CN103525920B - A kind of molecule marking method for seed selection Altai Sheep meat production and application thereof - Google Patents
A kind of molecule marking method for seed selection Altai Sheep meat production and application thereof Download PDFInfo
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Abstract
The present invention discloses a kind of molecule marking method for seed selection Altai Sheep meat production and application thereof, choose MyoD1 gene as the candidate gene affecting Altai Sheep meat yield, utilize the genetic construction of MyoD1 gene in PCR-SSCP technology for detection Altai Sheep kind, analyze and judge the correlationship of itself and sheep meat yield, by the DNA sequence dna of sheep MyoD1 gene, designed, designed two pairs of Auele Specific Primers, PCR-SSCP technology is utilized to carry out polymorphic detection to Altay, Xinjiang sheep MyoD1 gene, and carry out correlation analysis with meat-producing traits, thus find out the molecule marker with Altai Sheep meat yield significant correlation, the present invention is in order to improve the meat production of Altai Sheep, thus greatly accelerate to improve the speed recovering the excellent meat production of Altai Sheep, this is for raising peasants and herdsmen income, accelerate the significant and actual application value of the development of Xinjiang meat sheep industry.
Description
Invention field
The present invention relates to animal molecular genetic breeding technical field, be specifically related to utilize molecular biotechnology to find the effective molecule marker affecting Altay, Xinjiang sheep meat yield proterties, and then be applied to the technical field improving Altai Sheep meat production.
Background technology
Xinjiang is one of main pastoral area of China, there is abundant sheep variety resource, wherein Altai Sheep is large with physique, meat fat production performance is high and famous, has crude feed tolerance, kindly to trek, anti-severe cold, physique are solid, be suitable for features such as herding, are Xinjiang Local Excellent naked eyed test.But In Altay, xinjiang ethnic minority accounts for the overwhelming majority, reach nearly half a year winter, and the coldlyest reach-35--40 DEG C, because natural condition are severe, many improved seeds here cannot be survived.And Altai Sheep is exactly that formed by long-term nature and artificial selection, be namely adapted to local condition, have again the sheep variety of higher meat production, its live-weight reaches as high as 171 kilograms, and dressing percentage reaches 49-50% under so special condition.In addition, Altai Sheep delicious meat, and due to its grazing pasture natural pollution-free, produce organic mutton by Altai Sheep, squeeze into high-end market, increase the economic level of local peasants and herdsmen with this, so improve and make the life better.Visible, Altai Sheep is the main edible mutton sheep of local peasants and herdsmen and cultivation domestic animal, is the means of living and the production means.Due to traditional habit and the religious belief of Xinjiang region ethnic minority, the demand for mutton is very big, and mutton holds at high price in recent years.Therefore, have excellent meat production and the Altai Sheep deeply liked by local peasants and herdsmen, for solution Shish Kebab, supply falls short of demand, ethnic minority lives a stable life that well-to-do level is significant.
Altai Sheep is as Local Excellent kind; there is it unique, the available protecting of variety source should be carried out, but improve without seed selection due to long-term in recent years; its excellent product meat characteristic is not reasonably developed, and is greatly unfavorable for the development of local economy and Xinjiang meat sheep industry.
At present, modern technique means are used, in conjunction with GENERALIZATION OF MODERN BREEDING TECHNIQUE and traditional breeding way, Development in Xinjiang advantage Characteristic Stockbreeding, improves sheep improved variety degree, cultivates mutton sheep specific breed, improving mutton production level, is the effective means accelerating Xinjiang Sheep industry development.
The meat yield of sheep belongs to economic characters.In GENERALIZATION OF MODERN BREEDING TECHNIQUE, utilize candidate gene strategy, the efficiency of important economical trait seed selection can be increased substantially.So-called candidate gene strategy is exactly consider from physiology, biochemical angle, using the candidate gene of some functional genes as the corresponding production traits difference of impact, the DNA information that searching and trait phenotypes make a variation relevant in candidate gene is again as mark, the indirect foundation as choosing seeds in early days, for the method for breed improvement or breeding of new variety.Sichuan Province of China skin and flesh dual-purpose type goat new variety Nanjiang-Huang goat adopts molecular breeding method exactly, has only used the kind that 8 years were just bred as.
The myoblastic propagation of growth needs of muscle and differentiation, therefore the product meat power of animal and myofibroblasts quantity and grow closely related.There are some researches show, Myostatin Gene (Myogenic Determination gene, MyoD) family is the important gene family participating in molecular regulation control action kou in myogenesis process, can start and maintain Skeletal Muscle Cell differentiation and development and growth, the peculiar gene of muscle of activation stationary state is combined with the distinctive enhanser of muscle and jointly promotes to transcribe, impel some cell to break up to Skeletal Muscle Cell, closely related with the meat yield of the generation of myocyte, animal.Prior art describes Myostatin Gene (MyoD) and comprises 4 gene: MyoD1 (MyoD or myf3), Myogenin (MyoG or Myf4), Myf5, Myf-6 (herculin or MRF4).
At present, at home and abroad do not see and utilize candidate gene strategy to carry out research and the report of seed selection raising to Altai Sheep meat-producing traits, belong to blank completely.In addition, for using MyoD1 gene as affecting Stock genetics and breeding candidate gene, the report carrying out correlative study is more common in goat, ox and pig, in sheep, have no report, and in Altai Sheep class, applied research has no report completely.
Summary of the invention
For having no report in prior art specially for the state of the art of the molecule marking research of Altai Sheep meat yield significant correlation.The present invention is intended to find to provide a kind of molecular marking technique that utilizes the method affecting the effective molecule marker of Altay, Xinjiang sheep meat yield, improves Altai Sheep meat production for seed selection.
Technical scheme of the present invention: adopt candidate gene strategy, choose MyoD1 gene as the candidate gene affecting Altai Sheep meat yield, utilize molecular marking technique PCR-SSCP (Single Strand Conformation Polymorphism, single strand conformation polymorphism) detect the genetic construction of MyoD1 gene in Altai Sheep kind, from molecular level, scientific analysis and judge the correlationship of itself and sheep meat yield, by the DNA sequence dna (GenBank accession number: NM_001009390) of sheep MyoD1 gene, by known software Primer5.0 designed, designed two pairs of Auele Specific Primers, PCR-SSCP technology is utilized to carry out polymorphic detection to Altay, Xinjiang sheep MyoD1 gene, and carry out correlation analysis with meat-producing traits, thus find out the molecule marker with Altai Sheep meat yield significant correlation, in order to improve the meat production of Altai Sheep, thus greatly accelerate to improve the speed recovering the excellent meat production of Altai Sheep, this is for raising peasants and herdsmen income, accelerate the significant and actual application value of the development of Xinjiang meat sheep industry.
The invention provides a kind of molecule marking method for seed selection Altai Sheep meat production, concrete grammar step is as follows:
(1) mutating alkali yl of MyoD1 gene in Altai Sheep kind is found out: according to the DNA sequence dna of sheep MyoD1 gene, designed, designed two pairs of Auele Specific Primers, application PCR-SSCP (Single Strand Conformation Polymorphism, single strand conformation polymorphism) and polyacrylamide gel electrophoresis (Polyacrylamide Gel Electrophoresis, PAGE) technology, detect genetic construction and the polymorphism of MyoD1 gene-specific amplification fragment in Altai Sheep kind, filter out the gene fragment with polymorphism, namely there is Multi-genotype in this gene fragment, and through DNA sequencing, find out the mutating alkali yl causing gene amplification fragment to occur Multi-genotype.
(2) association analysis of mutating alkali yl and Altai Sheep meat yield is carried out, determine to affect the DNA molecular marker of Altai Sheep meat yield and carry out test of significance, study the difference of meat yield between polymorphic gene different genotype in Altai Sheep, judge that can this gene as the DNA molecular marker affecting Altai Sheep meat yield thus; Through test of significance, P < 0.01 time difference heteropole is remarkable; Significant difference during P < 0.05; During P > 0.05, difference is not remarkable; If significant difference or extremely remarkable, then illustrate cause gene amplification fragment to occur the mutating alkali yl of Multi-genotype is the gene mutation site of remarkably influenced Altai Sheep meat yield, this polymorphic gene can be used as the DNA molecular marker affecting Altai Sheep meat yield.
Concrete, the present invention provides a kind of molecule marking method for seed selection Altai Sheep meat production further in detail, and its concrete grammar step is as follows:
(1) Altai Sheep extracting genome DNA: gather Altai Sheep blood sample 5ml, extract DNA ,-20 DEG C of preservations; 0.8% agarose gel electrophoresis detects, and after the dyeing of bromination ingot, irradiates under gel imaging instrument ultraviolet lamp, observes size and the brightness of band, judges the extraction quality of DNA.
(2) Altai Sheep MyoD1 gene-specific primer design: the DNA sequence dna (GenBank accession number: NM_001009390) utilizing sheep MyoD1 gene, engineer's two pairs of Auele Specific Primers voluntarily, two pairs of specific primer sequences are respectively:
Primer 1:
Upstream primer F:5 '-CCGCTGTTTACTGTGGG-3 '
Downstream primer R:5 '-GCTGGTTTGGGTTGCT-3 '
Primer 2:
Upstream primer F:5 '-AGCAACCCAAACCAGC-3 '
Downstream primer R:5 ' ATGCCGTCGGAACAGT-3 '
(3) pcr amplification of Altai Sheep MyoD1 gene: according to two pairs of primers of design, two fragments of specific amplification Altai Sheep MyoD1 gene; PCR reaction system is: Mi x l0.0 μ l, and each 0.4 μ l of upstream and downstream primer, DNA profiling 1.5 μ l, add water to cumulative volume 20 μ l; Reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, annealing temperature 30s, 72 DEG C of extension lmin, and circulate 31 times, 72 DEG C extend 5mi n, 4 DEG C of preservations; Two PCR primer of specific amplification detect by l% agarose gel electrophoresis, after the dyeing of bromination ingot, irradiate under gel imaging instrument ultraviolet lamp, observe size and the brightness of amplified band, judge PCR primer amplification quality.
(4) SSCP detects: carry out SSCP detection to two PCR primer of Altai Sheep MyoDl gene respectively, and screening has the pcr amplified fragment of polymorphism; Get 3 μ l PCR primer to mix with the denaturing agent of 98% deionized formamide of 7 μ l, ice bath 10min after 98 DEG C of sex change 10min, make it to keep denatured state; PCR primer 8% non denatured polyacrylate hydrogel (polyacrylamide gel, PAG) 180V electrophoresis 20h after sex change, after the colour developing of silver dye, irradiate under gel imaging instrument white light, observe the size of band, determine whether polymorphism, have polymorphic judgement genotype.
(5) DNA sequencing, finds out Altai Sheep MyoD1 gene mutation site; There is during SSCP is detected the gene of polymorphism, the PCR primer of its different genotype is carried out DNA sequencing, in order to judge mutating alkali yl and site.
(6) mutating alkali yl and Altai Sheep meat yield carry out association analysis, determine the DNA molecular marker affecting Altai Sheep meat yield, and carry out test of significance to the whose body weight in Altai Sheep between polymorphic gene different genotype; Through test of significance, P < 0.01 time difference heteropole is remarkable; Significant difference during P < 0.05; During P > 0.05, difference is not remarkable; If significant difference or extremely remarkable, then illustrate cause gene amplification fragment to occur the mutating alkali yl of Multi-genotype is the gene mutation site of remarkably influenced Altai Sheep meat yield, this polymorphic gene can be used as the DNA molecular marker that impact determines Altai Sheep meat yield.
Meanwhile, the invention provides the application of molecule marking method in the meat production improving Altai Sheep for seed selection Altai Sheep meat production, be the Altai Sheep individuality of TT or TC for genotype, can eliminate; For the Altai Sheep individuality that genotype is CC, can continue to employ, carry out with this meat yield that seed selection improves Altay, Xinjiang sheep.
In the present invention, determining to affect in the DNA molecular marker of Altai Sheep meat yield, utilizing known biological analysis software SPSS17.0, carry out test of significance.
In the present invention, molecular marking technique PCR-SSCP (the Single Strand Conformation Polymorphism of application, single strand conformation polymorphism) and polyacrylamide gel electrophoresis (Polyacrylamide Gel Electrophoresis, PAGE) technology is all techniques well known means, is the common technique means that those of ordinary skill in the art adopt.
In the present invention, in the design of Altai Sheep MyoD1 gene-specific primer, by International Molecular bioinformation website NCBI (National Center for Biotechnology), retrieval obtains the DNA sequence dna (GenBank accession number: NM_001009390) of sheep MyoD1 gene, by known software Pr imer5.0, engineer's two pairs of Auele Specific Primers voluntarily, primer sequence send associated mechanisms to synthesize voluntarily.
By implementing the concrete summary of the invention of the present invention, following beneficial effect can be reached:
(1) in the present invention, although as mentioned above, in a kind of molecule marking method for seed selection Altai Sheep meat production of structure, relating in the technological step adopted utilizes known biological analysis software SPSS17.0 to carry out test of significance, molecular marking technique PCR-SSCP (the Single Strand Conformation Polymorphism of application, single strand conformation polymorphism) and polyacrylamide gel electrophoresis (Polyacrylamide Gel Electrophoresis, PAGE) technology, by in known software Pr imer5.0, although these technique means are all this area visible technique means, but carry out an Optimum combinatorial design for selecting prior art, utilizing candidate gene strategy, report is had no both at home and abroad to the research that Altai Sheep meat-producing traits carries out seed selection raising, in employing prior art, MyoD1 gene is as affecting Stock genetics and breeding candidate gene, for applicable sheep, particularly be suitable for the technology that Altai Sheep class meat proterties carries out seed selection raising aspect and have no report, because Altai Sheep kind is located in long-term low temperature, reach nearly half a year winter, and the coldlyest reach-35--40 DEG C, because natural condition are severe, many improved seeds here cannot be survived, and Altai Sheep is exactly under so special condition, formed by long-term nature and artificial selection, visible, molecule marking method for seed selection Altai Sheep meat production provided by the invention, Altai Sheep meat production is improved for seed selection there is important and far-reaching technical contribution and realistic function.
(2) the present invention is by specifically providing a kind of molecule marking method for seed selection Altai Sheep meat production and application thereof, two pcr amplified fragments of PCR-SSCP technology to Altay, Xinjiang sheep MyoD1 gene are utilized to carry out polymorphic detection, and carry out association analysis with meat-producing traits (body weight), thus find out the DNA molecular marker with Altai Sheep meat yield significant correlation, determine that superseded genotype is the Altai Sheep individuality of TT or TC, continue to employ the Altai Sheep individuality that genotype is CC, in order to improve the meat production of Altai Sheep, the meat yield that seed selection improves Altay, Xinjiang sheep is carried out with this, this has not only filled up the technological gap of this research field domestic and international, and for this improved seeds resource of protection Altai Sheep, improve peasants and herdsmen's income, accelerate the significant and actual application value of Xinjiang meat sheep industry development.
Accompanying drawing explanation
Fig. 1 is pcr amplification product electrophorogram, and in figure a, b, M is DL2000DNA Ma rke r, and swimming lane 1-10 is pcr amplification product.
Fig. 2 is 8% polyacrylamide gel electrophoresis figure, and in figure, swimming lane 2,3,5,6,8,9 is judged to be TT genotype, and swimming lane 1,7 is judged to be TC genotype, and swimming lane 4 is judged to be CC genotype.
Fig. 3 is DNA sequencing Comparative result figure, and in figure, Altai Sheep MyoD1 gene exists a base mutation T706C.
Embodiment
, for embodiment, the present invention is described below, but the present invention is not limited to following embodiment.Equipment in the present invention and material have:
The main raw material(s) adopted and related reagent etc.: Tutofusin tris (i.e. Tris-base) is provided by village alliance biology (BG05S); 2 × Taq PCR MasterMix is provided by Bo Maide biology (PC0912); DL2000DNA Marker is provided by village alliance biology (ZM404-1); EB is provided by the precious letter in Xinjiang.
Key instrument equipment: TG16-W High Speed microCentrifuges originates from Changsha Xiang Yi whizzer Instrument Ltd.; DYCZ-24F type electrophoresis chamber and DYY-6C type electrophoresis apparatus originate from Beijing Liuyi Instrument Factory; AL204-IC electronic balance originates from (Shanghai) Co., Ltd. of plum Teller-Tuo Le multiple instruments factory; JY04S gel imaging instrument originates from Jun Yi east, Beijing electrophoresis equipment company limited; Thermostat container water bath with thermostatic control vibrator originates from Beijing Sang Yi laboratory apparatus institute.
All raw and auxiliary materials, reagent and the instrument selected in the present invention, equipment are all well known selecting, but do not limit enforcement of the present invention, and other reagent more well known in the art and equipment are all applicable to the enforcement of the following embodiment of the present invention.
Embodiment one: the method for screening and Altai Sheep meat yield significant correlation DNA molecular marker
For the molecule marking method of seed selection Altai Sheep meat production, utilize the method for PCR-SSCP technology screening and Altai Sheep meat yield significant correlation DNA molecular marker, main inventive point is as follows:
One, extracting genome DNA
Gather Altai Sheep jugular vein blood 5ml to be measured, Sodium Citrate anti-freezing ,-20 DEG C of preservations;
With reference to " Molecular Cloning: A Laboratory guide ", extract genomic dna with phenol chloroform extraction method ,-20 DEG C of preservations.
Prepare 0.8% sepharose: agarose 0.8g, is dissolved in 100m10.5 × TBE, microwave-oven-heating, to fully dissolving, is poured into and is inserted with in the glue groove of comb after being down to room temperature, stand-by after cooled and solidified.
0.8% sepharose of preparation is utilized to carry out electrophoresis detection, voltage 100V, electric current 50mA, electrophoresis 30 minutes.After bromination ingot dyes 15 minutes, irradiate under gel imaging instrument ultraviolet lamp, observe size and the brightness of band, judge the extraction quality of DNA.
Two, pcr amplification
(1) design of primers and synthesis
According to the DNA sequence dna (GenBank accession number: NM001009390) of sheep MyoD1 gene, utilize software Primer5.0 independent design two pairs of primers, specific amplification MyoD1 gene, the sequence of two pairs of primers of design is respectively:
The upstream primer sequence F:5 '-CCGCTGTTTACTGTGGG-3 ' of primer 1, the downstream primer sequence R:5 '-GCTGGTTTGGGTTGCT-3 ' of primer 1; Amplified fragments size is 155bp.
The upstream primer sequence F:5 '-AGCAACCCAAACCAGC-3 ' of primer 2, the downstream primer sequence R:5 '-ATGCCGTCGGAACAGT-3 ' of primer 2, amplified fragments size is 218bp.
(2) pcr amplification reaction system and program optimization
According to two pairs of primers of design, two fragments of specific amplification Altai Sheep MyoD1 gene.PCR reaction system is: Mix10.0 μ l, and each 0.4 μ l of upstream and downstream primer, DNA profiling 1.5 μ l, add water to cumulative volume 20 μ l; Reaction conditions is: 94 DEG C of denaturation 5min, and 94 DEG C of sex change 30s, annealing temperature 30s, 72 DEG C of extension 1min, circulate 31 times, 72 DEG C extend 5min, 4 DEG C of preservations.
The annealing temperature of the pcr amplification reaction program of two pairs of primers is optimized.When other reaction conditionss are constant, PCR instrument arranges gradient to annealing temperature, gradient scope is 50 DEG C-60 DEG C, and namely average each plate hole transformation temperature is 1 DEG C.Detect through 1.5% agarose gel electrophoresis, observe brightness and the purity of amplified band, finally determine that the suitableeest annealing temperature of primer 1 is 52 DEG C, the suitableeest annealing temperature of primer 2 is 54 DEG C.
Two groups of PCR primer of specific amplification detect by 1.5% agarose gel electrophoresis, voltage 100V, electrophoresis 30min, after bromination ingot EB dyes 15min, irradiate under Bio-RAD gel imaging instrument ultraviolet lamp, observe size and the brightness of amplified band, judge PCR primer amplification quality.Result is see accompanying drawing 1, and accompanying drawing 1 is made up of figure a, b, schemes a and reflect the good situation of PCR primer amplification quality in accompanying drawing 1.
Three, SSCP detects
The preparation of (1) 8% non-denaturing polyacrylamide gel
Prepare 2 plate glue: 30% acrylamide soln 26.6ml, 5 × TBE20ml, 10%AP700 μ l, TEMED66 μ l, distilled water 52.8ml.At the uniform velocity pour into gel maker after stirring, and insert comb, after gelling is solid, slowly extract comb.
(2) pcr amplification product sex change
Get 3 μ l PCR primer, add 7 μ l denaturation buffer, 95% methane amide, 10mmol/L EDTA, 0.05% bromjophenol blue, 0.05% dimethylbenzene green grass or young crops, after brief centrifugation, in PCR instrument, 98 DEG C of sex change 10min, are placed on ice after taking-up immediately, whole loading after 10min.
(3) polyacrylamide gel electrophoresis
Appropriate 0.5 × tbe buffer liquid is added upper and lower groove, drives out of with the bubble of syringe by generation, and rinse loading wells; To close electrophoresis chamber lid by positive and negative electrode, after prerunning 30min, the PCR primer after drawing sex change with microsyringe adds loading wells; After point sample completes, voltage 180V, electric current 50mA, electrophoresis 20h.
(4) dye, develop the color:
From electrophoresis chamber, take out gel, after distilled water rinsing 2 times, add staining fluid (0.1% Silver Nitrate) 250ml, be put in shaking table lucifuge and shake 15min gently, pour out staining fluid;
Add nitrite ion (2% sodium hydroxide+0.1% formaldehyde) 250ml, submergence gel, shakes while observe, until gel manifests electrophoresis band; Pour out nitrite ion, use deionized water rinsing 2 ~ 3 times rapidly;
The imaging of Bio-RAD gel imaging system, carries out gene according to band quantity and position and sentences type.Result shows, the gene fragment that primer 1 increases exists polymorphism, and have three kinds of genotype: TT, TC and CC, result is see accompanying drawing 2; The gene fragment of primer 2 amplification does not have polymorphism.
Four, DNA sequencing
Choose the PCR primer 40 μ l of different genotype individuality in primer 1 amplification gene fragment, be sent to Shanghai biotechnology limited liability company and check order, utilize DNA MAN software analysis comparing dna sequence, find mutational site 706T/C, result is see accompanying drawing 3.
Five, association analysis
Utilize software SPSS17.0, carry out significance test of difference to the mean body weight (kg) between different genotype, result is as following table 1:
Table 1: the mean body weight (kg) between different genotype carries out significance test of difference table
Note: Mean ± sD represents mean+SD; Colleague's data, when shoulder is designated as different lowercase, represent significant difference (P < 0.05).
As can be seen from Table 1, in Altay, Xinjiang sheep, the population mean body weight of mutant homozygous type CC is significantly higher than wild homozygous TT2.52kg (P < 0.05).
This result shows, MyoD1 gene can be used as a molecule marker of Altay, Xinjiang sheep meat yield, can be used for the meat production that seed selection improves Altai Sheep.
Embodiment two: utilize the seed selection of molecule marker MyoD1 gene to improve Altai Sheep meat yield
Gather the blood sample 5ml of Altai Sheep individuality to be measured, extract genomic dna; Obtain the sequence of primer 1, carry out pcr amplification, wherein annealing temperature is 52 DEG C; Amplified production carries out SSCP detection after sex change; Genotype is judged with reference to accompanying drawing 2.
For the Altai Sheep individuality that genotype is TT or TC, can eliminate; For the Altai Sheep individuality that genotype is CC, can continue to employ, carry out with this meat yield that seed selection improves Altay, Xinjiang sheep.
As mentioned above, the present invention is in a kind of molecule marking method for seed selection Altai Sheep meat production of structure, relating in the technological step adopted utilizes known biological analysis software SPSS17.0 to carry out test of significance, molecular marking technique PCR-SSCP (the Single Strand Conformation Polymorphism of application, single strand conformation polymorphism) and polyacrylamide gel electrophoresis (Polyacrylamide Gel Electrophoresis, PAGE) technology, by in known software Pr imer5.0, although these technique means are all this area visible technique means, but utilizing candidate gene strategy, report is had no both at home and abroad to the research that Altai Sheep meat-producing traits carries out seed selection raising, for using MyoD1 gene as affecting Stock genetics and breeding candidate gene, adopt prior art for applicable sheep, particularly be suitable for the technology that Altai Sheep class meat proterties carries out seed selection raising aspect and have no report.Because Altai Sheep kind is located in long-term low temperature, reach nearly half a year winter, and the coldlyest reach-35--40 DEG C, because natural condition are severe, many improved seeds here cannot be survived, and Altai Sheep is exactly under so special condition, formed by long-term nature and artificial selection, visible, the molecule marking method for seed selection Altai Sheep meat production provided by the invention, improves Altai Sheep meat production for seed selection and has important and far-reaching technical contribution and realistic function.
By the molecule marking method for seed selection Altai Sheep meat production that above-described embodiment provides, two pcr amplified fragments of PCR-SSCP technology to Altay, Xinjiang sheep MyoD1 gene are utilized to carry out polymorphic detection, and carry out association analysis with meat-producing traits (body weight), thus find out the DNA molecular marker with Altai Sheep meat yield significant correlation, for the Altai Sheep individuality that genotype is TT or TC, can eliminate; For the Altai Sheep individuality that genotype is CC; can continue to employ; in order to improve the meat production of Altai Sheep; carry out with this meat yield that seed selection improves Altay, Xinjiang sheep, this is for this improved seeds resource of protection Altai Sheep, raising peasants and herdsmen income, the significant and actual application value of quickening Xinjiang meat sheep industry development.
Claims (1)
1. the application of molecule marking method in the meat production improving Altai Sheep of a seed selection Altai Sheep meat production, it is characterized in that, the application of molecule marking method in the meat production improving Altai Sheep of seed selection Altai Sheep meat production is specific as follows:
(1) Altai Sheep extracting genome DNA: gather Altai Sheep blood sample 5ml, extract DNA ,-20 DEG C of preservations; 0.8% agarose gel electrophoresis detects, and after the dyeing of bromination ingot, irradiates under gel imaging instrument ultraviolet lamp, observes size and the brightness of band, judges the extraction quality of DNA;
(2) Altai Sheep MyoD1 gene-specific primer design: the DNA sequence dna (GenBank accession number: NM_001009390) utilizing sheep MyoD1 gene, engineer's two pairs of Auele Specific Primers voluntarily, two pairs of specific primer sequences are respectively:
Primer 1:
Upstream primer F:5 '-CCGCTGTTTACTGTGGG-3 ';
Downstream primer R:5 '-GCTGGTTTGGGTTGCT-3 ';
Primer 2:
Upstream primer F:5 '-AGCAACCCAAACCAGC-3 '
Downstream primer R:5 '-ATGCCGTCGGAACAGT-3 ';
(3) pcr amplification of Altai Sheep MyoD1 gene: according to two pairs of primers of design, two fragments of specific amplification Altai Sheep MyoD1 gene; PCR reaction system is: Mix10.0 μ l, and each 0.4 μ l of upstream and downstream primer, DNA profiling 1.5 μ l, add water to cumulative volume 20 μ l; Reaction conditions is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, annealing temperature 30s, 72 DEG C of extension 1min, and circulate 31 times, 72 DEG C extend 5min, 4 DEG C of preservations; Two PCR primer of specific amplification detect by 1% agarose gel electrophoresis, after the dyeing of bromination ingot, irradiate under gel imaging instrument ultraviolet lamp, observe size and the brightness of amplified band, judge PCR primer amplification quality;
(4) SSCP detects: carry out SSCP detection to two PCR primer of Altai Sheep MyoD1 gene respectively, and screening has the pcr amplified fragment of polymorphism; Get 3 μ l PCR primer to mix with the denaturing agent of 98% deionized formamide of 7 μ l, ice bath 10min after 98 DEG C of sex change 10min, make it to keep denatured state; PCR primer 8% non denatured polyacrylate hydrogel (polyacrylamide gel, PAG) 180V electrophoresis 20h after sex change, after the colour developing of silver dye, irradiate under gel imaging instrument white light, observe the size of band, determine whether polymorphism, have polymorphic judgement genotype;
(5) DNA sequencing, finds out Altai Sheep MyoD1 gene mutation site T706C; There is during SSCP is detected the gene of polymorphism, the PCR primer of its different genotype is carried out DNA sequencing, in order to judge mutating alkali yl and site;
(6) mutating alkali yl and Altai Sheep meat yield carry out association analysis, determine the DNA molecular marker affecting Altai Sheep meat yield, and carry out test of significance to the whose body weight in Altai Sheep between polymorphic gene different genotype; Through test of significance, P < 0.01 time difference heteropole is remarkable; Significant difference during P < 0.05; During P > 0.05, difference is not remarkable; If significant difference or extremely remarkable, then illustrate cause gene amplification fragment to occur the mutating alkali yl of Multi-genotype is the gene mutation site of remarkably influenced Altai Sheep meat yield, this polymorphic gene can be used as the DNA molecular marker that impact determines Altai Sheep meat yield;
Superseded genotype is that the Altai Sheep of TT or TC is individual, continues to employ the Altai Sheep individuality that genotype is CC, carrys out with this meat yield that seed selection improves Altay, Xinjiang sheep.
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