CN101168777B - Method and kit for detecting Chinese holstein cattle produced milk property - Google Patents
Method and kit for detecting Chinese holstein cattle produced milk property Download PDFInfo
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Abstract
The invention discloses a method and a reagent box for detecting the milk production traits of the Chinese Holstein cow. The invention provides a detecting method of the milk production traits of the Chinese Holstein cow that whether the deoxyribose nucleotide of the Chinese Holstein cow from the 10433-10434th positions (namely, the 15-16th positions of the DGAT1 expressed region) of the (5) end in the GenBank Accession No.AJ318490 is AA or GC is detected, the gene type of the Chinese Holstein cow is determined, and then the milk production traits is determined through the gene type. The reagent box provided by the invention comprises the primer of the gene group fragment of the nucleotide used for the PCR application and contained with 10433-10434th positions (namely, the 15-16th positions of the DGAT1 expressed region) of the (5) end in the GenBank Accession No.AJ318490, restrictive endonuclease CfrI and PCR reagent and restrictive endonuclease buffer solution. The operation of the method of the invention and the reagent box is simple, quick and sensitive, and the result detected through the method and the reagent box is reliable, stable and accurate, therefore, the invention method and the reagent box have a broad application prospect in the cow breeding field.
Description
Technical field
The present invention relates to a kind of method and test kit that detects the Chinese holstein cattle milk production trait.
Background technology
Since the twentieth century end, the World Health Organization classifies dairy products occupancy volume per capita as the leading indicator of weighing national living standards of the people.Show according to latest information, the per capita milk amount in the world remains on about 100kg (2006), wherein developed country surpasses 200kg (France reaches 456kg) mostly, Japan in long-time from rise to present 70kg less than 10kg, contiguous Thailand, India etc. also all surpass 60kg, and China only is 6.8kg at present, is 1/15 of world average level.
Why the occupancy volume of dairy products is far below world average level per capita in China, and except the influence that is subjected to traditional diet and some other factor, most important reason is that the milk cow industry of China is also undeveloped, and especially the cattle breeding level is also relatively backward.Therefore need to strengthen scientific research input, to improve milk industry production efficiency to milk cow production breeding aspect.
Be accompanied by the development of Heredity theory, the cattle breeding technology is also selected to develop into breeding value from phenotypic number and is selected.Especially the improvement of breed that develops into of genetic generation of molecular amounts and theory and technology thereof provides new strategy, make the improvement of breed scholar progressively carry out the transition to manipulation quantitative character genotype from handling the quantitative character phenotype, transfer marker assisted selection (Marker-Assisted Selection to from selection by phenotype, MAS) (Soller andBeckmamn, 1983), finally realized molecular breeding.Marker assisted selection is on the basis of genome analysis, by the dna marker technology livestock and poultry quantitative trait loci is directly selected, or, remove unfavorable gene etc. by auxiliary the eliminating of mark, thereby reach the purpose that more effectively improves livestock and poultry by the auxiliary importing beneficial gene that infiltrates of mark.
Lipid in the milk mainly is triglyceride level (triglycerides), accounts for 98% of dairy fats.The main approach of synthetic glycerine three esters is Phosphoric acid glycerol esters diester approach.Diglyceride transacylase (acylCoA:diacylglycerol acyltransferase) is catalyzing glycerol three ester synthetic key enzymes in the final step reaction of Phosphoric acid glycerol esters diester approach, so the encoding gene DGAT1 of diglyceride transacylase has material impact to butterfat synthetic.Therefore, the relation of the DGAT1 gene being studied itself and growth traits and milk production trait as quantitative character has very big practice significance to the breeding of ox.
Summary of the invention
An object of the present invention is to provide a kind of method that detects the Chinese holstein cattle milk production trait.
The method of detection Chinese holstein cattle milk production trait provided by the present invention is that to detect Chinese holstein cattle be AA or GC from 5 ' end 10433-10434 position (being the 15-16 position of DGAT1 exon 8) deoxyribonucleotide in GenBank Accession No.AJ318490, determine the genotype of described Chinese holstein cattle, determine milk production trait by genotype then;
Wherein, described genotype is what to determine as follows: if be AA from 5 ' end 10433-10434 position (being the 15-16 position of DGAT1 exon 8) deoxyribonucleotide in GenBank Accession No.AJ318490, its homozygotic genotype is KK; If in GenBank Accession No.AJ318490 from 5 ' end 10433-10434 position (being the 15-16 position of DGAT1 exon 8) deoxyribonucleotide is GC, its homozygotic genotype is MM; Their heterozygote genotype is KM.
When described milk production trait was milk yield, the milk yield of MM genotype individuality was higher than the individual and KK genotype individuality of KM genotype, and KM genotype individuality is higher than KK genotype individuality; When described milk production trait was fat yield, the fat yield of KK genotype individuality was higher than the individual and MM genotype individuality of KM genotype, and KM genotype individuality is higher than MM genotype individuality; When described milk production trait was the milk-protein amount, the milk-protein amount of MM genotype individuality was higher than the individual and KK genotype individuality of KM genotype.
In actual applications, the definite milk production trait of the inventive method can be used as the assisted Selection mark of Chinese holstein cattle breeding.
Detecting Chinese holstein cattle described in the present invention is that AA or the method for GC specifically can be first pcr amplification and contain in GenBank Accession No.AJ318490 from the genomic fragment of 5 ' end 10433-10434 position (being the 15-16 position of DGAT1 exon 8) Nucleotide from 5 ' end 10433-10434 position (being the 15-16 position of DGAT1 exon 8) deoxyribonucleotide in GenBank Accession No.AJ318490, then amplified production is checked order or cuts amplified production with restriction endonuclease CffI enzyme.Wherein, in a pair of primer of pcr amplification, the nucleotide sequence of a primer is a sequence 1 in the sequence table, and the nucleotide sequence of another primer is a sequence 2 in the sequence table.
Wherein, cut described pcr amplification product with restriction endonuclease CffI enzyme, if having to the 201bp fragment, its genotype is the KK homozygote; If obtain 201bp, 178bp and three endonuclease bamhis of 23bp, its genotype is the KM heterozygote; If obtain 178bp and two fragments of 23bp, its genotype is the MM homozygote.
Another object of the present invention provides a kind of test kit.
Test kit provided by the present invention comprises that being used for pcr amplification contains primer, restriction endonuclease CffI, PCR reagent and the restriction endonuclease damping fluid from the genomic fragment of 5 ' end 10433-10434 position (being the 15-16 position of DGAT1 exon 8) Nucleotide from GenBank Accession No.AJ318490.In a pair of primer of pcr amplification, the nucleotide sequence of a primer is a sequence 1 in the sequence table, and the nucleotide sequence of another primer is a sequence 2 in the sequence table.
The method of detection Chinese holstein cattle milk production trait of the present invention and test kit can carry out the detection of milk production trait in early days at individuality, accelerate breeding process, efficiently improve the performance of giving milk of cows.Method of the present invention detects with conventional milk production trait and combines, and can improve the accuracy and the efficient of detection.The inventive method and test kit are easy and simple to handle, quick, sensitive, the reliable results of detection, stable, accurate.
Description of drawings
Fig. 1 is the agarose gel electrophoresis detected result of the individual poba gene group DNA of part.
Fig. 2 is the agarose gel electrophoresis figure of the individual pcr amplification product of part.
Swimming lane M:DNA molecular weight standard 100 ladder Marker, swimming lane 1 is to the 15:PCR product.
Fig. 3 is the individual enzyme cutting type of part figure as a result.
Swimming lane M is dna molecular amount standard MarkI, and swimming lane 7,10-11 are the KK genotype, and swimming lane 1,3-6,8-9,12-14 are the KM genotype, and swimming lane 2 is the MM genotype.
Fig. 4 is the sequencing result of the PCR product of three kinds of genotype individualities.Arrow is depicted as the mutational site.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Reagent:
(1) 50 * TAE (1L): Tris alkali 242g, glacial acetic acid 57.1ml, the EDTA of 0.5mol/L (pH=8.0) 100ml adds water and fully dissolves, and is fixed molten to 1L.
(2) 0.7% sepharoses: the adding of 0.7g agarose is contained in the triangular flask of 100ml 1 * TAE the microwave oven heating.
(3) 2.0% sepharoses: the adding of 2.0g agarose is contained in the triangular flask of 100ml 1 * TAE the microwave oven heating.
(4) 4.0% sepharoses: the adding of 4.0g agarose is contained in the triangular flask of 100ml 1 * TAE the microwave oven heating.
One, laboratory animal chooses
The offspring of 17 Chinese He Sitan bulls is chosen in 14 cattle farms under Beijing ternary group green lotus milk cattle cultivating center, and every bull daughter counts average out to 12-153 head, amounts to 1222 cow heads, and to the sampling of taking a blood sample respectively of this 1222 cow head.The sample distribution situation sees Table 1.
Table 1 test materials structure
| The cattle farm number | 14 |
| A bull number | 17 |
| Each lactation number of cows | On average: 87.3; Minimum: 2; At most: 206 |
| Every bull daughter number | On average: 71.9; Minimum: 12; At most: 153 |
Choose 305 days milk yield, fat yield, milk-protein amount, milk fat content and protein ratios as the research milk production trait.
Two, utilize test kit to detect the genotype of Chinese holstein cattle
1, the extraction of poba gene group DNA
1222 cow heads are carried out the extraction of poba gene group DNA respectively, and the genomic dna of extraction is as pcr template.Adopt a day root poba gene group DNA extraction test kit DP318, key step is as follows:
Clip 200 μ l blood clots shred in the 1.5ml centrifuge tube
↓
Add 500 μ l cell pyrolysis liquid CL, put upside down mixing, the centrifugal 1min of 10000rpm abandons supernatant, stays the nucleus precipitation, thoroughly can not repeat above step once as cracking
↓
In the nucleus precipitation that centrifuge tube is collected, add 200 μ l damping fluid GS, mixing
↓
Add 20 μ l Proteinase Ks
↓
Add 220 μ l damping fluid GB, fully put upside down mixing, about 56 ℃ of digestion 10min, put upside down mixing therebetween for several times to molten
It is limpid that liquid becomes
↓
Add 220 μ l dehydrated alcohols, fully put upside down mixing, flocks may appear in this moment
↓
Be poured into (adsorption column is put into the waste collection pipe) in the CB3 adsorption column the centrifugal 30s of 12000rpm
↓
Add 500 μ l protein liquid removal GD, the centrifugal 30s of 12000rpm abandons waste liquid
↓
Add 700 μ l rinsing liquid PW, the centrifugal 30s of 12000rpm abandons waste liquid
↓
The CB3 adsorption column is put back in the waste collection pipe the centrifugal 2min of 12000rpm
↓
Adsorption column CB3 is changed in the clean centrifuge tube, add 100 μ l elutriant TB (elutriant is in 60 ℃ of-70 ℃ of water-bath preheatings, better effects if), room temperature is placed 2-5min, and the centrifugal 30s of 12000rpm collects solution centrifugal
In the pipe
↓
The centrifugal solution that obtains is added in the adsorption column again, and room temperature is placed 2min, and the centrifugal 2min of 12000rpm prepares against utilization with 4 ℃ of preservations of the dna solution in the centrifuge tube.
The poba gene group DNA that extracts is detected with 0.7% agarose gel electrophoresis, and detected result shows that the DNA of extraction does not have degraded (Fig. 1) substantially.
2, utilize test kit to detect the genotype of Chinese holstein cattle
This test kit comprises: a pair of primer of pcr amplification, the nucleotide sequence of one of them primer are sequences 1 in the sequence table, and the nucleotide sequence of another primer is a sequence 2 in the sequence table; Other pcr amplification reagent; Restriction endonuclease CfrI and enzyme cutting buffering liquid thereof.
1) design of primers
Sequence (AJ318490) according to milk cow DGAT1 gene in the GenBank database, adopt OLIgo6.0 software design primer, primer sequence is respectively: 5 ' ctcgtagctttggcaggtaag3 ' is (sequence 1 in the sequence table) (F), and 5 ' aagttgagctcgtagcacagg3 ' is (sequence 2 in the sequence table) (R).This primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.
2) pcr amplification
The total system 20 μ l of pcr amplification reaction: template DNA (100ng/ μ l) 1 μ l; Primer (10pmol/ μ l), each 0.6 μ l; DNTP (2.5mmol/L), 1.6 μ l; TaqDNA polysaccharase (5U/ μ l), 0.1 μ l; 10 * PCR damping fluid (Mg
2+Free), 2 μ l; MgCl
2(25mmol/L), 1ul; Mend ultrapure water to 20ul.
Pcr amplification program: desire sex change 5min for 94 ℃ earlier; 94 ℃ of 45s then, 60 ℃ of 30s, 72 ℃ of 40s, totally 35 circulations; Last 72 ℃ are extended 10min.
Pcr amplification product carried out 2% agarose gel electrophoresis.The result shows that the pcr amplification of 1222 Chinese holstein cattles all obtains the dna fragmentation of 201bp clearly, pcr amplification product agarose gel electrophoresis result such as Fig. 2 that part is individual.
3) enzyme cutting type:
Endonuclease reaction system: 8 μ l pcr amplification products, 1 μ l CfrI restriction endonuclease (10U/ μ l), 1 μ l restriction endonuclease damping fluid (10 * buffer).37 ℃ of enzymes were cut 2 hours in the water-bath.Enzyme is cut product carry out 4% agarose gel electrophoresis detection (Fig. 3).
The result shows and has three kinds of banding patterns, is respectively 201bp, 178bp, 23bp, and wherein, the fragment of 23bp is too short, therefore can't show (Fig. 3) at Jiao Tushang.The respectively corresponding three kinds of genotype of three kinds of banding patterns: what will obtain has only the segmental genotype called after of 201bp KK type, have to two segmental genotype called after MM types of 178bp and 23bp, obtain three segmental genotype called after KM types of 201bp, 178bp and 23bp.
The KK genotype is 175 in these 1222 the test cows, 745 of KM genotype, 302 on MM type.
The pcr amplification product of all test cows directly checks order, and every cow is repeated 3 times, obtains the partial dna sequence of Chinese holstein cattle DGAT1 gene extron 8, analyzes (Fig. 4) with CHROMAS software.The result show the genotypic individuality of all KK in GenBank Accession No.AJ318490 from 5 ' end 10433-10434 position (being the 15-16 position of DGAT1 exon 8) deoxyribonucleotide all is AA; The genotypic individuality of all MM in GenBank Accession No.AJ318490 from 5 ' end 10433-10434 position (being the 15-16 position of DGAT1 exon 8) deoxyribonucleotide all is GC; The genotypic individuality of all KM not only contains AA but also contain GC from 5 ' end 10433-10434 position (being the 15-16 position of DGAT1 exon 8) deoxyribonucleotide in GenBank Accession No.AJ318490.Among Fig. 4, NN=AA or GC.
All test cows are repeated above-mentioned pcr amplification and RFLP restriction analysis, and twice experimental result is in full accord.
3, the correlation analysis of genotype and milk production trait
Adopt animal model that data are carried out match, by the MIXED process of SAS (8.2) software 5 milk production traits such as 305 days milk yield, fat yield, milk-protein amount, milk fat content and protein ratios and genotype are carried out association analysis, linear equation is as follows:
y＝μ+hys+b×M+G+a+e
Wherein: y=milk production trait (milk yield, fat yield, milk-protein amount, milk fat content, protein ratio) observed value
μ=colony's average
Hys=field season in year effect
The regression coefficient of b=concomitant variable M
M=calving monthly age effect
G=genotype effect
A=is additive genetic effect at random
E=random residual effect
Wherein
Under animal model, the A battle array is the additive inheritance correlation matrix between all animal individuals, and each element of A battle array calculates with following recursion formula:
S wherein
i(s
j) and d
i(d
j) be father and mother of individual i (j).
When utilizing following formula to calculate the A battle array, earlier all individualities in the pedigree number are listed as into one 3 tabulation (data file) with female number by individual number, father, when tabulating, should note:
1) should comprise that in individual row all are at father and female individuality of listing existing mistake;
2) in individual row, should guarantee that the offspring will never appear at its father and mother before, generally can sort, earlier birth preceding by the date of birth.
3) for ease of coding, the individual applications natural number is since 1 serial number.
According to mentioned above principle, use the additive inheritance relation conefficient between 1222 individualities of Fortran95 coding calculating, obtain the A battle array.σ
a 2Be the additive genetic variance of each proterties, the additive genetic variance of milk yield, fat yield, milk fat content, milk-protein amount, protein ratio is respectively 696800kg
2, 713.3 kg
2, 0.05091,585.2kg
2, 0.003363.G is read in the MIXED process.
Association analysis is the result show, the effect of DGAT1 gene pairs milk yield, fat yield and milk-protein amount all reaches extremely significantly (P＜0.01) level, to the effect of milk fat content and protein ratio not significantly (P＞0.05).
Analyzed of the influence of three kinds of genotype of DGAT1 gene by the Bonferroni multiple comparisons to milk production trait.The result shows: the milk yield of MM genotype individuality is significantly higher than KM and KK genotype individuality (P＜0.01), and KM genotype individuality is significantly higher than KK genotype individuality (P＜0.01); The individual fat yield of KK genotype is significantly higher than KM and MM genotype individuality (P＜0.01), and KM genotype individuality is significantly higher than MM genotype individuality (P＜0.01); The individual milk-protein amount of MM genotype is significantly higher than KM and KK genotype individuality; Different genotype influences difference not significantly (P＞0.05) (table 2) to milk fat content and protein ratio.
The least square average and the standard error of the milk production trait of table 2 DGAT1 gene different genotype
| Genotype | Milk yield (proofreading and correct 305d)/Kg LSM ± SE | Fat yield/Kg LSM ± SE | Milk fat content/% LSM ± SE | Milk-protein amount/Kg LSM ± SE | Protein ratio/% LSM ± SE |
| KK(175) | 8060.l3 A±98.87 | 339.87 A±4.55 | 4.238±3.9E-4 | 259.43 A±2.87 | 3.230±1.4E-4 |
| KM(745) | 8114.91 B±59.66 | 319.34 B±2.76 | 3.952±2.3E-4 | 259.35 A±1.74 | 3.207±0.8E-4 |
| MM(302) | 8521.70 C±78.60 | 314.48 C±3.61 | 3.694±3.1E-4 | 268.76 B±2.28 | 3.164±1.1E-4 |
Annotate: have different letter shoulder target mean value differences extremely significantly (p＜0.01) in the same column
4, DGAT1 gene and genotype effect analysis
Gene, genotype effect index and calculation formula are:
Wherein: a is a dominant effect; D is an additive effect;
Be the gene substitution average effect; X
KK, X
KM, X
MMBe corresponding gene type milk production trait least square average.
Milk production trait to the different genotype significant difference calculates gene and genotype effect, the result shows (table 3): the gene substitution average effect of milk yield, fat yield and milk-protein amount is respectively-248.739kg, 11.882kg and-5.158kg, all reach utmost point conspicuous level (P＜0.01), illustrate that from another point of view DGAT1 gene pairs milk production trait has material impact.By calculating and check additive effect, dominant effect and genotype effect show: genotype to the influence of milk production trait owing to additive effect causes, the dominant effect of different genotype is not remarkable, illustrates that the influence of DGAT1 gene pairs milk production trait can heredity.
Table 3 DGAT1 gene, genotype effect
| Milk production trait | The gene substitution average effect | The genotype effect | Additive effect | Dominant effect |
| Milk yield/Kg | -248.739** | -406.475** | -230.470** | -176.005 |
| Fat yield/Kg | 11.882** | 4.860** | 12.695** | -7.835 |
| Milk-protein amount/Kg | -5.158** | -9.410** | -4.665** | -4.745 |
Annotate: * * represents difference extremely significantly (p＜0.01)
Sequence table
<160>2
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
ctcgtagctttggcaggtaag 18
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
aagttgagctcgtagcacagg 18
Claims (1)
1. method that detects the Chinese holstein cattle milk production trait, be that to detect Chinese holstein cattle be AA or GC from 5 ' end 10433-10434 position deoxyribonucleotide in GenBankAccession No.AJ318490, determine the genotype of described Chinese holstein cattle, determine milk production trait by genotype then;
Described genotype is determined as follows: if be AA from 5 ' end 10433-10434 position deoxyribonucleotide in GenBank Accession No.AJ318490, its homozygotic genotype is KK; If in GenBank Accession No.AJ318490 from 5 ' end 10433-10434 position deoxyribonucleotide is GC, its homozygotic genotype is MM; Their heterozygote genotype is KM;
Described milk production trait is a milk yield; The milk yield of described MM genotype individuality is higher than the individual and KK genotype individuality of KM genotype, and KM genotype individuality is higher than KK genotype individuality;
Described detection Chinese holstein cattle in GenBank Accession No.AJ318490 from 5 ' end 10433-10434 position deoxyribonucleotide is that AA or the method for GC comprise the steps: that first pcr amplification contains in GenBank Accession No.AJ318490 from the genomic fragment of 5 ' end 10433-10434 position Nucleotide, cuts amplified production with restriction endonuclease CfrI enzyme then;
In a pair of primer of described pcr amplification, the nucleotide sequence of a primer is a sequence 1 in the sequence table, and the nucleotide sequence of another primer is a sequence 2 in the sequence table;
Described endonuclease reaction system of cutting amplified production with restriction endonuclease CfrI enzyme is: pcr amplification product 8 μ l, 10U/ μ l CfrI restriction enzyme 1 μ l, 10 * restriction endonuclease damping fluid, 1 μ l, 37 ℃ of enzymes were cut 2 hours in the water-bath, enzyme is cut product carry out the detection of 4% agarose gel electrophoresis, carry out genotype according to electrophoresis result and judge; If have to the 201bp fragment, its genotype is the KK homozygote; If when obtaining 201bp, 178bp and three endonuclease bamhis of 23bp, its genotype is the KM heterozygote; If when obtaining 178bp and two fragments of 23bp, its genotype is the MM homozygote.
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| CN101921849B (en) * | 2010-07-30 | 2012-11-21 | 中国农业大学 | Method for assisting to authenticate milk cows with different milk producing characteristics and special primer pairs used by same |
| CN102154525B (en) * | 2011-04-29 | 2013-09-11 | 贵州省畜牧兽医研究所 | Perlsucht PCR (polymerase chain reaction) quick diagnosis reagent kit |
| CN102747150B (en) * | 2012-06-08 | 2015-01-21 | 西北农林科技大学 | Method for detecting body sizes of Qinchuan cattle through using ANAPC13 gene |
| CN106957910B (en) * | 2017-02-22 | 2020-11-20 | 中国农业大学 | Method for identifying milk production traits of dairy cows based on CDKN1A gene and application thereof |
| CN107254525B (en) * | 2017-06-22 | 2020-08-25 | 陕西师范大学 | Method for evaluating milk quality based on cattle BAF60c gene locus mutation |
| CN112662788B (en) * | 2021-01-28 | 2022-08-19 | 武汉市农业科学院 | SNP marker related to milk production traits of Holstein cows in south China and application thereof |
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Non-Patent Citations (4)
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| 徐秀容.DGAT1和DGAT2基因多态性与牛部分经济性状相关性的研究.西北农林科技大学博士学位论文.2005,全文,尤其是摘要第1段和第2节,第38页引言第3段,第41页表3-2,1.2.5-1.2.8节,第46页第1段到第47页第2段和图3-5,第59页表3-8. * |
| 邹荣婕.中国荷斯坦奶牛DGAT1、LOX-1基因多态性与产奶性状之间的关系研究.西北农林科技大学硕士学位论文.2007,全文,尤其是摘要第1段和第3、5节,第37页2.4.3节到第38页2.4.5节,表2-6. * |
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