CN1233848C - Mutant chicken myostatin and detecting method of said gene and application in chicken breeding - Google Patents
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Abstract
突变型鸡Myostatin基因的检测方法在鸡育种中的应用，它涉及鸡遗传育种改良的生物技术。该突变型鸡的Myostatin基因的核苷酸多态性为：G→A(304位)，A→G(322位)，C→T(334位)，G→A(167位)，7263A→A。本发明的育种方法按照如下步骤进行：一、PCR引物的设计，二、鸡基因组DNA的提取；三、PCR扩增；四、SSCP分析；五、对被提取基因进行分析；六、根据各基因型的特性选择种鸡。本发明是一种准确、简单、迅速、直接检测Myostatin基因单核苷酸多态性方法，利用该方法可以对种鸡进行遗传标记辅助选择育种，从而可以从根本上解决传统遗传育种中由于对肉鸡生长速度过高追求，带来的问题。
The invention relates to the application of the detection method of mutant chicken Myostatin gene in chicken breeding, which relates to the biotechnology of chicken genetic breeding improvement. The nucleotide polymorphisms of the Myostatin gene of this mutant chicken are: G→A (position 304), A→G (position 322), C→T (position 334), G→A (position 167), 7263A→ a. The breeding method of the present invention is carried out according to the following steps: one, the design of PCR primers, two, the extraction of chicken genomic DNA; three, PCR amplification; four, SSCP analysis; five, the extracted gene is analyzed; six, according to each gene Type of traits for selecting breeders. The present invention is an accurate, simple, rapid and direct method for detecting the single nucleotide polymorphism of Myostatin gene, which can be used to carry out genetic marker-assisted selective breeding for breeders, thereby fundamentally solving the problems caused by traditional genetic breeding. Broiler growth rate is too high to pursue, which brings problems.
Description
Technical field: the present invention relates to the biotechnology of chicken genetic breeding improvement, be to diagnose the energy for growth of lipidosis of chicken abdomen and muscle by detecting myostatin (Myostatin) gene genotype specifically, thereby the seed selection of carrying out chicken breeding is used.
Background technology: China is the big country of a poultry production, first place, the birds, beasts and eggs turnout row world, poultry output row second place of the world.Yet still on the low side from China's poultry breeding fraction of coverage, good chicken kind, especially laying hen and Bai Yu fryer mainly are based on the import kind, so the overall benefit that herding is produced is not high.Because to the too high pursuit of growth of meat chicken speed, the problem of bringing is that the lipid content of fryer is more and more higher, quality worse and worse, the deposition excess fat, influenced feed conversion rate, laying hen produces and also exists the egg material than problem on the low side, and utilizes molecular genetics and marker assisted selection breeding method to combine, and provides best approach to solving top problem undoubtedly.For the fryer production of China, mainly contain the fast large-scale fryer and the production of high-quality chicken.Fast large-scale fryer kind is that the chicken kind by external import is main basically, and annual production is 2,000,000,000.And, have good growth momentum as the most characteristic high-quality chicken of China, begin to expand by the south of China to the whole nation.(Myostatin MSTN) claims GDF-8 (Growth Differentiation Factor 8) again to myostatin, is the new gene that at first clone from mice skeletal cDNA library (1997) such as McPherron.Protein homology proves that relatively myostatin belongs to a newcomer in TGF-β (Transforming Growth Factor β, the transforming growth factor-beta) superfamily but do not belong to the subfamily of having found.Further discover, myostatin is mainly expressed in the skeletal muscle of mouse, and utilize its function of gene knockout technical study to find, the skeletal muscle of MSTN clpp gene deratization is more than 3 times of normal wild type mouse, the muscle of its shoulder, buttocks is obviously loose, the skeletal muscle of whole health is all much bigger than the mouse of wild-type, and the weight of monolithic muscle is about 2～3 times of wild mouse, thereby has caused the increase of body weight.The number of mutant mice skeletal fiber exceeds 86% (P＜0.01) than wild mouse, shows the existing myocyte's of reason of mutant mice skeletal muscle hypertrophy hyperplasia, and myofibrillar hypertrophy is also arranged.But on other Growth Traits, do not find any phenotypic difference.Above result shows, MSTN plays crucial effect in the growing of regulation and control skeletal muscle.The function of MSTN has also obtained checking the blue ox of Belgium.Belgian blue ox through long-term hereditary and selection has very strong skeletal muscle, and is higher by 20% than other kinds.Molecular biological analysis to two flesh cattle breeds Belgium blue oxen (Belgian Blue) and Pierre Meng Teniu (Piedmontese) MSTN gene proves, the blue two flesh oxen of Belgium are at MSTN gene extron III disappearance 11bp, cause phase shift mutation, make and begin to stop translation from the 14th codon of disappearance back, consequently C-end only 7 amino acid obtain translating all the other 102 amino acid and all lose, can't produce myostatin with biologic activity.Usually could detect after need butchering for carcass proterties such as the abdomen fat of chicken and muscle, by the Myostatin gene genotype is detected diagnosis, just can know the ability of growing of chicken in early days growing, very meaningful for the seed selection of chicken.
Summary of the invention: the present invention develops a kind of Myostatin gene mononucleotide polymorphism method that accurately, simply, rapidly, directly detects, utilize this method to carry out the genetic marker assisted selection to kind of chicken, thereby can fundamentally solve in the traditional genetic breeding because to the too high pursuit of growth of meat chicken speed, the problem of bringing (as, the lipid content of fryer is more and more higher, quality worse and worse, the deposition excess fat, influenced feed conversion rate, laying hen produces and also to have the egg material than on the low side etc.).The nucleotide polymorphisms of the Myostatin gene of mutant chicken of the present invention is: G → A (304), A → G (322), C → T (334), G → A (167), 7263A → A.The concrete steps of the detection method of the Myostatin gene of mutant chicken of the present invention are:
The first step: PCR primer design
It is as follows to have designed 3 pairs of primers according to this laboratory clone's chicken Myostatin genom sequence:
P60 5′-TCCTATCAGGAAAACCTATC-3′
P61 5′-ACCTCAAGGAAAATTCTGAG-3′
P93 5′-CAACTTTCAGTAATAATGGAA-3′
P94 5′-TGATAGGTTTTCCTGATAGGTA-3′
P80 5′-CTAAACGTAGTAAAACAAAAGGCAGC-3′
P81 5′-AACATTTATTTACAAAATATTGATG-3′
Second step: the extraction of chicken genomic dna;
The 3rd step: pcr amplification, pcr amplification reaction system are 10 * PCR buffer2.5 μ l, 10mmol/LdNTPs 2 μ l, 20 μ mol/L upstream primers, 1 μ l, 20 μ mol/L downstream primers, 1 μ l, Taq enzyme 1.0U, 15ng/ μ l dna profiling 4.0 μ l, ddH
2O 13.3 μ l.The PCR program is 1 circulation of 94 ℃ of 5min; 94 ℃ of 40s, 57 ℃ of 40s, 72 ℃ of 40s totally 35 circulations; 1 circulation of 72 ℃ of 8min; 4 ℃ of insulations.
The 4th step: sscp analysis, 1 μ l PCR product and 5 μ l Loading buffer (98% methane amide, 0.025% tetrabromophenol sulfonphthalein, 0.025% dimethylbenzene green grass or young crops, 10mmol/L EDTA (pH8.0), 10% glycerine) mix, 98 ℃ of sex change 10min, ice bath 5min, (Acr: Bis=29: silver dyes colour developing after the 1) electrophoretic analysis (10V/cm, 10h) through 14% non-denaturing polyacrylamide gel.
The instrumented medication less investment easy and simple to handle, required of a small amount of extraction step of above-mentioned chicken genomic dna, the on-the-spot use is suitable for raising chickens; Above-mentioned PCR reaction system is a part of the present invention, is the optimization PCR method that detects chicken Myostatin gene mononucleotide polymorphism.
Result of the present invention:
One, utilize the method for the invention described above to find chicken Myostatin gene mononucleotide polymorphism.
Adopt primer P80/P81 to carry out PCR-SSCP and analyze, we find 3 kinds of genotype (Fig. 1).Finding to have in this fragment 3 Nucleotide that sudden change has taken place, is respectively G → A (304), A → G (322), and C → T (334), these sudden changes have caused Myostatin 5 '-control region to present polymorphism (Fig. 2).To have identical sequence with GenBank and be defined as the AA type, mutant is defined as the BB type.
Utilize primer P93/P94, to the sscp analysis of this PCR product, we find 3 kinds of genotype (Fig. 3).Two kinds of homozygous genotype fragments are reclaimed, clone and check order that relatively to find to have in this fragment 1 Nucleotide be that sudden change (Fig. 4) has taken place G → A (167).Equally, will have identical sequence with GenBank and be defined as the FF type, mutant is defined as the EE type.
Carry out PCR-SSCP with primer P80/P81 and analyze, we find 3 kinds of genotype (Fig. 5).Fragment to two kinds of homozygous genotypes reclaims, clones, checks order and finds that relatively the 7263rd Nucleotide A sports T in this fragment, thereby causes the polymorphism (Fig. 6) of Myostatin 3 '-control region.To have identical sequence with GenBank and be defined as the CC type, mutant is defined as the DD type.
Two. found the relation between Myostatin gene mononucleotide polymorphism and production performance
We to the Myostatin single nucleotide polymorphism found and birth weight, carcass is heavy, chest muscle is heavy, leg flesh is heavy, liver is heavy and abdomen fat heavily waits the relation of proterties to carry out the least square variance analysis.We find, the P60/61 loci gene type to 12 age in week abdomen fat weight, abdomen fat rate, birth weight, chest muscle rate influential (P＜0.05): the abdomen fat method of double differences of AA type and BB type different remarkable (P＜0.05); AA and AB type abdomen fat rate are significantly higher than BB type (P＜0.05); The birth weight of AA type is significantly higher than BB type (P＜0.05); AA type chest muscle rate is significantly higher than AB type (P＜0.05) (table 1).P93/94 site and chest muscle heavily have significant correlation (P＜0.05): the EF type has higher chest muscle heavy (table 2) than EE type.The P80/P81 site is relevant with chest muscle rate (P＜0.01) with chest muscle heavy (P＜0.05): the chest muscle method of double differences between CC type and the DD type different significantly (P＜0.05), and the chest muscle of CC type is heavy higher; Chest muscle rate variance heteropole between CC type, the DD type is (P＜0.01) significantly, CC type and CD type ask significant difference (P＜0.05), the CC type is than the chest muscle rate height of CD and DD type, and the chest muscle rate of CD type is also than the height (P＜0.05 (table 3) of DD type.
Table 1 P60/61 primer Myostatin different genotype is to the influence of birth weight, abdomen fat weight and abdomen fat rate
| Genotype | Number of individuals | Genotype frequency | Abdomen fat is heavy | Abdomen fat rate | Birth weight | The chest muscle rate |
| AA AB BB | 116 187 38 | 0.340 0.548 0.111 | 52.55± 3.38 a49.10± 3.32 ab43.34± 5.02 b | 0.0354± 0.0022 a 0.0353± 0.0022 a 0.0294± 0.0038 b | 30.79± 0.28 a 30.35± 0.21 ab 29.51± 0.45 b | 0.0613± 0.001 a0.0592± 0.001 b0.0586± 0.0015 ab |
Annotate: average is the alphabetical identical person's difference of same row not significantly (P＞0.05) relatively the time
Table 2 P93/94 primer Myostatin different genotype is to the influence of birth weight, abdomen fat weight and abdomen fat rate
| Genotype | Number of individuals | Genotype frequency | Chest muscle is heavy |
| EE EF FF | 170 131 40 | 0.499 0.384 0.117 | 86.70±3.32 b 93.23±3.50 a 90.16±4.56 ab |
Annotate: average is the alphabetical identical person's difference of same row not significantly (P＞0.05) relatively the time
Table 3 p80/81 primer Myostatin different genotype is to the influence of chest muscle weight and chest muscle rate
| Genotype frequency | Chest muscle is heavy | The chest muscle rate | ||
| CC CD DD | 213 126 6 | 0.617 0.365 0.017 | 95.37±1.57 a 89.93±2.12 ab 84.49±7.29 b | 0.0634±0.00058 a 0.0614±0.00078 b 0.0544±0.00269 c |
Annotate: average is the alphabetical identical person's difference of same row not significantly (P＞0.05) relatively the time
No matter be fast large fowl or high-quality a breed of chicken, muscle weighs and the muscle rate all is very important selection index, and directly can not realize its mensuration, the polymorphism mark of the chicken muscle proterties Myostatin gene that we invent, can be used as the selective marker of muscle proterties and fatty character in the fryer breeding, in the muscle growth speed that improves chicken, reach the purpose that reduces lipid content to a certain extent.Present method by improving the advantage of muscle rate, just can improve the speed of weight increment of colony as long as detect genotype.Detection method is simple, genotype is judged easily, therefore has promotional value.
The detection method of mutant chicken Myostatin gene of the present invention selects the concrete steps of the application in the breeding to be chicken:
The first step: PCR primer design
It is as follows to have designed 3 pairs of primers according to this laboratory clone's chicken Myostatin genom sequence:
P60 5′-TCCTATCAGGAAAACCTATC-3′
P61 5′-ACCTCAAGGAAAATTCTGAG-3′
P93 5′-CAACTTTCAGTAATAATGGAA-3′
P94 5′-TGATAGGTTTTCCTGATAGGTA-3′
P80 5′-CTAAACGTAGTAAAACAAAAGGCAGC-3′
P81 5′-AACATTTATTTACAAAATATTGATG-3′
Second step: the extraction of chicken genomic dna;
The 3rd step: pcr amplification, pcr amplification reaction system are 10 * PCR bufier, 2.5 μ l, 10mmol/LdNTPs 2 μ l, 20 μ mol/L upstream primers, 1 μ l, 20 μ mol/L downstream primers, 1 μ l, Taq enzyme 1.0U, 15ng/ μ l dna profiling 4.0 μ l, ddH
2O 13.3 μ l.The PCR program is 1 circulation of 94 ℃ of 5min; 94 ℃ of 40s, 57 ℃ of 40s, 72 ℃ of 40s totally 35 circulations; 1 circulation of 72 ℃ of 8min; 4 ℃ of insulations.
The 4th step: sscp analysis, 1 μ l PCR product and 5 μ l Loading buffer (98% methane amide, 0.025% tetrabromophenol sulfonphthalein, 0.025% dimethylbenzene green grass or young crops, 10mmol/L EDTA (pH8.0), 10% glycerine) mix, 98 ℃ of sex change 10min, ice bath 5min, (Acr: Bis=29: silver dyes colour developing after the 1) electrophoretic analysis (10V/cm, 10h) through 14% non-denaturing polyacrylamide gel.
The 5th step: 1, carry out PCR-SSCP and analyze whether the Mostatin gene is following genotype with primer P80/P81:
AA ATGGATTCCTCGTGTTTGCAATGTATTTATTACGTATT
BB ATAGATTCCTCGTGTTTGCAGTGTATTTATTATGTATT
AB ATA/GGATTCCTCGTGTTTGCAA/GTGTATTTATTAC/TGTATT
2, carry out PCR-SSCP with primer P93/P94 and analyze whether the Mostatin gene is following genotype:
EE ATTTTGATACTAAAGGGTCCAATAGTTA
FF ATTTTGATACTAGAGGGTCCAATAGTTA
EF ATTTTGATACTAA/GAGGGTCCAATAGTTA
3, carry out PCR-SSCP with primer P80/P81 and analyze whether the Mostatin gene is following genotype:
CC TTGCTACAGAAATTGTTAAAAAAC
DD TTGCTACAGATATTGTTAAAAAAC
CD TTGCTACAGAA/TATTGTTAAAAAAC
The 6th step: according to the kind chicken of each genotypic following characteristic selection needs,
Abdomen fat weight, abdomen fat rate, birth weight, chest muscle rate: AA＞AB＞BB
Chest muscle is heavy: EF＞FF＞EE
Chest muscle is heavy, chest muscle rate: CC＞CD＞DD.
Poultry has replaced beef becomes second-biggest-in-the-world consumption meat, according to the FAO statistics, the 1993-1995 annual global is pre-capita consumption poultry 9.1kg every year on average, and will increase to 13.7kg by 2005, the consumption of developing country will be increased to 10.2kg from 5.7kg, this wherein the growth in Asia be the fastest.China is the big country of a poultry production, birds, beasts and eggs, first place, the poultry turnout row world.Chicken and egg production have critical role in China's livestock industry is produced.Yet still on the low side from China's poultry breeding fraction of coverage, good chicken kind, especially laying hen and Bai Yu fryer mainly are based on the import kind, so the overall benefit that herding is produced is not high.Because to the too high pursuit of growth of meat chicken speed, the problem of bringing is that the lipid content of fryer is more and more higher, quality worse and worse, the deposition excess fat, influenced feed conversion rate, laying hen produces and also exists the egg material than problem on the low side, and utilizes molecular genetics and marker assisted selection breeding method to combine, and provides best approach to solving top problem undoubtedly.For the fryer production of China, mainly contain the fast large-scale fryer and the production of high-quality chicken.Fast large-scale fryer kind is that the chicken kind by external import is main basically, and annual production is 2,000,000,000, exports 400,000 tons, earns foreign exchange 700,000,000 dollars.High-quality chicken is that the seed selection of Chinese elite indigenous chicken kind forms, has fine and tender taste, the characteristics of delicious flavour, has good growth momentum, the production and consumption of high quality meat chicken is spread to all parts of the country from partial area, and section port is arranged, and year amount of delivering for sale reaches 1,500,000,000,3,000 ten thousand live chickens of year outlet, planting the fowl raising amount is 2,000 ten thousand covers.The tempo of whole nation high quality meat chicken production reaches and year increases 25%-30%, and market potential is huge.
A breed of chicken method of the present invention is a kind of Myostatin gene mononucleotide polymorphism method that accurately, simply, rapidly, directly detects, utilize this method to carry out the genetic marker assisted selection to kind of chicken, thereby can fundamentally solve in the traditional genetic breeding because to the too high pursuit of growth of meat chicken speed, the problem of bringing (as, the lipid content of fryer is more and more higher, quality worse and worse, the deposition excess fat, influenced feed conversion rate, laying hen produces and also to have the egg material than on the low side etc.).Because, no matter be fast large fowl or high-quality a breed of chicken, muscle weighs and the muscle rate all is very important selection index, and directly can not realize its mensuration, the polymorphism mark of the chicken muscle proterties Myostatin gene that we invent, can be used as the selective marker of muscle proterties and fatty character in the fryer breeding, in the muscle growth speed that improves chicken, reach the purpose that reduces lipid content to a certain extent.Present method by improving the advantage of muscle rate, just can improve the speed of weight increment of colony as long as detect genotype.Detection method is simple, genotype is judged easily, therefore has promotional value.
Description of drawings: Fig. 1 is that the SNPs of Myostatin gene 5 '-control region analyzes (P60/61), Fig. 2 is AA type and BB type Myostatin gene order comparison sheet (P60/P61), Fig. 3 is that the SNPs of chicken Myostatin gene 5 '-control region analyzes (P93/P94), Fig. 4 is EE type and EF type Myostatin gene order comparison sheet (P93/P94), Fig. 5 is that the SNPs of chicken Myostatin gene 3 '-control region analyzes (P80/P81), and Fig. 6 is CC type and DD type Myostatin gene order comparison sheet (P80/P81).
Embodiment one: the concrete steps of the detection method of the Myostatin gene of mutant chicken are:
The first step: PCR primer design
It is as follows to have designed 3 pairs of primers according to this laboratory clone's chicken Myostatin genom sequence:
P60 5′-TCCTATCAGGAAAACCTATC-3′
P61 5′-ACCTCAAGGAAAATTCTGAG-3′
P93 5′-CAACTTTCAGTAATAATGGAA-3′
P94 5′-TGATAGGTTTTCCTGATAGGTA-3′
P80 5′-CTAAACGTAGTAAAACAAAAGGCAGC-3′
P81 5′-AACATTTATTTACAAAATATTGATG-3′
Second step: a small amount of of chicken genomic dna is extracted, and present method instrumented medication less investment easy and simple to handle, required is convenient to on-the-spot use of raising chickens, and its concrete steps are as follows:
Get 20 μ l fresh bloods, add the ACD anti-freezing, add 600 μ l fowl lysates, adding Proteinase K to final concentration is 100-200 μ g/ml, 55 ℃ of digestion of mixing, 6～10hr no longer includes the heavy-gravity agglomerate in solution, solution is cooled to room temperature, add equal-volume phenol, put upside down centrifuge tube repeatedly, mix forming emulsion, 12 until two-phase, 000rpm, the centrifugal 10min of room temperature.Get supernatant, use equal-volume phenol again: chloroform, each extracting of chloroform 1 time.Get reset and add 1/10 volume NaAc (3M, pH5.2) and 2 times of volume dehydrated alcohol deposit D NA.DNA chosen be put in the 1.5ml centrifuge tube, wash twice with 70% ethanol.(attention can not be too dried) after the DNA drying is dissolved in an amount of TE or the sterilization distilled water.
The 3rd step: pcr amplification, this PCR reaction system is a part of the present invention, is the optimization PCR method that detects chicken Myostatin gene mononucleotide polymorphism, reactions steps is as follows:
The pcr amplification reaction system is 10 * PCR buffer, 2.5 μ l, 10mmol/L dNTPs 2 μ l, 20 μ mol/L upstream primers, 1 μ l, 20 μ mol/L downstream primers, 1 μ l, Taq enzyme 1.0U, 15ng/ μ lDNA template 4.0 μ l, ddH
2O 13.3 μ l.The PCR program is 1 circulation of 94 ℃ of 5min; 94 ℃ of 40s, 57 ℃ of 40s, 72 ℃ of 40s totally 35 circulations; 1 circulation of 72 ℃ of 8min; 4 ℃ of insulations.
The 4th step: sscp analysis, 1 μ l PCR product and 5 μ l Loading buffer (98% methane amide, 0.025% tetrabromophenol sulfonphthalein, 0.025% dimethylbenzene green grass or young crops, 10mmol/L EDTA (pH8.0), 10% glycerine) mix, 98 ℃ of sex change 10min, ice bath 5min, (Acr: Bis=29: silver dyes colour developing after the 1) electrophoretic analysis (10V/cm, 10h) through 14% non-denaturing polyacrylamide gel.
Silver staining method: electrophoresis is shut electrophoresis apparatus after finishing, and emits electrophoresis liquid, carefully takes off gel, places 70% ethanol, and the water-bath oscillator slowly shakes up fixedly 10-15min; Deionization washing glue 2 times, each 2min, flush away ethanol; With 200ml staining fluid (100ml silver dye liquor: NH
3H2O 1ml; 3.6%NaOH2.1ml; 20%AgNO
31.8ml; Add deionized water to 100ml) dyeing 30min.Deionization washing glue 3 times, each 2min, the staining fluid that flush away is unnecessary; With 200ml liquid (the 200ml liquid that develops the color: 1% Trisodium Citrate 1ml that develops the color; Formaldehyde 100ul; Add deionized water to 200ml) colour developing, about 10-30min when the intensity of being with as DNA is suitable, outwells colour developing liquid; Deionized water is washed unnecessary colour developing liquid off, and preservative film is sealed, scanography or preservation.
Embodiment two: a breed of chicken method:
The first step: PCR primer design
It is as follows to have designed 3 pairs of primers according to this laboratory clone's chicken Myostatin genom sequence:
P60 5′-TCCTATCAGGAAAACCTATC-3′
P61 5′-ACCTCAAGGAAAATTCTGAG-3′
P93 5′-CAACTTTCAGTAATAATGGAA-3′
P94 5′-TGATAGGTTTTCCTGATAGGTA-3′
P80 5′-CTAAACGTAGTAAAACAAAAGGCAGC-3′
P81 5′-AACATTTATTTACAAAATATTGATG-3′
Second step: the extraction of chicken genomic dna, get 20 μ l fresh bloods, add the ACD anti-freezing, add 600 μ l fowl lysates, adding Proteinase K to final concentration is 100-200 μ g/ml, 55 ℃ of digestion of mixing, 6～10hr, in solution, no longer include the heavy-gravity agglomerate, solution is cooled to room temperature, adds equal-volume phenol, put upside down centrifuge tube repeatedly, mix formation emulsion until two-phase, 12,000rpm, the centrifugal 10min of room temperature.Get supernatant, use equal-volume phenol again: chloroform, each extracting of chloroform 1 time.Get reset and add 1/10 volume NaAc (3M, pH5.2) and 2 times of volume dehydrated alcohol deposit D NA.DNA chosen be put in the 1.5ml centrifuge tube, wash twice with 70% ethanol.(attention can not be too dried) after the DNA drying is dissolved in an amount of TE or the sterilization distilled water.
The 3rd step: pcr amplification, pcr amplification reaction system are 10 * PCR buffer, 2.5 μ l, 10mmol/LdNTPs 2 μ l, 20 μ mol/L upstream primers, 1 μ l, 20 μ mol/L downstream primers, 1 μ l, Taq enzyme 1.0U, 15ng/ μ l dna profiling 4.0 μ l, ddH
2O 13.3 μ l.The PCR program is 1 circulation of 94 ℃ of 5min; 94 ℃ of 40s, 57 ℃ of 40s, 72 ℃ of 40s totally 35 circulations; 1 circulation of 72 ℃ of 8min; 4 ℃ of insulations.
The 4th step: sscp analysis, 1 μ l PCR product and 5 μ l Loading buffer (98% methane amide, 0.025% tetrabromophenol sulfonphthalein, 0.025% dimethylbenzene green grass or young crops, 10mmol/L EDTA (pH8.0), 10% glycerine) mix, 98 ℃ of sex change 10min, ice bath 5min, (Acr: Bis=29: silver dyes colour developing after the 1) electrophoretic analysis (10V/cm, 10h) through 14% non-denaturing polyacrylamide gel.
The 5th step: 1, carry out PCR-SSCP and analyze whether the Mostatin gene is following genotype with primer P80/P81:
AA ATGGATTCCTCGTGTTTGCAATGTATTTATTACGTATT
BB ATAGATTCCTCGTGTTTGCAGTGTATTTATTATGTATT
AB ATA/GGATTCCTCGTGTTTGCAA/GTGTATTTATTAC/TGTATT
2, carry out PCR-SSCP with primer P93/P94 and analyze whether the Mostatin gene is following genotype:
EE ATTTTGATACTAAAGGGTCCAATAGTTA
FF ATTTTGATACTAGAGGGTCCAATAGTTA
EF ATTTTGATACTAA/GAGGGTCCAATAGTTA
3, carry out PCR-SSCP with primer P80/P81 and analyze whether the Mostatin gene is following genotype:
CC TTGCTACAGAAATTGTTAAAAAAC
DD TTGCTACAGATATTGTTAAAAAAC
CD TTGCTACAGAA/TATTGTTAAAAAAC
The 6th step: according to the kind chicken of each genotypic following characteristic selection needs,
Abdomen fat weight, abdomen fat rate, birth weight, chest muscle rate: AA＞AB＞BB
Chest muscle is heavy: EF＞FF＞EE
Chest muscle is heavy, chest muscle rate: CC＞CD＞DD.
The chicken that present embodiment is selected to have BB, EF and CC type gene carries out breeding for kind of a chicken.This chicken has the advantages that abdomen fat rate is low, the chest muscle rate is high.
Myostatin gene of mutant chicken of the present invention and the contrast of the nucleotide sequence of the Myostatin gene in the GenBank gene pool are as follows:
One, the single nucleotide polymorphism in the Myostatin gene 5 ' promotor
Gene pool sequence A CTATTTCTGTGTAATATTAGAGTTAAGTAGCTCATGATATTCCTTAAAAATACCTC TCCTACTAAAAGTCTCTCAGTATCTAATTTGCT 90
Mutant sequence------------------------------------------------------------------------------------------90
Gene pool sequence GCCCAGGATTTTGCTGACTAGGCAAACTTGCTTTACTTAATAAGCGTCCACACTTC AGTAATGAATTTTGATACTA
GAGGGTCCAATAGT 180
Mutant sequence----------------------------------------------------------------------------A-------------180
Gene pool sequence TAGCACTTATAGTCACACGTGTACAAAATGTTTATTCCTGCTCACCTAGTACCTAT CAGGAAAACCTATCATGATTTTCTGAAATCTGAG 270
Mutant sequence------------------------------------------------------------------------------------------270
Gene pool sequence C TGCTTAATGCACGTGAACTGTTGAACAAGCAT
GGATTCCTCGTGTTTGCA
ATGTATTTATTA
TGTATTTTTTTCCCCTCCTCCTAACAG 360
Mutant sequence---------------------------------A-----------------G-----------C--------------------------360
Gene pool sequence A AATCCCTCAGAATTTTCCTTGAGGTAGTACAAACTTTCAGCCACAATAGTGATAGA ATCCTAAAGGAACCCTAAAAGAGAGCTCTGCCT 450
Mutant sequence------------------------------------------------------------------------------------------450
Gene pool sequence C AATTCATAGTCCAACTATGCGTTCAGTGTATATTTAAGAATGATAGTGCTGTCTTC CAGCACTGCTGCCCATAGTACTTGGAAATATAT 540
Mutant sequence------------------------------------------------------------------------------------------540
Gene pool sequence C CTTTCAGTATGTGAAGACGTATCCTCTACGAAGCCACCAGATAAATCAGTTCACCC TTGGCTGTAATCAAATGCTGTCTAGTGACTTGT 630
Mutant sequence------------------------------------------------------------------------------------------630
Gene pool sequence GATCGACAGGGCTTTAACCTCTGACAGCTAGATTCATTGTTGGGACAACAACCAAT CGTCGGTTTTGACGACATGAGCCTAATCAAAGTT 720
Mutant sequence------------------------------------------------------------------------------------------720
Gene pool sequence GGAGTATAAAAGCCCCCTTGGCATATATAAGGCACACCAGTGTGGCAAGCCGTCTC TCAGATTGCATTTGCTGTCCCGGATCTGTTTAGA 810
Mutant sequence------------------------------------------------------------------------------------------810
Gene pool sequence A CTGAAAGAAAAGGGGAAAGGGAGAGGGGGGGGAAAAAAGGCAAAAAGATGCAGTGA GGTGTAAGAAAAAATGCAAAAGCTAGCAGTCTA 900
Mutant sequence------------------------------------------------------------------------------------------900
Gene pool sequence TGTTTATATTTACCTGTTCATGCAGATCGCGGTTGATCCGGTGGCTCTGGATGGCA GTAG 960
Mutant sequence------------------------------------------------------------960
Two, the single nucleotide polymorphism of Myostatin gene 3 ' non-coding region
Gene pool sequence GGTGCTCATGAATTTTGGCTGACGTGAGACCCCTTCGATAAATTGTGGAAGCCACC AAAAAAAAAAGCTATATCCCCTCATCCATCTTTG 6450
Mutant sequence------------------------------------------------------------------------------------------6450
Gene pool sequence A AACTGTGAAATTACGTACGCTAGGCATTGCCAACATCCATATACTGTACAACTGTA CAGACCACATATATCACAACATGAGCTGCAGAA 6540
Mutant sequence------------------------------------------------------------------------------------------6540
Gene pool sequence TGTGAACTTAAAGACAGTAGAGTTACCTAAGGGCTGGCTTTGAAACAACGGACAAA GAAGCTACAATCAAAATCACCGGATTTAACAAAT 6630
Mutant sequence------------------------------------------------------------------------------------------6630
Gene pool sequence GGGTTTCTTACACTGTGAGGGAATCAATATTCAGTCATTCAGACACAAATTTATAT GCAGTTTTCAACATATGTTGGTAATCAAAAGTAA 6720
Mutant sequence------------------------------------------------------------------------------------------6720
Gene pool sequence GCTCCTTCTCCTCTGAGGACAGAAGGAGCGGGCTATTAAATCAACTTCTTCACAGC TACACTTAATATTGTATTTACAGCAAAATATATA 6810
Mutant sequence------------------------------------------------------------------------------------------6810
Gene pool sequence C TGGTAACGTATACACCACTACACATTACCACCAGAATCATCCTTGAACACTTGAAT ATATAGTGCAGAGTTATGATAAGATGAAATTCC 6900
Mutant sequence------------------------------------------------------------------------------------------6900
Gene pool sequence A CGTAAATGGACAAATCCTGAAGTTAGGGATGGTATAGTGTATTTAGCAGTGTTTCC ATTCCTTTTTTTCGTTAGTATGTTAGTAATCAA 6990
Mutant sequence------------------------------------------------------------------------------------------6990
Gene pool sequence TGGCAATGGTGCTACGTAAGCAGGCTGAGTAAATAGAAAGATAGTTATCTAAGTGA AAGAATTTAGAGTAATAATGAATTTGCCCTATCC 7080
Mutant sequence------------------------------------------------------------------------------------------7080
Gene pool sequence TCAGGTACACTATTCAACATTCACAAGAAAAGGATTTTTTTTAAACAAAAGGTGAA TAGTTTTCTAAACGTAGTAAAACAAAAGGCAGCA 7170
Mutant sequence------------------------------------------------------------------------------------------7170
Gene pool sequence C GGAAGTCTGATGTTCAAACCATAATCCCATATCGTAATCTGCCTTTGCAACGTTAC CGTTTGCACTATGATAAGCCAATGCAAATAGTT 7260
Mutant sequence------------------------------------------------------------------------------------------7260
Gene pool sequence GGTTGCTACAGA
AATTGTTAAAAAACCACTTTGATATAACTGACTTGTGTAATATGTATGCATCAATAT TTTGTAAATAAATGTTTATTT 7350
Mutant sequence------------T-----------------------------------------------------------------------------7350
Gene pool sequence TTTAATCTGTTCGTATACATTCATTACAAAAAAAGAGTAATGGTAACCTGTCCTTT AGTTTAATAAACAAACCCAGGTTTTCTCTTCATA 7440
Mutant storehouse row------------------------------------------------------------------------------------------7440
Gene pool sequence A CATAATTTCCTGGTTTTTAACAGTAATATGAAAGAACAGTGCAACAGAGTAAACCA CAGGTAGAGAATTATACCACATTTTGTATGTTT 7530
Mutant sequence------------------------------------------------------------------------------------------7530
Gene pool sequence GGTTTTTTTTTTTTAACATTGGCATGA 7557
Mutant sequence---------------------------7557
Claims (1)
1, the application of detection method in chicken breeding of mutant chicken Myostatin gene is characterized in that the first step: the PCR primer design:
It is as follows to have designed 3 pairs of primers according to this laboratory clone's chicken Myostatin genom sequence:
P605′-TCCTATCAGGAAAACCTATC-3′
P615′-ACCTCAAGGAAAATTCTGAG-3′
P935′-CAACTTTCAGTAATAATGGAA-3′
P945′-TGATAGGTTTTCCTGATAGGTA-3′
P805′-CTAAACGTAGTAAAACAAAAGGCAGC-3′
P815′-AACATTTATTTACAAAATATTGATG-3′
Second step: the extraction of chicken genomic dna;
The 3rd step: pcr amplification, pcr amplification reaction system are 10 * PCR buffer, 2.5 μ l, 10mmol/LdNTPs 2 μ l, 20 μ mol/L upstream primers, 1 μ l, 20 μ mol/L downstream primers, 1 μ l, Taq enzyme 1.0U, 15ng/ μ l dna profiling 4.0 μ l, ddH
2O 13.3 μ l; The PCR program is 1 circulation of 94 ℃ of 5min; 94 ℃ of 40s, 57 ℃ of 40s, 72 ℃ of 40s totally 35 circulations; 1 circulation of 72 ℃ of 8min; 4 ℃ of insulations;
The 4th step: sscp analysis, 1 μ l PCR product and 5 μ l Loading buffer mix, 98 ℃ of sex change 10min, ice bath 5min, silver dyes colour developing after 14% native polyacrylamide gel electrophoresis analysis;
The 5th step: (1), carry out PCR-SSCP with primer P80/P81 and analyze whether the Mostatin gene is following genotype:
AA ATGGATTCCTCGTGTTTGCAATGTATTTATTACGTATT
BB ATAGATTCCTCGTGTTTGCAGTGTATTTATTATGTATT
AB ATA/GGATTCCTCGTGTTTGCAA/GTGTATTTATTAC/TGTATT
(2), carry out PCR-SSCP with primer P93/P94 and analyze whether the Mostatin gene is following genotype:
EE ATTTTGATACTAAAGGGTCCAATAGTTA
FF ATTTTGATACTAGAGGGTCCAATAGTTA
EF ATTTTGATACTAA/GAGGGTCCAATAGTTA
(3), carry out PCR-SSCP with primer P80/P81 and analyze whether the Mostatin gene is following genotype:
CC TTGCTACAGAAATTGTTAAAAAAC
DD TTGCTACAGATATTGTTAAAAAAC
CD TTGCTACAGAA/TATTGTTAAAAAAC
The 6th step: according to the kind chicken of each genotypic following characteristic selection needs, abdomen fat weight, abdomen fat rate, birth weight, chest muscle rate: AA＞AB＞BB
Chest muscle is heavy: EF＞FF＞EE
Chest muscle is heavy, chest muscle rate: CC＞CD＞DD.
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