CN103173531B - Application of bactericidal/permeability-increasing protein gene in serving as common disease resistance genetic marker of pigs - Google Patents
Application of bactericidal/permeability-increasing protein gene in serving as common disease resistance genetic marker of pigs Download PDFInfo
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Abstract
The invention relates to a genetic marker for screening the combined gene model of the fourth and tenth exon variation sites of a common disease resistance BPI (bactericidal/permeability increasing protein) gene of pigs, and belongs to the technical fields of genetic breeding and molecular marker assisted selection of pigs. The method comprises the following steps of: extracting DNA (deoxyribonucleic acid) of a pig genome of a sample to be tested, and performing PCR (polymerase chain reaction) amplification on the pig genome; analyzing the PCR product of the exon 4 through SSCP (single strand conformation polymorphism), and performing enzyme digestion on the PCR product of the exon 10 through restriction enzyme HpaII, wherein when the gene model of the pig body to be tested is a CDAB model, the pig body is strong in common disease resistance. According to the application, a molecular genetic marker with higher efficiency and accuracy is provided to molecular marker assisted selection of the pig disease resistance breeding work, and an effective molecular marker breeding means is provided for improving the common disease resistance and immune response capability of piglets. The detection method is simple in operation, low in cost and high in accuracy, can realize automatic direct detection, and takes a magnificent effect in pig breeding.
Description
Technical field
The invention belongs to pig genetics and breeding and Molecular Marker Assisted Selection Technology field, be specifically related to screening and the application of the general disease resistance genetic marker of pig.
Background technology
Along with popularizing of intensive farm way to manage, in pig industry is produced, the harm of disease is very easily produced and is brought heavy losses to livestock industry.In the U.S., the expense of paying due to swine disease every year exceeds 1,500,000,000 dollars.This huge financial loss is mainly because pig death, production efficiency reduce, medical expenses increase and product loss causes.Stress working condition under, selection that important economical trait is applied is pressed, and also can cause the increase of disease incident.Disease can make the Efficiency Decreasing of selecting, and the heredity that affects the production traits improves.At present, to the measure of control of livestock and poultry, mainly to adopt vaccine inoculation and Antibiotics pharmacological agent, but, because germ type is various and it is fast to change, the use of vaccine is always ineffective, abusing antibiotic can cause livestock product to carry a large amount of left drugs simultaneously, become medicine food (Drug-food), the mankind's health is produced to detrimentally affect, present many countries start import prohibition medicine food and the application of restriction antibiotic in livestock and poultry treatment one after another, therefore in breeding high-yield variety, find a safe and reliable and economically viable livestock and poultry disease control and prevention approach, oneself is the common pressing problem of being concerned about of scientist and the herding producer.
In order to obtain more diseases prevention and treatment method, must be well understood that immune disease-resistance system and the pathogenesis of animal.The generation of disease is generally interactional result between the genetic background of animal and its environment of living in.If a certain animal is to a kind of disease-susceptible humans in heredity, envrionment conditions (comprising conventional diseases prevention and treatment method) may be only effective to the control part of disease.One can replace disease conventional control method is to carry out selection breeding for improving domestic animal disease resistance.Heredity disease resistance has comprised the disease-resistant system of immunity of organism and interactional many aspects thereof, thereby very complicated.Fundamentally carry out at present the genetics of resistance Mechanism Study of pig, carry out breeding for disease resistance, caused the great attention of Chinese scholars.
Pathogenic agent, in infectious process, can run into 3 road defense mechanisms, i.e. epithelium defense mechanism, nonspecific defense mechanism and specificity defense mechanism.In the time that individuality is subject to pathogen infection, can transfers the defense mechanism of this 3 aspect and be resisted.Whether pig falls ill and depends on and infect and the interactional result of defensive enginery, and the pig that defensive enginery is strengthened just shows nature disease resistance.Disease resistance can be divided into special disease resistance and general disease resistance by the difference of hereditary basis.Special disease resistance refers to the resistance of pig to certain specified disease or pathogenic agent, and this resistance is controlled by mainly in a key-gene site, also can be subject to some extent other site (comprising regulator) and such environmental effects simultaneously.Existing result of study shows, the inherent mechanism of special disease resistance is the interior existence of host or lacks certain molecule or its variant, this can not only determine allosome identification and specificity simplified reaction, can also determine the special attachments power of pathogenic agent, can pathogenic agent be bred after entering in body in vivo, cause host to fall ill.General disease resistance is not limited to anti-a certain pathogenic agent, and it is subject to the combined influence of polygene and environment, and the antigenic specificity of pathogenic agent is minimum on general disease resistance impact, does not even affect at all, and this disease resistance has embodied the total defense function of body to disease.
Research discovery, the pathogenesis of many swine diseases is relevant with the genetic background of pig itself, and may there is resistance wholly or in part to some diseases in pig.In a broad sense, the generation of any disease is all the coefficient result of nature-nurture factor, and nearly all disease is all relevant with heredity.Say narrowly, inherited disease is because genetic material in sexual cell or zygote has occurred due to the variation in structure or function.Therefore, in the long run/term, adopt genetic method to improve pig from hereditary basis and the general resistance of disease is there is to the effect of effecting a permanent cure.And general resistance is to maintain the prerequisite that pig survives and produces in poor environment, therefore cultivating the pig kind with general resistance becomes one of the major subjects in breeding field, and the genetic marker of screening and the general disease resistance of definite pig has become cultivates the task of top priority and the key point with general resistance pig kind.
Cytokine has been brought into play important complementary effect in immune response process, and IL-2 can promote the propagation of T, B cell, aspect antitumor, toxinicide and immunomodulatory, is bringing into play important effect; IL-4 can induce the expression of MHC-class Ⅱ antigens, and strengthens it to tumour cell and parasitic lethal; IL-6 can promote the expression of IL-2 and acceptor thereof, in immune response regulation and hematopoiesis, plays an important role; IL-10 is an anti-inflammatory factor, participates in the autarcetic adjusting of body; IL-12 mainly works in cellular immunization and the cell-mediated antineoplastic immune of I type; IFN-Beta can suppress the synthetic of viral protein, the increase of induction MHC-class Ⅰ antigens, thereby the immunologic function of adjustment body.The level of above-mentioned important cytokine is the important symbol of immune response ability and general disease resistance.
Bactericidal power/permeability Enhancin (bactericidal/permeability-increasing protein, BPI) be people and mammiferous endogenous cationic protein, mainly be present in the AG of polymorphonuclear leukocyte (Ploymorphneclear leukocytes, PMNs).BPI is except killing G-bacterium, in and intracellular toxin or lipopolysaccharides (lipopolysaccharide, LPS) outside activity, also have conditioning functions, promote complement activation, strengthen the conditioning functions engulfed, suppress vasculogenesis, suppress a series of biological functions such as inflammatory mediator discharges, antimycotic and protozoon, in the natural defence of animal body, played very important effect.Having bioactive BPI is the natural inhibition of LPS, and LPS can the activated mononuclear mononuclear phagocyte system release cells factor (as TNFa, IL6, IL2 etc.), cause systemic inflammatory response, therefore BPI gene can remote effect partial immunity index level, be to zoologize the important candidate gene of breeding for disease resistance.
Both at home and abroad about the polymorphism of pig BPI gene and to the rarely seen report of the relevant research of the resistance/susceptibility of disease, in United States Patent (USP) net, there is one section of sequence about pig BPI gene, long 1452bp, clearly do not annotate, now by its called after patent BPI sequence (United States, Kind Code:A1, Patent Application:20040234980).In the public information of this patent, Christopher etc. are in York, hampshire, Du Luoke, long white, great Bai, wild boar and Meishan pig, detect that BPI gene extron 4 and 10 exists respectively Ava II and HpaII enzyme to cut polymorphism, show that by challenge test its genotype is relevant with the susceptibility of pig Salmonellas, and BPI gene is asserted to the sick breeding candidate gene of anti-salmonella.
Summary of the invention
The present invention is molecule marker and detection method and the application as the general disease resistance of pig by BPI gene extron 4 and 10 polymorphic site combination gene types.
The method that detects this molecule marker comprises extraction and the pcr amplification thereof of the pig genomic dna of testing sample, the PCR product of exon 4 is cut through the enzyme of restriction enzyme Hpa II through the PCR product of sscp analysis and exons 10, when pig idiotype to be measured is CDAB type (the PCR product of exon 4 occurs that through sscp analysis the PCR product of 4 bands and exons 10 cuts out existing 445bp/304bp/142bp band through restriction enzyme Hpa II enzyme), it is the individuality that general disease resistance is strong.Wherein, the primer A of exon 4 is as shown in SEQ ID NO.1 and SEQ ID NO.2; The primer B of exons 10 is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The present invention provides effective molecule marker for permanently improve the general disease resistance of piglet and immunne response ability hereditary predisposition.
Advantage applies of the present invention exists:
1) the present invention, for the molecular marker assisted selection of pig disease resistant breeding work provides a molecular genetic marker for efficiently and accurately more, provides a kind of effective molecular marker breeding means for improving the general disease resistance of piglet.The general disease resistance of pig is carried out to marker assisted selection by this genetic marker, pig resistance is started with and is carried out breeding for disease resistance, the immunne response ability that improves pig, can more effectively solve a lot of diseases of pig as the problem of respiratory tract disease and environmental interaction, fundamentally reduces the sickness rate of many diseases.
2) use the combination gene type of 2 variant sites to serve as a mark, gene pyramiding effect is not the simple addition of each site effect, combined pattern effect, higher than individual gene type effect, is improved the molecular breeding of piglet disease-resistant ability and is laid a good foundation for acceleration, will accelerate breeding process.
3) detection method of the present invention is simple to operate, and expense is comparatively cheap, and accuracy is high, and can realize the direct-detection of automatization, and the present invention will play a great role in the breeding of pig.
Brief description of the drawings
Fig. 1 is the sscp analysis figure of BPI gene the 4th exon pcr amplification product; Wherein the 1st swimming lane is DD genotype; 2-3 swimming lane is CC genotype; 4-5 swimming lane is CD genotype.
Fig. 2 is that BPI gene exon10 enzyme is cut rear 3 kinds of genotypic polyacrylamide gel electrophoresis figure; Wherein M is pUC19DNA/MSp I (HapII) marker, and the the the 2nd, 6,8,11,12 swimming lanes are AA genotype; 4th, 10 swimming lanes are BB genotype, and the the the 1st, 3,5,7,10 swimming lanes are AB genotype.
Embodiment
One, utilize BPI gene the 4th exon and exon10 variant sites mark to the too pig core group complete detection genotype of reviving.
Each individuality is adopted the about 1.0g of ear tissue piece, and it is for subsequent use that the Eppendoff pipe of putting into 1.5mL is fetched Yangzhou University's Animal Genetics, Breeding and Reproduction key discipline laboratory in ice chest.Phenol-chloroform method extracts DNA routinely.The DNA extracting measures its content and purity with nucleic acid-protein determinator, and-20 DEG C save backup.
Find 21 chromosomal mapview of pig by the BLAST of NCBI, through blast searchagainst pig sequences, look for one section of refseq sequence (nonredundant) BPI mRNA sequence EF436278 and select high-throughput test database (HTGS), thereby find a homology to reach 99% sequence, this section is the high-throughput cycle tests that comprises BPI complete sequence, sequence number is FP339579.2, but does not annotate.Then by the spidey on NCBI carry out nonredundant mRNA therewith high-throughput result comparison just can show that BPI has the full detail of 15 exons, then choose the sequences such as exon 4 and 10, utilize Primer Premier5.0 software to carry out design of primers (table 1).
Table 1BPI gene extron 4 and 10 design of primers
PCR reaction system: genomic dna (100ng μ L-1) 1.0 μ L, 10 × buffer B uffer(Mg2+) 2.0 μ L, dNTP mixture 1.5 μ L, the each 1.0 μ L of primer (10pmolL-1), Taq enzyme (5U μ L-1) 0.2 μ L, adds sterile purified water and complements to 20 μ L.Pcr amplification condition is: 94 DEG C of denaturation 5min, and 94 DEG C of sex change 30s, annealing 30s(annealing temperature is in table 1), 72 DEG C are extended 30s, carry out altogether 30 circulations, and then 72 DEG C are extended 8min, finally put into 4 DEG C and save backup.
Exon 4PCR-SSCP analyzes: carry out pcr amplification with the primer of design, 1% agarose gel electrophoresis, expanding fragment length with expect that clip size is consistent, and there is no non-specific band, can carry out sscp analysis.Get 7 μ L sample-loading buffers and 4 μ L PCR products mixed, 98 DEG C of sex change 15min, ice bath 10min.Then the sex change compound sample of ice bath is is all clicked and entered in 12% non-sex change polyacrylamine gel (Acr:Bis=29:1), 110V electrophoresis spends the night, and silver dyes colour developing.PCR-SSCP found that, BPI gene extron 4 detects 3 kinds of genotype, what be defined as respectively that 4 bands appear in CC, CD, DD(is CD type, and what occur the 1st and the 3rd this 2 band in the middle of 4 bands be CC type, and what occur the 2nd and the 4th middle this 2 band of 4 bands is DD type) (Fig. 1).
Exons 1 0PCR-RFLP analyzes: endonuclease reaction system is 10 μ L, comprises PCR product 3 μ L, restriction enzyme Hpa II (5U/ μ L) 0.2 μ L, 10 × Buffer, 1 μ L, distilled water 5.8 μ L, put after 37 DEG C of isothermal reaction 3h, dye analysis through 10% polyacrylamide gel electrophoresis silver.BPI gene extron 10 can, by complete degestion, produce AA type (445bp), BB type (304bp/142bp) and 3 kinds of bands of AB type (445bp/304bp/142bp) (Fig. 2) after the digestion of restriction enzyme Hpa II.
Two, the mensuration of the general anti-disease ability of pig (important cytokine)
To 98 35 age in days weanling pig precaval vein blood samplings, conventional separation of serum, uses pig ELISA test kit (R & D company, the U.S.) to carry out the mensuration of immunne response index.Process serum sample in strict accordance with test kit specification sheets, and according to explanation, standard substance are done to 5 concentration dilution gradients, detect absorbancy (OD value) at microplate reader 450nm wavelength place, then calculate the concentration of IL-2, IL-4, IL-6, IFN-beta, IL-10, IL-12 in serum according to typical curve.
Three, BPI gene the 4th, 10 exon polymorphisms and and general anti-disease ability between relationship analysis and resistance marker determine
Revive too PCR-SSCP and the PCR-RFLP banding pattern of pig BPI exon 4 and 10 are added up, analyzed combination genotype according to the banding pattern type of same individual BPI-4 and BPI-10, and carry out the calculating (table 2) of genotype and corresponding gene frequency
Table 2 revive too genotype frequency and the gene frequency (n=98) of pig BPI-4 and BPI-10
Note: χ
2 0.05(2)=5.991, χ
2 0.01(2)=9.21.
Respectively the different genotype to revive too pig BPI-4 and BPI-10 site and partial immunity index level carry out association analysis, result shows, the different genotype in BPI-4 site is on interleukin-6(IL6) there is a remarkably influenced, the different genotype in BPI-10 site is on interleukin-12(IL12) there is a remarkably influenced, the result of multiple comparisons shows, the IL6 level of the CD genotype individuality in BPI-4 site is significantly higher than CC and DD genotype individuality (P < 0.05), the IL12 level of the AB genotype individuality in BPI-10 site is significantly higher than AA and BB genotype individuality (P < 0.05, table 4).
The table 3 too impact of pig BPI-4 different genotype on immune indexes level of reviving
Letter is not all significant difference (P<0.05)
The table 4 too impact of pig BPI-10 different genotype on immune indexes level of reviving
Letter is not all significant difference (P<0.05)
Affect at single exon on the basis of immune indexes horizontal analysis, different genotype to BPI-4 and BPI-10 combines, utilize combined pattern analysis of variance model to analyze the impact of different combined patterns on immune indexes level, result shows that combined pattern is to IL2, IL6 and IL12 level have remarkably influenced (P < 0.05), as shown in Table 5, the immune indexes of BPI combination gene type and the pig too of reviving is same has certain dependency, the IL6 content of combination gene type CDAB is significantly higher than genotype CCAB(P < 0.05), IL12 content is significantly higher than genotype DDAA, and the IL6 content of combination gene type CDAB and IL12 content is highest level in 6 kinds of combination gene types, the genotypic IL6 content of CD and AB and IL12 content have been exceeded respectively simultaneously.
The table 5 too impact of pig BPI4-BPI10 combined pattern on immune indexes level of reviving
IL-2 can promote the propagation of T, B cell, aspect antitumor, toxinicide and immunomodulatory, is bringing into play important effect; IL-4 can induce the expression of MHC-class Ⅱ antigens, and strengthens it to tumour cell and parasitic lethal; IL-6 can promote the expression of IL-2 and acceptor thereof, in immune response regulation and hematopoiesis, plays an important role; IL-12 mainly works in cellular immunization and the cell-mediated antineoplastic immune of I type; IL-12 mainly promotes propagation and the lethal effect of T cell and NK cell, has vital role in antineoplastic immune, and IL-12 also can, by promoting the Differentiation and proliferation of Th1 cell, play a role in prevention malaria infects.The colony that the horizontal content of IL-6, IL12 is higher has higher disease resistance, effective genetic marker that therefore can be using CDAB as the general disease resistance of individuality.
Sequence table
Claims (1)
1. bactericidal power/permeability Enhancin gene extron 4 and 10 polymorphic site combination gene types application in molecular marker breeding as the general disease resistance genetic marker of pig; Specifically comprise the following steps:
(1) the ear tissue DNA of extraction sample to be tested, for subsequent use;
(2) pcr amplification: taking the DNA of step (1) as template, carry out respectively pcr amplification with following primer A and primer B,
Primer A:
Upstream primer is: 5 '-TCAGGTTGGTTACCGCAGAG-3 '
Downstream primer is: 5 '-ACCCTGTTGATGTGGCTTCT-3 '
Primer B:
Upstream primer is: 5 '-CCCAACATGGAGATGCAGTTC-3 '
Downstream primer is: 5 '-CAATGAATCAATGAGCACACC-3 ';
(3) pcr amplification product of primer A is analyzed by SSCP method, the pcr amplification product of primer B dyes analysis through polyacrylamide gel electrophoresis silver after cutting with restriction enzyme Hap II enzyme, selects primer A amplified production to occur that 4 bands and while primer B amplified production occur that the sample of 445bp/304bp/142bp band is the individuality that general disease resistance is strong.
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| US20040234980A1 (en) * | 2001-05-31 | 2004-11-25 | Tuggle Christopher K. | Genetic markers for improved disease resistance in animals (bpi) |
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Non-Patent Citations (2)
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| 11个猪群体BPI基因第10外显子Hpa II遗传变异分析;朱璟等;《遗传育种》;20111231;第47卷(第23期);摘要、第14页右栏-第15页左栏第2段 * |
| 猪BPI基因部分cSNPs检测及其对蛋白结构功能的影响;吴正常等;《畜牧兽医学报》;20131231;第44卷(第7期);1000-1007 * |
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