CN102899395A - Breed selection method for improving mastitis resistance of dairy cow and use thereof - Google Patents
Breed selection method for improving mastitis resistance of dairy cow and use thereof Download PDFInfo
- Publication number
- CN102899395A CN102899395A CN2012102033585A CN201210203358A CN102899395A CN 102899395 A CN102899395 A CN 102899395A CN 2012102033585 A CN2012102033585 A CN 2012102033585A CN 201210203358 A CN201210203358 A CN 201210203358A CN 102899395 A CN102899395 A CN 102899395A
- Authority
- CN
- China
- Prior art keywords
- gene
- pcr
- seq
- cow
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a breed selection method for improving mastitis resistance of a dairy cow and a use thereof, and belongs to the technical field of animal genetic breeding. The breed selection method comprises the following steps of 1, providing a dairy cow mastitis-susceptible/resistant alpha-2 macroglobulin (A2M) candidate gene by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and isobaric tags for relative and absolute quantitation (iTRAQ), 2, analyzing alternative splicing by reverse transcription-polymerase chain reaction (RT-PCR) and cloning sequencing, and simultaneously, detecting single nucleotide polymorphism (SNPs) of the dairy cow mastitis-susceptible/resistant A2M candidate gene by a gene direct sequencing technology, and 3, analyzing the relationship between the alternative splicing and the SNPs by software, simultaneously, carrying out association analysis of a somatic cell score (SCS) and an A2M genetype, determining that a SNPs locus is a functional locus, and selecting a favorable allele individuals as a mastitis-resistant dairy cow breed. Through the breed selection method as a genetic breeding technology, mastitis resistance of a dairy cow is improved; a drug use amount is reduced; milk quality and safety are improved; and an economic loss is reduced.
Description
Technical field
The present invention relates to the Animal Genetics technical field, specifically a kind of implementation method and application thereof that improves the mastitis-resisting ability of milk cow.
Background technology
General, mazoitis is one of the most common in the raising dairy cattle, the most complicated disease, also is to cause milk cattle cultivating already to lose maximum a kind of disease.At present, the whole world has 2.6457 hundred million cow heads (FAO, 2010) approximately, and 1/3 milk cow trouble mazoitis is arranged every year approximately.According to estimates, about 95% case is to be infected by streptococcus aureus (Staph.aureus), suis (Strep.uberis) and intestinal bacteria (E.coli) to cause, the annual loss that causes because of mazoitis is up to 35,000,000,000 dollars (Wellenberg etc., 2002).About the history in existing more than 100 year of research of mastadenitis of cow pathogeny and control, and obtained significant progress.Yet mastadenitis of cow is a kind of very complicated disease, although many countries all implement comprehensive preventive health measures to various mazoitiss, it is a kind of disease that is subjected to multifactor impact, is difficult to control.At present, the sickness rate that control and the preventive measures of mazoitis are not reduced mazoitis on the whole, and also mazoitis also is the main reason (Villli, 2001) that microbiotic used during milk cow produced.Therefore, we reduce the sickness rate of mazoitis in the urgent need to inquiring into other approach, select to improve a kind of best long term policy (Burton etc., 2001) that the mastadenitis of cow resistance is considered to solve the mazoitis problem by heredity.If by the genetic breeding means, can make milk cow strengthen the resistance of mazoitis, the usage quantity of minimizing medicine is improved milk qualities safety, reduces financial loss, and this is perplexing the difficult problem of world's development of dairy industry for a long time to be expected to fundamentally to solve mazoitis.The heritability of milk cow resistance is very low, based on the genetic improvement poor effect of Phenotypic Selection.And the milk cow molecular breeding can be realized the early stage selection of milk cow, greatly shortens the generation interval of milk cow genetic improvement, saves the seed selection cost, improves efficiency of selection.The molecule marker that has in the gene can affect expression regulation, gene function of gene etc. such as single nucleotide mutation, the phenotype of individuality is had important impact.Therefore, be well worth doing based on the molecular breeding technology of functional molecular marker assisted selection.
α-2-macroglobulin (A2M) gene is positioned at No. 5 karyomit(e)s of ox, and full length gene 48.3kbp comprises 37 exons and 36 introns, transcribes the mRNA of rear generation 4.9kbp.A2M is as a kind of upper conservative non-specific broad-spectrum protease inhibitor of evolving, suppress to invade the parasite of body and multiple protein enzyme and the toxin that pathogenic agent discharges, reduce it to host's destruction, strengthen host's disease resistance (Armstrong PB, Quigley JP.1999.Alpha2-macroglobulin:an evolutionarily conserved arm of the innate immune system.Dev Comp Immunol.23,375-90).Complement component 3 (C3) in it and the complement system, Complement component C4 (C4) and C5 (C5) all belong to α-macroglobulin family.
Variable shearing is a kind of important post-transcriptional control mechanism, can improve the diversity of gene product.Variable shearing is Eukaryotic a kind of basic and important regulatory mechanism, the function of its meticulous coordination gene, the quantitative expression of efficient regulatory gene and the variation of protein function, make the transcription product of a gene in the different etap, under noble cells and the physiological status, by different cut modes, can obtain different mRNA and translation product, thereby affect the mode of connection between protein and its each part, the activity of enzyme, the change that distributes in space structure and the cell is to the differentiation of cell, grow, physiological function and pathological state are all significant.A gene can produce multiple mRNA by variable shearing, produces multiple proteins, but for few known to its regulation mechanism.Exon montage enhanser (ESE) has been brought into play very important effect as causing one of cis acting original paper of variable shearing in the process that produces variable shearing.Mononucleotide polymorphic (SNPs) is one of transgenation, can cause the unusual shearing of Pre-mRNA, comprises exon skipping, intron retention etc.Recently, many report proof SNPs cause gene to produce the major cause of unusual variable shearing.(Montgomery?SB，Sammeth?M，Gutierrez-Arcelus?M，Lach?RP，Ingle?C，Nisbett?J，Guigo?R，Dermitzakis?ET.2010.Transcriptome?genetics?using?second?generation?sequencing?in?a?Caucasian?population.Nature.464，773-777)。Reported that many unusual variable shearings and people's numerous disease are related.Up to the present do not have ox A2M gene to have variable shearing and cause the report of the functional SNPs of variable shearing, do not have the report of A2M gene function SNPs and milk cow mastitis-resisting disease yet.
Summary of the invention
Technical assignment of the present invention is to solve the deficiencies in the prior art, and a kind of implementation method and application thereof that improves the mastitis-resisting ability of milk cow is provided.
Technical scheme of the present invention realizes in the following manner: a kind of seed choosing method that improves the mastitis-resisting ability of milk cow, and its relative with isotopic labeling by real time fluorescent quantitative RT-qPCR to method absolute quantitation iTRAQ provides a mastadenitis of cow susceptible/resistance A2M candidate gene; Analyze variable shearing by reverse transcription RT-PCR and cloning and sequencing, simultaneously by the single nucleotide polymorphism SNPs of gene direct Sequencing technology for detection to this gene, utilize the relation of the variable shearing of software analysis and SNPs, simultaneously somatocyte scoring SCS and A2M genotype are carried out association analysis, judge that the SNPs site is functional site, select favourable aflelotype unity to reserve seed for planting as milk cow mastitis-resisting ability.
Improve the seed choosing method of the mastitis-resisting ability of milk cow, the steps include:
A: screening A2M gene is as mastadenitis of cow susceptible/resistance candidate gene, a1: A2M mRNA is carried out quantitative fluorescent PCR, the record result, a2: utilize isotopic labeling method relative and absolute quantitation iTRAQ A2M albumen to be carried out relative quantification, the record result;
B: utilize reverse transcription RT-PCR amplified production electrophoresis and cloning and sequencing to analyze variable shearing;
The functional SNP site of c:A2M gene is differentiated;
The analysis of d:SNPs and variable shearing relation.
3, a kind of seed choosing method that improves the mastitis-resisting ability of milk cow according to claim 2 is characterized in that a: screen the A2M gene as mastadenitis of cow susceptible/resistance candidate gene,
A1: A2M mRNA is carried out the quantitative fluorescent PCR step is:
Select at first respectively 3 normal and 3 mammary tissues of suffering from mazoitis, utilize
Premix Ex TaqTM II (Dalian Bao Bio-Engineering Company, China) and
480 II (Roche Diagnostics) instrument carries out real-time fluorescence quantitative PCR (RT-qPCR), relative expression's level of gene with housekeeping gene β-actin (sequence number: NM_173979.3) do confidential reference items, the experiment triplicate;
20 μ l RT-qPCR amplification systems: 10.0 μ l
Premix Ex TaqTM II (2 *), 0.4 μ M upstream and downstream primer:
A2M-F：SEQ?ID?NO.1，A2M-R：SEQ?ID?NO.2；
Or
β-actinF：SEQ?ID?NO.3，β-actin-R：SEQ?ID?NO.4；
2.0μl?A2M?cDNA(＜100ng)
Or
β-actin plasmid DNA, 6.4 μ l distilled water;
Amplification reaction condition: 94 ℃ of 5min; Then 94 ℃ of 15s, 56 ℃ of 15s, this step is totally 40 circulations; Last 70 ℃ of 5s,
The result that analysis of fluorescence is quantitative, relatively A2M mRNA in the mammary tissue of suffering from the mazoitis milk cow expression amount and the expression amount in the normal mammary tissue;
A2: utilize isotopic labeling relatively and the method for absolute quantitation iTRAQ is carried out the relative quantification step to A2M albumen and is:
Utilize isotopic labeling method relative and absolute quantitation iTRAQ mammary tissue A2M albumen normal to 3 and 3 trouble mazoitiss to carry out relative quantification, the albumen reference database of ox is UniProt ID:Q9H0H5, and the result of iTRAQ relatively suffers from mazoitis and organizes A2M expressing quantity and normal tissue expression amount;
B: the step of utilizing reverse transcription RT-PCR amplified production electrophoresis and cloning and sequencing to analyze variable shearing is:
Take the cDNA of cow mammary gland tissue extraction as template, according to A2M gene cDNA sequence (NCBI reference sequences: NM_001109795.1) design special primer amplification A2M cDNA fragment, special primer upstream primer A2MF sequence is: SEQ ID NO.9, downstream primer A2MR sequence is: SEQ IDNO.10
25 μ l PCR system reacted constituents comprise: DNA (100ng/ μ l) 1 μ l, and A2MF, each 0.5 μ l of A2MR primer (10 μ mol/L), 2 * Taq PCR MasterMix (gloomy biotech firm of Beijing Novi), 12.5 μ l, double distilled water is mended to 25 μ l,
The PCR program is: 94 ℃ of 4min, and 94 ℃ of 30s then, 64.9 ℃ of 30s, 72 ℃ of 30s, totally 35 circulations, 72 ℃ of 10min,
The PCR product found that except 4623bp PCR purpose band to also have 4 clearly bands, to being connected to behind these 4 zone purifications with 1% agarose gel electrophoresis
Carrier is found 2 kinds of new variable shear-forms after the order-checking, be labeled as A2M-AS1 and A2M-AS2;
The functional SNP site of c:A2M gene differentiates that step is:
According to causing the variable background context data of shearing mechanism of gene, with reference to ox A2M gene order (sequence number: NC_007303.4) among the NCBI, take milk cow DNA as template, near the dna sequence dna the variable clipped position occurs in design F1/R1 and F2/R2 primer amplification:
Primer sequence F1 is: SEQ ID NO.5; R1 is: SEQ ID NO.6;
Primer sequence F2 is: SEQ ID NO.7; R2 is: SEQ ID NO.8;
25 μ l PCR systems, the PCR reaction, then to the product direct Sequencing, use the SeqMan software analysis sequencing result in the DNAStar software package, found two SNPs sites at 29 and 37 exons of A2M gene: c.3535A＞T and c.4520T＞C (NCBI reference sequences: NM_001109795.1); Wherein, c.3535A＞T is the mononucleotide site that is positioned at amplified fragments SEQ ID NO.12 642bp place; C.4520T＞C is positioned at the mononucleotide site at amplified fragments SEQ ID NO.14 526bp place,
2 SNPs sites selecting to differentiate on the A2M gene order adopt the method for direct Sequencing to carry out the SNP gene type in 338 china holstein cows individualities, adopt SAS8.1 software to carry out correlation analysis, by the correlation analysis result, AA and the TT genotype type of finding respectively these 2 sites have higher SCS, phenotype according to these 2 genotype and SCS is relevant, filter out mazoitis resistance/susceptible functional molecular marker, by to the Id judgement of milk cow, method by marker assisted selection, but seed selection mazoitis Resistant Individuals is cultivated the milk cow new lines of mastitis-resisting;
The analysis of d:SNPs and variable shearing relation:
In order to analyze the check order SNPs of discovery and the relation of variable shearing, utilize ESEfinder3.0 that these 2 SNP are analyzed, find that these two SNP are positioned to shear enhanser ESE, wherein, c.3535A＞T suddenlys change has increased the combination of two kinds of shear proteins of SC35 and SRp40, c.4520T＞C suddenlys change has increased the combination of SRp40 shear protein
Simultaneously milk cow DNA is checked order, and extract their corresponding total RNA, utilize RT-PCR and cloning and sequencing technical Analysis c.3535 and the c.4520 genotype in site and the relation of unusual variable spliced body.
The designed pcr amplification primer of the different variable spliced bodies of pair for amplification milk cow A2M, it is characterized in that upstream primer A2MF sequence is: SEQ ID NO.9, downstream primer A2MR sequence is: SEQ ID NO.10, the product size of the unusual variable spliced body A2M-AS1 of its pcr amplification is 1242bp, sequence such as SEQ IDNO.16; The product size of unusual variable spliced body A2M-AS2 is 3607bp, sequence such as SEQ IDNO.17.
A kind ofly affect nucleotide sequence and the functional mononucleotide site thereof that mastadenitis of cow susceptible/resistance A2M gene can produce unusual variable spliced body A2M-AS1, it is characterized in that its sequence such as SEQID NO.12, and the mononucleotide site at amplified fragments 642bp place is A or T.
A kind ofly affect nucleotide sequence and the functional mononucleotide site thereof that mastadenitis of cow susceptible/resistance A2M gene can produce unusual variable spliced body A2M-AS2, it is characterized in that its sequence such as SEQID NO.14, and the mononucleotide site at amplified fragments 526bp place is T or C.
Described nucleotide sequence and the application of functional mononucleotide site in the mastadenitis of cow resistance breeding thereof.
Affect the application of A2M gene function molecule marker site in the improvement of mastadenitis of cow genetics of resistance of mastadenitis of cow susceptible/resistance.
The beneficial effect that the present invention compared with prior art produces is:
The present invention can realize the mazoitis resistance of milk cow individuality or colony is selected in early days by the Protocols in Molecular Biology means, changes existing according to Phenotypic Selection poor effect situation, accuracy and the reliability that can significantly provide the mastadenitis of cow resistance to select.
The present invention can increase the distribution of mazoitis resistant gene in colony by the genetic breeding means, can increase milk cow colony to the resistance of mazoitis, reducing sickness rate, and the usage quantity of minimizing medicine is improved milk qualities safety, reduces financial loss.
The present invention has differentiated 2 functional SNPs sites, can cause milk cow to produce 2 kinds of variable spliced bodies of A2M gene unconventionality, detect the genotype in these 2 sites of individual milk cow A2M gene by the molecular biology correlation technique, and after measuring (DHI) data relation analysis with milk cow production performance, select favourable genotype individuality to reserve seed for planting, can improve the ratio of A2M mazoitis resistant gene type individuality in colony, reduce the sickness rate of mastadenitis of cow disease, reduce financial loss.The present invention provides a kind of new method for the mazoitis genetics of resistance improvement of milk cow.
Description of drawings
The differential expression of Fig. 1 A2M gene in normal and mazoitis tissue, A:mRNA differential expression B: protein diversity is expressed.
The PCR product of Fig. 2 A2M cDNA amplification is in the detection of 1% agarose.
The pattern diagram of the variable spliced body A2M-AS1 of Fig. 3 and A2M-AS2.
Fig. 4 c.3535A＞T sudden change before and after the variation of ESE.
Fig. 5 c.4520T＞the gene sequencing peak figure of ox is carried in the C site.
Fig. 6 c.4520T＞C sudden change before and after the variation of ESE.
Embodiment
The below is described in detail below a kind of implementation method and application thereof that improves the mastitis-resisting ability of milk cow of the present invention.
Purpose one of the present invention is the method that method relative with isotopic labeling by real-time fluorescence quantitative PCR (RT-qPCR) and absolute quantitation (iTRAQ) provides a mastadenitis of cow susceptible/resistance A2M candidate gene; By RT-PCR and cloning and sequencing new discovery 2 kinds of variable shearings, simultaneously by 2 SNPss of gene direct Sequencing technology for detection to this gene, utilize the relation of the newfound 2 kinds of variable shearings of ESEfinder 3.0 software analysis and SNPs, simultaneously association analysis has been carried out in somatocyte scoring (SCS) and A2M genotype, found that these 2 SNPs sites are functional site.Select favourable aflelotype unity to reserve seed for planting, can improve the mastitis-resisting ability of milk cow, improve milk yield and milk quality.
Two of purpose of the present invention is the A2M gene function molecule marker site application in the improvement of mastadenitis of cow genetics of resistance that affect mastadenitis of cow susceptible/resistance.
The method that purpose one realizes is specific as follows:
1, screening A2M gene is as mastadenitis of cow susceptible/resistance candidate gene
1.1A2M mRNA carries out quantitative fluorescent PCR
Select at first respectively 3 normal and 3 mammary tissues of suffering from mazoitis, utilize
Premix Ex TaqTM II (Dalian Bao Bio-Engineering Company, China) and
480 II (Roche Diagnostics) instrument carries out real-time fluorescence quantitative PCR (RT-qPCR).Relative expression's level of gene is with housekeeping gene β-actin (sequence number: NM_173979.3) do confidential reference items.The experiment triplicate.20 μ l RT-qPCR amplification systems: 10.0 μ l
Premix Ex TaqTM II (2 *), 0.4 μ M upstream and downstream primer (A2M-F:SEQ ID NO.1, A2M-R:SEQ ID NO.2 or β-actinF:SEQ ID NO.3, β-actin-R:SEQ ID NO.4), 2.0 μ l A2M cDNA (＜100ng) or β-actin plasmid DNA, 6.4 μ l distilled water.Amplification reaction condition is as follows: 94 ℃ of 5min; Then 94 ℃ of 15s, 56 ℃ of 15s, this step is totally 40 circulations; Last 70 ℃ of 5s.
The result of fluorescent quantitation shows that the expression of A2M mRNA in the mammary tissue of suffering from the mazoitis milk cow is significantly higher than normal mammary tissue, sees Fig. 1.
1.2A2M albumen carries out relative quantification
Utilize isotopic labeling method relative and absolute quantitation (iTRAQ) mammary tissue A2M albumen normal to 3 and 3 trouble mazoitiss to carry out relative quantification.The albumen reference database of ox is UniProt ID:Q9H0H5.
The result of iTRAQ shows that suffering from mazoitis organizes the A2M expressing quantity to raise 3.5 times nearly compared with normal tissue, sees Fig. 1.
2, utilize RT-PCR amplified production electrophoresis and cloning and sequencing analysis
Take the cDNA of cow mammary gland tissue extraction as template, according to the A2M gene cDNA sequence of having reported (NCBI reference sequences: NM_001109795.1) design special primer amplification A2M cDNA fragment.Special primer upstream primer A2MF sequence is: SEQ ID NO.9, downstream primer A2MR sequence is: SEQID NO.10.
25 μ l PCR system reacted constituents comprise: DNA (100ng/ μ l) 1 μ l, and A2MF, each 0.5 μ l of A2MR primer (10 μ mol/L), 2 * Taq PCR MasterMix (gloomy biotech firm of Beijing Novi), 12.5 μ l, double distilled water is mended to 25 μ l.The PCR program is: 94 ℃ of 4min; Then 94 ℃ of 30s, 64.9 ℃ of 30s, 72 ℃ of 30s, totally 35 circulations; 72 ℃ of 10min.
The PCR product found that except 4623bp PCR purpose band to also have 4 clearly bands, to being connected to behind these 4 zone purifications with 1% agarose gel electrophoresis
Carrier is found 2 kinds of new variable shear-forms after the order-checking, called after A2M-AS1 and A2M-AS2, and shear mode is seen Fig. 3.
3, the functional SNP site of A2M gene is differentiated
According to causing the variable background context data of shearing mechanism of gene, with reference to ox A2M gene order (sequence number: NC_007303.4) among the NCBI, take milk cow DNA as template, near the dna sequence dna the variable clipped position occurs in design F1/R1 and F2/R2 primer amplification.Primer sequence F1 is: SEQ ID NO.5; R1 is: SEQ ID NO.6.Primer sequence F2 is: SEQ ID NO.7; R2 is: SEQ ID NO.8.
Described in the 25 μ l PCR systems such as above-mentioned 2, then to the product direct Sequencing, use the SeqMan software analysis sequencing result in the DNAStar software package.The applicant has found two SNPs sites at 29 and 37 exons of A2M gene: c.3535A＞and T and c.4520T＞C (NCBI reference sequences: NM_001109795.1).Wherein, c.3535A＞T is the mononucleotide site that is positioned at amplified fragments SEQ ID NO.12 642bp place.C.4520T＞C is positioned at the mononucleotide site at amplified fragments SEQ ID NO.14 526bp place.
2 SNPs sites selecting to differentiate on the A2M gene order adopt the method for direct Sequencing to carry out the SNP gene type in 338 china holstein cows individualities.Adopt SAS8.1 software to carry out correlation analysis (table 1).By the correlation analysis result, the applicant finds that respectively the AA in these 2 sites and TT genotype type have higher SCS, and concrete analysis sees Table 1.Phenotype according to these 2 genotype and SCS is relevant, filter out mazoitis resistance/susceptible functional molecular marker, by to the Id judgement of milk cow, by the method for marker assisted selection, but seed selection mazoitis Resistant Individuals is cultivated the milk cow new lines of mastitis-resisting.
Table 1 A2M genotype and somatic number phenotype correlation analysis
4, the analysis of SNPs and variable shearing relation
In order to analyze the check order SNPs of discovery and the relation of variable shearing, utilize ESEfinder 3.0 that these 2 SNP are analyzed, find that these two SNP are positioned to shear enhanser (ESE).Wherein, c.3535A＞T suddenlys change has increased the combination (Fig. 4) of two kinds of shear proteins of SC35 and SRp40.Report is arranged, shear protein SC35 can cause unusual variable shearing (Gabut M, Min é M, Marsac C, Brivet M, Tazi J, Soret be SR protein SC35 is responsible for aberrant splicing of the Elalpha pyruvate dehydrogenase mRNA in a case of mental retardation with lactic acidosis.Mol Cell Biol.25,3286-94. J.2005.The).C.4520T＞C suddenlys change has increased the combination (Fig. 5) of SRp40 shear protein.
Simultaneously milk cow DNA is checked order, and extract their corresponding total RNA.Utilize RT-PCR and cloning and sequencing technical Analysis c.3535 and the c.4520 genotype in site and the relation of unusual variable spliced body sees table 2 and table 3 for details.
Table 2 is loci gene type and the variable relation of shearing A2M-AS1 of generation c.3535
Table 3 is loci gene type and the variable relation of shearing A2M-AS2 of generation c.4520
The method that purpose two realizes is specific as follows:
The present invention utilizes the information in 2 functional SNPs sites of A2M gene, adopts the molecular biology correlation technique to differentiate milk cow in the genotype in these 2 sites, selects to have the genotype individuality of mazoitis resistance.
The present invention has differentiated 2 functional SNPs sites, can cause milk cow to produce 2 kinds of variable shearings of A2M gene unconventionality, detect the genotype in these 2 sites of individual milk cow A2M gene by the molecular biology correlation technique, and after measuring (DHI) data relation analysis with milk cow production performance, select favourable genotype individuality to reserve seed for planting, can improve the ratio of A2M mazoitis resistant gene type individuality in colony, reduce the sickness rate of mastadenitis of cow disease, reduce financial loss.The present invention provides a kind of new method for the mazoitis genetics of resistance improvement of milk cow.
Utilize 228 china holstein cowses as laboratory animal, these milk cow blood samples are from the good precious cattle farm of Qingdao high yield and Jinan.228 oxen have the DHI data.
Adopt the method for direct dna sequencing that c.3535 the A2M gene is being carried out the SNP gene type at 228 above-mentioned cow heads in the site.In the somatotype process, amplified fragments the 642nd base A of F1/R1 primer is wild-type, and sequence is SEQ ID NO.12; Amplified fragments the 642nd base T is mutant, and sequence is SEQID NO.13.Simultaneously the genotype in this SNP site and the SCS phenotype in the DHI record are carried out the cognation statistical study, see Table 4.Simultaneously the ox DNA of some amount checked order, and extracted their corresponding mRNA.Utilize RT-PCR and cloning and sequencing the technical Analysis c.3535 genotype in site and the relation of variable shearing A2M-AS1, see Table 5.
Table 4 A2M genotype and somatic number phenotype correlation analysis
Table 5 is loci gene type and the variable relation of shearing A2M-AS1 of generation c.3535
Analytical results show the SNP site c.3535A＞T, A allelotrope is resistant gene, T allelotrope is tumor susceptibility gene, the frequency of genotypes AA individuality has resistivity to mastadenitis of cow; This site is respectively the needed excellent genes type individuality of selection and improvement that AA type individuality is the sick milk cow of mastitis-resisting.
Utilize 192 china holstein cowses as laboratory animal, these milk cow blood samples are from the good precious cattle farm of Qingdao high yield and Jinan.The DHI data logging of these 192 oxen is complete.
Adopt the method for direct dna sequencing that c.4520 the A2M gene is being carried out the SNP gene type at 192 above-mentioned cow heads in the site.In the somatotype process, amplified fragments the 526th base T of F2/R2 primer is wild-type, and sequence is SEQ ID NO.14; Amplified fragments the 526th base C is mutant, and sequence is SEQID NO.15.Simultaneously to this SNP site carry out behind the somatotype with the DHI record in the SCS phenotype carry out the cognation statistical study, see Table 5.Simultaneously ox DNA is checked order, and extract their corresponding mRNA.Utilize RT-PCR and cloning and sequencing the technical Analysis c.4520 genotype in site and the relation of variable shearing A2M-AS2, see Table 6.
Table 5 A2M genotype and somatic number phenotype correlation analysis
Table 6 is loci gene type and the variable relation of shearing A2M-AS2 of generation c.4520
Analytical results show the SNP site c.4520T＞C, T allelotrope is resistant gene, C allelotrope is tumor susceptibility gene, genotype TT individuality has resistivity to mastadenitis of cow.This site is respectively the needed excellent genes type individuality of selection and improvement that TT type individuality is the sick milk cow of mastitis-resisting.
Claims (8)
1. seed choosing method that improves the mastitis-resisting ability of milk cow is characterized in that relative with isotopic labeling by real time fluorescent quantitative RT-qPCR and method absolute quantitation iTRAQ provides a mastadenitis of cow susceptible/resistance A2M candidate gene; Analyze variable shearing by reverse transcription RT-PCR and cloning and sequencing, simultaneously by the single nucleotide polymorphism SNPs of gene direct Sequencing technology for detection to this gene, utilize the relation of the variable shearing of software analysis and SNPs, simultaneously somatocyte scoring SCS and A2M genotype are carried out association analysis, judge that the SNPs site is functional site, select favourable aflelotype unity to reserve seed for planting as milk cow mastitis-resisting ability.
2. a seed choosing method that improves the mastitis-resisting ability of milk cow is characterized in that the steps include:
A: screening A2M gene is as mastadenitis of cow susceptible/resistance candidate gene, a1: A2M mRNA is carried out quantitative fluorescent PCR, the record result, a2: utilize isotopic labeling method relative and absolute quantitation iTRAQ A2M albumen to be carried out relative quantification, the record result;
B: utilize reverse transcription RT-PCR amplified production electrophoresis and cloning and sequencing to analyze variable shearing;
The functional SNP site of c:A2M gene is differentiated;
The analysis of d:SNPs and variable shearing relation.
3. a kind of seed choosing method that improves the mastitis-resisting ability of milk cow according to claim 2 is characterized in that a: screening A2M gene is as mastadenitis of cow susceptible/resistance candidate gene,
A1: A2M mRNA is carried out the quantitative fluorescent PCR step is:
Select at first respectively 3 normal and 3 mammary tissues of suffering from mazoitis, utilize
PremixEx TaqTM II (Dalian Bao Bio-Engineering Company, China) and
480 II (Roche Diagnostics) instrument carries out real-time fluorescence quantitative PCR (RT-qPCR), relative expression's level of gene with housekeeping gene β-actin (sequence number: NM_173979.3) do confidential reference items, the experiment triplicate;
20 μ l RT-qPCR amplification systems: 10.0 μ l
Premix Ex TaqTM II (2 *), 0.4 μ M upstream and downstream primer:
A2M-F：SEQ?ID?NO.1，A2M-R：SEQ?ID?NO.2；
Or
β-actinF：SEQ?ID?NO.3，β-actin-R：SEQ?ID?NO.4；
2.0μl?A2M?cDNA(＜100ng)
Or
β-actin plasmid DNA, 6.4 μ l distilled water;
Amplification reaction condition: 94 ℃ of 5min; Then 94 ℃ of 15s, 56 ℃ of 15s, this step is totally 40 circulations; Last 70 ℃ of 5s,
The result that analysis of fluorescence is quantitative, relatively A2M mRNA in the mammary tissue of suffering from the mazoitis milk cow expression amount and the expression amount in the normal mammary tissue;
A2: utilize isotopic labeling relatively and the method for absolute quantitation iTRAQ is carried out the relative quantification step to A2M albumen and is:
Utilize isotopic labeling method relative and absolute quantitation iTRAQ mammary tissue A2M albumen normal to 3 and 3 trouble mazoitiss to carry out relative quantification, the albumen reference database of ox is UniProt ID:Q9H0H5, and the result of iTRAQ relatively suffers from mazoitis and organizes A2M expressing quantity and normal tissue expression amount;
B: the step of utilizing reverse transcription RT-PCR amplified production electrophoresis and cloning and sequencing to analyze variable shearing is:
Take the cDNA of cow mammary gland tissue extraction as template, according to A2M gene cDNA sequence (NCBI reference sequences: NM_001109795.1) design special primer amplification A2M cDNA fragment, special primer upstream primer A2MF sequence is: SEQ ID NO.9, downstream primer A2MR sequence is: SEQ IDNO.10
25 μ l PCR system reacted constituents comprise: DNA (100ng/ μ l) 1 μ l, and A2MF, each 0.5 μ l of A2MR primer (10 μ mol/L), 2 * Taq PCR MasterMix (gloomy biotech firm of Beijing Novi), 12.5 μ l, double distilled water is mended to 25 μ l,
The PCR program is: 94 ℃ of 4min, and 94 ℃ of 30s then, 64.9 ℃ of 30s, 72 ℃ of 30s, totally 35 circulations, 72 ℃ of 10min,
The PCR product found that except 4623bp PCR purpose band to also have 4 clearly bands, to being connected to behind these 4 zone purifications with 1% agarose gel electrophoresis
Carrier is found 2 kinds of new variable shear-forms after the order-checking, be labeled as A2M-AS1 and A2M-AS2;
The functional SNP site of c:A2M gene differentiates that step is:
According to causing the variable background context data of shearing mechanism of gene, with reference to ox A2M gene order (sequence number: NC_007303.4) among the NCBI, take milk cow DNA as template, near the dna sequence dna the variable clipped position occurs in design F1/R1 and F2/R2 primer amplification:
Primer sequence F1 is: SEQ ID NO.5; R1 is: SEQ ID NO.6;
Primer sequence F2 is: SEQ ID NO.7; R2 is: SEQ ID NO.8;
25 μ l PCR systems, the PCR reaction, then to the product direct Sequencing, use the SeqMan software analysis sequencing result in the DNAStar software package, found two SNPs sites at 29 and 37 exons of A2M gene: c.3535A＞T and c.4520T＞C (NCBI reference sequences: NM_001109795.1); Wherein, c.3535A＞T is the mononucleotide site that is positioned at amplified fragments SEQ ID NO.12 642bp place; C.4520T＞C is positioned at the mononucleotide site at amplified fragments SEQ ID NO.14 526bp place,
2 SNPs sites selecting to differentiate on the A2M gene order adopt the method for direct Sequencing to carry out the SNP gene type in 338 china holstein cows individualities, adopt SAS8.1 software to carry out correlation analysis, by the correlation analysis result, AA and the TT genotype type of finding respectively these 2 sites have higher SCS, phenotype according to these 2 genotype and SCS is relevant, filter out mazoitis resistance/susceptible functional molecular marker, by to the Id judgement of milk cow, method by marker assisted selection, but seed selection mazoitis Resistant Individuals is cultivated the milk cow new lines of mastitis-resisting;
The analysis of d:SNPs and variable shearing relation:
In order to analyze the check order SNPs of discovery and the relation of variable shearing, utilize ESEfinder3.0 that these 2 SNP are analyzed, find that these two SNP are positioned to shear enhanser ESE, wherein, c.3535A＞T suddenlys change has increased the combination of two kinds of shear proteins of SC35 and SRp40, c.4520T＞C suddenlys change has increased the combination of SRp40 shear protein
Simultaneously milk cow DNA is checked order, and extract their corresponding total RNA, utilize RT-PCR and cloning and sequencing technical Analysis c.3535 and the c.4520 genotype in site and the relation of unusual variable spliced body.
4. the designed pcr amplification primer of the different variable spliced bodies of pair for amplification milk cow A2M, it is characterized in that upstream primer A2MF sequence is: SEQ ID NO.9, downstream primer A2MR sequence is: SEQ IDNO.10, the product size of the unusual variable spliced body A2M-AS1 of its pcr amplification is 1242bp, sequence such as SEQ ID NO.16; The product size of unusual variable spliced body A2M-AS2 is 3607bp, sequence such as SEQ ID NO.17.
5. one kind affects nucleotide sequence and the functional mononucleotide site thereof that mastadenitis of cow susceptible/resistance A2M gene can produce unusual variable spliced body A2M-AS1, it is characterized in that its sequence such as SEQID NO.12, and the mononucleotide site at amplified fragments 642bp place is A or T.
6. one kind affects nucleotide sequence and the functional mononucleotide site thereof that mastadenitis of cow susceptible/resistance A2M gene can produce unusual variable spliced body A2M-AS2, it is characterized in that its sequence such as SEQID NO.14, and the mononucleotide site at amplified fragments 526bp place is T or C.
7. arbitrary described nucleotide sequence and the application of functional mononucleotide site in the mastadenitis of cow resistance breeding thereof in 1,2,3,4,5 in the claim.
8. affect the application of A2M gene function molecule marker site in the improvement of mastadenitis of cow genetics of resistance of mastadenitis of cow susceptible/resistance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210203358.5A CN102899395B (en) | 2012-06-20 | 2012-06-20 | Breed selection method for improving mastitis resistance of dairy cow and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210203358.5A CN102899395B (en) | 2012-06-20 | 2012-06-20 | Breed selection method for improving mastitis resistance of dairy cow and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102899395A true CN102899395A (en) | 2013-01-30 |
| CN102899395B CN102899395B (en) | 2014-12-10 |
Family
ID=47571882
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210203358.5A Expired - Fee Related CN102899395B (en) | 2012-06-20 | 2012-06-20 | Breed selection method for improving mastitis resistance of dairy cow and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102899395B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107012248A (en) * | 2017-05-16 | 2017-08-04 | 北京市畜牧总站 | A kind of molecular labeling for detecting mastitis for milk cows resistance and application thereof |
| CN108034734A (en) * | 2018-02-01 | 2018-05-15 | 中国农业大学 | A kind of method and its dedicated kit for screening different protein ratio milk cows |
| CN109182504A (en) * | 2018-09-29 | 2019-01-11 | 南京农业大学 | Mastadenitis of cow key SNPs site rs20438858 and 2b-RAD Genotyping and analysis method |
| CN109182538A (en) * | 2018-09-29 | 2019-01-11 | 南京农业大学 | Mastadenitis of cow key SNPs site rs88640083 and 2b-RAD Genotyping and analysis method |
| CN109182505A (en) * | 2018-09-29 | 2019-01-11 | 南京农业大学 | Mastadenitis of cow key SNPs site rs75762330 and 2b-RAD Genotyping and analysis method |
| CN110338138A (en) * | 2019-06-19 | 2019-10-18 | 山东省农业科学院奶牛研究中心 | A kind of Mycoplasma bovis infects animal model constructing method and its application of cavy |
| CN111549124A (en) * | 2020-06-17 | 2020-08-18 | 黑龙江八一农垦大学 | Molecular marker for detecting staphylococcus aureus mastitis of dairy cows and application thereof |
| CN114525346A (en) * | 2022-03-01 | 2022-05-24 | 天津市农业科学院 | SNP molecular marker related to somatic cell number of sheep milk and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008039257A2 (en) * | 2006-06-13 | 2008-04-03 | Monsanto Technology Llc | Single nucleotide polymorphisms and use of same in selection of dairy cattle |
| CN101693923A (en) * | 2009-11-09 | 2010-04-14 | 山东奥克斯生物技术有限公司 | HSP70A1A gene SNP loci, application and kit for selecting heat-resistant cows |
-
2012
- 2012-06-20 CN CN201210203358.5A patent/CN102899395B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008039257A2 (en) * | 2006-06-13 | 2008-04-03 | Monsanto Technology Llc | Single nucleotide polymorphisms and use of same in selection of dairy cattle |
| CN101693923A (en) * | 2009-11-09 | 2010-04-14 | 山东奥克斯生物技术有限公司 | HSP70A1A gene SNP loci, application and kit for selecting heat-resistant cows |
Non-Patent Citations (4)
| Title |
|---|
| 《Journal of Dairy Research》 19920831 Liisa K. Rantamaki等 Isolation and characterization of alpha2-macroglobulin from mastitis milk 273－285 1、2 第59卷, 第3期 * |
| IRELAND,J.L.等: "NM_001109795.1", 《NCBI》 * |
| LIISA K. RANTAMAKI等: "Isolation and characterization of α2-macroglobulin from mastitis milk", 《JOURNAL OF DAIRY RESEARCH》 * |
| 刘梅等: "MBL1基因第一内含子与第二外显子多态性与中国荷斯坦牛乳腺炎和乳品质的关联分析", 《中国农业科学》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107012248A (en) * | 2017-05-16 | 2017-08-04 | 北京市畜牧总站 | A kind of molecular labeling for detecting mastitis for milk cows resistance and application thereof |
| CN107012248B (en) * | 2017-05-16 | 2020-10-27 | 北京市畜牧总站 | Molecular marker for detecting cow mastitis resistance and application thereof |
| CN108034734A (en) * | 2018-02-01 | 2018-05-15 | 中国农业大学 | A kind of method and its dedicated kit for screening different protein ratio milk cows |
| CN109182504A (en) * | 2018-09-29 | 2019-01-11 | 南京农业大学 | Mastadenitis of cow key SNPs site rs20438858 and 2b-RAD Genotyping and analysis method |
| CN109182538A (en) * | 2018-09-29 | 2019-01-11 | 南京农业大学 | Mastadenitis of cow key SNPs site rs88640083 and 2b-RAD Genotyping and analysis method |
| CN109182505A (en) * | 2018-09-29 | 2019-01-11 | 南京农业大学 | Mastadenitis of cow key SNPs site rs75762330 and 2b-RAD Genotyping and analysis method |
| CN109182538B (en) * | 2018-09-29 | 2022-01-04 | 南京农业大学 | Method for genotyping and analyzing key SNPs sites rs88640083 and 2b-RAD of dairy cow mastitis |
| CN110338138A (en) * | 2019-06-19 | 2019-10-18 | 山东省农业科学院奶牛研究中心 | A kind of Mycoplasma bovis infects animal model constructing method and its application of cavy |
| CN110338138B (en) * | 2019-06-19 | 2021-04-06 | 山东省农业科学院奶牛研究中心 | Animal model construction method for guinea pig infected by mycoplasma bovis and application thereof |
| CN111549124A (en) * | 2020-06-17 | 2020-08-18 | 黑龙江八一农垦大学 | Molecular marker for detecting staphylococcus aureus mastitis of dairy cows and application thereof |
| CN114525346A (en) * | 2022-03-01 | 2022-05-24 | 天津市农业科学院 | SNP molecular marker related to somatic cell number of sheep milk and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102899395B (en) | 2014-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102899395B (en) | Breed selection method for improving mastitis resistance of dairy cow and use thereof | |
| Li et al. | Host genetics influence the rumen microbiota and heritable rumen microbial features associate with feed efficiency in cattle | |
| US20090269741A1 (en) | Method for assessing traits selected from longissimus dorsi peak force, intramuscular fat, retail beef yield and net feed intake in bovine animals | |
| CN102016031B (en) | Identification of hypertension susceptibility gene group | |
| CN112662788B (en) | SNP marker related to milk production traits of Holstein cows in south China and application thereof | |
| US20230287502A1 (en) | Piezo Type Mechanosensitive Ion Channel Component 1 (PIEZO1) Variants And Uses Thereof | |
| Magotra et al. | Polymorphism in DNA repair gene BRCA1 associated with clinical mastitis and production traits in indigenous dairy cattle | |
| KR101083213B1 (en) | Diagnosis method of meat quality using DNA marker associated with marbling score in Hanwoo | |
| Lemay et al. | Bleomycin-induced pulmonary fibrosis susceptibility genes in AcB/BcA recombinant congenic mice | |
| Chen et al. | Eight SNVs in NF-κB pathway genes and their different performances between subclinical mastitis and mixed Chinese Holstein cows | |
| CN102899396B (en) | Core promoter for influencing cow mastitis infectibility/resistance HMGB3 gene and functional molecule mark and application | |
| CN113774154B (en) | Method for screening bovine body high mutation related molecular marker and application thereof | |
| Huriletemuer et al. | Gene polymorphisms and related risk factors in Mongolian hypertensive stroke patients | |
| Samzadeh et al. | Association of G/A polymorphism, rs266882, in AREI region of the prostate-specific antigen gene with prostate cancer risk and clinicopathological features | |
| Yaghmaei et al. | Endothelial nitric oxide synthase gene Glu298Asp polymorphism and risk of preeclampsia in South East of Iran | |
| Fu et al. | Investigation of JAK1 and STAT3 polymorphisms and their gene–gene interactions in nonspecific digestive disorder of rabbits | |
| KR101289484B1 (en) | Method of genetic test for diagnosis of marbling trait in Korean cattle | |
| KR101676089B1 (en) | Polymorphism biomarker for predicting prognosis in lung cancer patients and the method for predicting prognosis using the same | |
| KR102063486B1 (en) | Association of RNF213 single nucleotide polymorphism with the risk of Moyamoya disease in a Korean population | |
| KR101289576B1 (en) | Development of molecular marker related to meat quantity in Hanwoo | |
| CN105420402B (en) | A kind of and the relevant molecular labeling of milk cow somatic cell score and its application | |
| KR101307087B1 (en) | Diagnosis method of high quality meat using the SNP marker in Hanwoo | |
| KR20110038490A (en) | Development of dna marker associated with higher meat quantity and diagnosis method of high meat quantity producing hanwoo using the dna marker | |
| US20150344952A1 (en) | Dna markers for beef tenderness in cattle | |
| Dai et al. | Association between EPAS1 gene single nucleotide polymorphisms and risk and prognosis of renal clear cell carcinoma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141210 Termination date: 20150620 |
|
| EXPY | Termination of patent right or utility model |