CN105039334B - The IGF2 gene molecule markers of Jin Nanniu for differentiating that growth performance is excellent a kind of and its application - Google Patents
The IGF2 gene molecule markers of Jin Nanniu for differentiating that growth performance is excellent a kind of and its application Download PDFInfo
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Abstract
The invention discloses a kind of IGF2 gene molecule markers SNP and its application for being used to differentiate the excellent Jin Nanniu of growth performance, by carrying out Genotyping to the SNP site of Shanxi south ox IGF2 genes, the growth traits of genotypic results and Jin Nanniu is associated analysis using the GLM processes of SAS9.2 softwares and finds that body weight, Body steep length and the bust pole of CC types individual are significantly higher than TT types individual (P<0.01), the body weight of CC types individual and bust are significantly higher than CT types individual (P<0.05).The progress molecular breeding that is found to be in the site provides new mark；The method of the high Jin Nanniu Dominant varieties of identification growth performance provided by the invention is accurately and reliably, convenient and easy, and SNP marker provided by the invention is not limited by age, sex etc., available for early stage seed selection, accelerates breeding process.
Description
Technical field
The present invention relates to biology field, more particularly to a kind of IGF2 bases for the Jin Nanniu for differentiating that growth performance is excellent
Because molecular marker SNP, SNP primer, SNP kit and discrimination method.
Background technology
Growth traits is the main economic characters of beef cattle, and it mainly includes body height, body weight, Body steep length, bust, abdominal circumference, waist
The indexs such as pin is wide, point of the buttocks is wide and hip cross is high.Growth traits is the quantitative character controlled by polygenes seat simultaneously, and phenotype is selected
Select that genetic progress is slow, the determination for influenceing the key-gene of growth traits is cultivated China's Chinese native cattle breeds to meat direction and has weight
Want meaning.
SNP (SNP) is the human genome research center scholar by Massachusetts Institute Technology in 1996
A kind of genetic marker that Lander is proposed, is primarily referred to as in genomic level as the DNA caused by the variation of single nucleotide acid
Sequence polymorphism.The polymorphism that SNP is shown relates only to the variation of single base, the form of expression have conversion, transversion, insertion and
Missing etc..Sequencing, single-strand conformation polymorphism PCR, RFLP PCR and
The technologies such as flight time mass spectrum can realize SNP detection.SNP as new genetic marker be widely used to the assignment of genes gene mapping,
Clone, genetic breeding and multifarious research.
IMA-IGF2BP3-001 (insulin-like growth factor 2) is also known as growth regulators, be by
The protein of 67 amino acid residue compositions, there is Cell differentiation inducing activity, promote mitosis, embry ogenesis, regulation and control body life
The effect of long development.Forefathers' research has shown that IGF2 plays an important role during the skeletal muscle regeneration of ox.
The content of the invention
It is an object of the invention to provide a kind of IGF2 gene molecule marks for being used to differentiate the excellent Jin Nanniu of growth performance
Remember SNP and its application.
The present invention is achieved by the following technical solutions：
The invention provides a kind of IGF2 gene molecule marker SNP for being used to differentiate the excellent Jin Nanniu of growth performance, institute
The nucleotide sequence of molecular labeling is stated as shown in SEQ ID NO.1, the base at the 71st site is C or T, and base is C's herein
Jin Nanniu growth performance is significantly higher than the Jin Nanniu that base herein is T.
Present invention also offers the primer pair for detecting the molecular marker SNP, nucleotide sequence are as follows：
Forward primer F：5 '-TGGCAGCGACTGCTACTATTG-3 ',
Reverse primer R：5 '-GAGCACTCCAAAGGCAGCTTT-3 ',
SNP parting extension primers：5’-AGTCGAGAGCCTGGGGCACCAG-3’.
The primer pair specific amplification goes out SEQ ID NO:Nucleotide sequence shown in 1.
Present invention also offers a kind of kit for being used to differentiate the excellent Jin Nanniu of growth performance, the kit includes
In primers F, primer R, SNP serotype specific primer, Premix TaqTM, ExoI, FastAP, ExoI buffer, Snapshot Mix
One or more, wherein Premix TaqTM are made up of dNTPs, archaeal dna polymerase, Mg2+, PCR buffer solution.
Present invention also offers a kind of method that the high Jin Nanniu of growth performance is identified using SNP marker, including following step
Suddenly：
(1) Jin Nanniu to be detected genomic DNA is extracted；
(2) genomic DNA obtained using the first step is entered performing PCR using the primers F described in claim 2 and R and expanded as template
Increase, obtain the purpose fragment of 203bp on the ox IGF2 genes of Shanxi south；PCR reaction systems are 25 μ L, wherein 100ng/ μ L genomes
DNA profiling 1 μ L, 10pmol/ μ L primers Fs and R each 1 μ L, 12.5 μ L Premix TaqTM, 9.5 μ L ddH2O, PCR reaction conditions
For：95℃10min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations；72℃10min；4℃forever；
(3) SnaPshot technology for detection PCR primers are utilized, PCR primer is purified first：Take PCR primer ExoI and
FastAP is purified, and mainly removes the remaining primer in reaction product with ExoI, and remaining DNTP in reaction is removed with FastAP,
Reaction system is PCR primer 3ul, ExoI 0.2ul, FastAP 0.8ul, ExoI buffer 0.7ul, moisturizing to 7ul, is reacted
Condition is 37 DEG C of 15min, 80 DEG C of 15min；Then extension is carried out：System is purified pcr product 2ul, Snapshot
Mix reagent 1ul, extension primer mixing 2ul, moisturizing to 6ul, condition is 96 DEG C of 1min；96 DEG C of 10sec, 52 DEG C of 5sec, 60 DEG C
30sec, 30 circulations；1ul extension products are taken, add 10ul loadings loading, 95 DEG C of denaturation 3min, immediately ice-water bath, upper sequencing
Instrument, if in amplified production sequence at 71bp being C, Shanxi south to be measured Bos is in the high advantage individual of growth performance.
Present invention also offers following any applications：
(1) application of the above-mentioned molecular marker SNP in the high Jin Nanniu Dominant varieties of identification growth performance；
(2) application of the above-mentioned primer pair in the high Jin Nanniu Dominant varieties of identification growth performance；
(3) application of the mentioned reagent box in the high Jin Nanniu Dominant varieties of identification growth performance；
(4) application of the above method in the high Jin Nanniu Dominant varieties of identification growth performance.
The principle of the invention and beneficial effect：
The present invention utilizes the GLM mistakes of SAS9.2 softwares by carrying out Genotyping to the SNP site of Shanxi south ox IGF2 genes
The growth traits of genotypic results and Jin Nanniu is associated analysis and finds individual body weight, Body steep length and the bust of CC types by journey
Pole is significantly higher than TT types individual (P<0.01), the body weight of CC types individual and bust are significantly higher than CT types individual (P<0.05).The position
The progress molecular breeding that is found to be of point provides new mark.
The method of the high Jin Nanniu Dominant varieties of identification growth performance provided by the invention is accurately and reliably, convenient and easy, and
And SNP marker provided by the invention is not limited by age, sex etc., available for early stage seed selection, accelerate breeding process.
Brief description of the drawings
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.
Fig. 1 is three kinds of genotype SnaPshot sequencing and typing figures of Shanxi south ox in the present invention, wherein (a) is CT types, (b) is CC
Type, (c) are TT types；
Fig. 2 is three kinds of genotype sequencer maps of Shanxi south ox in the present invention.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
The acquisition and identification of the IGF2 gene molecule marker related to Jin Nanniu growth traits of embodiment 1
The extraction of 1.1 Shanxi south bovine blood genomic DNAs to be measured
Tail vein gathers Shanxi south to be measured bovine blood, and room temperature 3-4 hours treat blood clotting, now blood be divided into serum and
Sludged blood two parts, serum are in clear yellow, and sludged blood is kermesinus, only contains DNA in leucocyte in bovine blood, is present in
In sludged blood.
Genomic DNA is extracted from sludged blood using Tiangeng blood/cell/tissue genome DNA sample extracts kit,
Comprise the following steps that：
It is put into eye scissors clip 0.2-0.3mL sludged blood in sterilized 2mL round bottom centrifuge tubes, adds 500 μ L's
Cell pyrolysis liquid CL；
Clot is fully homogenized using hand-held Syrup-homogenizing instrument, 15s is vibrated, 12000rpm centrifugation 1min, discards upper strata kermesinus
Supernatant；
700 μ L cell pyrolysis liquid CL are added again, and fully vibration makes lower sediment scatter suspensions, and 12000rpm is centrifuged
1min, abandoning supernatant；
200 μ L buffer solution GS are added, is fully vortexed and is fully suspended to precipitation；
20 μ L Proteinase Ks and 250 μ L buffer solution GB are added, are fully mixed；
Centrifuge tube is sealed with sealed membrane and is put into digestion 3-4 hours in 56 DEG C of hybrid heaters, to ensure fully to digest, digestion
During need it is reverse mix for several times, final solution becomes limpid transparent, brief centrifugation；
Add 200 μ L and ice absolute ethyl alcohol, turn upside down and mix 15s, now it is possible that flocculent deposit；
Previous step resulting solution is transferred in adsorption column CB3, adsorption column is put into collecting pipe, 12000rpm centrifugation 30s, is abandoned
The waste liquid gone in collecting pipe, adsorption column is put into collecting pipe；
500 μ L deproteinized buffer solution GD are added into adsorption column CB3,2min is stood, 12000rpm centrifugation 30s, discards receipts
Waste liquid in collector, adsorption column is put into collecting pipe；
700 μ L rinsing liquids PW, 12000rpm centrifugation 30s are added into adsorption column CB3, discard the waste liquid in collecting pipe, will
Adsorption column is put into collecting pipe；
500 μ L rinsing liquids PW, 12000rpm centrifugation 30s are added into adsorption column CB3, discard the waste liquid in collecting pipe；
Adsorption column is put into collecting pipe, 12000rpm centrifugation 2min discard waste liquid, and adsorption column is placed in into room temperature places number
Minute, remaining ethanol on the adsorption column that thoroughly volatilizees；
Adsorption column is transferred into a new centrifuge tube, adds the TE buffer solutions of 100 μ L, 56 DEG C of preheatings, room temperature is placed
5min, 12000rpm centrifuge 2min, and genomic DNA is then present in centrifuge tube.
The amplification of 1.2 purpose fragments
According to the ox IGF2 gene orders provided on NCBI, primer, including forward primer F are designed using oligo 6：5’-
TGGCAGCGACTGCTACTATTG-3 ' and reverse primer R：5 '-GAGCACTCCAAAGGCAGCTTT-3 ', with genome in 1.1
DNA is that template enters performing PCR amplification, and purpose fragment sequence as shown in SEQ ID NO.1, SNP site is located at 71bp, herein base
For C or T.
PCR reaction systems are 25 μ L, wherein 100ng/ μ L genomic DNA templates 1 μ L, 10pmol/ μ L primers Fs and each 1 μ of R
L, 12.5 μ L Premix TaqTM, 9.5 μ L ddH2O.
PCR reaction conditions are：95℃10min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations；72℃
10min；4℃forever.
1.3 Genotyping
Genotyping is carried out using SnaPshot technologies, is comprised the following steps that：
PCR primer purifies：Take the PCR primer obtained in 1.2 to be purified with ExoI and FastAP, mainly removed with ExoI anti-
The remaining primer in product is answered, remaining DNTP in reaction is removed with FastAP.
Reaction system is PCR primer 3ul, ExoI 0.2ul, FastAP 0.8ul, ExoI buffer 0.7ul, and moisturizing is extremely
7ul。
Reaction condition is 37 DEG C of 15min, 80 DEG C of 15min.
Extension：System is that purified pcr product 2ul, Snapshot Mix reagent 1ul, extension primer have mixed 2ul,
Moisturizing is to 6ul.
Condition is 96 DEG C of 1min；96 DEG C of 10sec, 52 DEG C of 5sec, 60 DEG C of 30sec, 30 circulations；
1ul extension products are taken, add 10ul loadings loading, 95 DEG C of denaturation 3min, immediately ice-water bath, upper sequenator.
According to sequencing result interpretation genotype, as shown in figure 1, Fig. 1 (a) is CT types, Fig. 1 (b) is CC types, and Fig. 1 (c) is TT
Type.
The association analysis of the SNP site of embodiment 2 and Jin Nanniu growth traits
SNP partings, IGF2 genes are carried out to 356 Jin Nanniu of Shanxi Province, China difference Shanxi south ox plant in the present embodiment
Genotyping result is as shown in table 1 at the 71bp of sequence as shown in SEQ ID NO.1.
The association analysis between genotype and growth traits is carried out with the GLM processes of SAS9.2 softwares, model is as follows：
Yijl=μ+gi+hj+al+eijl,
Wherein, yijl is the phenotypic number of growth traits；μ is population mean；Gi is genotype effects；Hj is field-effect；al
For age effect；Eijl is random residual.
Gene frequency and gene frequency of the SNP genotype of table 1 in studied colony
As shown in Table 1, the homozygous number of individuals of CC is significantly higher than TT types, and C allele is protogene, and the side's of card adaptability is examined
Test χ 2=1.08<χ 20.05 (1)=3.84, desired value is not notable with observation difference, illustrates that the SNP site is in colony
Hardy-Weinberg poised states.
The SNP genotype of table 2 and Jin Nanniu growth traits association analysis
Note：Different capitalizations represent that difference is extremely notable, and different lowercase letter indication differences are notable
As shown in Table 2, influence of the genotype to growth traits reaches the level of signifiance, the body weight of CC types individual, Body steep length and
Bust pole is significantly higher than TT types individual (P<0.01), the body weight of CC types individual and bust are significantly higher than CT types individual (P<0.05).
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Claims (6)
1. applications of the Jin Nanniu IGF2 gene molecule markers SNP in differentiating that growth performance is excellent, the growth performance is body
Weight, Body steep length and bust, sequence is CACCAGC on the IGF2 genes that the molecular marker SNP is Jin NanniuCACGTC is corresponding
Site, the base at underscore is C or T, and it is T's that the growth performance for the Jin Nanniu that base is C, which is significantly higher than base herein, herein
Jin Nanniu.
2. test right requires the primer pair of 1 molecular marker SNP, it is characterised in that：Nucleotide sequence is as follows：
Forward primer F：5 '-TGGCAGCGACTGCTACTATTG-3 ',
Reverse primer R：5 '-GAGCACTCCAAAGGCAGCTTT-3 ',
SNP parting extension primers：5’-AGTCGAGAGCCTGGGGCACCAG-3’.
A kind of 3. kit for being used to differentiate the excellent Jin Nanniu of growth performance, it is characterised in that：Including described in claim 2
Primer pair.
A kind of 4. kit for being used to detect sow embryo survival according to claim 3, it is characterised in that：Also include
One or more of mixtures, described in PremixTaqTM, ExoI, FastAP, ExoIbuffer and SnapshotMix
PremixTaqTM is made up of dNTPs, archaeal dna polymerase, Mg2+, PCR buffer solution.
A kind of 5. method that the high Jin Nanniu of growth performance is identified using SNP marker, it is characterised in that：Comprise the following steps：
(1) Jin Nanniu to be detected genomic DNA is extracted；
(2) genomic DNA obtained using the first step enters performing PCR amplification as template using the primers F described in claim 2 and R,
Obtain the purpose fragment of 203bp on the ox IGF2 genes of Shanxi south；PCR reaction systems are 25 μ L, wherein 100ng/ μ L genomic DNA moulds
Plate 1 μ L, 10pmol/ μ L primers Fs and R each 1 μ L, 12.5 μ LPremixTaqTM, 9.5 μ LddH2O, PCR reaction conditions are：95℃
10min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 30sec, 35 circulations；72℃10min；4℃forever；
(3) SnaPshot technology for detection PCR primers are utilized, PCR primer is purified first：Take PCR primer ExoI and FastAP
Purifying, the remaining primer in reaction product mainly is removed with ExoI, remaining DNTP in reaction, reactant are removed with FastAP
It is the moisturizing to 7ul for PCR primer 3ul, ExoI0.2ul, FastAP0.8ul, ExoIbuffer0.7ul, reaction condition is 37 DEG C
15min, 80 DEG C of 15min；Then extension is carried out：System is purified pcr product 2ul, SnapshotMix reagent 1ul, is prolonged
Extend thing mixing 2ul, moisturizing to 6ul, and condition is 96 DEG C of 1min；96 DEG C of 10sec, 52 DEG C of 5sec, 60 DEG C of 30sec, 30 circulations；
1ul extension products are taken, add 10ul loadings loading, 95 DEG C of denaturation 3min, immediately ice-water bath, upper sequenator, if amplified production
It is C at 71bp in sequence, then Shanxi south to be measured Bos is in the high advantage individual of growth performance.
6. following any applications：
(1) application of the molecular marker SNP described in claim 1 in the high Jin Nanniu Dominant varieties of identification growth performance；
(2) application of the primer pair described in claim 2 in the high Jin Nanniu Dominant varieties of identification growth performance；
(3) claim 3 or kit described in claim 4 in the high Jin Nanniu Dominant varieties of identification growth performance should
With；
(4) application of the method described in claim 5 in the high Jin Nanniu Dominant varieties of identification growth performance.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005007881A2 (en) * | 2003-07-22 | 2005-01-27 | Sheila Marie Schmutz | Improving production characteristics of cattle |
| CN101137747A (en) * | 2005-03-01 | 2008-03-05 | 国家细胞科学中心 | A composition for creating an artificial bone-marrow like environment and use thereof |
| CN103290123A (en) * | 2013-05-28 | 2013-09-11 | 西北农林科技大学 | Detecting method and kit of cattle IGF2 (Insulin-like Growth Factor 2) gene mononucleotide polymorphism |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005007881A2 (en) * | 2003-07-22 | 2005-01-27 | Sheila Marie Schmutz | Improving production characteristics of cattle |
| CN101137747A (en) * | 2005-03-01 | 2008-03-05 | 国家细胞科学中心 | A composition for creating an artificial bone-marrow like environment and use thereof |
| CN103290123A (en) * | 2013-05-28 | 2013-09-11 | 西北农林科技大学 | Detecting method and kit of cattle IGF2 (Insulin-like Growth Factor 2) gene mononucleotide polymorphism |
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