CN104357548A - Molecular marking method for indicating common premunition of sheep - Google Patents
Molecular marking method for indicating common premunition of sheep Download PDFInfo
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Abstract
The invention relates to the field of animal molecular genetics, and relates to the technology of genetics and breeding and marker-assisted selection of sheep. A molecular marking method for indicating common premunition of sheep comprises the following steps: designing a pair of primers according to the indoleamine 2,3-dioxygenase 1 (IDO1) gene sequence of sheep; extracting genomic DNAs (deoxyribonucleic acids) from the blood of the sheep, and utilizing the primer INF and the primer INR to carry out PCR (Polymerase Chain Reaction) amplification on the genomic DNAs of the sheep; using restriction enzyme Nhe I for digestion by PCR amplification products, carrying out electrophoretic separation on digestion products through sepharose gel, and carrying out genotype decision according to the result of the electrophoretic separation; constructing a genotype effect statistic model; dividing the group of the sheep into three types according to genotype, and selecting AA genotype individuals for grouping so as to complete molecular marking for the common premonition of sheep. The molecular marking method for indicating the common premonition of sheep is convenient in operation, economic and practical, and is more effective and accurate, so that the accuracy of strain selection can be improved.
Description
Technical field
The invention belongs to animal molecular genetics field, belong to sheep genetic breeding and Molecular Marker Assisted Selection Technology, be specifically related to screening and the application of the general disease resistance genetic marker of sheep, particularly relate to a kind of molecule marking method indicating the general disease resistance of sheep.
Background technology
Current, the main purpose of Sheep Breeding is according to specific breeding objective, and the specific production performances such as abundant lifting is meat, Mao Yong, to obtain maximum production economy benefit.Along with popularizing of intensive farm mode, and the raising of seed selection degree, sheep important economical trait is applied to the increasing of Selective Pressure, sheep disease resistance decreases, and causes the increase of disease incident, especially communicable disease.At present, vaccine inoculation and antibiotics treatment are mainly adopted to the measure of control of livestock and poultry, but, due to germ wide variety and variation is very fast, immunization, pharmacological agent, sanitary partition from etc. means of prevention can not fully effective control disease popular, antibiotic abuse also can cause animal products to carry a large amount of left drug, causes food contamination, detrimentally affect is produced to the health of the mankind, has a strong impact on Modern Animal Husbandry health, sustainable development.According to statistics, the financial loss caused by disease accounts for the 12-15% of the livestock industry output value, and this huge financial loss mainly causes due to sheep death, production efficiency reduction, medical expenses increase, overhead charges increase and product loss.Therefore, while cultivation high yield sheep variety, find that one safe and reliable and economically viable diseases prevention and treatment controlling soil moist has been the task of top priority.
The generation of disease is generally interactional result between the genetic background of animal and its environment, if sheep exists susceptibility at genetic aspect to some disease, the methods for the treatment of of employing may not cure the disease that envrionment conditions causes completely.When this, the effect of breeding for disease resistance just seems particularly important, and the disease resistance for sheep is carried out selection breeding and become a kind of more efficiently method replacing conventional treatment.Result of study finds different varieties, with the hereditary difference that there is disease resistance between kind Different Individual, this hereditary difference is caused by heritable variation, thus constitutes the hereditary basis that body selects disease resistance.Therefore, can select for sheep disease resistance by modern molecular genetic thremmatology method, to improve immunizing power and the disease resistance of body.
Disease resistance is also by the acting in conjunction of h and E, and disease resistance can be divided into general disease resistance and special disease resistance according to the difference of hereditary basis.General disease resistance is not limited to a certain pathogenic agent, and it is by polygene and the common impact of environment, and the antigenic specificity of pathogenic agent is very little on the impact of general disease resistance.Special disease resistance refers to the resistance of sheep to certain specified disease or pathogenic agent, mainly control by a major gene, simultaneously also may the impact by other gene locus (comprising controlling element) and environmental factors in various degree, special disease resistance can be measured by the direct challenge test of specific disease substance.Because general disease resistance embodies the total defense ability of body to disease, be the prerequisite that maintenance body is survived and produced in poor environment, therefore determine that the genetic marker of the general disease resistance of sheep has become the key factor of cultivating and having general disease resistance sheep.Research shows, the overall immunity function improving body can improve general disease resistance, and therefore, the inherent immunity of body just seems particularly important in the general disease resistance of raising.
Animal defines fairly perfect immunity system in long-term evolutionary process, and to resist infection and the invasion and attack of exotic disease pathogenic microorganism, simultaneously the cell of aging death in purged body, maintains stable machine state.Research shows, immunne response can be used as the indirect indexes of disease resistance, the individuality that immunizing power is strong shows stronger disease resistance, and multidirectional seed selection is carried out on the basis of clear and definite immune character hereditary property, is expected to cultivate the stronger sheep variety of general disease resistance (being).Cytokine plays important booster action in immune response, and the secretion level of cytokine is the important symbol of immune response ability and general disease resistance.Research shows, IFN-α and IFN-γ has immunomodulatory and antiviral, antineoplastic action, plays a significant role, directly can reflect the immunological status under animal standard state in the reaction of animal innate immunity.IgG is the immunoglobulin (Ig) that in humans and animals serum, content is the highest, and not only content is high but also the time length is long in animal body, can play the immunologic competences such as antibacterial, antiviral, toxinicide.IL-1 β is the cytokine that Monocyte-macrophages produces, and participates in immune response and inflammatory reaction.IL-2 is SCIF main, the strongest in body, it is the core substance in Organism immunoregulation network, in cell and humoral immunization, all there is important regulating effect, propagation and the differentiation of T cell and B cell can be promoted, promote the expression of cytokine profiles and acceptor thereof.IL-5 can promote B cell proliferation and differentiation, the synthesis of induction IgA.IL-6 can promote the expression of IL-2 and acceptor thereof, plays an important role in regulating in immunne response, acute phase reaction, hematopoiesis.IL-8 has chemotaxis and inflammatory reaction effect, chemotactic and activation neutrophil leucocyte and chemotactic basophilic granulocyte.IL-10 is an anti-inflammatory factor, participates in the body natural immunity and regulates, can suppress scavenger cell, Th1 cell secretion of cytokines, promote B cell proliferation and antibody tormation, promote thymus gland and proliferation of mast cells.IL-12 works in coordination with IL-2 and promotes cytotoxic T cell (TC), NK and LaK cytodifferentiation, induction Th1 cell, suppresses Th2 cell and promotes that B cell Ig produces, and works in cellular immunization and the cell-mediated antineoplastic immune of I type.
Indoleamine 2,3 dioxygenases (indoleamine-2,3-diogenase, IDO) are the enzymes that a kind of cell includes heme, being uniquely can indole ring oxicracking in catalysis tryptophan modules beyond liver, be along the catabolic rate-limiting enzyme of kynuric acid approach.In recent years, research finds, the express cell of IDO or its associated metabolites is mainly distributed in the T cell district of thymic medulla and secondary lymphatic organ, and expresses all specifically on scavenger cell or dendritic cell (dendritic cells, DC).IDO wide expression is in immunity system, comprise immunological tolerance or the immunologic privileged sites such as thymus gland, placenta, gastrointestinal mucosal, anterior chamber of the eye, epididymis, special on some inhibition antigen presenting cells up-regulated, as the subgroup of some mononuclear macrophage and dendritic cell (DC).In the research of transplantation immunity, IDO all has immunoregulation effect to T cell, B cell, NK cell, scavenger cell and dendritic cell.The expression of IDO can reduce the immune response of body; Cell protection and tissue grafts; participate in immunologic tolerance in allograft, in maintenance periphery immunological tolerance, autoimmune disorder, organ transplantation, tumour immunity and immunity of pregnancy, play important regulating effect.
IDO is distributed widely in people and other mammal extrahepatic tissues, and in low expression level under standard state, in inflammation or course of infection, the expression of IDO significantly increases.Cytokine interferon gamma (IFN-γ) is the strongest IDO induced expression thing, and tumor necrosis factor alpha (TNF-α), tumor necrosis factor β (TNF-β) and lipopolysaccharides (LPS) also can induce the generation of IDO to a certain extent.
At present, mainly carry out the correlative study of the resistance/susceptibility of mankind IDO gene polynorphisms and disease both at home and abroad, proved that IDO gene rs9657182 site is relevant with the dysthymia disorders that Caucasia humanIFN-α induces.IDO gene IVS3+562del C, IVS6+61G → A and IVS9+2431G → A site mutation is not the reason causing Iranian to repeat spontaneous abortion.The sudden change of IDO gene affects Treg function, can bring out autoimmune disorder.And the correlative study of the resistance/susceptibility of IDO gene polynorphisms and sheep disease is not reported.
In recent years, the Recent Progresses In The Development of gene SNP detection method and pertinent instruments is very fast, has reported and has established multiple SNP detection method.Taqman method is that probe mark cost is higher, causes testing cost also higher for SNP site variation design specific probe.DHPLC technology and mass spectroscopy all need to use high performance liquid chromatograph and mass spectrograph, apparatus expensive, are difficult to large-scale application.Single-strand conformation polymorphism analysis (SSCP) technology is difficult to the needs meeting automatization, is difficult to large-scale application.Allele specific oligonucleotide oligonucleotide hybridization (ASO) seldom uses at present.DNA chip is widely applied due to advantages such as high-throughputs in SNP detects, but when carrying out multidigit point and detect simultaneously condition be difficult to control, repeatable poor, easily there is false positive and false negative result.Micrometering sequence, as a kind of mutation analysis based on array, is also faced with the key issues such as the variation how reducing false negative rate and target nucleotide sequences composition at present.High temperature conjunction enzyme detection reaction (ligase detection reaction, LDR) is a kind of novel gene pleiomorphism detecting method in recent years, reliable results, but also needs further checking.SNaPshot method, based on the typing method of fluorescent mark single-basic extension principle, is applicable to 10-30 SNP site analysis usually.DNA sequencing method is relatively accurate, and price is more expensive.High resolving power melting curve method (HRM) is the SNP research tool the method for rising in recent years, and without the need to designing probe, cost is low, but result precision is lower.And restriction fragment length polymorphism (RFLP) detection accuracy is high, expense is low, is particularly suitable for the detection of unit point.Therefore, this patent, for IDO1 gene polynorphisms site, sets up the PCR-RFLP method for selecting molecular marker of the general disease resistance of a kind of energy Accurate Prediction sheep.
Summary of the invention
The object of the present invention is to provide a kind of easy to operate, economical and practical, more effectively, the molecule marking method of the general disease resistance of sheep accurately, for the raising general disease resistance of sheep and immunne response ability provide a kind of effective molecular marker breeding means, can assist-breeding adaptability and the strong high-quality kind sheep of disease resistance, accelerate breeding process, improve the accuracy of seed selection.
The present invention is achieved through the following technical solutions:
Indicate a molecule marking method for the general disease resistance of sheep, its feature mainly comprises the following steps:
(1) described according to sheep indoleamine 2,3-dioxygenase 1 (IDO1) gene order design pair of primers:
INF:5’-TGCTGCTGAATTTCTCCAGG-3’；
INR:5’-CACCTTGGATTGCCGGCTTGCAGGAATCATGATGTA
TAG-3’；
For meeting the detection analysis of force-RFLP, according to restriction enzyme Nhe I recognition sequence GCTAGC, force to introduce GC base mutation in downstream primer INR, namely AA two base mutations of IDO1 gene coding region 1076 and 1077 are GC base, the mutating alkali yl introduced shows with frame table, thus forms Nhe I forced-RFLP-PCR detection analysis.The primer I NF of IDO1 gene extron 9 is as shown in SEQ ID NO:1 in sequence table, and primer I NR is as shown in SEQ ID NO:2 in sequence table;
(2) from Blood In Sheep, extract genomic dna, utilize above-mentioned primer I NF and primer I NR to carry out pcr amplification to the genomic dna of sheep;
(3) pcr amplification product restriction enzyme Nhe I enzyme is cut, digestion products carries out electrophoretic separation through sepharose, genotype judgement is carried out according to electrophoretic separation result, the standard that genotype judges as: electrophoresis presents a band, size is 232bp, then undergo mutation in site, sheep IDO1 gene coding region 1072, and PCR primer can not being limited property restriction endonuclease Nhe I incision, by its called after AA genotype; Electrophoresis presents two bands, and size is 232bp and 191bp, then site, sheep IDO1 gene coding region 1072 is in heterozygous state, and PCR primer can not be cut by being limited property restriction endonuclease Nhe I completely, by its called after AG genotype; Electrophoresis presents a band, and size is 191bp, then site, sheep IDO1 gene coding region 1072 is without sudden change, and PCR primer can be cut, by its called after in GG genotype by being limited property restriction endonuclease Nhe I completely;
(4) experimentally Sheep Populations feature, build genotype effects statistical model: Y=μ+G+B+A+S+F+e, wherein μ is colony's average, G is genotype effects, and B is variety effect, and A is age effect, S is sex-effects, and F is the other effect in field, and e is random error; Utilize JMP4.0 statistical software that genotype effects and sheep immune indexes proterties are carried out association analysis, and estimate the least square average of proterties, analytical results shows, IFN-α, IL-2 and IL-12 level pole significant correlation (P<0.01) in the genotype effects that IDO1 gene coding region 1072 site mutation causes and sheep peripheral blood; The result of multiple comparisons shows, IFN-α and the IL-2 water mean pole of AA and AG genotype individuals are significantly higher than GG genotype individuals, and the IL-12 level pole of AA genotype individuals is significantly higher than AG and GG genotype individuals;
(5) according to genotype, Sheep Populations is divided into three types, selects AA genotype individuals to carry out cohort, thus realize the molecular mark of the general disease resistance of sheep, namely complete the molecule marking method of the general disease resistance of sheep;
Wherein in step (2), pcr amplification condition is 94 DEG C of sex change 5min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations; 72 DEG C extend 5min; 4 DEG C of insulations.
The base sequence that wherein in step (3), restriction enzyme Nhe I identifies is GCTAGC.
Described agarose gel electrophoresis is separated digestion products, and the sepharose concentration used is 2.5%-3%.
The present invention is for providing effective molecule marker from the breeding for disease resistance aspect raising general disease resistance of sheep and immunne response.
Advantage applies of the present invention exists:
(1) the present invention be the molecular marker assisted selection of sheep breeding for disease resistance work provide one more efficient, molecular genetic marker accurately, a kind of effective molecular marker breeding means are provided for improving the general disease resistance of sheep, by this genetic marker, marker assisted selection is carried out to the general disease resistance of sheep, carry out the breeding for disease resistance of sheep, improve the immunne response ability of sheep, more effectively can reduce the generation of sheep heredopathia, reduce the sickness rate of many diseases, improve the environmental compatibility of sheep, increase and herd and the sheep survival rate under confined farming condition.
(2) use 1 variant sites as mark, application forced PCR-RFLP is main detection method, have that cost is low, practical, accuracy is high, more easy-operating advantage, for the disease-resistant molecular genetic breeding work accelerating to improve sheep is laid a good foundation, thus accelerate breeding process.
(3) detection method of the present invention is simple to operate, and testing cost is low, and accuracy is high, and can realize the direct-detection of automatization, and the present invention plays a significant role in the breeding for disease resistance of sheep.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of IDO1 gene PCR product; Wherein M is pUC19DNA/Msp I (Hpa II) marker, C swimming lane is blank; 1-7 swimming lane is the individual IDO1 gene PCR product of different sheep.
Fig. 2 is that IDO1 gene PCR product is cut through restriction enzyme Nhe I enzyme, the agarose gel electrophoresis figure of digestion products; Wherein M is pUC19DNA/Msp I (Hpa II) marker, the 1st, 3,7,9,12, and 15 swimming lanes are GG genotype; 4th, 8,10,14 swimming lanes are AG genotype; 2nd, 5,6,11,13,16 swimming lanes are AA genotype.
Embodiment
Embodiment: embodiment is described in more detail the present invention below:
One, the detection analysis in sheep IDO1 gene the 9th Varia nt in exon site
1, sample collecting
Disposable syringe is adopted to extract the blood of about 1mL from sheep jugular vein, inject through autoclaving and the aseptic EDTA of about 200 μ L2% (Ethylene diamine tetraacetic acid is housed, in the 1.5mL centrifuge tube of EDTA) antithrombotics, shake up gently, record sheep number ,-20 DEG C save backup.
2, medicine and enzyme
Tutofusin tris (Tris), Sigma Chemicals Co; The saturated phenol of Tris, EDTA-Na
2ancient cooking vessel state biotech development center, Beijing; Proteinase K (Proteinase K), MERCK Co; DNTP (dATP; DTTP; DCTP; DGTP), Taq enzyme (supporting 10 × Taq buffer, 25mmol/L Mg
2+), the precious biotech firm in Dalian; Agarose (Agarose H), pUC19DNA/Msp (Hpa II) Marker, Sangon Biotech (Shanghai) Co., Ltd.; Restriction enzyme Nhe I, Fermentas (MBI) company.
3, key instrument
Icycler PCR instrument (Bio-Rad company), Gel2000 gel imaging system, Ultrospec1000 ultraviolet spectrophotometer, Sigma3K30 high speed freezing centrifuge, Milli-Q ultrapure water instrument, Bio-Rad voltage stabilizing electrophoresis apparatus and supporting electrophoresis chamber.
4, the Design and synthesis of primer
According to sheep IDO1 gene (GenBank accession number: NM_001141953) the 9th exon sequence and restriction enzyme Nhe I recognition sequence GCTAGC, design upstream primer INF and downstream primer INR, primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd.:
INF:5’-TGCTGCTGAATTTCTCCAGG-3’；
INR:5’-CACCTTGGATTGCCGGCTTGCAGGAATCATGATGTAGCTAG-3’。
5, a small amount of of ovine genome DNA is extracted
Method one:
(1) get 20 μ L anticoagulated bloods, add 500 μ L hemocyte lysates, adding Proteinase K to final concentration is 100-200 μ g/mL, mixes 55 DEG C of digestion 12h, until no longer include the agglomerate of thickness in solution.
(2) solution is cooled to room temperature, adds 5M NaCl to final concentration 1.5M, mixing 10min.Add equal-volume phenol/chloroform, repeatedly put upside down centrifuge tube mixing 10min.
(3) 12,000rpm, the centrifugal 10min of room temperature.Get supernatant, add equal-volume chloroform mixing 10min.
(4) 12,000rpm, the centrifugal 10min of room temperature.Get supernatant 2 times of volume dehydrated alcohol precipitation DNA.
(5) DNA is chosen be put in 1.5mL centrifuge tube, wash 1 time with 70% ethanol.
(6) 7,500rpm, the centrifugal 5min of room temperature, abandons supernatant.
(7) will be dissolved in after DNA drying in 200 μ L TE.
Method two:
(1) 20 μ L whole bloods are added in the 1.5mL centrifuge tube that 700 μ L1 × SET are housed, mix gently.
(2) SDS of Proteinase K (10mg/mL) to final concentration 100-200 μ g/ μ L and 10% is added to final concentration 0.5%, 55 DEG C of digestion 12h.
(3) after digestion completely, add the saturated phenol of isopyknic Tris, put upside down back and forth, make it mix.
The centrifugal 10min of (4) 12,000rpm, carefully moving into upper strata aqueous phase in another centrifuge tube with cutting off most advanced and sophisticated suction nozzle, discarding organic phase.Repeat the third and fourth step once.
(5) in aqueous phase, isopyknic phenol, chloroform, primary isoamyl alcohol mixed solution (volume ratio is 24:23:1) is added, mixing 10min.12,000rpm, centrifugal 10min, shift out aqueous phase to another centrifuge tube.
(6) in aqueous phase, add isopyknic chloroform, primary isoamyl alcohol mixed solution (23:1), put upside down mixing 10min, 12,000rpm back and forth, centrifugal 10min, shifts out aqueous phase to another centrifuge tube.
(7) in aqueous phase, add the dehydrated alcohol of 1/10 volume NaAc (3M, pH5.2) and 2 times volume, put upside down back and forth, precipitation DNA.
(8) DNA is chosen be put in 1.5mL centrifuge tube, wash 1 time with 70% ethanol.
The centrifugal 5min of (9) 7,500rpm.Carefully outwell ethanol in pipe, will be upside down on filter paper, allow ethanol flow to end, be placed in air drying.
(10) add the TE of 200 μ L, put in 50 DEG C of water-baths the dissolving DNA that spends the night.Be stored in after dissolving-20 DEG C for subsequent use.
Method three:
Operate according to Ezup pillar genome DNA extraction test kit specification sheets (SK8252) of Sangon Biotech (Shanghai) Co., Ltd. or the genome DNA extracting reagent kit specification sheets of other biological technology company.
6, PCR reaction
(1) with ovine genome DNA for template carries out pcr amplification, goal gene PCR primer size is 232bp.In pcr amplification product, ovine genome sequence is as shown in SEQ3.
Following solution or reagent is comprised in 25 μ L reaction systems:
(2) above-mentioned solution mixed and carry out PCR reaction by following condition.
94 DEG C of sex change 5min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations; 72 DEG C extend 5min.
(3), after reaction terminates, use 2% sepharose to carry out electrophoresis detection to PCR primer (5 ~ 10 μ L), electrophoresis result is shown in Fig. 1.
7, the rflp analysis of IDO1 gene
Application Fermentas (MBI) restriction enzyme Nhe I carries out enzyme and cuts, and the enzyme system of cutting is 10 μ L, reaction system and condition as follows:
Above-mentioned reaction solution is mixed, in 37 DEG C of waters, digests 3 ~ 5h, after digestion, total overall reaction liquid is carried out 2.5% agarose gel electrophoresis, detect PCR primer enzyme and cut effect and segment polymorphism.
8, the analysis in IDO1 genovariation site
The PCR primer of IDO1 gene 232bp is cut through restriction enzyme Nhe I enzyme, be separated through 2.5% agarose gel electrophoresis, detect the base transition variant sites C.1072G>A in IDO1 gene extron, according to restriction enzyme digestion and electrophoresis collection of illustrative plates, different genotype sheep individuality is named, be respectively AA genotype (232bp), AG genotype (232bp and 191bp) and GG genotype (191bp) three kinds of genotype (Fig. 2).
Two, the mensuration of the general anti-disease ability of sheep:
Gather 343 10 monthly age lamb precaval vein blood 2mL of different varieties health, wherein Suffolk 62 (26 ♂, 36 ♀), polyembryony Suffolk 118 (36 ♂, 82 ♀), Kazakh sheep 64 (25 ♂, 39 ♀), sheep 59 (13 ♂, 46 ♀), polyembryony Suffolk and Kazakh sheep F1 hybrid lamb 40 are (hereinafter referred to as Saha F1; 7 ♂, 33 ♀).Ordinary method separation of serum, in the centrifugal 15min of 2500r/min, gets supernatant packing.Application sheep IgG, IFN-α, IFN-γ, IL-1 β, IL-2, IL-5, IL-6, IL-8, IL-10 and IL-12ELISA detection kit (upper sea blue base bio tech ltd, China) carry out immunne response index determining, in strict accordance with test kit specification sheets process serum sample, standard substance are done 5 concentration dilution gradients, at microplate reader (Model550, U.S. Bio-Rad) 450nm wavelength place detection absorbancy (OD value), then IgG in serum is calculated according to typical curve, IFN-α, IFN-γ, IL-1 β, IL-2, IL-5, IL-6, IL-8, the concentration of IL-10 and IL-12.
Three, sheep IDO1 gene the 9th exon polymorphism and and general anti-disease ability between relationship analysis and resistance marker determine.
Respectively statistical study is carried out to Suffolk, polyembryony Suffolk, Kazakh sheep, sheep and Saha F1 sheep IDO1 gene the 9th exon PCR-RFLP banding pattern, and carry out the calculating (table 1) of genotype frequency and gene frequency.
The genotype frequency in table 1 sheep IDO1 gene C .1072G>A site and gene frequency
Note: χ
2 0.05(2)=5.991, χ
2 0.01(2)=9.21.
Experimentally Sheep Populations feature, build genotype effects statistical model: Y=μ+G+B+A+S+F+e, wherein μ is colony's average of proterties, G is genotype effects, and B is variety effect, and A is age effect, S is sex-effects, and F is the other effect in field, and e is random error.Utilize JMP4.0 statistical software to carry out association analysis to the different genotype effect of sheep IDO1 gene and partial immunity index character respectively, and estimate the least square average of proterties.Analytical results shows (see table 2), IFN-α, IL-2 and IL-12 level pole significant correlation (P<0.01) in the genotype effects that IDO1 gene coding region 1072 site mutation causes and sheep peripheral blood, does not cause the noticeable change of IFN-γ, IgG, IL-6, IL-1 β, IL-5, IL-8 and IL-10 content in peripheral blood.The result of multiple comparisons shows, IFN-α and the IL-2 water mean pole of AA and AG genotype individuals are significantly higher than GG genotype individuals (P<0.01), and the IL-12 level pole of AA genotype individuals is significantly higher than AG and GG genotype individuals (P<0.01).
Table 2 sheep IDO1 gene different genotype is on the impact of immune indexes
Note: colleague's lowercase difference represents that difference extremely significantly (P<0.01).
IFN-α has immunomodulatory and antiviral, antineoplastic action, plays a significant role, directly can reflect the immunological status under animal standard state in the reaction of animal innate immunity.IL-2 is SCIF main, the strongest in body, it is the core substance in Organism immunoregulation network, in cell and humoral immunization, all there is important regulating effect, propagation and the differentiation of T cell and B cell can be promoted, promote the expression of cytokine profiles and acceptor thereof.IL-12 can work in coordination with IL-2 and promote cytotoxic T cell (TC), NK and LaK cytodifferentiation, induction Th1 cell, suppresses Th2 cell and promotes that B cell Ig produces, and works in cellular immunization and the cell-mediated antineoplastic immune of I type.The colony that in sheep peripheral blood, IFN-α, IL-2 and IL-12 level content is higher has higher general disease resistance, therefore, IDO1 gene can be used as one of main candidate of the general disease resistance of sheep, AA genotype can be used as the individual effective genetic marker of general disease resistance, for predicting and identify the sheep that general disease resistance is stronger, and can be used for the stronger Sheep Populations of the general disease resistance of fast assembling.
Claims (5)
1. indicate a molecule marking method for the general disease resistance of sheep, its feature mainly comprises the following steps:
(1) according to sheep indoleamine 2,3-dioxygenase 1 (IDO1) gene order design pair of primers:
INF:5’-TGCTGCTGAATTTCTCCAGG-3’；
INR:5’-CACCTTGGATTGCCGGCTTGCAGGAATCATGATGTAGCTAG-3’；
(2) from Blood In Sheep, extract genomic dna, utilize above-mentioned primer I NF and primer I NR to carry out pcr amplification to the genomic dna of sheep;
(3) pcr amplification product restriction enzyme Nhe I enzyme is cut, digestion products carries out electrophoretic separation through sepharose, genotype judgement is carried out according to electrophoretic separation result, the standard that genotype judges as: electrophoresis presents a band, size is 232bp, then undergo mutation in site, sheep IDO1 gene coding region 1072, and PCR primer can not being limited property restriction endonuclease Nhe I incision, by its called after AA genotype; Electrophoresis presents two bands, and size is 232bp and 191bp, then site, sheep IDO1 gene coding region 1072 is in heterozygous state, and PCR primer can not be cut by being limited property restriction endonuclease Nhe I completely, by its called after AG genotype; Electrophoresis presents a band, and size is 191bp, then site, sheep IDO1 gene coding region 1072 is without sudden change, and PCR primer can be cut, by its called after in GG genotype by being limited property restriction endonuclease Nhe I completely;
(4) experimentally Sheep Populations feature, build genotype effects statistical model: Y=μ+G+B+A+S+F+e, wherein μ is colony's average, G is genotype effects, and B is variety effect, and A is age effect, S is sex-effects, and F is the other effect in field, and e is random error; Utilize JMP4.0 statistical software that genotype effects and sheep immune indexes proterties are carried out association analysis, and estimate the least square average of proterties, analytical results shows, IFN-α, IL-2 and IL-12 level pole significant correlation (P<0.01) in the genotype effects that IDO1 gene coding region 1072 site mutation causes and sheep peripheral blood; The result of multiple comparisons shows, IFN-α and the IL-2 water mean pole of AA and AG genotype individuals are significantly higher than GG genotype individuals, and the IL-12 level pole of AA genotype individuals is significantly higher than AG and GG genotype individuals;
(5) according to genotype, Sheep Populations is divided into three types, selects AA genotype individuals to carry out cohort, thus realize the molecular mark of the general disease resistance of sheep, namely complete the molecule marker of the general disease resistance of sheep.
2. the molecule marking method of the general disease resistance of indication sheep as claimed in claim 1, is characterized in that wherein the middle pcr amplification condition of step (2) is 94 DEG C of sex change 5min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations; 72 DEG C extend 5min; 4 DEG C of insulations.
3. the molecule marking method of the general disease resistance of indication sheep as claimed in claim 1 or 2, is characterized in that the base sequence that wherein the middle restriction enzyme Nhe I of step (3) identifies is GCTAGC.
4. the molecule marking method of the general disease resistance of indication sheep as claimed in claim 1 or 2, it is characterized in that described agarose gel electrophoresis is separated digestion products, the sepharose concentration used is 2.5% ~ 3%.
5. the molecule marking method of the general disease resistance of indication sheep as claimed in claim 3, it is characterized in that described agarose gel electrophoresis is separated digestion products, the sepharose concentration used is 2.5% ~ 3%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104928386A (en) * | 2015-06-21 | 2015-09-23 | 甘肃农业大学 | Molecular marker from RPS23 gene for sheep immune traits and its application |
| CN114058714A (en) * | 2021-12-03 | 2022-02-18 | 新疆畜牧科学院畜牧业质量标准研究所（新疆维吾尔自治区种羊与羊毛羊绒质量安全监督检验中心） | Goat hair color related molecular marking method and application thereof |
| CN117051132A (en) * | 2023-10-11 | 2023-11-14 | 中国农业科学院北京畜牧兽医研究所 | Sheep SNP molecular marker and application thereof in sheep brucellosis resistance character detection |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928386A (en) * | 2015-06-21 | 2015-09-23 | 甘肃农业大学 | Molecular marker from RPS23 gene for sheep immune traits and its application |
| CN104928386B (en) * | 2015-06-21 | 2017-11-21 | 甘肃农业大学 | Molecular labeling and its application of the RPS23 genes as sheep immune character |
| CN114058714A (en) * | 2021-12-03 | 2022-02-18 | 新疆畜牧科学院畜牧业质量标准研究所（新疆维吾尔自治区种羊与羊毛羊绒质量安全监督检验中心） | Goat hair color related molecular marking method and application thereof |
| CN117051132A (en) * | 2023-10-11 | 2023-11-14 | 中国农业科学院北京畜牧兽医研究所 | Sheep SNP molecular marker and application thereof in sheep brucellosis resistance character detection |
| CN117051132B (en) * | 2023-10-11 | 2024-01-30 | 中国农业科学院北京畜牧兽医研究所 | Sheep SNP molecular marker and application thereof in sheep brucellosis resistance character detection |
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