CN102719545A - Identification method of cattle excellent superovulation character molecular marker by hypoxia-inducible factor and application thereof - Google Patents
Identification method of cattle excellent superovulation character molecular marker by hypoxia-inducible factor and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of animal gene engineering, and in particular relates to clone of an HIF3alpha gene segment of a cattle excellent superovulation character related gene by utilizing a specific primer and an application thereof in cattle marker-assisted selection. The length of the nucleotide sequence of the acquired HIF3alpha gene segment is 379bp; an A->G base mutation exists in the 278thbp of the segment, which causes generation of Alw44I-RFLP restriction fragment polymorphism; and the mutation site is used as a molecular marker, and is detected by using restriction enzyme Alw44I, and the detection result and the cattle excellent superovulation character are related. Analysis results prove that the superovulation characters of individuals with different gene types are remarkably different. The identification method has the characteristics that partial segment of the HIF3alpha gene related to the cattle excellent superovulation character is acquired by utilizing the specific primer, and the segment is subjected to polymorphism analysis by utilizing a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method, so that a molecular marker and an application thereof are provided to cattle marker-assisted selection.
Description
Technical field
The invention belongs to animal gene engineering technology field, the clone who is specifically related to ox hypoxia inducible factor 3 alpha gene fragments with and application in ox superovulation proterties marker assisted selection.
Background technology
(Mapletoft RJ etc. such as Mapletoft; Assisted reproductive technologies in cattle:a review.Rev Sci Technol 2005; 24:393-403) report; Superovulation is one of the most significant technology in the mankind and animal embryo fields of implantation, is employed for many years.This technology is the important component part of ox Embryo Production and embryo transfer in the worldwide especially.Have above 500,000 pieces of embryos through this technology acquisition every year in the worldwide.In ox embryo transfer process, the morula of more options normal development (Morula) class above available embryo (fine morula and blastaea) transplant.Therefore, utilize this technology to obtain more available embryo, ox Embryo Production and transplanting are had great importance.
Mapletoft etc. (Mapletoft R J etc., Recent advances in the superovulation in cattle.Reprod Nutr Dev 2002,42:601-611) report, inherited genetic factors possibly be one of factor that influences the superovulation effect.(Fauser B C etc. such as Fauser; Predictors of ovarian response:progress towards individualized treatment in ovulation induction and ovarian stimulation.Hum Reprod Update 2008; 14:1-14) report, the mononucleotide genetic polymorphism of candidate gene possibly become the first-selected factor of prediction ovary response.
Maynard M A etc. report (Maynard M A etc.; Multiple splice variants of the human HIF-3 α locus are targets of the von Hippel-Lindau E3 ubiquitin ligase complex.J Biol Chem.2003,278:11032-11040) hypoxia inducible factor 3 α (HIF-3 α) have very high expression amount in people's placenta and cardiac muscle.Genbacev O etc. report (Genbacev O etc.; Human cytotrophoblast expression of the von Hippel-Lindau protein is down-regulated during uterine invasion in situ and upregulated by hypoxia in vitro.Dev.Biol.2001; 233:526-536) because very limited through the amount of oxygen that placenta offers fetus through maternal blood; The embryo just is exposed under the low-oxygen environment gradually; At this moment HIF-3 α can not degrade again, and accumulates in performance biological action in the nucleus in a large number.(Toshiharu Yamashita etc. such as Toshiharu Yamashita; Abnormal Heart Development and Lung Remodeling in Mice Lacking the Hypoxia-Inducible Factor-Related Basic Helix-Loop-Helix PAS Protein NEPAS.Molecular and cellular Biology.2008; 4 (28): 1285-1297) also report; In embryo and newborn stage, significant variation also takes place in the HIF-3 alpha expression.(Formenti F etc. such as Formenti F; " Regulation of human metabolism by hypoxia-inducible factor.Proc.Natl.Acad.Sci.U.S.A.107 (28): 12722-12727) proof is in Mammals, and disappearance HIF can cause the death of fetus.Simultaneously, it also plays central role in regulating human body metabolism.
Therefore, hypoxia inducible factor 3 α (HIF3A) gene can be chosen to be candidate gene, and its mononucleotide genetic polymorphism possibly provide molecule marker for the assisted Selection of animal reproduction and production performance.
Summary of the invention
The objective of the invention is to clened cows hypoxia inducible factor 3 α (HIF3A) gene fragment; Identify its specific mutational site with detection method, for the ox marker-assisted breeding provides a kind of significant molecule marker as Niu Youliang superovulation trait related gene polymorphum.
The present invention realizes through following technical scheme:
A kind of 379bp sequence of passing through ox superovulation trait related gene hypoxia inducible factor 3 α (HIF3A) of Auele Specific Primer and the acquisition of PCR (Polymerase Chain Reaction) method is shown in table SEQ ID NO:1.
Just like the base mutation of the described C278-G278 of table SEQ ID NO:1, cause the generation of Alw44I-RFLP (Restriction Fragment Length Polymorphism) polymorphum during the DNA of the HIF3A gene fragment that obtains is following.
A kind of screening is applicable to the molecular marker method that Niu Youliang superovulation proterties is relevant, prepares according to following steps:
A pair of through design specific primers, forward primer HIF3A-F:5 '-CTGGGCAGTTGCTACTGTTCCTAT-3 ' and reverse primer HIF3A-R:5 '-AGTCCCGTCCAGGATTGGT-3 ' extract genomic dna from bovine blood, carry out pcr amplification.Because the 278th base mutation that has C-G of the segmental dna sequence dna of PCR product causes the generation of Alw44I-RFLP polymorphum, utilizes restriction enzyme A lw44I can carry out the detection in this mutational site.
The segmental enzyme of PCR product is cut product 2% agarose gel electrophoresis capable of using and is detected, with different genotype shown in the detected result and the association analysis of ox superovulation proterties.The individuality that possesses the special genes type will obtain better superovulation effect.
Below the present invention is explained in detail:
One, the clone of ox hypoxia inducible factor 3 α (HIF3A) gene fragment
Utilize biological software Oligo6.0 to design a pair of Auele Specific Primer.Set up the PCR reaction conditions, particular case is following:
HIF3A-F:5 '-CTGGGCAGTTGCTACTGTTCCTAT-3 ' (forward primer)
HIF3A-R:5 '-AGTCCCGTCCAGGATTGGT-3 ' (reverse primer)
For obtaining good result quickly, the present invention selects 2 * Bench Top of Biomiga company
TMTaq Master mix carries out pcr amplification.Concrete reaction system is: 2 * Master Mix (provides in the product packing box, wherein contains Taq DNA Polymerase 0.05 units/ μ l, 4mM MgCl
2, 0.4mM dNTPs) and 10.0 μ l, each 0.5 μ l of forward primer and reverse primer (concentration is for being 10pmol/ μ l), genomic dna 0.5 μ l (containing 10-50ngDNA), two heat up in a steamer water 8.5 μ l.The PCR reaction conditions is: 94 ℃ of preparatory sex change 5 minutes, and 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, totally 35 circulations, 72 ℃ of last extensions 5 minutes.
Two, the foundation of RLFP diagnostic method
Obtain the specific amplified fragment (SEQ ID NO:1) of 379bp with primer HIF3A-F and HIF3A-R amplification cow genome group DNA.Sequencing result is found, in this 379bp fragment, because there is the sudden change of C → G in the 278bp place, thereby causes the generation of Alw44I restriction enzyme site (G^TGCAC), wherein, is the polymorphum point of contact of Alw44I enzyme between 278-279bp.Pcr amplification product is cut through the Alw44I enzyme and is produced three kinds of genotype, can obviously differentiate different banding patterns through 2% agarose gel electrophoresis.Wherein the CC type has only 379bp one band, and the CG type contains 379bp, 278bp, 101bp three bands, and the GG type has 101bp and 278bp two bands (Fig. 2).
Three, mark property association analysis
With the experimental population that the applicant was had is that experimental subjects is carried out the measurement of superovulation proterties, utilizes SPSS19.0 software to carry out different genotype and interindividual proterties association analysis.
Effect of the present invention is as follows:
1, the clone of ox HIF3A Gene Partial dna sequence dna
Pcr amplification product is shown as the specific PCR product through 2% agarose gel electrophoresis detected result, and is as shown in Figure 1.The PCR product is reclaimed order-checking, and the result shows that product length is 379bp.Sequencing result shows that this fragment 278bp place exists C, two allelotrope of G, and the peak that partly checks order is as shown in Figure 3.
2, be directed against the foundation of ox HIF3A Gene Partial dna fragmentation PCR-RFLP diagnostic method
The 379bp specific amplified fragment that the primer amplified cow genome group DNA that designs with the present invention obtains.The result of order-checking finds can cause Alw44I restriction enzyme site (G^TGCAC) in the sudden change that this 379bp fragment exists, and wherein the 278bp-279bp place is a restriction enzyme site.Amplified production is cut through restriction enzyme A lw44I enzyme can produce three kinds of genotype, i.e. CC type, CG type and GG type, and concrete picture is as shown in Figure 2.
3, mark property is carried out association analysis
The association analysis result of the extension increasing sequence Alw44I polymorphic site of ox HIF3A gene and ox superovulation proterties shows among the present invention, and some superovulation proterties of the pairing individuality of this restriction enzyme site different genotype exist significantly or extremely significant difference.
Description of drawings
Fig. 1 is that the HIF3A gene amplification product is through 2% agarose gel electrophoresis detected result.Wherein the M swimming lane is standard molecular weight Marker, and the 1-14 swimming lane is 14 PCR products to be detected.
Fig. 2 is that the Alw44I enzyme that 2% agarose gel electrophoresis detects 20 HIF3A gene amplification products randomly drawing is cut the result.Wherein the M swimming lane is standard molecular weight Marker. Swimming lane 11,15,20 is that the GG type is individual; Swimming lane 1,2,3,4,7,9,10,12,13,14,16,17,18,19 is that the AG type is individual; Swimming lane 5,6,8 is that the GG type is individual.
Fig. 3 is the cloning and sequencing peak figure of sudden change position in the HIF3A Gene Partial dna sequence dna that increases of the present invention.Arrow indication position is the base mutation position.
Fig. 4 is 2% a sepharose detected result of pcr amplification product among the embodiment.Wherein the M swimming lane is standard molecular weight Marker, and swimming lane 1-22 is a PCR product to be detected.
Fig. 5 is 2% a sepharose detected result of endonuclease reaction product among the embodiment.Wherein the M swimming lane is standard molecular weight Marker, and swimming lane 1-22 is that 22 enzymes to be detected are cut product.Swimming lane 7,15,17 is that the CC type is individual; Swimming lane 1,2,3,4,5,6,8,9,10,11,12,13,14,16,18,19,21 is that the CG type is individual; Swimming lane 20,22 is that the GG type is individual.
Embodiment
The present invention describe particular content in detail in order more to be expressly understood with embodiment.
The present invention is a template with the cow genome group DNA that extracts, and design specific primers is a pair of, and the partial dna sequence of clened cows HIF3A gene utilizes the PCR-RFLP method, uses restriction enzyme A lw44I that the pcr amplification reaction product is carried out enzyme and cuts.Enzyme is cut as a result somatotype and different genotype and superovulation proterties are carried out association analysis, for the marker assisted selection of ox provides molecule marker.
One, the clone of ox HIF3A Gene Partial dna fragmentation
Utilize 1 pair of Oligo6.0 software design Auele Specific Primer, for guaranteeing good primer quality, in the present invention, primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and amplified production length is 379bp, and primer sequence is as follows:
HIF3A-F:5 '-CTGGGCAGTTGCTACTGTTCCTAT-3 ' (forward primer)
HIF3A-R:5 '-AGTCCCGTCCAGGATTGGT-3 ' (reverse primer)
For obtaining good result comparatively fast, preferably, the present invention selects 2 * Bench Top of Biomiga company
TMTaqMaster mix carries out pcr amplification.Concrete reaction system is: 2 * Master Mix (provides in the product packing box, wherein contains Taq DNA Polymerase 0.05 units/ μ l, 4mM MgCl
2, 0.4mM dNTPs) and 10.0 μ l, each 0.5 μ l of forward primer and reverse primer (concentration is for being 10pmol/ μ l), genomic dna 0.5 μ l (containing 10-50ngDNA), two heat up in a steamer water 8.5 μ l.The PCR reaction conditions is: 94 ℃ of preparatory sex change 5 minutes, and 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, totally 35 circulations, 72 ℃ of last extensions 5 minutes.
Two, the foundation of PCR-RFLP method
1, the detection of PCR product
The specificity product that utilizes designed primer HIF3A-F of the present invention and HIF3A-R that cow genome group DNA cloning is obtained; Detect through 2% agarose gel electrophoresis; The result is as shown in Figure 1, and wherein the M swimming lane is standard molecular weight Marker, and the 1-14 swimming lane is 14 PCR products to be detected.
2, be directed against the foundation of ox HIF3A Gene Partial dna fragmentation PCR-RFLP diagnostic method
Utilize Auele Specific Primer HIF3A-F and HIF3A-R amplification cow genome group DNA that the present invention designs, obtain the specific amplified fragment (SEQ ID NO:1) of 379bp.Sequencing result is found, in this 379bp fragment, if there is the sudden change of C → G in the 278bp place, can cause the generation of Alw44I restriction enzyme site (G^TGCAC), wherein, is the polymorphum point of contact of Alw44I enzyme between 278-279bp.Pcr amplification product is cut through the Alw44I enzyme and is produced three kinds of genotype, can obviously differentiate different banding patterns through 2% agarose gel electrophoresis.As shown in Figure 2, wherein the CC type has only 379bp one band, and the CG type contains 379bp, 278bp, 101bp three bands, and the GG type has 278bp and 101bp two bands.
Restriction enzyme A lw44I can select voluntarily.For reaching efficient, high speed effect, select the Alw44I restriction endonuclease of MBI Fermentas Life Science (MBI company) for use among the present invention.PCR product endonuclease reaction system is 16 μ l, and wherein 10 * Buffer Tango [provides in the restriction endonuclease packing box, contains 330mM Tris-acetate (PH7.9); 100mM magnesium acetate, 660mM potassium acetate, 1mg/ml BSA] 1.6 μ l; PCR product 5.0 μ l, restriction enzyme A lw44I 1.0 μ l (total amount 10U provides in the restriction endonuclease packing box); Two heated up in a steamer water 8.4 μ l, with the even back 37 ℃ of waters bath with thermostatic control of sample mix 8 hours.Water bath with thermostatic control is got 10 μ l enzymes and is cut product after finishing, and detects enzyme with 2% agarose gel electrophoresis and cuts the result, record genotype detection result.
Three, mark property association analysis
With the experimental population that the applicant was had is that experimental subjects is carried out the measurement of superovulation proterties, utilizes SPSS19.0 software to carry out different genotype and interindividual proterties association analysis.
The association analysis of the extension increasing sequence Alw44I polymorphic site of ox HIF3A gene and ox superovulation proterties among the present invention; Genotype detection result shows; The different superovulation proterties that Different Individual had comprise that significant difference analytical results (least square mean standard error) is seen table 1 between unfertilized embryo number, degeneration embryo number, available embryo number, embryo's sum, morula number, the blastaea number.
Significant difference analytical results between the individual superovulation proterties of table 1 different genotype
| Ultra row's proterties | The CC type is individual | The CG type is individual | The GG type is individual |
| Unfertilized embryo number | 0.80±0.197 a | 1.70±0.198 ab | 2.23±0.316 b |
| The degeneration embryo number | 2.03±0.461 a | 2.75±0.212 ab | 3.97±0.491 B |
| Available embryo number | 7.95±0.728 a | 8.03±0.488 a | 10.12±1.025 a |
| Embryo's sum | 10.78±1.036 a | 12.44±0.551 a | 16.32±1.225 B |
| The morula number | 7.00±0.618 a | 7.70±0.459 a | 9.87±1.059 b |
| The blastaea number | 0.35±0.377 | 0.41±0.077 | 0.45±0.147 |
Annotate: the shoulder marker tape has different letter representation significant differences (P＜0.05); Have different capitalizations and represent difference extremely significantly (P＜0.01).
Analytical results shows that some superovulation proterties of the pairing individuality of this restriction enzyme site different genotype exist significantly or extremely significant difference.It is individual that the individual superovulation proterties of GG type generally is superior to the CC type, though existing negative effect aspect unfertilized embryo number and the degeneration embryo number two, for Embryo Production, can obtain more morula and blastaea and have more actual application value.Therefore, during by superovulation individual, select GG type individuality will help obtaining more available embryo selected.
Embodiment
Selected at random 22 healthy individuals of handling through superovulation in the cow colony of Jilin black wool ox industry ltd.Gather blood and carry out extracting genome DNA.Utilize designed primer of the present invention, PCR reaction system and condition to carry out pcr amplification.Get 2 * Master Mix of 10.0 μ l, each 0.5 μ l of forward primer and reverse primer (concentration is for being 10pmol/ μ l), genomic dna 0.5 μ l (containing 10-50ngDNA), two heat up in a steamer water 8.5 μ l.The PCR reaction conditions is: 94 ℃ of preparatory sex change 5 minutes, and 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, totally 35 circulations, 72 ℃ of last extensions 5 minutes.
Amplified production detects through 2% agarose gel electrophoresis, and the result is shown as the specific PCR product.As shown in Figure 4, wherein the M swimming lane is standard molecular weight Marker, and swimming lane 1-22 is a PCR product to be detected.The PCR product is carried out endonuclease reaction by the RFLP method of the present invention's design.PCR product endonuclease reaction system is 16 μ l, 10 * Buffer Tango, 1.6 μ l wherein, and PCR product 5.0 μ l, restriction enzyme A lw44I 1.0 μ l, two heat up in a steamer water 8.4 μ l, with evenly back 37 ℃ of waters bath with thermostatic control 8 hours of sample mix.For improving endonuclease reaction efficient, can whenever reaction tubes be taken out at a distance from 1 hour, the part evaporated liquid gently is thrown to the pipe bottom.Water bath with thermostatic control is got 10 μ l enzymes and is cut product after finishing, and detects enzyme with 2% agarose gel electrophoresis and cuts the result.As shown in Figure 5, wherein the M swimming lane is standard molecular weight Marker, and swimming lane 1-22 is that enzyme to be detected is cut product.Swimming lane 7,15,17 is that the CC type is individual; Swimming lane 1,2,3,4,5,6,8,9,10,11,12,13,14,16,18,19,21 is that the CG type is individual; Swimming lane 20,22 is that the GG type is individual.
With these 22 experiment samples is that experimental subjects is carried out the proterties association analysis, utilizes SPSS19.0 software to carry out different genotype and interindividual proterties association analysis.
Genotype detection result shows that the significant difference analytical results of the superovulation average between the pairing individuality of different genotype (least square value and standard error) is seen table 4.
Significant difference analytical results between the individual shearing force of table 2 different genotype
| Ultra row's proterties | The CC type is individual | The CG type is individual | The GG type is individual |
| Unfertilized embryo number | 0.33±0.333 | 1.43±0.441 | 1.60±0.678 |
| The degeneration embryo number | 1.33±0.667 | 2.07±0.539 | 3.60±1.364 |
| Available embryo number | 2.67±0.333 a | 5.00±0.989 a | 8.40±2.421 b |
| Embryo's sum | 4.00±0.577 a | 8.43±1.118 b | 13.60±1.400 B |
| The morula number | 2.33±0.333 | 4.79±0.921 | 7.80±2.267 |
| The blastaea number | 0.33±0.333 | 0.29±0.163 | 0.60±0.600 |
Annotate: least square mean standard error shoulder marker tape has different letter representation significant differences (P＜0.05) in the table
Have different capitalizations and represent difference extremely significantly (P＜0.01).
Analytical results shows, in the individual superovulation proterties of GG type, though that unfertilized embryo number and degeneration embryo number are higher than the CC type is individual, in other proterties, especially aspect available embryo number and embryo's sum, the individual remarkable or utmost point of GG type is significantly higher than CC type individuality.It is the beneficial gene type that is beneficial to Embryo Production.
Claims (2)
1. a pair of Auele Specific Primer that is used to identify ox hypoxia inducible factor 3 α (HIF3A) gene fragment is characterized in that the sequence of said primer is following:
CCR9-F:5 '-CTGGGCAGTTGCTACTGTTCCTAT-3 ' (forward primer)
CCR9-R:5 '-AGTCCCGTCCAGGATTGGT-3 ' (reverse primer)
2. the application of the described primer of claim 1 in Niu Youliang superovulation proterties table and assisted Selection.
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| CN104651523A (en) * | 2015-03-09 | 2015-05-27 | 吉林大学 | Applications of single-stranded nucleotide sequence of INHA gene as animal superovulation molecule marker, and detection method |
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| CN105132579A (en) * | 2015-10-15 | 2015-12-09 | 吉林大学 | Application of zinc finger protein 33B gene for serving as cattle superovulation character molecular marker |
| CN109735627A (en) * | 2019-02-25 | 2019-05-10 | 吉林大学 | Application of the BTBD9 gene as ox superfecundation trait molecular marker |
| CN109994153A (en) * | 2019-04-09 | 2019-07-09 | 山东省农业科学院奶牛研究中心 | A kind of method and its application for screening ox high altitude hypoxia adaptation molecular labeling |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104651523A (en) * | 2015-03-09 | 2015-05-27 | 吉林大学 | Applications of single-stranded nucleotide sequence of INHA gene as animal superovulation molecule marker, and detection method |
| CN104878124A (en) * | 2015-06-12 | 2015-09-02 | 广西壮族自治区兽医研究所 | Multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus) |
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| CN109735627A (en) * | 2019-02-25 | 2019-05-10 | 吉林大学 | Application of the BTBD9 gene as ox superfecundation trait molecular marker |
| CN109735627B (en) * | 2019-02-25 | 2022-04-19 | 吉林大学 | Application of BTBD9 gene as bovine superovulation trait molecular marker |
| CN109994153A (en) * | 2019-04-09 | 2019-07-09 | 山东省农业科学院奶牛研究中心 | A kind of method and its application for screening ox high altitude hypoxia adaptation molecular labeling |
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