CN101693923A - HSP70A1A gene SNP loci, application and kit for selecting heat-resistant cows - Google Patents
HSP70A1A gene SNP loci, application and kit for selecting heat-resistant cows Download PDFInfo
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Abstract
The invention relates to SNP loci and a detecting method for selecting heat-resistant cows, wherein the SNP loci are basic groups of a Number 6400 locus, a Number 6600 locus and a Number 6601 locus of an HSP70A1A gene of a cow. By extracting genome DNA of the cow, heat shock protein 70A1A (HSP70A1A) gene haplotype combination of the cow is measured so as to forecast heat resistance of the cow: when the HSP70A1A gene haplotype combination is H2H3, the heat resistance of the cow is the strongest; when an HSF1 gene haplotype combination is H2H4, the heat resistance of the cow is the poorest. The detecting method has the advantages of expounding the correlation between the three polymorphic loci of the HSP70A1A gene and the heat resistance of the cow for the first time. The invention provides the method for forecasting the heat resistance of the cow, further provides a kit applied to the method and supplies a novel molecular marker method for marker-assisted selection and selective breeding for the cow.
Description
Technical field
The present invention relates to a kind of SNP site of screening heat-resistant cows, specifically utilize the 6400th, 6600 of HSP70A1A genes and 6601 bit base polymorphisms prediction milk cow thermotolerance, belong to the domestic animal technical field of molecular biology.
Background technology
Cow heat stress refers to when milk cow is subjected to surpassing the excessive temperature stimulation of thermoregulation ability own, acts on hypophysis-interrenal system and causes the nonspecific defense reaction of body and the general indication of specificity obstacle.The dairy bread that China raises is the He Sitanniu that derives from Northern Europe mostly, this kind performance height of giving milk, build is big, area of dissipation is little relatively, the sweat gland function is undeveloped, can not utilize the skin heat radiation well, and the heat in galactopoiesis and metabolic processes is big, and the food consumption of this kind milk cow is big, and feed ferments in cud and produces a large amount of heats.Temperature, strain, lactation stage, parity etc. also have certain influence to the milk cow thermotolerance.Along with global warming, China's summer high temperature time extends gradually, make ox particularly cow in summer heat stress takes place easily.The milk cow body is in the environment of heat stress for a long time, can cause the immunity degradation of body.High temperature has reduced the ability of body cell immunity and humoral immunization to some extent, helps the breeding of multiplying of pathogenic micro-organism, and milk cow is reduced disease resistance.Thereby mazoitis, retention of placenta and endometritis increase.Heat stress can cause the milk cow metabolic disturbance, and food consumption reduces, and production performance descends, and reproductivity reduces, and immunizing power reduces, and diseases such as easy infection mazoitis, retention of placenta, endometritis can cause heatstroke when serious, even dead.Cow heat stress has become present puzzlement China's milk cattle cultivating No.1 difficult problem already.The suitableeest life temperature of milk cow is-1 ℃-15 ℃.More than 21 ℃, 0.6 ℃ of cow feeding amount of the every rising of temperature decline 1.4kg causes body nutrition intake deficiency, 1 ℃ of the every rising of temperature this moment, milk yield decline 0.18kg-0.68kg.It is reported, press continuous high temperature and calculated in 90 days, ten thousand cattle farms that milk yield is medium, the financial loss that causes because of heat stress just reaches 36.5 ten thousand yuan (Qu Junmei, the flat .2005 of Li Wen).
At present, red corpuscle potassium is the evaluation milk cow thermotolerance index of relatively generally acknowledging.The research work of red corpuscle potassium comes from sheep, Evans (Evans.1954) has found the difference between Britain's kind sheep individuality first, and be divided into high potassium (HK) and two types of low potassium (LK), subsequently, the adult same individuality of the ox red corpuscle potassium content in different times of proof is constant relatively, and the LK type adapt to easily heat and the arid environment (Evans, Mounib.1957).Evans (1963) under this enlightenment, begin one's study Brahmin bull, shorthorn, Africander and hereford cow etc. and hybrid thereof, find that thermotolerance the strongest Africander and Brahmin's bull red corpuscle potassium content are minimum, so think that it can be used as the stable on heating reliability index of ox.Comberg (1969) has proved this result with 3 pairs with the twin calf of ovum He Sitan, and it is not remarkable to illustrate twin internal difference, and each is to there are differences.Mu Yuyun (1993) etc. at the verification experimental verification in Nanjing heat-resisting milk cow and thermo-labile milk ORBC potassium content significant difference under the same environmental conditions.Recent study shows that red corpuscle potassium and thermotolerance are negative correlation, is not subjected to the restriction at sex and age, and the heritability height is measured simply, and the repetition rate height can be used as thermotolerance genetic marker .1993 such as () Shi Binlin.Different other, different parity of He Sitanniu, different calving month, red corpuscle potassium content difference was not remarkable, prove that the red corpuscle potassium content is basicly stable, was not subjected to nutrition, physiology and such environmental effects, determined (Mu Yu cloud .1990) by inherited genetic factors.This result of study with Cao Jingbao (1997), Lai Dengming (1997), Liu Yuqing (1997) etc. is consistent.
Ritossa in 1962 finds that first genetic transcription strengthens in thermal environment, and Tissiereso in 1974 utilizes SDS-PAGE to separate and obtains one group of new protein-HSP that thermal stress reaction produces.Subsequently, discover that HSP70 is the most important histone matter of HSP family.Heat shock protein 70 (Heat shock protein 70, HSP70) participate in the thermal stress reaction of animal as of paramount importance molecular chaperones, participate in that nascent protein is folding, the reparation of transhipment, injury protein matter, assemble proteinic degraded unusually by its molecular chaperones effect, many important physiological dispositions such as antigen presentation, thus normally and under the stressed condition play a significant role organism.Studies show that in a large number HSP70 family is relevant with stable on heating acquisition, it can make thermoinducible metaprotein recover state of nature (Bosch etc., 1988 as early as possible; Morimoto etc., 1990; Sanchez etc., 1993; Maloyan etc., 1999).Animal is when heat stress, and HSP70 is induced (Bao Endong etc., 2004) rapidly in the body, strengthens along with HSP70 expresses, and body strengthens (Krobitsch etal., 2000) to the tolerance of heat; Change the HSP70 gene over to normal cell, the cell heat hardiness strengthens.Zhen etc. (2006) discover that the sudden change of gene influences the thermotolerance of animal, have the thermotolerance height of the animal of some specific gene type.
Along with the genetic development of molecular amounts, association analysis becomes a kind of reliable method of research function candidate gene, promptly utilize candidate genes polymorphism to set up correlation model and carry out statistical study, thereby find the molecule marker of a certain proterties of remarkably influenced with the index that a certain proterties can be described.Can be used as the reliability index of mastadenitis of cow level as the somatocyte of milk cow scoring (SCS), so people carry out the molecule marker that association analysis just can obtain remarkably influenced mastadenitis of cow resistance by functional gene polymorphism and SCS.Utilize this method, people have obtained the molecule marker of a large amount of milk-proteins, butterfat and milk yield in milk cow.Liu Qinghua, Liang Xuewu (2006) utilize the PCR-SSCP technical study to find that HSP70A1A gene 5 ' flanking region AC genotype resistance toheat is higher than other genotype.Chen Qiang (2007) infers that the A gene of HSP70A1A gene may be heat-resisting gene, and the B gene is thermo-labile gene.Therefore, we further study the relation of HSP70A1A gene SNP and the heat-resisting proterties of milk cow, determine the heat-resisting haplotype combination of HSP70A1A gene.
Summary of the invention
Purpose of the present invention proposes a kind of SNP site that is used to screen the HSP70A1A gene of heat-resistant cows;
Second purpose of the present invention is to propose to utilize the method for above-mentioned HSP70A1A gene SNP site haplotype combination screening heat-resistant cows;
The 3rd purpose of the present invention is, by the relation of above-mentioned haplotype combination and the heat-resisting proterties of milk cow, and then provides a kind of test kit of new screening heat-resistant cows, for the marker assisted selection of milk cow provides new molecule marker, can carry out the early screening of milk cow.
For achieving the above object, invention thinking of the present invention is: the haplotype combination H2H3 (GCG/CTA) of a new milk cow HSP70A1A gene of proposition, be that detection milk cow the 6400th deoxyribonucleotide in GenBank Accession NoAY149619.1 is G or C, the 6600th deoxyribonucleotide is C or T, the 6601st deoxyribonucleotide is G or A, determine the haplotype combination of described milk cow, determine heat-resisting proterties by haplotype combination then in these 3 sites.
The concrete technical scheme of the present invention is as follows:
A kind of SNP site of screening heat-resistant cows, described SNP site are the 6400th of milk cow HSP70A1A gene, the 6600th and the 6601st bit base.
To be G/C, the 6600th be G/A for C/T and the 6601st to the 6400th of described milk cow HSP70A1A gene.
A kind of method of screening heat-resistant cows; described method is: extract the milk cow genomic dna; measure the 6400th, 6600 of milk cow HSP70A1A genes and 6601 bit base polymorphisms, according to the thermotolerance of the 6400th, 6600 of milk cows and 6601 s' haplotype combination prediction milk cow.
When the 6400th, 6600 of described milk cow HSP70A1A genes and 6601 gene haplotype combination were the GCG/CTA genotype, the milk cow thermotolerance was the strongest.When the 6400th, 6600 of described milk cow HSP70A1A genes and 6601 gene haplotype combination were the GCG/CTG genotype, the milk cow thermotolerance was the poorest.
Described haplotype combination is determined as follows: if the 6400th deoxyribonucleotide is G in GenBank Accession NoAY149619.1, its homozygotic genotype is GG; If the 6400th deoxyribonucleotide is C, its homozygotic genotype is CC; Their heterozygote genotype is GC.As the 6600th deoxyribonucleotide is C, and its homozygotic genotype is CC; As the 6600th deoxyribonucleotide is T, and its homozygotic genotype is TT; Their heterozygote genotype is CT.As the 6601st deoxyribonucleotide is G, and its homozygotic genotype is GG; As the 6601st deoxyribonucleotide is A, and its homozygotic genotype is AA; Their heterozygote genotype is GA.Find 6 haplotype combination, i.e. H1H1 (GCA/GCA), H1H2 (GCA/GCG), H2H2 (GCG/GCG), H2H3 (GCG/CTA), H2H4 (GCG/CTG) and H4H4 (CTG/CTG) after 3 Sites Combination.
The primer of above-mentioned screening heat-resistant cows has the base sequence shown in sequence table SEQ ID NO.2 and the SEQ ID NO.3.Described primer can specific amplification goes out to contain sequence shown in the SEQ ID NO.1 of HSP70A1A gene.
A kind of test kit that screens heat-resistant cows is characterized in that, described test kit is that 100 oxen detect dosage, and the storage temperature of test kit is-20 ℃, and described primer has the base sequence shown in sequence table SEQ ID NO.2 and the SEQ IDNO.3; Test kit is composed as follows:
120 μ L, 10 * PCR damping fluid;
24μL?10mM?dNTP；
10 μ L 5unit/ μ L Taq archaeal dna polymerases;
Each 24 μ L of the upstream and downstream primer of 10mM;
25mmol/L?MgCl
2?75μL；
The 1ml pure water.
Described test kit also comprise with the 6400th, 6600 of HSP70A1A genes and 6601 sudden change bonded probes and identification HSP70A1A gene in the 6400th, the restriction enzyme of 6600 and 6601 sudden changes.
Described sudden change is selected from following single nucleotide polymorphism:
6400 G → C; 6600 C → T; 6601 G → A;
Wherein, the nucleotide position numbering is based on No.AY149619.1.
The present invention proposes the 6400th of HSP70A1A gene, the 6600th and the 6601st the new application that SNP base site is used to prepare the test kit that screens heat-resistant cows.
In actual applications, the heat-resisting proterties determined of the present invention can be used as the assisted Selection mark of cattle breeding.The thermotolerance of H2H3 (GCG/CTA) haplotype combination individuality is higher than described other haplotype combination individualities, and the thermotolerance of H2H4 (GCG/CTG) haplotype combination individuality is lower than described other haplotype combination individualities.
H2H3 (GCG/CTA) haplotype combination of detection milk cow HSP70A1A gene provided by the invention comprises the steps:
(1) utilize conventional phenol/chloroform extraction process to extract genomic dna, it is standby that the DNA sample is diluted into 50ng/ μ l.
(2) whether there is following single nucleotide polymorphism in the PCR-SSCP technology for detection amplified production:
6400 G → C; 6600 C → T; 6601 G → A;
Wherein, the nucleotide position numbering is based on GenBank call number: AY149619.1.
(3) at 3 sudden change design milk cow HSP70A1A gene-specific primers, carry out the PCR reaction, obtain the amplified production of specific fragment (shown in sequence table SEQ ID NO:1), described amplified production length is 549bp.
(4) polymorphism of 6400 mononucleotide polymorphic site G/C of PCR-SSCP technology for detection amplified production, 6600 mononucleotide polymorphic site C/T and 6601 mononucleotide polymorphic site G/A.The electrophoretogram of PCR-SSCP is if obtain band 1,3, and its haplotype combination is H1H1; If obtain band 1,2,3 and 4, its haplotype combination is H1H2; If obtain band 2,4, its haplotype combination is H2H2; If obtain band 1,3,5 and 6, its haplotype combination is H2H3; If obtain band 2,4,5 and 6, its haplotype combination is H2H4; If obtain band 5,6, its haplotype combination is H4H4.
(5) be divided into from identifying 6 kinds of haplotype combination, to those skilled in the art, the construction process of haplotype can change as required, as long as be fit to detect haplotype of the present invention.
Advantage of the present invention and beneficial effect:
(1) identify 3 mutational sites 6400 of milk cow HSP70A1A genes, 6600 and 6601, and isolation identification goes out a haplotype combination H2H3,, prove new SNP and haplotype combination through international genome database and document patent retrieval.
(2) through the heat-resisting proterties of 549 Chinese holstein cattle individualities of above-mentioned haplotype combination and milk cows is carried out correlation analysis, haplotype combination H2H3 that new isolation identification goes out and the heat-resisting proterties significant correlation of milk cow, the early screening that can be used for milk cow, thereby the attenuating feeding cost increases value-added content of product.
(3) test kit of being invented only just can detect 3 mutational sites with a SSCP electrophoresis, and is simple and convenient, can be used for early detection to improve detection efficiency, reduces the detection cost.
Description of drawings
Fig. 1 is the different haplotype combination PCR-SSCP of milk cow HSP70A1A gene somatotype result;
Fig. 2 is the different haplotype combination sequencing results of milk cow HSP70A1A gene.
Embodiment
The present invention is directed to Chinese holstein cattle HSP70A1A gene and carried out deep research, find 3 new SNPs sites, analyze at the haplotype combination that these 3 SNPs sites are formed, wherein statistic analysis result shows, the red corpuscle potassium concn of the haplotype combination H2H3 of milk cow HSP70A1A gene significantly is lower than H2H4, haplotype combination H2H3 is heat-resisting haplotype combination, the heat-resistance coefficient of H2H3 haplotype combination individuality is significantly higher than H2H4 and H4H4 haplotype combination (P＜0.05), its milk yield rate of descent significantly is lower than heat labile H2H4 haplotype combination individuality, further specifies it and is heat-resisting haplotype.Therefore this haplotype combination can be used as the specific haplotypes combination that detects the heat-resisting proterties of milk cow, is used for auxiliary seed selection of mark and the apolegamy of milk cow.Finished the present invention on this basis.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989).
Determining of embodiment 1 haplotype combination and mutational site
The present invention adopt PCR-SSCP and PCR sequencing technologies to milk cow HSP70A1A transgenation carry out examination, analyze and the sequencing result comparison by the SSCP electrophoretic band, determined 6400,6600 and 6601 3 mutational sites, 6 kinds of haplotype combination.
1.1 (He Sitan bovine jugular vein blood used in the present invention is taken from mass-producing cattle farm, Shandong Province to collection milk bovine jugular vein blood, comprises the good treasured in Jinan, new garden difficult to understand, Qingdao, Tai'an gold orchid.Be the direct sources and the primary source of the used blood sample of the present invention), the ACD anti-freezing utilizes conventional phenol/chloroform extraction process to extract genomic dna, and it is standby that the DNA sample is diluted into 50ng/ μ L.Totally 540 of experiment oxen are from the mass-producing diary farm in the Shandong Province.
1.2 design of primers:
According to the Genbank accession number is the gene order of the HSP70A1A of AY149619.1, and with PrimerPremier5.0 software design primer, the sequence of designed primer, the fragment length of amplified production see Table 1.
The used primer of table 1PCR amplification
1.3PCR amplification
The reaction system of 12 μ L: template (50ng/ μ l) 1 μ L, the dNTP 0.24 μ L of 10mM, 10 * PCRbuffer, 1.2 μ L, each 0.24 μ L of 10 μ mol/L upstream and downstream primers, 25mmol/l MgCl
20.75 μ L, Taq enzyme 0.5U.
The PCR reaction conditions: 95 ℃ of sex change 5min, 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ are extended 7min, 16 ℃ of preservations.
1.4SSCP analyze and order-checking
Behind 95 ℃ of sex change 10min of PCR product, put on the ice chest immediately and carry out point sample after putting into-20 ℃ of refrigerator 10min, 10% polyacrylamide gel electrophoresis, electrophoretic buffer is 1 * TBE, 150V, 12h, silver dyes colour developing, observe the PCR-SSCP electrophoretogram and take pictures the polymorphism of discriminatory analysis HSP70A1A gene.
As shown in Figure 1, be the different haplotype combination PCR-SSCP of milk cow HSP70A1A gene somatotype result.The electrophoretogram of PCR-SSCP is found 6 kinds of haplotype combination altogether, is respectively H1H1 (having electrophoretic band 1,3), H1H2 (having electrophoretic band 1,2,3 and 4), H2H2 (having electrophoretic band 2,4), H2H3 (having electrophoretic band 1,3,5 and 6), H2H4 (having electrophoretic band 2,4,5 and 6) and H4H4 (having electrophoretic band 5,6).
After the PCR product that every kind of haplotype combination is got 3 individualities respectively was purified, the interpretation and the SNP that carry out sequence with ABI 3770 dna sequencing instrument confirmed.As shown in Figure 2, be the different haplotype combination sequencing results of HSP70A1A gene, sequencing analysis shows that the mononucleotide situation of these 6 kinds of haplotype combination in 3 sites is H1H1:GCA/GCA, H1H2:GCA/GCG, H2H2:GCG/GCG, H2H3:GCG/CTA, H2H4:GCG/CTG and H4H4:CTG/CTG.Finding 4 kinds of haplotypes altogether at the HSP70A1A gene, is respectively H1 (GCA), H2 (GCG), H3 (CTG) and H4 (CTA), haplotype frequency difference 0.2787,0.4065,0.1361 and 0.1787.Those of ordinary skills are known, and the construction process of above-mentioned haplotype can change as required, as long as be fit to detect haplotype of the present invention.
Embodiment 2HSP70A1A gene haplotype combination is stable on heating relevant with milk cow
The mensuration of red corpuscle potassium concn: oxtail venous blood collection 5ml, 3000r/min is centrifugal, and 10min discards blood plasma.Add 5ml physiological saline in red corpuscle, the centrifugal 8min of 7000r/min repeats to give a baby a bath on the third day after its birth time, accurately inhales the 0.5ml red corpuscle, injects small test tube, and label refrigeration send academy of agricultural sciences, Shandong Province centralab with atomic absorption spectrophotometer red corpuscle potassium content then.
Heat-resistance coefficient is that Rhoad (1944) foundes mensuration, formula is as follows: the mean body temperature that heat-resistance coefficient=100-18 (BT-38.3 ℃) records when wherein BT is for test, 38.3 ℃ be the normal body temperature of ox, 18 for body temperature changes the coefficient of changing into fundamental unit, and 100 for keeping the complete efficient of body temperature in normal body temperature.
The ANOVA-Linear Models program of SAS8.2 (Statistical Analysis System) software is adopted in the least square variance analysis, set up following model, red corpuscle potassium concentration and genotype and haplotype are carried out least square analysis and test of significance, and its model is:
Y
ijkl＝μ+G
i+E
j+S
k+P
l+e
ijkl
Y in the formula
IjklBe the record value of each and every one body surface type of i, μ is colony's mean value, G
iBe i individual genetic effect value, Ej is the environmental effect value, S
kBe the fixed effect in season, P
lBe the fixed effect of parity, e
IjkBe potential difference at random.As shown in table 2.
The least square analysis of table 2 milk cow HSP70A1A gene haplotype combination and the every index of thermotolerance (least square mean value standard error, LSM ± SE)
Annotate: the level of difference between the different letters in same site, a, b:(P＜0.05); A, B:(P＜0.01).
As shown in Table 2, the red corpuscle potassium concn of milk cow HSP70A1A gene H2H3 haplotype combination individuality significantly is lower than H2H4 (P＜0.05), there are some researches show that the milk cow concentration of blood kalium becomes remarkable negative correlation with resistance toheat, red corpuscle potassium concn low heat resistant more can be strong more, and H2H3 is heat-resisting haplotype combination.The heat-resistance coefficient of H2H3 haplotype combination individuality is significantly higher than H2H4 and H4H4 haplotype combination (P＜0.05), the milk yield rate of descent of H2H3 haplotype combination individuality significantly is lower than heat labile H2H4 haplotype combination individuality (P＜0.01), and further specifying H2H3 is heat-resisting haplotype combination.This has the potential using value in milk cow seed selection and breed improvement.In view of the above, the breeding work person can instruct the early screening of ox, thereby reduces feeding cost, increases value-added content of product.As seen, milk cow HSP70A1A gene haplotype combination has important application prospects in auxiliary seed selection of the mark of milk cow and apolegamy.Therefore this haplotype combination can be used as the specific haplotypes combination that detects the heat-resisting proterties of milk cow.
Embodiment 3 detection kit
Based on the test kit of HSP70A1A gene haplotype combination label screening milk cow, as described in embodiment 1,6400 G → C among the AY149619.1,6600 C → T and 6601 G → haplotype combination that A sudden change makes up and the heat-resisting proterties of milk cow are closely related.Therefore, can increase and detect based on this 3 SNP sites design HSF1 gene-specific primers.
Preparation detects the stable on heating test kit of milk cow (100 times), shown in the table 3 composed as follows:
Table 3
Concrete composition and consumption are: 120 μ L, 10 * PCR damping fluid, 24 μ L 10mM dNTP, 10 μ L (5unit/ μ L) Taq archaeal dna polymerase, each 24 μ L of the upstream and downstream primer of 10mM, 25mmol/L MgCl
275 μ L, the 1ml pure water.
Gather bovine blood, extract total DNA, react by embodiment 1 described method.PCR primer in the test kit is diluted to 1 μ mol/L, is that template is carried out the PCR reaction with above-mentioned test kit with the DNA that is extracted.The PCR reaction conditions: 95 ℃ of sex change 5min, 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ are extended 7min, 16 ℃ of preservations.By embodiment 1 described method the amplified production haplotype combination is confirmed.
The applying detection test kit detects 100 cow heads, and it is the H2H3 haplotype that 28 oxen are arranged, and is heat resistant type (heat-resistance coefficient＞80, the milk yield rate of descent is lower than 25%) and 27 oxen are arranged in these 28 oxen, so test kit accuracy of the present invention can reach more than 96%.
According to marking method provided by the invention, if with 6400 C among the AY149619.1,6600 and 6601 sudden change bonded probes or directly order-checking also can detect the 6400th G → C, 6600 C → T and 6601 G → A sudden changes among the AY149619.1.
The present invention has the illustration of practicality:
1) detection method of HSP70A1A gene pleiomorphism of the present invention can be used for analyzing 3 SNPs sites on No. 23 chromosomal HSP70A1A genes of ox, is applied to the stable on heating early diagnosis of ox, is beneficial to cultivate heat-resisting cattle breeds.
2) nucleotide sequence and the thermotolerance related locus of the detection HSP70A1A gene pleiomorphism set up of the present invention, but highly sensitive, be applied to the test kit of ox thermotolerance gene diagnosis specifically.
As mentioned above, reach a conclusion, the HSP70A1A gene is at the polymorphism and the ox thermotolerance tool significant correlation of the 6400th C, 6600 and 6601 bit bases.Therefore, according to the present invention, measure this polymorphism and can be used for carrying out gene diagnosis.
The present invention has narrated the relevant new mutant site of HSP70A1A gene thermotolerance, and a kind of method of the HSP70A1A of mensuration gene pleiomorphism is provided.And, according to the present invention, only need a small amount of DNA sample just to be enough to measure 3 loci polymorphisms of HSP70A1A gene.As a result, the invention provides a kind of gene diagnosis method of measuring the relevant haplotype combination of ox thermotolerance.
Sequence table
＜110〉Ox Biological Tech. Co., Ltd., Shandong
＜120〉a kind ofly be used to screen stable on heating HSP70A1A gene haplotype combination of milk cow and application thereof
<160>3
<210>1
<211>549
<212>DNA
＜213〉ox (Bos Taurus, bovine)
<400>1
TAAAGCCACC?ACGATGTTTC?AGCCAAAGTT?GAGAAAAACC?ACTGTACCAG?AGGCTATTCC?60
TGATTATTTT?TTAGAGGTAT?GGGGACAAGA?GAGAGGGAGG?AGAGGAAGGT?GTTAGTGCTT?120
CCAGGAGCAG?CAGCTGGTGG?CAAGGGATTT?TCTGCTGTGA?ATCAAAGCTG?GAAAGGCCTT?180
AAAGTGTTGA?TTCACTGGCA?GCAGTAGTAA?TGCTAAACTT?TAGGGAAATT?GCCACCAACA?240
GCTTAATAGG?CCAAAAAAGA?GAATTCCTGA?AGCTGGGTCA?ATTAATTTCA?TGAGTCCTCA?300
CTAGGGAAAT?GGCTCATTAG?AAAGCAACAT?CAGGGCGTCA?TTTCCTGGCC?CTCCAGCGGT?360
TAGGACTCAG?TGCTTTCATT?GCCAGGGCCT?GGGTTCGATC?CCTGGTGGAA?GTAAAATCCC?420
ACAAGCAGTG?AGGCTTAGCC?AAAGTAATTA?ACACAGAACT?TACAAAAAAA?AGATATGAGC?480
CATGGAGGAG?TTCGCTTGCT?TAGGAGCCTT?GCCCATGGAG?AATTGTGGAT?GATTGCGGTG?540
GAACCTTCA 549
<210>2
<211>18
<212>DNA
＜213〉synthetic
<400>2
TAAAGCCACC?ACGATGTT 18
<210>3
<211>20
<212>DNA
＜213〉synthetic
<400>3
GGGGTAAGTC?TGGTTACGAC 20
Claims (10)
1. a SNP site of screening heat-resistant cows is characterized in that, described SNP site is the 6400th of milk cow HSP70A1A gene, the 6600th and the 6601st bit base.
2. the SNP site of screening heat-resistant cows according to claim 1 is characterized in that, to be G/C, the 6600th be G/A for C/T and the 6601st to the 6400th of described milk cow HSP70A1A gene.
3. method of screening heat-resistant cows; it is characterized in that; described method is: extract the milk cow genomic dna; measure the 6400th, 6600 of milk cow HSP70A1A genes and 6601 bit base polymorphisms, according to the thermotolerance of the 6400th, 6600 of milk cows and 6601 s' haplotype combination prediction milk cow.
4. the method for screening heat-resistant cows according to claim 3 is characterized in that, when the 6400th, 6600 of described milk cow HSP70A1A genes and 6601 gene haplotype combination were the GCG/CTA genotype, the milk cow thermotolerance was the strongest.
5. according to the method for claim 3 or 4 described screening heat-resistant cows, it is characterized in that when the 6400th, 6600 of described milk cow HSP70A1A genes and 6601 gene haplotype combination were the GCG/CTG genotype, the milk cow thermotolerance was the poorest.
6. a primer that screens heat-resistant cows is characterized in that, described primer has the base sequence shown in sequence table SEQ ID NO.2 and the SEQ ID NO.3.
7. the primer of screening heat-resistant cows according to claim 6 is characterized in that, described primer can specific amplification goes out to contain sequence shown in the SEQ ID NO.1 of HSP70A1A gene.
8. a test kit that screens heat-resistant cows is characterized in that, described test kit is that 100 oxen detect dosage, and the storage temperature of test kit is-20 ℃, and test kit comprises:
120 μ L, 10 * PCR damping fluid;
24μL 10mM?dNTP；
10 μ L 5unit/ μ L Taq archaeal dna polymerases;
Each 24 μ L of the upstream and downstream primer of 10mM;
25mmol/L?MgCl
2?75μL；
The 1ml pure water.
Described primer has the base sequence shown in sequence table SEQ ID NO.2 and the SEQ ID NO.3.
9. the test kit of screening heat-resistant cows according to claim 8, it is characterized in that, described test kit also comprise with the 6400th, 6600 of HSP70A1A genes and 6601 sudden change bonded probes and identification HSP70A1A gene in the 6400th, the restriction enzyme of 6600 and 6601 sudden changes.
10.HSP70A1A the 6400th, the 6600th and the 6601st SNP base site of gene is used to prepare the application of screening heat-resistant cows test kit.
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| CN102899395A (en) * | 2012-06-20 | 2013-01-30 | 山东省农业科学院奶牛研究中心 | Breed selection method for improving mastitis resistance of dairy cow and use thereof |
| CN102899320A (en) * | 2011-07-30 | 2013-01-30 | 华中农业大学 | Molecule marker for cow oxidative stress correlated character detection, and applications thereof |
| CN103421772A (en) * | 2012-12-18 | 2013-12-04 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to heat-proof character of cow, and application |
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| CN113462789A (en) * | 2021-08-11 | 2021-10-01 | 江苏省农业科学院 | SNP locus combination for heat stress tolerance identification of Holstein cattle and chip preparation |
| CN114854872A (en) * | 2022-04-15 | 2022-08-05 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker related to goat heat-resistant character and application thereof |
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