036.2A CN201610825036A CN106480174B CN 106480174 B CN106480174 B CN 106480174B CN 201610825036 A CN201610825036 A CN 201610825036A CN 106480174 B CN106480174 B CN 106480174B
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包文斌
戴超辉
吴圣龙
吴森
吴嘉韵
黄小国
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Yangzhou University
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Abstract

The invention discloses a kind of for the genetic molecule labeling method of pig disease resistant breeding and application.The present invention is the molecule labelling method and application for using Large White FUT2 genetic transcription initiation codon -84bp polymorphic site as breeding for disease resistance.The method for detecting the molecular labeling includes the extraction of the pig genomic DNA of sample to be tested and the PCR amplification of FUT2 genetic transcription upstream from start codon sequence, the pcr amplification product of translation initiation codon upstream sequence is through sscp analysis, it is then the advantageous individual of breeding for disease resistance when individual pig to be tested genotype is TT type (occur the 2nd and the 3rd band in 3 bands).The present invention provides effective molecular labeling permanently to improve Large White weanling pig E. coli resistance and Some Economic Traits from heredodiathesis.Assisted Selection is marked when with the genetic marker to Large White boar breeding, carry out breeding for disease resistance, the ability that the anti-Escherichia coli of weanling pig are infected can be improved, fundamentally reduce the disease incidence of diarrhoeal diseases, while the newborn piglet number and number pigs weaned of boar can be improved.

Description

It is a kind of for the genetic molecule labeling method of pig disease resistant breeding and application
Technical field
The invention belongs to pig genetics and breeding and Molecular Marker Assisted Selection Technology fields, and in particular to one kind is disease-resistant for pig The genetic molecule labeling method of breeding and application.
Background technique
Grice diarrhoea and oedema are the most common a kind of diseases of large-scale pig farm, bring huge economic damage to pig breeding industry It loses.Enterotoxigenic escherichia coil (Enterotoxigenic Escherichia coli, ETEC 18) is nascent and wean is young The most common and most important pathogen of diarrhea of pigs disease, wherein F18 Escherichia coli (E.coli F18) are that most universal, harm occurs again One of maximum Escherichia coli cause of disease.Studies have shown that can E.coli F18, which cause a disease, directly depends on Small Intestine of Piglets mucosal epithelium Whether there is or not receptors corresponding with its adhesin for cell brush border.
The researchs such as Snoeck find that α-(1,2) fucosyl transferase 1 (FUT1 gene coding) is E.coli F18 by the bodily form At key enzyme, catalysis H-2 blood group antigens be that receptor forms main glucide.The researchs such as Meijerink are further It was found that there are the mutational sites G/A at FUT1 gene M 307, the important genetic marker of E.coli F18 can be used as.But Coddens etc. passes through thin-layered chromatography (TLC), negative ion mass spectrum, proton according to this genetic marker of 307 site of FUT1 gene M The technologies such as NMR spectrum, the ABH blood group antigens for participating in regulation to FUT1 enzyme are verified, and find E.coli F18 receptor For FUT2 gene coding α-(1,2) fucosyl transferase 2 be catalyzed 1 type H antigen, and not FUT1 gene coding α-(1, 2) the 2 type H antigens that fucosyl transferase 1 is catalyzed.In addition, the early periods such as Bao Wenbin use chip of expression spectrum, ball series-sheath is found Glycolipid biosynthesis pathway may have important regulating and controlling effect to Soviet Union's too pig weanling pig E.coli F18 resistance, and preliminary true Determining FUT2 gene is the key gene in ball series-glycosyl sphingolipid biosynthesis pathway.
Summary of the invention
It is an object of the invention to provide a kind of for the genetic molecule labeling method of pig disease resistant breeding and application.It is specific to use greatly Molecule labelling method and application of the white pig FUT2 genetic transcription initiation codon -84bp polymorphic site as breeding for disease resistance.
Realizing the technical solution of the object of the invention is:
A kind of genetic molecule labeling method for pig disease resistant breeding, comprising the following steps:
1) screening and determination of pig FUT2 genetic mutation site
Extract the ear tissue DNA of sample to be tested；Using the sample gene to be tested group DNA comprising FUT2 gene as template, carry out PCR amplification, the DNA profiling of PCR amplification use hybrid dna, and every 5 individual DNA samples construct altogether 10 as 1 pond DNA The pond DNA is as amplification template, upstream and downstream primer sequence are as follows:
Upstream primer are as follows: 5 '-CACAGCCATGCAAGACAAGG-3 '
Downstream primer are as follows: 5 '-AAAGAAGAATGGGGAGCCGA-3 '；
Sequencing result carries out Blast comparison, and discovery does not have variation position in the code area of FUT2 gene amplification fragment Point, but there are 1 SNP sites at Gene A TG upstream from start codon -84bp；
2) it is marked using FUT2 Gene A TG upstream from start codon -84bp to pig core group complete detection genotype
Extracting genomic DNA to each individual sample pig is that template carries out PCR amplification, and amplified fragments include that ATG starting is close The PCR primer of numeral upstream 195bp sequence, target fragment length 203bp, upstream and downstream primer sequence are as follows:
Upstream primer are as follows: 5 '-AGGGGAAACACACCCTCAAAA-3 '
Downstream primer are as follows: 5 '-GGGAGAACAGGGAGCGGTTA-3 '；
3) amplified production is analyzed with SSCP method, and primer extension product is selected to occur the among 3 bands the 2nd and the 3rd The sample of this 2 band is the advantageous individual in breeding for disease resistance；
4) analysis and resistance mark are associated between FUT2 gene -84bp (C → T) mutation and F18 E. coli resistance The determination of note.
Wherein, sample to be tested selects Large White, and each individual takes ear tissue sample about 1.0g, is put into the centrifuge tube of 1.5mL It is interior, set -20 DEG C of storages.Genomic DNA is extracted using conventional phenol-chloroform method, it is micro with NanoDrop-1000 after ultrapure water dissolution Nucleic acid analyzer measures its concentration and purity, is diluted to 100mg/ μ L with ultrapure water according to its concentration, -20 DEG C save backup.
The PCR amplification system: 10.0 μ L of genome hybrid dna (100ng/ μ L) 1.0 μ L, PCR Master-Mix, on Each 1.0 μ L of downstream primer (10pmol/L) F1/R1, adds ddH2O complements to 20 μ L；Condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of changes Property 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 50s, 35 circulation, 72 DEG C of extension 10min, last 4 DEG C save backup.
The present invention is that Large White weanling pig E. coli resistance and part economy are permanently improved from heredodiathesis Character provides effective molecular marker breeding means.
Advantages of the present invention is embodied in:
1) present invention provides more an efficiently and accurately, letter for the molecular marker assisted selection of Large White breeding for disease resistance work The molecular genetic marker of easy row provides one kind to the resistivity and Some Economic Traits of colibacillosis to improve piglet Effective molecular marker breeding means.Assisted Selection is marked with E. coli resistance of the genetic marker to pig, it can be effective The piglet diarrhoea problem in actual production is alleviated on ground, while the Some Economic Traits of pig can be improved.
2) molecule breeding is carried out using the genetic marker, not only accelerates breeding process, but also the immune of Large White is answered Answering ability and general premunition does not have any detrimental effect.
3) detection method of the invention is easy to operate, and expense is more cheap, and accuracy is high, and can realize the direct of automation Detection, the present invention will play a great role in the breeding for disease resistance of Large White.
Detailed description of the invention
Fig. 1 is FUT2 genetic transcription upstream from start codon sequence PCR-SSCP banding pattern.
Fig. 2 is that peak figure is sequenced in FUT2 genetic transcription upstream from start codon series jump.
Fig. 3 is that the susceptible type of Escherichia coli determines result (A: reference culture 107/86；B:rE.coli1534；C: PnirBMisL-fedF (photo uses oil mirror camera lens, is shot with 1000 times of magnification ratios)).
Fig. 4 is that E. coli resistance type determines result (A: reference culture 107/86；B:rE.coli1534；C: The Disability (photo uses oil mirror camera lens, is shot with 1000 times of magnification ratios) of pnirBMisL-fedF).
Specific embodiment
For a better understanding of the present invention, technical solution of the present invention is illustrated below by specific embodiment.
The screening and determination of 1 Large White FUT2 genetic mutation site of embodiment
All Large Whites (242) come from Changzhou health and happiness Farming Ltd. (national live pig core breeding station).Per each and every one Body takes ear tissue sample about 1.0g, is put into the centrifuge tube of 1.5mL, sets -20 DEG C of storages.Base is extracted using conventional phenol-chloroform method Because of a group DNA, its concentration and purity are measured with NanoDrop-1000 trace dna analyzer after ultrapure water dissolution, according to its concentration It is diluted to 100mg/ μ L with ultrapure water, -20 DEG C save backup.According to GenBank announce pig FUT2 gene order (accession number: U70881.2) (5 ' → 3 ') design primer F:F1:CACAGCCATGCAAGACAAGG/R:AAAGAAGAATGGGGAGCCGA.PCR The DNA profiling of amplification uses hybrid dna, and every 5 individual DNA samples construct altogether 10 ponds DNA as expansion as 1 pond DNA Increase template.PCR amplification system: 10.0 μ L of genome hybrid dna (100ng/ μ L) 1.0 μ L, PCR Master-Mix, upstream and downstream Each 1.0 μ L of primer (10pmol/L) F1/R1, adds ddH2O complements to 20 μ L；Condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 50s, 35 circulations, 72 DEG C of extension 10min, last 4 DEG C save backup.PCR amplification produces After object is detected through 1% agarose gel electrophoresis, it is double to be routed directly to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's progress Blast comparison is carried out to sequencing analysis, and according to sequencing result, discovery is not deposited in the code area of FUT2 gene amplification fragment In variant sites, but there are 1 SNP sites at Gene A TG upstream from start codon -84bp.
Embodiment 2 is marked using FUT2 Gene A TG upstream from start codon -84bp to Large White core group complete detection base Because of type.
The PCR primer that amplified fragments include ATG initiation codon upstream 195bp sequence is designed, Primer is utilized Premier5.0 software carries out design of primers.Amplimer, F:5 '-AGGGGAAACACACCCTCAAAA-3 ' (SEQ ID NO.1)；R:5 '-GGGAGAACAGGGAGCGGTTA-3 ' (SEQ ID NO.2), target fragment length 203bp.Primer is by Shanghai The synthesis of Sheng Gong bioengineering Co., Ltd.
PCR amplification is carried out by template of the genomic DNA of extraction.PCR amplification system: genome hybrid dna (100ng/ μ L each 1.0 μ L of) 1.0 μ L, PCR Master-Mix, 10.0 μ L, upstream and downstream primer (10pmol/L) F1/R1, adds ddH2O is complemented to 20μL；Condition are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 50s, 35 recycle, and 72 DEG C Extend 10min, last 4 DEG C save backup.
SSCP detection: 3 μ LPCR products are taken to mix with 8 μ L denaturants, then 98 DEG C of denaturation 15min, ice bath 15min take 10 μ L is added in polyacrylamide gel (Acr:Bis=29:1) by the denaturation aggregate sample of ice bath, 120V electrophoresis 11h.Benefit Use 0.1%AgNO3Silver staining is carried out, is then developed the color using 1.5%NaOH.
According to PCR-SSCP testing result, chooses different genotype individual and the raw work biotechnology service in Shanghai is transferred to have Limit company ABI PRISM 377DNA automatic sequencer carries out bidirectional sequencing analysis.
PCR-SSCP as a result, it has been found that, detect 3 kinds of genotype, be respectively defined as TT, CT, CC (occur 3 bands be CT Type, the 1st and the 3rd this 2 band occurred among 3 bands is CC type, the 2nd and the 3rd this 2 band among 3 bands occurs and is TT type) (Fig. 1), determine that there are C → T to be mutated (Fig. 2) in ATG initiation codon upstream -84bp really through direct Sequencing.
The genotype frequency that FUT2 gene purpose mutational site is calculated according to Hardy-Weinberg equilibrium law, is as a result shown in Table 1, CC genotype is preponderant genotype in Large White group, and genotype frequency reaches 0.653；Allele C is advantage equipotential Gene, gene frequency 0.773.Through the comptibility test of card side, which deviates Hardy-Weinberg equilibrium state.
1 Large White FUT2 gene -84bp (C → T) mutated-genotype of table and gene frequency distribution
Note: χ2Indicate whether different genotype distribution meets Hardy's Weinberg equilibrium: χ20.05 (1)=3.84, χ20.01 (1)=6.63
The adhesion assay of 3 Small Intestine of Piglets epithelial cell of embodiment determines individual Escherichia coli F18 bacterial strain resistance/neurological susceptibility
Before and after weaned piglet, and most easy infection Escherichia coli F18 bacterial strain and show age grades on the 35th of symptom of diarrhea Section, which is butchered, takes duodenum, jejunum intestinal segment 15cm to be used for the preparation of intestinal epithelial cell,
It is micro- by Univ Pennsylvania USA's veterinary college to produce F18ab pili reference culture 107/86 (O139:K12:H1) Biology laboratory give.Can induce expression F18ac pili operon containing fed full genome recombination coliform rE.coli1534 by This laboratory constructs and saves, the recombination bacillus coli pnirBMisL- of energy surface secreting, expressing F18ab pili FedF subunit FedF is also constructed by this laboratory.The bacterium of anaerobic induction is centrifuged repeatedly with PBS and is washed 3 times, adjustment bacterial concentration to about 1 × 109CFU/ml.The recombinant bacterium bacterium solution 0.5ml for taking above-mentioned wild mushroom or inducing expression respectively, it is sweet to add final concentration of 1% wherein Dew sugar mixes after 37 DEG C of effect 30min with 0.5ml intestinal epithelial cell, then at 37 DEG C of incubation 30min, 1000r/min centrifugations 5min, with taking 50 μ l drops to spontaneously dry after PBR solution suspension on clean slide, flame is fixed, and dyes 3-5min with methylene blue, Oil mirror observes result.
The intestinal epithelial cell of weanling pig is as can be with expression F18ab pili reference culture 107/86, inducing expression Adhesion (figure occurs for the recombination bacillus coli rE.coli1534 and recombination bacillus coli pnirBMisL-fedF of inducing expression 3), it is defined as Escherichia coli F18 bacterial strain responsive type；And as Small Intestine of Piglets epithelial cell sticks above-mentioned 3 plants of Escherichia coli Attached ability is almost nil (Fig. 4), then is defined as resistance type.
The relationship analysis and resistance of 4 Large White FUT2 gene -84bp (C → T) of embodiment mutation and F18 E. coli resistance The determination of label
Large White FUT2 gene -84bp (C → T) is mutated 3 kinds of genotype individuals Escherichia coli F18 bacterial strain resistance/neurological susceptibilities It is analyzed, 3 kinds of genotype individuals Escherichia coli F18 bacterial strain resistance/neurological susceptibility frequencies are shown in Table 2；As it can be seen that big in TT genotype The frequency of enterobacteria F18 bacterial strain resistance type individual is 88.5%, is higher than CT genotype (38.5%) and CC significant or extremely significantly The individual of genotype (15.4%).Large White FUT2 gene -84bp (C → T) mutation T T genotype can be determined as anti-large intestine The beneficial gene type of bacillus.
23 kinds of genotype individuals Escherichia coli F18 bacterial strain resistance/neurological susceptibility frequency meters of table
The measurement of 5 pig important economical trait of embodiment
80 piglets are randomly selected in 35 age in days of weaned piglet, vena cava anterior blood sampling 5ml utilizes pig multiple cytokine to examine Test agent box (Procarta Immunoassay Kits) (Affymetrix, USA) is important to part in above-mentioned sample serum thin Intracellular cytokine (IL-2, IL-4, IL-6, IL-10, IL-12, IFN-α, IFN-β, IFN-γ) level is measured, concrete operations step Suddenly it is carried out in strict accordance with specification.
Great Bai Suprapubic arch sling 200 of 6 tires of farrowing or more are randomly selected, the reproductive performance and growth of each sow are arranged Energy data, the impact analysis for subsequent hereditary variation to Some Economic Traits.In 185-195 age in days, B ultrasound living body measurement great Bai Eye muscle thickness, the thickness of backfat (thickness of backfat on the left of sow at the 10th rib) of pig, weigh in.Record reproductive performance in detail is relatively stable The 3rd~5 tire number born alive, number pigs weaned, born weight, weight of weaning litter." 100kg weight age in days " and " 100kg living body back Fat thickness " corrects as follows respectively: age in days-(actual measurement weight -100)/CF1, CF1=is (real when 100kg weight age in days=measurement Survey weight/measurement age in days) × 1.715 (sows)；The 100kg thickness of backfat=actual measurement the thickness of backfat × CF2, in which: CF2=A/ { A+ [B × (actual measurement weight -100)] }, the A value and B value of large white sow are respectively 13.706,0.119.Every economy of 3 parity is referred to Mark data statistics processing is averaged, the analysis data as economic characters.
6 Large White FUT2 gene -84bp (C → T) of embodiment mutation and its relationship analysis between important economical trait
Based on 8 kinds of cellular immunity factors between the genotype individuals of 3 kinds of the site FUT2 gene -84bp (C → T) in peripheral blood It is horizontal significant difference (table 3) is not present, show the site for immune response ability and general premunition without conspicuousness Influence.
The important immune factor in part is horizontal between table 3FUT2 gene different genotype individual
This test picks 200 (CC:130, CT:46 and TT:24) Suprapubic arch sling determination part point economy altogether Shape data.The results are shown in Table 4 for analysis, and on newborn piglet number and number pigs weaned index, TT > CT > CC genotype is presented Trend, and the newborn piglet number and number pigs weaned of TT genotype individuals are significantly higher than CC genotype (P < 0.05)；Birth weight, It corrects in the indexs such as 100Kg weight age in days, eye muscle thickness and the correction 100Kg thickness of backfat, 3 kinds of genotypic differences are not significant.Show TT Genotype is also advantageous that genotype for important reproductive trait newborn piglet number and number pigs weaned, this may be with TT base Because type has stronger premunition related.
Part important economical trait between table 4FUT2 gene different genotype individual
Note: the numerical value that upper right indicates different letters represents significant difference (P < 0.05).
SEQ ID No:1
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Claims (4)

1. a kind of genetic molecule labeling method for the anti-colibacillosis breeding of pig, which comprises the following steps:
1) the ear tissue DNA of sample to be tested is extracted；
2) it is marked using -84 bp of FUT2 Gene A TG upstream from start codon to pig core group complete detection genotype
PCR amplification is carried out by template of the DNA of extraction, amplified fragments include SNP at -84 bp of ATG initiation codon upstream Point, 195 bp of expanding fragment length, upstream and downstream primer sequence are as follows:
Upstream primer are as follows: 5 '-AGGGGAAACACACCCTCAAAA-3 '
Downstream primer are as follows: 5 '-GGGAGAACAGGGAGCGGTTA-3 '；
3) amplified production is analyzed with SSCP method: as shown in Figure of description Fig. 1 label 1,3 bands occur is CT type, As shown in Fig. 1 label 2,3, the 1st and the 3rd this 2 band occurred among 3 bands is CC type, as shown in Fig. 1 label 4, occurs 3 The the 2nd and the 3rd this 2 band among band is TT type, selects the sample of TT type for the advantageous individual in breeding for disease resistance.
2. genetic molecule labeling method according to claim 1, which is characterized in that sample to be tested selects Large White, each Individual takes about 1.0 g of ear tissue sample, is put into the centrifuge tube of 1.5 mL, sets -20 DEG C of storages；It is mentioned using conventional phenol-chloroform method Genomic DNA is taken, its concentration and purity are measured with NanoDrop-1000 trace dna analyzer after ultrapure water dissolution, according to it Concentration is diluted to 100 mg/ μ L with ultrapure water, and -20 DEG C save backup.
3. genetic molecule labeling method according to claim 1, which is characterized in that the PCR amplification system: genome is mixed Conjunction DNA concentration is 100 ng/ μ 10.0 μ L of L, 1.0 μ L, PCR Master-Mix, and upstream and downstream primer F1/R1 concentration is 10 Each 1.0 μ L of pmol/L, adds ddH2O complements to 20 μ L；Condition are as follows: 95 DEG C of 5 min of initial denaturation, 95 DEG C of 30 s of denaturation, 63 DEG C are moved back 30 s of fire, 72 DEG C of 50 s of extension, 35 circulations, 72 DEG C of 10 min of extension, last 4 DEG C save backup.
4. genetic molecule labeling method as claimed in any one of claims 1-3 is marked in the anti-colibacillosis breeding process of pig Remember the application of assisted Selection.
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CN109971840A (en) * 2019-04-11 2019-07-05 扬州大学 A kind of genetic molecular marker method and application for pig disease resistance breeding

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