CN102154266A - Chicken heat resistance-associated molecular marker and identification method and use thereof - Google Patents
Chicken heat resistance-associated molecular marker and identification method and use thereof Download PDFInfo
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- CN102154266A CN102154266A CN2010102833454A CN201010283345A CN102154266A CN 102154266 A CN102154266 A CN 102154266A CN 2010102833454 A CN2010102833454 A CN 2010102833454A CN 201010283345 A CN201010283345 A CN 201010283345A CN 102154266 A CN102154266 A CN 102154266A
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Abstract
The invention discloses a chicken heat resistance-associated molecular marker and an identification method and use thereof. The molecular marker is G-141A which is positioned in a 5' flanking region in a chicken heat stress protein 90-beta gene. In the invention, the polymerase chain reaction (PCR) amplification of the 5' flanking region in the chicken heat stress protein 90-beta gene, the product of the amplification is subjected to sequencing and single nucleotide polymorphism (SNP) screening, the screened SNP is subjected to PCR-restriction fragment length polymorphism(RFLP) in a large population, the obtained typing result and the heat resistance property index of the population are subjected to association analysis, an SNP locus which is associated obviously is initially determined as heat resistance property auxiliary selection molecular marker, and chicken of a GG genotype has high heat resistance in a high-temperature environment.
Description
Technical field
The present invention relates to genetically engineered and genetic breeding field, be specifically related to a kind of molecule marker relevant and authentication method and application with the chicken thermotolerance.
Background technology
Molecular marker assisted selection is the important application of genetically engineered in modern poultry breeding, pass through molecular marker method, can not only shorten the breeding time limit, significantly reduce the manpower and materials consumption of breeding, the diversity of molecule marker more makes the application potential of molecule marker in improvement of breed improve greatly in addition.High temperature season causes that easily chicken produces heat stress, causes growing slowly, and immunizing power reduces, and M ﹠ M raises, and produces for raising chickens of intensification and brings enormous economic loss.Mainly take aggregate measures such as aeration-cooling, diet regulating and controlling to alleviate the thermal stress reaction of chicken in producing at present, but this can not fundamentally solve the influence of heat stress to present poultry farming.Therefore, it is one of important research direction of chicken genetic breeding from now on that approach by breeding improves individual hot adaptability itself, the difficulty of measuring in view of the chicken resistance toheat is big, cost is high, and adopting the method for molecular marker assisted selection is the important means that reaches this purpose.
Heat stress resistance Candidate Gene Study mainly concentrates on heat stress proteins family (HSP) at present, difference according to the molecular weight size, heat stress proteins family can be divided into HSP110, HSP90, HSP70, HSP60, HSP47 and small molecules HSP, and these albumen are respectively by the gene transcript expression of correspondence.Wherein by the heat stress proteins 90 Chang Zuowei molecular chaperoneses of Hsp90 gene transcript expression, in bringing into normal play the process of function, signal transducer works.HSP90 when cell generation stress reaction, can with those owing to the protein-interacting that environmental stimulus changes self conformation, it is suitable folding and prevent the albumen non-specific aggregation to guarantee that albumen carries out, thereby keeps the normal activity of cell.HSP90 β is a kind of of HSP90, is the necessary albumen of cell normal growth, and the expression amount in the heat stress process in heart, liver, renal tissue is extremely significantly to be increased.
At present at the stable on heating molecule marking research of chicken seldom, the thermotolerance molecule marker that finds at the 5 ' flanking region of chicken Hsp90-b of the present invention belongs to first and finding.
Summary of the invention
The objective of the invention is to according to the deficiencies in the prior art, a kind of molecule marker relevant with the chicken thermotolerance is provided.
Another purpose of the present invention is to provide the authentication method of the above-mentioned molecule marker relevant with the chicken thermotolerance.
A further object of the invention is to provide the application of the above-mentioned molecule marker relevant with the chicken thermotolerance.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
In the poultry breeding chicken thermotolerance is difficult to carry out effectively directly select at present.The present invention is by analyzing the influence of chicken heat shock protein 90-beta gene (Hsp90-b) 5 ' flanking region G-141A site different genotype to the important heat-resisting proterties of chicken under the hot conditions, adopt Econ1-RFLP to carry out polymorphism and differentiate, for the thermotolerance that improves chicken provides an effective molecule marker.
For achieving the above object, the present invention adopts following technical measures:
A kind of molecule marker G-141A relevant with the chicken thermotolerance is positioned at 5 ' flanking region of chicken heat shock protein 90-beta gene.
A kind of method of utilizing molecule marker G-141A that the chicken thermotolerance is identified comprises the steps:
(1) extracting of chicken genomic dna;
(2) pcr amplification of 5 ' flanking region of chicken heat shock protein 90-beta gene;
(3) amplified production enzyme cutting type.
Extracting chicken genomic dna is extractive from chicken blood described in the step (1), and chicken blood is anticoagulation, and antithrombotics is EDTA or heparin sodium.Wherein, the extracting of chicken blood genomic dna comprises following concrete steps:
30ul anticoagulation+470ul 1 * SET+12.5ul 20% SDS+5ul Proteinase K mixing, 55 ℃ of water-bath digested overnight (annotating: add in order).
Add the saturated phenol 500ul of Tris, shake up, centrifugal.
Get supernatant in clean 1.5ml centrifuge tube, add the saturated phenol of 500ul Tris, shake up, centrifugal.
Get supernatant in clean 1.5ml centrifuge tube, add 500ul chloroform-primary isoamyl alcohol (23:1), shake up, centrifugal.
Get supernatant in clean 1.5ml centrifuge tube, add dehydrated alcohol (20 ° of C) 1000ul, shake up, place 20～30min in-20.
Abandon supernatant, add 1000ul 75% ethanol, shake up, centrifugal.
Abandon supernatant, oven dry adds an amount of TE, and 55 ° of C water-baths are spent the night.
As a kind of preferred version, the used primer sequence of pcr amplification described in the step (2) is shown in SEQ ID NO:1 ~ 2; The reaction system of described pcr amplification is that reaction system is 50 ~ 100ul, Mix accounts for reaction system 50 volume %, each 10 ~ 20pmol of upstream primer and downstream primer, and the Taq enzyme accounts for reaction system 0.75 volume %, chicken genomic dna 0.1 ~ 1ug adds the sterilization distilled water to reacting cumulative volume.
The response procedures of described pcr amplification is:
As a kind of preferred version, the endonuclease reaction system of enzyme cutting type described in the step (3) is PCR product 8 ~ 10 μ L, restriction endonuclease 3 ~ 5U, 10 * damping fluid is 2 times of restriction endonuclease volume, 37 ℃ of water-baths are spent the night, agarose gel electrophoresis detection enzyme with 2.5wt% is cut product, judges individual genotype according to electrophoretic band, and wherein the AA genotype is the band of 498bp; The GG genotype is two bands that are respectively 291bp and 207bp; The AG genotype is three bands that are respectively 498bp, 291bp and 207bp.Described restriction endonuclease is
Econ 
Compared with prior art, the present invention has following beneficial effect:
It is simple that the method applied in the present invention has condition, and price is low, advantages such as confidence level height.Therefore, the present invention is used to select hot adaptability to plant chicken preferably in the breeding to have vital role at the scene.
Description of drawings
Fig. 1 is site G-141A restriction enzyme digestion and electrophoresis figure, and wherein M is DL2000 Marker, is respectively 2000,1000,750,500,250 from top to bottom, 100bp.
Embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
Embodiment
Respectively indoor environment temperature when being 35 ℃ and 15 ℃ under the wing the yellow blood of 333 short pin of vein collection be distributed into three pipes: non-anticoagulation 1～2mL, serum is used to measure T3 and Kendall compound; Anticoagulation 1～2mL is used to measure H/L, and CD
3+, CD
4+Anticoagulation 1-2ml is used for the extracting of genomic dna.The direct centrifugal absorption serum of wherein non-anticoagulation utilizes enzyme-linked immunosorbent assay T3 and Kendall compound; Anticoagulation is used to measure H/L, dyes compound staining with Rui Shi and Jim Sa blood smear is dyeed, and microscopy carries out cell divide by the leukocyte differential count method of Alfred, and counts and calculate by the method for Campo; CD
3+, CD
4+Utilize flow cytometer to detect.
The genomic dna of 666 blood samples of extracting, each sample is got 10ul, makes each sample DNA diluent respectively, is template with the DNA diluent, prepares reaction system according to the PCR reaction system of applying for a patent explanation, and carries out pcr amplification by the respective reaction program.Carry out enzyme cutting type through the good product of detected through gel electrophoresis amplification, enzyme is cut design sketch such as Fig. 1, and enzyme is cut product and detected 3 kinds of genotype, is respectively GG(291bp and 207bp), AG(498bp, 291bp and 207bp) and AA(498bp); The heat-resisting proterties of somatotype result and mensuration is carried out correlation analysis, analytical results such as table 1:
Table 1 site G-141A and heat-resisting proterties association analysis result
Annotate: the different lowercase alphabet differentials of same column different significantly (P＜0.05) in the same site in the table, different capitalizations are represented difference extremely significantly (P＜0.01).
(15 ℃) G-141A and CD under the normal temperature state
3+Significant correlation (P＜0.05), the AA genotype is significantly higher than GG and GA genotype; G-141A and H/L significant correlation (P＜0.05), the GG genotype significantly is lower than the GA genotype.(35 ℃) G-141A and T3 utmost point significant correlation (P＜0.01) under the stress situation, the AA genotype utmost point is significantly higher than GG and GA genotype.
Within the specific limits, T3 is high more, and the thermotolerance of body is poor more.In the present case: behind the heat stress, T3 obviously raises, polymorphic site G-141A and T3 utmost point significant correlation (P＜0.01), AA genotype (45.96 ± 8.93) utmost point is significantly higher than GG(17.48 ± 3.04) and GA(19.69 ± 3.52) genotype, the GA utmost point is significantly higher than GG.Show that GG genotype chicken has better heat-resisting.
Within the specific limits, H/L is low more, and body resistance is high more.In the present case, under the normal temperature state, G-141A and H/L significant correlation (P＜0.05), GG genotype (0.50 ± 0.04) significantly is lower than GA genotype (0.62 ± 0.04) and AA genotype (0.63 ± 0.10).
CD3 is relevant with body's immunity, the low more disease resistance that is unfavorable for body more of its value, and in the present case behind the heat stress, peripheral blood CD
3+The T cell content obviously descends, illustrate body be subjected to temperatures involved after immune level reduce.(15 ℃) G-141A and CD under the normal temperature state
3+Significant correlation (P＜0.05), AA genotype (4.36 ± 0.46) is significantly higher than GG(3.36 ± 0.17) and GA genotype (3.29 ± 0.20).Therefore, although AA genotype immunizing power is significantly higher than GG and GA genotype, but because AA genotype number of individuals seldom, not representative, though and the GG genotype is not remarkable with GA genotype differences, but the GG genotype is higher than the GA genotype, so the immunizing power of GG genotype chicken still might be than the genotypic chicken height of GA.
SEQUENCE?LISTING
＜110〉Agricultural University Of South China
＜120〉a kind of molecule marker relevant and authentication method and application with the chicken thermotolerance
<130>
<160> 2
<170> PatentIn?version?3.2
<210> 1
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 1
ggtcgcgtgg?aactctctgg?aa 22
<210> 2
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 2
gtcggaggcg?ttggagatga?g 21
Claims (9)
1. a molecule marker relevant with the chicken thermotolerance is characterized in that described molecule marker is G-141A, is positioned at 5 ' flanking region of chicken heat shock protein 90-beta gene.
2. the authentication method of the described molecule marker relevant with the chicken thermotolerance of claim 1 is characterized in that comprising the steps:
(1) extracting of chicken genomic dna;
(2) pcr amplification of 5 ' flanking region of chicken heat shock protein 90-beta gene;
(3) amplified production enzyme cutting type.
3. according to the authentication method of the described molecule marker relevant with the chicken thermotolerance of claim 2, it is characterized in that extracting chicken genomic dna is extractive from chicken blood described in the step (1), chicken blood is anticoagulation, and antithrombotics is EDTA or heparin sodium.
4. according to the authentication method of the described molecule marker relevant of claim 2, it is characterized in that the used primer sequence of pcr amplification described in the step (2) is shown in SEQ ID NO:1 ~ 2 with the chicken thermotolerance.
5. according to the authentication method of the described molecule marker relevant of claim 2 with the chicken thermotolerance, the reaction system that it is characterized in that pcr amplification described in the step (2) is 50 ~ 100ul, Mix accounts for reaction system 50 volume %, each 10 ~ 20pmol of upstream primer and downstream primer, the Taq enzyme accounts for reaction system 0.75 volume %, chicken genomic dna 0.1 ~ 1ug adds the sterilization distilled water to reacting cumulative volume.
6. according to the authentication method of the described molecule marker relevant of claim 2, it is characterized in that the response procedures of pcr amplification described in the step (2) is with the chicken thermotolerance:
。
7. according to the authentication method of the described molecule marker relevant of claim 2 with the chicken thermotolerance, the endonuclease reaction system that it is characterized in that enzyme cutting type described in the step (3) is PCR product 8 ~ 10 μ L, restriction endonuclease 3 ~ 5U, 10 * damping fluid is 2 times of restriction endonuclease volume, 37 ℃ of water-baths are spent the night, agarose gel electrophoresis detection enzyme with 2.5wt% is cut product, judges individual genotype according to electrophoretic band, and wherein the AA genotype is the band of 498bp; The GG genotype is two bands that are respectively 291bp and 207bp; The AG genotype is three bands that are respectively 498bp, 291bp and 207bp.
8. according to the authentication method of the described molecule marker relevant of claim 7, it is characterized in that described restriction endonuclease is with the chicken thermotolerance
Econ 
9. described molecule marker the application in the screening of thermotolerance chicken breed relevant of claim 1 with the chicken thermotolerance.
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| CN104342490A (en) * | 2013-08-02 | 2015-02-11 | 中国农业大学 | Method for detecting chicken beard traits |
| KR20150055582A (en) * | 2013-11-11 | 2015-05-21 | 경상대학교산학협력단 | Biomaker gene and diagnostic composition specific for environmental stress of poultry, and Prediction method of stress possibility, egg production using the same |
| CN110643717A (en) * | 2019-10-28 | 2020-01-03 | 东北农业大学 | Molecular marking method for predicting and identifying cock heart growth and development |
| CN112189628A (en) * | 2020-10-21 | 2021-01-08 | 福建农林大学 | Method for judging cold and heat property degree of plant food |
| CN114831076A (en) * | 2022-05-19 | 2022-08-02 | 华中农业大学 | Method and device for judging heat resistance of chicken |
| CN114875161A (en) * | 2022-06-16 | 2022-08-09 | 江苏省家禽科学研究所 | Molecular marker related to low-temperature tolerance of chicken, primer combination and corresponding breeding method |
| CN114921570A (en) * | 2022-06-02 | 2022-08-19 | 江苏省家禽科学研究所 | Molecular marker related to high-temperature tolerance of chicken and application thereof |
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2010
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| CN104342490B (en) * | 2013-08-02 | 2016-12-28 | 中国农业大学 | A kind of method detecting chicken beard character |
| CN104342490A (en) * | 2013-08-02 | 2015-02-11 | 中国农业大学 | Method for detecting chicken beard traits |
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| KR20150055582A (en) * | 2013-11-11 | 2015-05-21 | 경상대학교산학협력단 | Biomaker gene and diagnostic composition specific for environmental stress of poultry, and Prediction method of stress possibility, egg production using the same |
| KR101590035B1 (en) | 2013-11-11 | 2016-02-03 | 경상대학교산학협력단 | Biomaker gene and diagnostic composition specific for environmental stress of poultry, and Prediction method of stress possibility, egg production using the same |
| CN110643717B (en) * | 2019-10-28 | 2023-04-14 | 东北农业大学 | Molecular marking method for predicting and identifying cock heart growth and development |
| CN110643717A (en) * | 2019-10-28 | 2020-01-03 | 东北农业大学 | Molecular marking method for predicting and identifying cock heart growth and development |
| CN112189628A (en) * | 2020-10-21 | 2021-01-08 | 福建农林大学 | Method for judging cold and heat property degree of plant food |
| CN112189628B (en) * | 2020-10-21 | 2022-05-31 | 福建农林大学 | Method for judging cold and heat property degree of plant food |
| CN114831076A (en) * | 2022-05-19 | 2022-08-02 | 华中农业大学 | Method and device for judging heat resistance of chicken |
| CN114831076B (en) * | 2022-05-19 | 2023-01-13 | 华中农业大学 | Method and device for judging heat resistance of chicken |
| CN114921570A (en) * | 2022-06-02 | 2022-08-19 | 江苏省家禽科学研究所 | Molecular marker related to high-temperature tolerance of chicken and application thereof |
| CN114921570B (en) * | 2022-06-02 | 2023-10-13 | 江苏省家禽科学研究所 | Molecular marker related to chicken high temperature tolerance and application thereof |
| CN114875161A (en) * | 2022-06-16 | 2022-08-09 | 江苏省家禽科学研究所 | Molecular marker related to low-temperature tolerance of chicken, primer combination and corresponding breeding method |
| CN114875161B (en) * | 2022-06-16 | 2024-02-09 | 江苏省家禽科学研究所 | Molecular marker related to chicken low temperature tolerance, primer combination and corresponding breeding method |
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Application publication date: 20110817 |