CN102827835A - Genetic marker of pig immunity molecules, and its application - Google Patents
Genetic marker of pig immunity molecules, and its application Download PDFInfo
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Abstract
The invention relates to a genetic marker of pig immunity molecules, and its application, and concretely relates to a pig miR-155 precursor flanking sequence SNP which is the genetic maker of the pig immunity trait and is a base sequence represented by SEQ ID No.1 in a sequence table. There is a base mutation C158-T158 at the 158bp of the SEQ ID No.1 sequence in the sequence table, and the base mutation causes the HhaI-RFLP polymorphism. The genetic marker is used for exploiting diagnosis methods or kits, so pigs carrying favorable allelic genes are selected through utilizing the methods in breeding plans, thereby a good selection effect can be reached.
Description
Technical field
The invention belongs to animal molecular genetic marker preparing technical field, be specifically related to a kind of preparation and application that indicates and identify the molecule marker of pig immunity with the miR-155 precursor flanking sequence.
Background technology
In modern times under the intensive farm mode, disease particularly transmissible disease become the restriction pig industry healthy, continue, the principal element of stable development.Though through take to strengthen feeding and management, the condition of improving environmental sanitation, drug application and vaccine is treated and measure such as prevention makes disease obtain certain control; But in the long run; Adopt the breeding measure to improve the swinery disease resistance, just can make pig industry really away from the puzzlement of disease from hereditary angle.Individual immunity power is to weigh the important indicator of animal disease resistant ability; Illustrate the genetic mechanism of immune character, excavate key gene, seek feasible molecule marker on this basis with great breeding value; And use aborning, become the research of molecule breeding for disease resistance emphasis it
The fowl poultry immune proterties is the quantitative character that receives controlled by multiple genes, and the research to pig molecule breeding for disease resistance at present mainly concentrates on following three aspects:
(1) QTL of the disease-resistant proterties of pig research.
Discover that at SSC9 5,6,13 exist PRV susceptible or resistance QTL; At SSC1,8 exist influence quantity of leucocyte, hematocrit, interleukin II (interleukin2, IL2) and the QTL of immune indexes such as IFN-(interferon α, IFN α) secretory volume; The susceptible or the resistance QTL that have Sarcocystis miescheriana at SSC7; At SSC5,7,8,12,13 exist red corpuscle proterties QTL.Nearest discovering finds that there are the QTL of control immune character in pig 1,2-4,6-8,12,15,16 and No. 18 karyomit(e)s.
(2) Candidate Gene Study of the disease-resistant proterties of pig.
Research at present is clear that swine escherichia coli K88 and F18 acceptor gene.Domestic scholars has also been carried out research to a certain degree to this genoid.In addition; Select the gene relevant with immunne response; As Interferon, rabbit (iterferon, IFN) and acceptor gene, swine leukocyte antigen complex body (swine leukocyte antigen, SLA) and antigen presentation gene, interleukin-(interleukin; IL), Toll appearance acceptor (Tolllike receptors; TLRs), natural resistance is relevant hugely bites albumen 1 (natural resistance-associated macrophage protein1, NRAMP1) gene such as gene and Mx1 is studied as the candidate gene of immune character accordingly.
(3) sick (resisting) former inductive host gene is expressed (spectrum) research.
Petry etc. have measured the express spectra with immunogene in the lung tissue of pig breeding and each strain individuality after breathing syndrome virus is handled different strain pigs and the bronchial lymph nodes; Find that (interleukin8 IL8) possibly be a candidate resistance gene to interleukin 8.Zhao etc. utilize gene chip that the lung tissue gene expression profile of normal pig and Salmonella infection pig is studied, and discovery transglutaminase gene family gene (transglutaminase family genes, TFG); IFNs induced gene (GBP1, GBP2, C1S; C1R, MHC2TA, PSMB8; TAP1, TAP2) differential expression before and after infecting such as grade is remarkable.In order to seek new immune character candidate gene, the investigator utilizes mRNA differential display technique and biochip technology from pig immunocyte and organ, to isolate a collection of new gene both at home and abroad.At present, these differential genes carry out as the correlative study of breeding for disease resistance candidate gene.
Though the research of pig molecule breeding for disease resistance has obtained certain achievement, the candidate gene of identifying at present can't satisfy the needs of breeding for disease resistance.The localized resource family of disease-resistant proterties QTL cost is high, difficulty is big because structure is used for, and therefore, the genetic marker of utilizing the candidate gene method to excavate disease-resistant proterties will be the important content of pig molecule breeding for disease resistance research.
The miRNA of first found participation immunoregulation of miR-155.Gene knockout discovers that the miR-155 knock out mice is compared with normal mouse, and the bacterial-infection resisting ability, antibody titers is low, the t cell responses level is low.Further research is sent out, and knocks out miR-155 and causes the activity of other gene to change, and can not carry out effective immunoreation, and autoimmunization and infection take place easily.
Further investigation to miR-155 finds that this gene is brought into play important regulation in immunity system.MiR-155 participates in the regulation and control of inherent immunity (innate immune) cells whose development.For example, miR-155 causes the granulocyte dysmaturity through acting on SHIP 1 (Src homology-2 domain-containing inositol 5-phosphatase 1).In inherent immunity is replied; MiR-155 target FADD (Fas-associated death domain protein); IKK ε (I κ B kinase ε) and Ripk1 (receptor interacting serine-threonine kinase 1), and then finely regulating immunne response.In addition, miR-155 also plays a role in the differentiation and development of acquired immunity (acquired immune) cell and function are regulated.The propagation that miR-155 comes modulating T cell through downward modulation SOCS 1 (suppressor of cytokine signaling 1); Reduce IgG 1 antibody horizontal through downward modulation AICD (activation-induced cytidine deaminase), and then the type conversion of regulation and control B cell.
The miR-155 gene plays a significant role in the mammalian immune regulation and control, but does not see the report that utilizes pig miR-155 gene to seek the pig immune trait related molecular marker at present.Given this, we select the candidate gene of this gene as pig disease resistant breeding, have cloned this gene and flank region thereof, utilize this sequence to carry out polymorphism analysis and proterties association analysis, in the hope of obtaining the pig immune trait related molecular marker.
Summary of the invention
The object of the present invention is to provide a kind of detection method of seeking pig miR-155 gene mutation site and gene pleiomorphism, just indicate and identify the molecule marking method of pig immunity with the miR-155 gene.
The objective of the invention is to realize like this: the invention provides the genome nucleotide sequence of the 454bp that comprises pig miR-155 gene and flank region thereof, comparing through the genome nucleotide sequence of Different Individual provides the place's SNP (SNP) that is positioned at pig 454bp amplified fragments inside.
Be specially the genetic marker of a boar miR-155 precursor flanking sequence SNP as pig immune trait, it is a base sequence shown in the sequence table SEQ ID No.1.At sequence table SEQ ID NO.1 sequence 158bp place a base mutation C158-T158 is arranged, and cause the HhaI-RFLP polymorphum.
The step for preparing this gene fragment is following:
(1) selecting each one of people pig, landrace and duroc is test materials, from pig blood, extracts DNA;
(2) download the precursor sequence and the mature sequence of pig miR-155 gene from miRBase, utilize ENSEMBL DB (Sscrofa9) to carry out the pre-miRNA chromosomal localization, with precursor sequence to 5 ' and 3 ' flanking region go on foot respectively move 600bp and 300bp after download sequence.According to download sequence design primer to as follows
F：5′-CTACCCACTTCGACTTATGAC-3′，
R：5′GGAACATCCCAGTGACCAG-3′；
(3) pcr amplification;
(4) PCR product purification, clone, order-checking.
(5) sequence alignment analysis.
The invention provides restriction fragment length polymorphism (RFLP) the genotyping method of identifying the above-mentioned sequence 158bp C/T of place variation.Its step comprises: pcr amplification is carried out in (1) in the pig genomic dna; (2) pcr amplified fragment HhaI enzyme cutting type and detection.
The present invention further provides and has utilized definite different genotype individuality of HhaI PCR-RFLP method and the correlationship between characters of number born.
Can be used to according to the method for the invention be developed to diagnostic method or test kit, thereby in breeding plan, utilize these methods to select to carry favourable allelic pig, and then reach and select effect preferably.
Description of drawings
Fig. 1: be pig miR-155 that is cloned and the flank region sequence thereof that is used for the detection molecules mark among the present invention, square frame is labeled as the mutational site of base among the figure, and the shadow zone of sequence head and the tail is that primer is right.
Fig. 2: the SNP site that different pig kinds are found behind sequence alignment, use the square frame mark.
Fig. 3: sepharose concentration is 1.5%; Swimming lane M is DNAMarker DL2,000; Swimming lane 1 is represented the primer amplification fragment, and clip size is 454bp.
Fig. 4: agarose gel concentration is 1.5%; Swimming lane M is DNAMarker DL2,000; The 2-4 swimming lane is the TT genotype, 454bp; 1 and 6 swimming lanes are the TC genotype, 295bp, 159bp and 454bp; 5 swimming lanes are the CC genotype, 295bp and 159bp.
Embodiment
For example the present invention is further specified below.
Embodiment 1: the acquisition of gene fragment and the foundation of pleiomorphism detecting method
I pig miR-155 gene and the amplification of flank region dna sequence dna thereof
(1) design of primers
Selecting place of china kind people pig and west kind landrace, duroc is test materials; Download the precursor sequence and the mature sequence of ssc-miR-155 gene from miRBase; Utilize ENSEMBL DB (Sscrofa9) to carry out the pre-miRNA chromosomal localization, with precursor sequence to 5 ' and 3 ' flanking region go on foot respectively move 600bp and 300bp after download sequence.According to download sequence design primer, primer sequence is following:
Forward primer F:5 '-CTACCCACTTCGACTTATGAC-3 '
Reverse primer R:5 '-GGTTCATCCCAGTGACCAG-3 '
In 3 pig kind genomic dnas (being respectively people pig, landrace and duroc), carry out pcr amplification with this primer, the PCR reaction system is 25 μ l, and wherein template DNA is 50ng; DNTPs concentration is 200 μ mol/L; Every primer concentration is 0.4 μ mol/L, and the Taq archaeal dna polymerase of 2U (Biostar International, Canada); 12.5 μ l buffer adds deionized water to TV 25 μ l; PCR response procedures: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 50s, 58 ℃ of annealing 45s, 72 ℃ of extensions 1min, totally 35 circulations then; Last 72 ℃ are extended 10min.The PCR product detects (Fig. 3) through 1.5% agarose gel electrophoresis, has obtained 454bp specific amplification fragment with primer amplification, is used for reclaiming order-checking.
(2) purifying of PCR product, clone and order-checking
The concrete operations step is following:
The purifying of PCR product: under uv lamp, contain the segmental gel of purpose, put into 1.5mL Ependorff pipe, use PCR product purification test kit purified pcr product then from the cutting-out of low melting-point agarose gel; According to this test kit specification sheets operation; Concrete steps are the S1 liquid that adds 300 μ L in the gel of every 100mg, melt fully in 65 ℃ of incubation 10min to gel, change the S1/DNA mixture over to the recovery post; The centrifugal 30s of 9200g makes slurries extrude through Minicolumn.Waste liquid in the following pipe is outwelled, and the W1 liquid that adds 500 μ L again is to pipe, and the centrifugal 15s of 9200g outwells the waste liquid in the pipe.Add the W1 liquid of 500 μ L again, leave standstill 1min, the centrifugal 30s of 9200g takes off Minicolumn and packs in the 1.5mlEpendorff pipe, adds aqua sterilisa or the TE liquid of 25 μ L, leaves standstill after the 1min, and the centrifugal 1min of 9200g is stored in the Ependorff pipe with eluted dna.
Ligation: purified pcr product is connected with pMDl8-T Vector, and the ligation TV is 10 μ L, comprising 2 * Rapid Ligation Bufer, and 2.5 μ L; Vector (50ng), 0.5 μ L; Reclaim DNA, 7-8 μ L.Bullet is beaten and is mixed a little, and perhaps 4 ℃ of connections are spent the night more than 16 ℃ of connection 1h.
The preparation of competent cell: the single colony inoculation of DH5 α of picking is in 2mL LB from 37 ℃ of fresh flat boards of having cultivated 16-20h; In 37 ℃ of shaking culture 3h; Switching 1mL bacterium liquid continues at 37 ℃ of about 4h of shaking culture in the saline bottle that contains 30mL LB, treats that when OD600 reaches 0.3-0.4 saline bottle being put ice bath from the shaking table taking-up cools off 10-15min; Then bacterium liquid is changed in the centrifuge tube in 4 ℃ 4; The centrifugal 10min of 000g is inverted centrifuge tube to abandon clean nutrient solution with collecting cell, ices the CaCl of the 0.1mol/L of precooling with 10mL
2Resuspended deposition, ice bath 30min repeats 4 ℃ 4, and the centrifugal 10min of 000g once ices the CaCl of the 0.1mol/L of precooling with 4ml
2Resuspended deposition, it is subsequent use to put 4 ℃ of preservations.
Transform: in the 1.5mL centrifuge tube of sterilization, add 100 μ L competent cells, add on ice and connect product 5 μ L, inhale with liquid-transfering gun and beat evenly ice bath 30min; Centrifuge tube is placed 42 ℃ circulator bath (not vibrations), rapid ice bath 2min behind the heat shock 90s; In centrifuge tube, add 400 μ L LB nutrient solutions again, bathe recovery 45-60min in 37 ℃ of shaking tables (200rpm/min) temperature; Centrifugal, remove the part supernatant, the bacterium liquid of getting after 100 μ L recover is distributed on the flat board that contains Amp, paves; After treating that liquid fully absorbs, be inverted plate, in 37 ℃ of cultivations 14-16 hour, observation had or not bacterium colony to grow;
The evaluation and the order-checking of positive colony: the bacterial plaque that the picking conversion is good from flat board inserts the 1.5mL centrifuge tube that contains 1mLLB and in 37 ℃ of joltings cultivates about about 8h.With this bacterium liquid is template, selects for use M13 (biotechnology ltd in Shanghai provides) order-checking universal primer (annealing temperature 58-60 ℃) to carry out pcr amplification.Electrophoresis detection, the bacterium liquid of picking positive colony is sent to order-checking, and sequencing is accomplished by the precious biotechnology in Dalian ltd.
(3) dna sequence dna comparison
The PCR product sequence of different pig kinds is carried out sequence alignment (Fig. 2) through ClusterW software.Above-mentioned amplified fragments size is 454bp, and the C/T variation that is positioned at this segmental 158bp place has caused the HhaI restriction enzyme site (polymorphum (Fig. 1) of GCG ↓ C).In view of the above, adopt HhaI PCR-RFLP method to carry out the SNP somatotype.
II PCR-RFLP diagnostic method is set up
Getting 8.5 μ l PCR products adds 0.5 μ l (10U/ μ l) restriction enzyme and 1 μ l, 10 * buffer and (contains 10 * BSA), 37 ℃ of HhaI enzymes and cut 4h, get 5 μ l enzymes and cut product and detect with agarose electrophoresis, under uv lamp, observe and the record enzyme is cut the result.This amplified fragments size is 454bp; The HhaI enzyme is cut pleomorphism site and is positioned at this segmental 158bp place; When the 158bp place is the C base; Can produce the restriction enzyme site (GCGC) of restriction enzyme HhaI, thus 2 fragments (295bp and 159bp) can be produced behind the HhaI digestion PCR product, with its called after CC genotype; When 158bp place variant sites is the T base (GTGC), then there is not the HhaI restriction enzyme site, can produce 1 fragment (454bp) behind the HhaI digestion PCR product, with its called after TT genotype; When variant sites site, 158bp place is the T/C heterozygosis, can produce 3 fragments (454bp, 295bp and 159bp) behind the HhaI digestion PCR product, with its called after TC genotype (Fig. 4).
Embodiment 2: the present invention clone's the distribution of molecule marker in different pig kinds detects
In order to confirm the distribution of molecule marker in different pig kinds, selecting people pig, landrace, Large White and duroc is test materials.The HhaI PCR-RFLP method that adopts embodiment 1 to be set up detects gene frequency and the genotype frequency of this marker site in different pig kinds.Visible by table 1, in people pig and Large White, there are 3 kinds of genotype; In landrace, has only the AA genotype; In duroc, have only two kinds of genotype of AA and AB.B allelotrope is the highest at place of china kind people pig medium frequency, is 0.47; Gene frequency is lower in the kind of west, is 0.45 in the Large White wherein, and duroc is 0.02, is 0 in the landrace.In detection pig kind, Large White is external introduced variety, but the same with people pig, and genotype distributes and compares balance.
Table 1 miR-155 gene flanking region SNP site allelotrope and the distribution of genotype frequency in different pig kinds
Embodiment 3: the present invention clone's the application of molecule marker in the association analysis of immunity-associated cell factor expression amount
In order to confirm whether miR-155 gene flank region polymorphum is relevant with the pig immune trait phenotypic difference, and selecting 194 German landraces of health and cross-bred pig thereof is test materials.Utilize the Real-time PCR method to detect the mRNA expression amount of each individual 6 immune factor (INFG, IL2, TNFA, IL10, IL6 and IL4).
The HhaI PCR-RFLP method that adopts embodiment 1 to be set up is carried out polymorphum and is detected; And analysis pig miR-155 gene flank region HhaI PCR-RFLP different genotype and pig INFG, IL2, TNFA; IL10, the correlationship of the mRNA expression amount of 6 genes such as IL6 and IL4.Adopt SAS statistical software (SAS Institute Inc, Version 8.0) GLM program to carry out single mark variance analysis, adopt the REG program to calculate additive effect of gene and dominant effect simultaneously, and carry out test of significance, the model that adopts is:
Y
ijkl＝μ+G
i+B
j+Y
k+S
l++e
ijkl
Y
IjBe the proterties phenotypic number, μ is a MV, G
iFor the genotype effect (comprises additive effect of gene and dominant effect; Additive effect is represented TT, TC and CC genotype respectively with-1,0 and 1, and dominant effect is with 1, and-1 and 1 represents TT, TC and CC genotype respectively); B
j, Y
k, S
jBe fixed effect, be respectively kind, year, seasonal effect; e
IjklBe the residual error effect.
The statistic analysis result of different genotype and immune factor expression amount is seen table 2.Can find out, HhaI PCR-RFLP genotype not simultaneously, there are significant difference in IL10 and IL6 expression amount.Can find out that from table 1 the individual IL10 expression amount of CC genotype is higher than TC type (P＜0.05) and TT type individual (P＜0.01); Additive effect of gene is (P＜0.01) significantly; The individual IL6 expression amount of CC type is significantly higher than TC type (P＜0.01) and TT type individual (P＜0.01), and additive effect of gene is (P＜0.01) significantly.
Statistics shows that CC type gene individuality has higher IL10 and IL6 expression amount.Therefore in breeding, select genotypic physical efficiency of CC to improve immunizing power.
The statistical analysis table of table 2 miR-155 gene HhaI-RFLP genotype and immune factor expression amount
Annotate: above numerical value is least square mean standard error, and target letter identical table differential is different not remarkable on the numerical value, letter not simultaneously, capitalization representes that difference is extremely remarkable, the lowercase alphabet differential is different significantly; The additive effect negative value representes that C allelotrope reduces the proterties phenotypic number, and its subscript * representes p＜0.05, and * * representes p＜0.01.
Claims (5)
1. pig immunity molecular genetic marker, it is a base sequence shown in the sequence table SEQ ID No.1.
2. pig immunity molecular genetic marker, it is characterized in that: there is a base mutation C158-T158 at sequence table SEQ ID NO.1 sequence 158bp place, and causes the HhaI-RFLP polymorphum.
3. a kind of pig immunity molecular genetic marker according to claim 1; It is characterized in that: described C158-T158 base mutation; The method that is used for detecting this sudden change of base may further comprise the steps: extract genomic dna from pig blood; The design primer, pcr amplification, PCR product HhaI PCR-RFLP (Restriction Fragments Length Polymorphism) genotyping; Wherein, the primer sequence that is used for pcr amplification is a base sequence shown in SEQ ID NO.2 and the SEQ ID NO.3.
4. each described a kind of pig immunity molecular genetic marker application in the pig assisted selection of claim 1-2.
5. according to claim 3 pig immunity molecular genetic marker, it is characterized in that: described primer is to the application in the pig molecule mark assisted selection.
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| CN105368922A (en) * | 2014-08-27 | 2016-03-02 | 国立屏东科技大学 | Method for identification of pig immunological characteristics by means of single nucleotide polymorphism marker combination and application thereof |
| CN105420382A (en) * | 2015-12-28 | 2016-03-23 | 华中农业大学 | Pig blood parameter property related microRNA molecular marker and application thereof |
| CN106755449A (en) * | 2017-01-04 | 2017-05-31 | 佛山科学技术学院 | One SNP marker related to porcine cytokine IFN γ and preparation method thereof |
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| CN102167737A (en) * | 2010-12-29 | 2011-08-31 | 中国农业科学院北京畜牧兽医研究所 | Swine immune trait related protein and encoding gene as well as application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105368922A (en) * | 2014-08-27 | 2016-03-02 | 国立屏东科技大学 | Method for identification of pig immunological characteristics by means of single nucleotide polymorphism marker combination and application thereof |
| CN105368922B (en) * | 2014-08-27 | 2019-07-26 | 国立屏东科技大学 | The method and its application of combination identification pig immunological characteristic are marked using mononucleotide polymorphism |
| CN105420382A (en) * | 2015-12-28 | 2016-03-23 | 华中农业大学 | Pig blood parameter property related microRNA molecular marker and application thereof |
| CN106755449A (en) * | 2017-01-04 | 2017-05-31 | 佛山科学技术学院 | One SNP marker related to porcine cytokine IFN γ and preparation method thereof |
| CN106755449B (en) * | 2017-01-04 | 2020-03-17 | 佛山科学技术学院 | SNP molecular marker related to porcine cytokine IFN gamma and preparation method thereof |
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Application publication date: 20121219 |