CN102899395B - 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
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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 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 raising dairy cattle, the most complicated disease, is also to cause the maximum a kind of disease of milk cattle cultivating industry loss.At present, approximately there are 2.6457 hundred million cow heads (FAO, 2010) in the whole world, approximately has 1/3 milk cow trouble mazoitis every year.According to estimates, about 95% case is to be infected and caused by streptococcus aureus (Staph.aureus), suis (Strep.uberis) and intestinal bacteria (E.coli), the loss causing because of mazoitis is every year up to 35,000,000,000 dollars (Wellenberg etc., 2002).About the research history of existing more than 100 year 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 subject to multifactor impact, is difficult to control.At present, the control of mazoitis and preventive measures are not reduced on the whole to the sickness rate of mazoitis, and mazoitis is also the main reason (Villli, 2001) that during milk cow produces, microbiotic is used.Therefore, we reduce the sickness rate of mazoitis in the urgent need to inquiring into other approach, by heredity, select to improve a kind of best long term policy (Burton etc., 2001) that mastadenitis of cow resistance is considered to solve mazoitis problem.If by genetic breeding means, can make milk cow strengthen the resistance of mazoitis, the usage quantity of minimizing medicine, improves milk qualities safety, reduces financial loss, and this is perplexing a difficult problem for world's development of dairy industry to be for a long time expected to fundamentally to solve mazoitis.The heritability of milk cow resistance is very low, the genetic improvement poor effect based on Phenotypic Selection.And milk cow molecular breeding can be realized the early stage selection of milk cow, greatly shorten the generation interval of milk cow genetic improvement, save seed selection cost, improve efficiency of selection.The molecule marker having in gene, can affect expression regulation, gene function of gene etc. such as single nucleotide mutation, individual phenotype is had to important impact.Therefore, the molecular breeding technology based on functional molecular marker assisted selection is well worth doing.
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 its destruction to host, 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 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 efficient quantitative expression of regulatory gene and the variation of protein function, make the transcription product of a gene in the different etap, under noble cells and 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 distributing in space structure and cell, differentiation to 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), as causing one of cis acting original paper of variable shearing, has been brought into play very important effect in the process that produces variable shearing.Mononucleotide polymorphic (SNPs) is one of transgenation, can cause the abnormal shearing of Pre-mRNA, comprises exon skipping, intron retention etc.Recently, many report proof SNPs cause gene to produce the major cause of abnormal 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 abnormal variable shearings and people's numerous disease are related.Up to the present there is no the variable shearing of ox A2M gene and cause the report of the functional SNPs of variable shearing, there is no 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 mastadenitis of cow susceptible/resistance A2M candidate gene; By reverse transcription RT-PCR and cloning and sequencing, analyze variable shearing, by gene direct Sequencing technology for detection, arrive the single nucleotide polymorphism SNPs of this gene simultaneously, utilize the relation of the variable shearing of software analysis and SNPs, somatocyte scoring SCS and A2M genotype are carried out to association analysis simultaneously, judgement SNPs site is functional site, selects favourable aflelotype unity to reserve seed for planting as milk cow mastitis-resisting ability.
The seed choosing method that improves 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 to quantitative fluorescent PCR, record result, a2: utilize isotopic labeling method relative and absolute quantitation iTRAQ to carry out relative quantification to A2M albumen, 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 A2M gene as mastadenitis of cow susceptible/resistance candidate gene,
A1: A2M mRNA is carried out to quantitative fluorescent PCR step is:
Select 3 normal oxen first respectively with 3 mammary tissues of suffering from mazoitis oxen, utilize
premix Ex TaqTM II (Dalian Bao Bio-Engineering Company, China) and
480II (Roche Diagnostics) instrument carries out real-time fluorescence quantitative PCR (RT-qPCR), the housekeeping gene β-actin for relative expression's level of gene (sequence number: NM_173979.3) do internal reference, experiment in 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, the relatively expression amount of A2M mRNA in the mammary tissue of suffering from mazoitis milk cow and the expression amount in normal mammary tissue;
A2: utilize isotopic labeling relatively and the method for absolute quantitation iTRAQ is carried out relative quantification step to A2M albumen and is:
Utilize isotopic labeling method relative and absolute quantitation iTRAQ to carry out relative quantification to the mammary tissue A2M albumen with 3 trouble mazoitis oxen of 3 normal oxen, the albumen reference database of ox is UniProtID:Q9H0H5, and the result of iTRAQ is relatively suffered from mazoitis and organized 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:
The cDNA of cow mammary gland tissue extraction of take is 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 ID 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, redistilled water is mended to 25 μ l,
PCR program is: 94 ℃ of 4min, and 94 ℃ of 30s then, 64.9 ℃ of 30s, 72 ℃ of 30s, totally 35 circulations, 72 ℃ of 10min,
1% agarose gel electrophoresis for PCR product, found that except 4623bp PCR object band, also has 4 bands clearly, to being connected to after these 4 zone purifications
-T Easy carrier, finds 2 kinds of new variable shear-forms after order-checking, be labeled as A2M-AS1 and A2M-AS2;
The functional SNP site of c:A2M gene differentiates that step is:
According to the background context data that causes that the variable shearing of gene is machine-processed, with reference to ox A2M gene order (sequence number: NC_007303.4) in NCBI, take milk cow DNA as template, there is near the DNA sequence dna variable clipped position 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, PCR reaction, then to product direct Sequencing, use the SeqMan software analysis sequencing result in DNAStar software package, on 29 and 37 exons of A2M gene, found two SNPs sites: 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,
Select 2 SNPs sites differentiating in A2M gene order in 338 china holstein cows individualities, to adopt the method for direct Sequencing to carry out SNP gene type, adopt SAS8.1 software to carry out correlation analysis, by correlation analysis result, AA and the TT genotype of finding respectively these 2 sites have higher SCS, relevant according to the phenotype of these 2 genotype and SCS, filter out mazoitis resistance/susceptible functional molecular marker, by to the Id judgement of milk cow, by the method for marker assisted selection, can seed selection mazoitis Resistant Individuals, cultivate the milk cow new lines of mastitis-resisting,
The analysis of d:SNPs and variable shearing relation:
In order to analyze the SNPs of order-checking discovery and the relation of variable shearing, utilize ESEfinder3.0 to analyze these 2 SNP, find that these two SNP are positioned to shear enhanser ESE, wherein, c.3535A > T sudden change has increased the combination of two kinds of shear proteins of SC35and SRp40, c.4520T > C sudden change has increased the combination of SRp40 shear protein
Milk cow DNA is checked order simultaneously, 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 abnormal 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 abnormal variable spliced body A2M-AS1 of its pcr amplification is 1242bp, and sequence is as SEQ ID NO.16; The product size of abnormal variable spliced body A2M-AS2 is 3607bp, and sequence is as SEQ ID NO.17.
A kind ofly affect nucleotide sequence and the functional mononucleotide site thereof that mastadenitis of cow susceptible/resistance A2M gene can produce abnormal variable spliced body A2M-AS1, it is characterized in that its sequence is 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 abnormal variable spliced body A2M-AS2, it is characterized in that its sequence is as SEQ ID NO.14, and the mononucleotide site at amplified fragments 526bp place is T or C.
The application in mastadenitis of cow resistance breeding of described nucleotide sequence and functional mononucleotide site thereof.
The application of the A2M gene function molecule marker site that affects mastadenitis of cow susceptible/resistance in the improvement of mastadenitis of cow genetics of resistance.
The beneficial effect that the present invention compared with prior art produced is:
The present invention, by Protocols in Molecular Biology means, can realize the mazoitis resistance of milk cow individuality or colony is selected in early days, changes existing according to Phenotypic Selection poor effect situation, accuracy and the reliability that can significantly provide mastadenitis of cow resistance to select.
The present invention, by genetic breeding means, can increase the distribution of mazoitis resistant gene in colony, can increase the resistance of milk cow colony to mazoitis, reduces sickness rate, reduces the usage quantity of medicine, improves 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, by molecular biology correlation technique, detect the genotype in these 2 sites of individual milk cow A2M gene, and select favourable genotype individuality to reserve seed for planting after measuring (DHI) data relation analysis with milk cow production performance, 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 milk bovine mastitis genetics of resistance improvement.
Accompanying drawing explanation
The differential expression of Figure 1A 2M 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 is the variation of > T sudden change front and back ESE c.3535A.
Fig. 5 c.4520T > C carries in site the gene sequencing peak figure of ox.
Fig. 6 is the variation of > C sudden change front and back ESE c.4520T.
Embodiment
Below a kind of implementation method and application thereof that improves the mastitis-resisting ability of milk cow of the present invention is described in detail below.
Object 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 mastadenitis of cow susceptible/resistance A2M candidate gene; By RT-PCR and cloning and sequencing new discovery 2 kinds of variable shearings, by gene direct Sequencing technology for detection, arrive 2 SNPs of this gene simultaneously, utilize the relation of the newfound 2 kinds of variable shearings of ESEfinder3.0 software analysis and SNPs, somatocyte scoring (SCS) and A2M genotype have been carried out to association analysis simultaneously, 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 object of the present invention is the application in the improvement of mastadenitis of cow genetics of resistance of A2M gene function molecule marker site that affect mastadenitis of cow susceptible/resistance.
The method that object 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 3 normal oxen first respectively with 3 mammary tissues of suffering from mazoitis oxen, utilize
premix Ex TaqTM II (Dalian Bao Bio-Engineering Company, China) and
480II (Roche Diagnostics) instrument carries out real-time fluorescence quantitative PCR (RT-qPCR).(the sequence number: NM_173979.3) do internal reference of housekeeping gene β-actin for relative expression's level of gene.Experiment in 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 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) to carry out relative quantification to the mammary tissue A2M albumen with 3 trouble mazoitis oxen of 3 normal oxen.The albumen reference database of ox is UniProt ID:Q9H0H5.
The result of iTRAQ shows that suffering from mazoitis organizes 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
The cDNA of cow mammary gland tissue extraction of take is 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: SEQ ID 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, redistilled water is mended to 25 μ l.PCR program is: 94 ℃ of 4min; Then 94 ℃ of 30s, 64.9 ℃ of 30s, 72 ℃ of 30s, totally 35 circulations; 72 ℃ of 10min.
1% agarose gel electrophoresis for PCR product, found that except 4623bp PCR object band, also has 4 bands clearly, to being connected to after these 4 zone purifications
-T Easy carrier, finds 2 kinds of new variable shear-forms after order-checking, called after A2M-AS1 and A2M-AS2, and shear mode is shown in Fig. 3.
3, the functional SNP site of A2M gene is differentiated
According to the background context data that causes that the variable shearing of gene is machine-processed, with reference to ox A2M gene order (sequence number: NC_007303.4) in NCBI, take milk cow DNA as template, there is near the DNA sequence dna variable clipped position 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, described in above-mentioned 2, then to product direct Sequencing, are used the SeqMan software analysis sequencing result in DNAStar software package.Applicant has found two SNPs sites on 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.
Select 2 SNPs sites differentiating in A2M gene order in 338 china holstein cows individualities, to adopt the method for direct Sequencing to carry out SNP gene type.Adopt SAS8.1 software to carry out correlation analysis (table 1).By correlation analysis result, applicant finds that respectively the AA in these 2 sites and TT genotype have higher SCS, and concrete analysis is in Table 1.Relevant according to the phenotype of these 2 genotype and SCS, filter out mazoitis resistance/susceptible functional molecular marker, by the Id judgement of milk cow, by the method for marker assisted selection, can seed selection mazoitis Resistant Individuals, cultivate the milk cow new lines of mastitis-resisting.
Table 1A2M genotype and somatic number phenotype correlation analysis
4, the analysis of SNPs and variable shearing relation
In order to analyze the SNPs of order-checking discovery and the relation of variable shearing, utilize ESEfinder3.0 to analyze these 2 SNP, find that these two SNP are positioned to shear enhanser (ESE).Wherein, c.3535A > T sudden change has increased the combination (Fig. 4) of two kinds of shear proteins of SC35and SRp40.There is report, shear protein SC35 can cause abnormal variable shearing (Gabut M, Min é M, Marsac C, Brivet M, Tazi J, Soret is SR protein SC35is responsible for aberrant splicing of the Elalpha pyruvate dehydrogenase mRNA in a case of mental retardation with lactic acidosis.Mol Cell Bio1.25,3286-94. J.2005.The).C.4520T > C sudden change has increased the combination (Fig. 5) of SRp40 shear protein.
Milk cow DNA is checked order simultaneously, 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 abnormal variable spliced body, refers to table 2 and table 3.
The relation of table 2c.3535 loci gene type and the variable shearing of generation A2M-AS1
The relation of table 3c.4520 loci gene type and the variable shearing of generation A2M-AS2
The method that object two realizes is specific as follows:
The present invention utilizes the information in 2 functional SNPs sites of A2M gene, adopts molecular biology correlation technique to differentiate that milk cow is 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, by molecular biology correlation technique, detect the genotype in these 2 sites of individual milk cow A2M gene, and select favourable genotype individuality to reserve seed for planting after measuring (DHI) data relation analysis with milk cow production performance, 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 milk bovine mastitis genetics of resistance improvement.
Utilize 228 china holstein cowses as laboratory animal, these milk cow blood samples are from Qingdao high yield and Jia Bao cattle farm, Jinan.228 oxen have DHI data.
Adopt the method for direct DNA sequencing c.3535 at 228 cow heads above-mentioned, to carry out SNP gene type in site to A2M gene.In somatotype process, the amplified fragments of F1/R1 primer the 642nd base A, is wild-type, and sequence is SEQ ID NO.12; Amplified fragments the 642nd base T is saltant type, and sequence is SEQID NO.13.SCS phenotype in the genotype in this SNP site and DHI record is carried out to cognation statistical study, in Table 4 simultaneously.The ox DNA of some amount is checked order simultaneously, and extract 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, in Table 5.
Table 4A2M genotype and somatic number phenotype correlation analysis
The relation of table 5c.3535 loci gene type and the variable shearing of generation A2M-AS1
Analytical results shows c.3535A > T of SNP site, and A allelotrope is resistant gene, and T allelotrope is tumor susceptibility gene, and 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 Qingdao high yield and Jia Bao cattle farm, Jinan.The DHI data logging of these 192 oxen is complete.
Adopt the method for direct DNA sequencing c.4520 at 192 cow heads above-mentioned, to carry out SNP gene type in site to A2M gene.In somatotype process, the amplified fragments of F2/R2 primer the 526th base T is wild-type, and sequence is SEQ ID NO.14; Amplified fragments the 526th base C is saltant type, and sequence is SEQ ID NO.15.Simultaneously to this SNP site carry out after somatotype with DHI record in SCS phenotype carry out cognation statistical study, in Table 5.Ox DNA is checked order simultaneously, 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, in Table 6.
Table 5A2M genotype and somatic number phenotype correlation analysis
The relation of table 6c.4520 loci gene type and the variable shearing of generation A2M-AS2
Analytical results shows c.4520T > C of SNP site, and T allelotrope is resistant gene, and C allelotrope is tumor susceptibility gene, and 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 (2)
1. improve a seed choosing method for the mastitis-resisting ability of milk cow, it is characterized in that relative with isotopic labeling by real time fluorescent quantitative RT-qPCR to method absolute quantitation iTRAQ provides mastadenitis of cow susceptible/resistance A2M candidate gene; By reverse transcription RT-PCR and cloning and sequencing, analyze variable shearing, by gene direct Sequencing technology for detection, arrive the single nucleotide polymorphism SNPs of this gene simultaneously, utilize the relation of the variable shearing of software analysis and SNPs, somatocyte scoring SCS and A2M genotype are carried out to association analysis simultaneously, judgement SNPs site is functional site, select favourable aflelotype unity to reserve seed for planting as milk cow mastitis-resisting ability, the steps include:
A: screening A2M gene is as mastadenitis of cow susceptible/resistance candidate gene, a1: A2M mRNA is carried out to quantitative fluorescent PCR, record result, a2: utilize isotopic labeling method relative and absolute quantitation iTRAQ to carry out relative quantification to A2M albumen, 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;
Wherein:
A: screen A2M gene as mastadenitis of cow susceptible/resistance candidate gene,
A1: A2M mRNA is carried out to quantitative fluorescent PCR step is:
Select 3 normal oxen first respectively with 3 mammary tissues of suffering from mazoitis oxen, utilize
premix Ex TaqTM II and
480II instrument carries out real-time fluorescence quantitative PCR, and relative expression's level of gene is done internal reference with housekeeping gene β-actin, and experiment in triplicate;
20 μ l RT-qPCR amplification systems: 10.0 μ l
premix Ex TaqTM II, 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；
2.0μlA2M cDNA
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, the relatively expression amount of A2M mRNA in the mammary tissue of suffering from mazoitis milk cow and the expression amount in normal mammary tissue;
A2: utilize isotopic labeling relatively and the method for absolute quantitation iTRAQ is carried out relative quantification step to A2M albumen and is:
Utilize isotopic labeling method relative and absolute quantitation iTRAQ to carry out relative quantification to the mammary tissue A2M albumen with 3 trouble mazoitis oxen of 3 normal oxen, the albumen reference database of ox is UniProt ID:Q9H0H5, and the result of iTRAQ is relatively suffered from mazoitis and organized 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:
The cDNA of cow mammary gland tissue extraction of take is template, and according to A2M gene cDNA sequence design special primer amplification A2M cDNA fragment, special primer upstream primer A2MF sequence is: SEQ ID NO.9, and downstream primer A2MR sequence is: SEQ ID NO.10,
25 μ l PCR system reacted constituents comprise: DNA1 μ l, and A2MF, each 0.5 μ l of A2MR primer, 2 * Taq PCR MasterMix, 12.5 μ l, redistilled water is mended to 25 μ l,
PCR program is: 94 ℃ of 4min, and 94 ℃ of 30s then, 64.9 ℃ of 30s, 72 ℃ of 30s, totally 35 circulations, 72 ℃ of 10min,
1% agarose gel electrophoresis for PCR product, found that except 4623bp PCR object band, also has 4 bands clearly, to being connected to after these 4 zone purifications
-T Easy carrier, finds 2 kinds of new variable shear-forms after order-checking, be labeled as A2M-AS1 and A2M-AS2;
The functional SNP site of c:A2M gene differentiates that step is:
According to the background context data that causes that the variable shearing of gene is machine-processed, with reference to ox A2M gene order in NCBI, take milk cow DNA as template, there is near the DNA sequence dna variable clipped position in design F1/Ri 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, PCR reaction, then to product direct Sequencing, use the SeqMan software analysis sequencing result in DNAStar software package, on 29 and 37 exons of A2M gene, found two SNPs sites: c.3535A > T and c.4520T > C; 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,
Select 2 SNPs sites differentiating in A2M gene order in 338 china holstein cows individualities, to adopt the method for direct Sequencing to carry out SNP gene type, adopt SAS8.1 software to carry out correlation analysis, by correlation analysis result, AA and the TT genotype of finding respectively these 2 sites have lower SCS, relevant according to the phenotype of these 2 genotype and SCS, filter out mazoitis resistance/susceptible functional molecular marker, by to the Id judgement of milk cow, by the method for marker assisted selection, can seed selection mazoitis Resistant Individuals, cultivate the milk cow new lines of mastitis-resisting,
The analysis of d:SNPs and variable shearing relation:
In order to analyze the SNPs of order-checking discovery and the relation of variable shearing, utilize ESEfinder3.0 to analyze these 2 SNP, find that these two SNP are positioned to shear enhanser ESE, wherein, c.3535A > T sudden change has increased the combination of two kinds of shear proteins of SC35and SRp40, c.4520T > C sudden change has increased the combination of SRp40 shear protein
Milk cow DNA is checked order simultaneously, 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 abnormal variable spliced body.
2. a kind of seed choosing method that improves the mastitis-resisting ability of milk cow according to claim 1, it is characterized in that: the designed pcr amplification primer of the different variable spliced bodies of amplification milk cow A2M, upstream primer A2MF sequence is: SEQ ID NO.9, downstream primer A2MR sequence is: SEQ ID NO.10, the product size of the abnormal variable spliced body A2M-AS1 of its pcr amplification is 1242bp, and sequence is as SEQ ID NO.16; The product size of abnormal variable spliced body A2M-AS2 is 3607bp, and sequence is as SEQ ID NO.17.
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