CN103911374A - Molecular marker related to butter-fat percentage character of milk cow and application thereof - Google Patents

Abstract

The invention provides an animal molecular marker and in particular relates to cloning of a TRAPPC9 gene segment of a milk cow and an application of the TRAPPC9 gene segment as a molecular marker. A nucleotide sequence of the molecular marker is shown in SEQ ID No.1 in a sequence table, and one T204-G204 base group at No.204th site is mutated, so that the butter-fat percentage character of the milk cow is polymorphic.

Description

The molecule marker relevant to Milk Fat Percentage in Dairy Cows proterties and application thereof

Technical field

The present invention relates to animal molecular marker field, specifically, relate to the application of a kind of TRAPPC9 gene relevant to Milk Fat Percentage in Dairy Cows proterties as molecule marker.

Background technology

Butterfat is a kind of natural high quality fat being present in milk, and than other fat, milk is more easily digested, and its digestibility can reach 98%.In milk-producing, the nutritive value of the content higher milk of butterfat in milk is also higher.At present, milk fat content is to weigh an important indicator of milk quality, and the height of its content is directly connected to the economic benefit that milk cow produces.

Along with the development of molecular genetics, Protocols in Molecular Biology and quantitative genetics, molecular genetic marker and marker assisted selection are widely applied in Animal Breeding, and have obtained huge achievement.Snapshot sequencing technologies is a kind of typing method based on fluorescent mark single-basic extension principle, also claim little order-checking, mainly for the SNP somatotype project of middle isoflux, its cardinal principle is that extension primer and the PCR product template of Sequenase, four kinds of fluorescently-labeled ddNTP, next-door neighbour's polymorphic site are mixed by certain system, base of primer extension stops, detect through sequenator, can learn the base kind Inner that participates in reaction according to the color at peak, thereby judgement sample is in the genotype in this site.The method has that somatotype is accurate, flux is high, be not subject to the advantage such as SNP loci polymorphism characteristic limitations and number of samples restriction.

Translocator particle composites 9 (TRAPPC9, trafficking protein particle complex9) gene is the relevant gene of the recessive mental retardation dysnoesia of huamn autosomal, the albumen translocator particle composites 9 of its coding participates in endoplasmic reticulum to the vesica transfer of golgi body and the differentiation of neurocyte, the albumen of its coding is also called NIBP(NIK and IKK β-binding protein), participate in film bubble transportation, there is the function of enhance TNF-α activation NF-κ B signal path simultaneously.NF-κ B is important nuclear factor, participates in regulation and control lots of genes and expresses, and is bringing into play important biological function in cells survival, propagation, differentiation and apoptotic process.About TRAPPC9 gene pleiomorphism, the impact of milk fat content is not also reported at present.

Summary of the invention

In order to solve problems of the prior art, the object of this invention is to provide a kind of molecule marker relevant to Milk Fat Percentage in Dairy Cows proterties and application thereof.

In order to realize the object of the invention, first the present invention provides a kind of molecule marker relevant to Milk Fat Percentage in Dairy Cows proterties, its nucleotide sequence, as shown in sequence table SEQ ID No.1, has 1 T204-G204 base mutation at the 204th, causes Milk Fat Percentage in Dairy Cows proterties to occur polymorphism.

Further, the site of described base mutation is positioned at the 57bp place of the 7th intron of TRAPPC9 gene.

The present invention also provides the primer pair for the described molecule marker that increases, and its nucleotide sequence is as follows:

Forward primer F:5 '-TCTTCTCTTTTGACCCTACAG-3 ';

Reverse primer R:5 '-GGTTTCAAGTTCTGACTCCAG-3 '.The present invention also provides the application of aforementioned molecule marker in china holstein cows milk fat content marker assisted selection.

Further, described application comprises the steps:

1) get the genomic dna of milk cow to be detected;

2) take genomic dna as template, utilize described primer pair to carry out pcr amplification, obtain the object fragment of 274bp on china holstein cows TRAPPC9 gene;

3) utilize SnaPshot technology for detection PCR product, if 204bp place is T in amplified production sequence, milk cow to be measured belongs to the china holstein cows Dominant variety that milk fat content is high.

Wherein, described step 2) in PCR reaction system be 25 μ L, wherein 100ng/ μ L genomic dna template 1 μ L, the each 1 μ L of 10pmol/ μ L primers F and R, 12.5 μ L Premix TaqTM, 9.5 μ L ddH ₂o.

Wherein, described step 2) in PCR reaction conditions be: 95 ℃ of denaturation 10min; 94 ℃ of sex change 30s, 60 ℃ of renaturation 30s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ are extended 10min; Be cooled to 4 ℃ of maintenances.

Wherein, in described step 3), utilize SnaPshot technology for detection PCR product, concrete steps are: get step 2) obtain PCR product 3ul purify with ExoI and FastAP, mainly to remove the residue primer in reaction product with ExoI, with remaining DNTP in FastAP removal reaction, after purifying, utilize SNP somatotype to extend primer and extend, after extending, upper machine checks order.If as shown in sequencing result figure as left in Fig. 1, be TT type, milk cow to be measured belongs to the china holstein cows advantage individuality of high milk fat content, occurs that i.e. TG or GG type, is common individuality as shown in figure and right figure in Fig. 1.

The present invention is by carrying out gene type to the SNP site of china holstein cows TRAPPC9 gene, and the milk fat content utmost point that utilizes the GLM process of SAS9.1 software that the milk fat content of gene type result and milk cow is carried out to association analysis discovery TT type individuality is significantly higher than GG type individuality (P<0.05).Being found to be of this site carried out molecular breeding provides new mark.

Further, the nucleotides sequence of described SNP somatotype extension primer is classified as:

5’-TTTTTTTTTTTTTTTTAGCAAAACCAGATGGACTTGTG-3’。

The present invention also provides the test kit that detects the high milk fat content Dominant variety of china holstein cows, and described test kit comprises one or more in forward primer F, reverse primer R, SNP somatotype extension primer and Premix TaqTM, ExoI, FastAP, ExoI buffer, Snapshot Mix;

Wherein Premix TaqTM is by dNTPs, archaeal dna polymerase, Mg ²⁺, PCR damping fluid composition.

Beneficial effect of the present invention is:

SNP molecule marker provided by the present invention site is found first, and milk fat content is had to utmost point remarkably influenced in china holstein cows cows, provides new material and scientific basis for carrying out china holstein cows milk fat content marker assisted selection.

SNP molecule marker provided by the invention is not subject to the restriction such as age, sex, can be used for early stage seed selection, accelerates breeding process; Detection method accurately and reliably, convenient and easy.

Accompanying drawing explanation

Fig. 1 is three kinds of genotype SnaPshot sequencing and typing figure of china holstein cows in the present invention; Wherein, left figure is TT type, and middle figure is TG type, and right figure is GG type (genotype of peak figure is by color differentiating conventionally, and its Green is TT type, and black is GG type).

Fig. 2 is three kinds of genotype sequencer maps; Be respectively TT type, TG type and GG type.

Embodiment

Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If without specified otherwise, the technique means in following embodiment is conventional means in this area, and material therefor is all buied from conventional reagent company.

Acquisition and the evaluation of the SNP mark that embodiment 1 is relevant to china holstein cows milk fat content proterties

The extraction of 1.1 china holstein cows poba gene group DNA to be measured

Tail vein gathers china holstein cows blood to be measured, and normal temperature is placed and within 3-4 hour, treated blood coagulation, and now blood is divided into serum and sludged blood two portions, serum is clear yellow, sludged blood is garnet, only has white corpuscle to contain DNA in bovine blood, is present in sludged blood.

Utilize day root blood/cell/tissue genome DNA sample to extract test kit and from sludged blood, extract genomic dna, concrete steps are as follows:

Put into sterilized 2mL round bottom centrifuge tube with the sludged blood of eye scissors clip 0.2-0.3mL, add the cell pyrolysis liquid CL of 500 μ L;

Utilize hand-held Syrup-homogenizing instrument by abundant clot homogenate, vibration 15s, the centrifugal 1min of 12000rpm, discards upper strata garnet supernatant liquor;

Again add 700 μ L cell pyrolysis liquid CL, fully vibration makes the lower sediment suspension of scattering, the centrifugal 1min of 12000rpm, abandoning supernatant;

Add 200 μ L damping fluid GS, fully vortex to precipitation fully suspends;

Add 20 μ L Proteinase Ks and 250 μ L damping fluid GB, fully mix;

Centrifuge tube is sealed to put in 56 ℃ of hybrid heaters with sealed membrane digest 3-4 hour, for guaranteeing abundant digestion, need to put upside down mix for several times in digestive process, final solution becomes limpid transparent, briefly centrifugal;

Add 200 μ L to ice dehydrated alcohol, turn upside down and mix 15s, now may occur flocks;

Previous step gained solution is proceeded in adsorption column CB3, and adsorption column is put into collection tube, and the centrifugal 30s of 12000rpm, discards the waste liquid in collection tube, and adsorption column is put into collection tube;

In adsorption column CB3, add 500 μ L Deproteinization damping fluid GD, leave standstill 2min, the centrifugal 30s of 12000rpm, discards the waste liquid in collection tube, and adsorption column is put into collection tube;

In adsorption column CB3, add 700 μ L rinsing liquid PW, the centrifugal 30s of 12000rpm, discards the waste liquid in collection tube, and adsorption column is put into collection tube;

In adsorption column CB3, add 500 μ L rinsing liquid PW, the centrifugal 30s of 12000rpm, discards the waste liquid in collection tube;

Adsorption column is put into collection tube, and the centrifugal 2min of 12000rpm, discards waste liquid, adsorption column is placed in to room temperature and places several minutes, the ethanol of remnants on the adsorption column that thoroughly volatilizees;

Adsorption column is proceeded to a new centrifuge tube, add the TE damping fluid of 100 μ L56 ℃ preheatings, room temperature is placed 5min, the centrifugal 2min of 12000rpm, and genomic dna is present in centrifuge tube.

The amplification of 1.2 object fragments

According to the ox TRAPPC9 gene order providing on NCBI, utilize oligo6 design primer, comprise forward primer F:5 '-TCTTCTCTTTTGACCCTACAG-3 ' and reverse primer R:5 '-GGTTTCAAGTTCTGACTCCAG-3 ', carry out pcr amplification take genomic dna in 1.1 as template, object fragment is sequence as shown in SEQ ID No.1, SNP site is positioned at 204bp place, and base is T or G herein.

PCR reaction system is 25 μ L, wherein 100ng/ μ L genomic dna template 1 μ L, the each 1 μ L of 10pmol/ μ L primers F and R, 12.5 μ L Premix Taq ^tM, 9.5 μ L ddH2O.

PCR reaction conditions is: 95 ℃ of 10min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 30sec, 35 circulations; 72 ℃ of 10min; 4 ℃ of maintenances.

1.3 gene type

Utilize SnaPshot technology to carry out gene type, concrete steps are as follows:

PCR product purification: getting the PCR product ExoI and the FastAP that obtain in 1.2 and purify, is mainly to remove the residue primer in reaction product with ExoI, with remaining DNTP in FastAP removal reaction.

Reaction system is PCR product 3ul, ExoI0.2ul, and FastAP0.8ul, ExoI buffer0.7ul, moisturizing is to 7ul.

Reaction conditions is 37 ℃ of 15min, 80 ℃ of 15min.

Extension: system is purified pcr product 2ul, Snapshot Mix reagent 1ul, extends primer mixing 2ul, and moisturizing is to 6ul.

Condition is 96 ℃ of 1min; 96 ℃ of 10sec, 52 ℃ of 5sec, 60 ℃ of 30sec, 30 circulations;

Get 1ul extension products, add 10ul loading loading, 95 ℃ of sex change 3min, ice-water bath immediately, upper sequenator.

According to Snapshot sequencing result interpretation genotype, as shown in Figure 1, be reverse primer owing to extending primer, therefore the left figure of sequencing result is TT type individuality, middle figure is GG type individuality, right figure is TG type individuality.

For the accuracy of checking somatotype, after somatotype, get sample segment and carry out pcr amplification, amplified production is delivered to order-checking company and check order, sequencing result as shown in Figure 2: be respectively TT type, TG type and GG type, sequencing result is consistent with somatotype result, somatotype result is described accurately and reliably.

The association analysis of embodiment 2SNP site and china holstein cows milk fat content

In the present embodiment, 332 china holstein cowses of the different dairy cow farms in Northern Part of China are carried out to SNP somatotype, TRAPPC9 gene 204bp punishment type result of sequence as shown in SEQ ID NO.1 is as shown in table 1.

Use the GLM process of SAS9.1 software to carry out the association analysis between genotype and milk fat content, model is as follows:

y _ijl=μ+g _i+h _j+p _l+e _ijl，

Wherein, y _ijlfor the phenotypic number of milk production trait; μ is population mean; g _ifor genotype effect; h _jfor season in field year effect; p _lfor parity effect; e _ijlfor random residual.

Gene frequency and the gene frequency of table 1SNP genotype in institute's Research Group

As shown in Table 1, the homozygous number of individuals of CC is significantly higher than TT type, C allelotrope is protogene, the side's of card comptibility test χ 2=2.06< χ 20.05 (1)=3.84, expected value and observed value difference are not remarkable, illustrate this SNP site in colony in Hardy-Weinberg equilibrium state.

Table 2SNP genotype and the association analysis of china holstein cows milk fat content

Note: different lowercase alphabets show significant difference, i.e. P<0.05.

As shown in Table 2, genotype reaches utmost point conspicuous level to the impact of milk fat content, and TT type milk fat content is significantly higher than GG type (P<0.05).

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (10)

1. a molecule marker relevant to Milk Fat Percentage in Dairy Cows proterties, is characterized in that, its nucleotide sequence, as shown in sequence table SEQ ID No.1, has 1 T204-G204 base mutation at the 204th, causes Milk Fat Percentage in Dairy Cows proterties to occur polymorphism.

2. molecule marker according to claim 1, is characterized in that, the site of described base mutation is positioned at the 57bp place of the 7th intron of TRAPPC9 gene.

3. for the primer pair of molecule marker described in the claim 1 that increases, it is characterized in that, its nucleotide sequence is as follows:

Forward primer F:5 '-TCTTCTCTTTTGACCCTACAG-3 ';

Reverse primer R:5 '-GGTTTCAAGTTCTGACTCCAG-3 '.

4. the application of molecule marker claimed in claim 1 in china holstein cows milk fat content marker assisted selection.

5. application according to claim 4, is characterized in that, comprises the steps:

1) get the genomic dna of milk cow to be detected;

2) take genomic dna as template, utilize primer pair described in claim 3 to carry out pcr amplification, obtain the object fragment of 274bp on china holstein cows TRAPPC9 gene;

3) utilize SnaPshot technology for detection PCR product, if 204bp place is T in amplified production sequence, milk cow to be measured belongs to the china holstein cows Dominant variety that milk fat content is high.

6. application according to claim 5, is characterized in that, described step 2) in PCR reaction system be 25 μ L, wherein 100ng/ μ L genomic dna template 1 μ L, the each 1 μ L of 10pmol/ μ L primers F and R, 12.5 μ L Premix TaqTM, 9.5 μ L ddH2O.

7. application according to claim 5, is characterized in that, described step 2) in PCR reaction conditions be: 95 ℃ of denaturation 10min; 94 ℃ of sex change 30s, 60 ℃ of renaturation 30s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ are extended 10min; Be cooled to 4 ℃ of maintenances.

8. application according to claim 5, it is characterized in that, in described step 3), utilize SnaPshot technology for detection PCR product, concrete steps are: get step 2) obtain PCR product 3ul purify with ExoI and FastAP, after purifying, utilize SNP somatotype to extend primer and extend, after extending, upper machine checks order.

9. application according to claim 8, is characterized in that, described SNP somatotype extends the nucleotides sequence of primer and classifies as: 5 '-TTTTTTTTTTTTTTTTAGCAAAACCAGATGGACTTGTG-3 '.

10. one kind is detected the test kit of the high milk fat content of china holstein cows, it is characterized in that, described test kit comprises: one or more in forward primer F, reverse primer R, SNP somatotype extension primer and Premix TaqTM, ExoI, FastAP, ExoI buffer, Snapshot Mix;

Wherein Premix TaqTM is by dNTPs, archaeal dna polymerase, Mg ²⁺, PCR damping fluid composition.