CN106521001B - The detection method and its application of Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism - Google Patents

The detection method and its application of Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism Download PDF

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CN106521001B
CN106521001B CN201611193760.4A CN201611193760A CN106521001B CN 106521001 B CN106521001 B CN 106521001B CN 201611193760 A CN201611193760 A CN 201611193760A CN 106521001 B CN106521001 B CN 106521001B
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陈宏�
董冬
魏雪锋
白跃宇
黄永震
蓝贤勇
雷初朝
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Abstract

The invention discloses the detection methods and its application of a kind of Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism, using Qinchuan Cattle complete genome DNA to be measured as template, using primer pair P2 as primer, and PCR amplification Qinchuan Cattle microRNA-320a-1 genetic fragment；Pcr amplification product is digested with restriction enzyme PvuII and then agarose gel electrophoresis is carried out to the amplified fragments after digestion；The single nucleotide polymorphism of Qinchuan Cattle microRNA-320a-1 gene rs518926539:C > T is identified according to agarose gel electrophoresis results.Detection method provided by the invention is that the marker assisted selection for carrying out the meat growth traits of Qinchuan Cattle using the relationship of microRNA-320a-1 gene SNP and growth traits is laid a good foundation, and is conducive to quickly establish the excellent Qinchuan Cattle population of genetic resources.

Description

The detection method of Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism and It is applied
Technical field
The invention belongs to field of biotechnology, are related to the detection of gene mononucleotide polymorphism (SNP), in particular to a kind of The method for detecting Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism.
Background technique
Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) is biological genome DNA sequence dna In due to caused by the replacement of single nucleotide acid (A/T/C/G) make a variation.Studies have shown that monokaryon glycosides occurs for genomic dna sequence Acid replacement, the amino acid of the change or coding protein that will cause DNA sequence dna changes, to make the biology of different genotype Body surface reveals different characters.
MicroRNAs is the non-coding RNA that one kind that biological genome DNA transcription generates is about 22nt, studies have shown that The mode that microRNAs plays a role is the mRNA base pair complementarity with downstream target gene, make the mRNA degrade or translate by To inhibition, to influence the character of organism.Currently, focus mostly on the research of microRNA-320a gene in the mankind, it is main to wrap It includes the transfer of the gene and cancer cell and infects equal correlative studys, have no that heredity of the microRNA-320a gene on ox becomes Different research.Ox microRNA-320a gene includes 2 hypotypes, is microRNA-320a-1 and microRNA-320a- respectively 2, the approach that the two hypotypes play a role is identical.
The research in the field of ox microRNA-320a-1 gene genetic variation at present is deficient, the function in the gene polymorphic site It can study and its hereditary variation is associated with Growth Traits (such as: weight, body height, body length, chest breadth, chest depth and bust character) Research is still blank.
Summary of the invention
The purpose of the present invention is to provide a kind of detections of Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism Method and its application accelerate the foundation with Quality and economy character Qinchuan Cattle population.
In order to achieve the above objectives, the invention adopts the following technical scheme:
It is to draw with primer pair P2 using the Qinchuan Cattle complete genome DNA to be measured comprising microRNA-320a-1 gene as template Object, PCR amplification Qinchuan Cattle microRNA-320a-1 genetic fragment；With restriction enzyme PvuII digestion pcr amplification product it Afterwards, then to the amplified fragments after digestion agarose gel electrophoresis is carried out；Qinchuan Cattle is identified according to agarose gel electrophoresis results The single nucleotide polymorphism in the site microRNA-320a-1 gene (rs518926539:C > T)；
The primer pair P2 are as follows:
Upstream primer: AACTCCCACGTTGCGTCCAGC 21nt；
Downstream primer: GCTCCCCTCCGCCTTCTCTT 20nt.
The pcr amplification reaction program are as follows:
94 DEG C of initial denaturation 2min；94 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 15s, 30~36 recycle；72℃ Extend 5min.
The agarose gel electrophoresis is the agarose gel electrophoresis that mass concentration is 3%.
Described agarose gel electrophoresis results identification Qinchuan Cattle microRNA-320a-1 gene (rs518926539:C > T) the single nucleotide polymorphism in site are as follows: CC genotypic expression is 188bp band；CT genotypic expression be 188bp, 168bp and 20bp band；TT genotypic expression is 168bp and 20bp band；Wherein, TT genotype is mutant homozygous genotype.
The invention has the following beneficial technical effects:
Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism detection method provided by the invention, for The site (rs518926539:C > T) is sported the mutation of T by C, introduces base mispairing by 3 ' ends of the upstream primer in design, Construct the cleavage site identified by restriction enzyme PvuII.When there is no mutation, PCR amplification microRNA- Restriction enzyme Pvu II recognition site can not be formed after 320a-1 gene near mismatch site；When sporting T by C, Pvu II recognition site can be formed after PCR amplification microRNA-320a-1 gene.By agarose gel electrophoresis can it is accurate, Quickly detect microRNA-320a-1 gene mononucleotide polymorphism: CC genotypic expression is 188bp band；CT genotype Show as 188bp, 168bp and 20bp band；TT genotypic expression is 168bp and 20bp band.It can be to Qinchuan Cattle group The variation of the allele and genotype frequency in the site microRNA-320a-1 gene (rs518926539:C > T) is monitored.
Simultaneously as polymorphism and the Qin Chuan in the site Qinchuan Cattle microRNA-320a-1 gene (rs518926539:C > T) The growth traits such as ox weight, body length, hip cross height are interrelated, and detection method provided by the invention is to utilize microRNA- The marker assisted selection (MAS) that the relationship of 320a-1 gene SNP and growth traits carries out the meat growth traits of Qinchuan Cattle is established Basis can quickly establish the excellent Qinchuan Cattle population of genetic resources.
Detailed description of the invention
Fig. 1 is serotype specific primer (primer pair P2) mentality of designing schematic diagram；
Fig. 2 is to identify Qinchuan Cattle microRNA-320a-1 gene after mispairing introduces the PCR product digestion of PvuII restriction enzyme site The electrophoresis detection result of single nucleotide polymorphism at the site (rs518926539:C > T)；Wherein DL500 is Marker column.
Fig. 3 is the different genotype of SNP at the site Qinchuan Cattle microRNA-320a-1 gene (rs518926539:C > T) (CC, CT, TT) sequencer map.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples, it is described be explanation of the invention and It is not to limit.
The present invention passes through the mononucleotide to the site Qinchuan Cattle microRNA-320a-1 gene (rs518926539:C > T) Polymorphism is detected, and for use in the marker assisted selection of the meat growth traits of Chinese Qinchuan Cattle, quickly establishes genetic resources Excellent Qinchuan Cattle population.
1, the design of PCR primer of the Qinchuan Cattle microRNA-320a-1 gene containing (rs518926539:C > T) mutation
Hereford cow (GCF_000003055.6) sequence announced with NCBI is reference, is designed using Primer 5.0 The PCR primer comprising Qinchuan Cattle microRNA-320a-1 gene and its each 200bp of upstream and downstream can be expanded to P1, primer sequence It arranges as follows:
Upstream primer F1 (SEQ.ID.NO.1): CCGAGCCCAGCCTAGAGC 18nt；
Downstream primer R1 (SEQ.ID.NO.2): CGCACCCCTTCGCACCCA 18nt；
With above-mentioned primer pair Qinchuan Cattle genome amplification, the 488bp of Qinchuan Cattle microRNA-320a-1 gene can be expanded Segment, wherein including the Sequence of mutating alkali yl.
Due to that, without natural restriction enzyme site, cannot be detected by direct enzyme cutting at this SNP site, and if engineer CAGA (sequence as shown in Fig. 1 underscore) mispairing is CAGC by PCR primer mispairing, when there is no mutation, PCR amplification Restriction enzyme PvuII recognition site can not be formed after microRNA-320a-1 gene near mismatch siteAnd when sporting T by C, PvuII recognition site can be formed after PCR amplification microRNA-320a-1 geneSite SNP polymorphism can thus be detected.Designed PCR amplification primer pair P2 are as follows:
Upstream primer F2 (SEQ.ID.NO.3): TTCTCCCACGTTGCGTCCAGC 21nt；
Downstream primer R2 (SEQ.ID.NO.4): GCTCCCCTCCGCCTTCTCTT 20nt.
Wherein, the A of upstream primer 21bp is by mispairing at C；The genetic fragment of primer pair P2 amplification is expanded in primer pair P1 Genetic fragment inside, size is 188bp, and the electrophoresis detection after digestion parting is as shown in Figure 2.
2, with the microRNA-320a-1 genetic fragment of primer pair P2 PCR amplification Qinchuan Cattle to be measured
(1) Qinchuan Cattle sample collection and extracting genome DNA
1) acquisition of Qinchuan Cattle sample
The present invention is specifically using the population of the excellent Qin Chuan cattle breeds of China as test object, and specific collecting sample is shown in Table 1: Shan Western Qinchuan Cattle (N=227), acquisition time in June, 2014；
The acquisition of 1. Qinchuan Cattle sample of table
2) separation, extraction of blood sample genomic DNA
A) blood sample (predominantly haemocyte) thaw at RT is freezed, 500 μ L to 1.5mL Eppendorf centrifuge tubes is shifted, adds Enter isometric PBS liquid, mix well, 12000r/min is centrifuged 10min (4 DEG C), discards supernatant liquid, repeats the above steps to supernatant Liquid is transparent, precipitating is in faint yellow；
B) 500 μ L of DNA extraction buffer is added in centrifuge tube, shakes, is detached from haemocyte precipitating and is centrifuged tube wall, 37 DEG C water-bath 1h；
C) 10 μ L Proteinase Ks (20mg/mL) are added and mix, 55 DEG C overnight to clarification.
D) reaction solution is cooled to room temperature, adds 500 μ L of Tris- saturated phenol, it is mild to shake centrifuge tube 20min, make it sufficiently It mixes；4 DEG C, 12000r/min is centrifuged 10min, and supernatant is transferred in another 1.5mL centrifuge tube, is repeated once；
E) chlorination imitates 500 μ L, mixes well 20min, and 4 DEG C, 12000r/min is centrifuged 10min, supernatant is transferred to another In 1.5mL centrifuge tube；
F) chlorination is imitative, 500 μ L of isoamyl alcohol mixed liquor (24:1), mixes well 20min, and 4 DEG C, 12000r/min centrifugation Supernatant is transferred in another 1.5mL centrifuge tube by 10min；
G) add the NaAc buffer of 0.1 times of volume and the ice-cold dehydrated alcohol of 2 times of volumes, be mixed by inversion, until white Flocculent deposit be precipitated, -20 DEG C of 30~60min of preservation；
H) 4 DEG C, 12000r/min is centrifuged 10min, discards supernatant liquid, is precipitated 2 times with 70% ice cold ethanol rinsing DNA；
I) 4 DEG C, 12000r/min is centrifuged 10min, abandons supernatant, makes ethyl alcohol volatilization clean at room temperature；
J) DNA after drying is dissolved in the TE liquid of 80~100 μ L, and 4 DEG C save until DNA is completely dissolved, and 0.8% agarose is solidifying Gel electrophoresis detects its quality, -80 DEG C of preservations.
(2) PCR amplification
PCR reaction system is using mixing sample-adding method, the i.e. quantity of various components and 1 according to needed for each reaction system The number of the reaction of PCR needed for secondary response, calculates the total amount of various reactive components, is added to 1 1.5mL or 2.0mL centrifugation Guan Zhong mixes well rear brief centrifugation, then is dispensed into each 200 μ L Eppendorf PCR pipe, and template DNA is then added, PCR amplification is carried out after brief centrifugation again；
PCR reaction system is shown in Table 2:
Table 2.PCR reaction system
2×Taq PCR MasterMix 12.5μL
Upstream primer F2 (10pmol/L) 0.5μL
Downstream primer R2 (10pmol/L) 0.5μL
DNA profiling (50ng/ μ L) 1.0μL
ddH2O Supply 25 μ L
PCR response procedures:
3, Pvu II is digested the microRNA-320a-1 genetic fragment of PCR amplification
(1) Pvu II endonuclease reaction digestion system (20 μ L): 17 μ L PCR products, 10 × M buffer, 2 μ L, Pvu II (15U/ μ L) is 1 μ L；
(2) it is digested condition: being digested overnight in 37 DEG C of constant incubators.
4, agarose gel electrophoresis is analyzed after Pvu II digests PCR product
(1) agarose gel electrophoresis
The Ago-Gel of production 3%, 120V electrophoresis 50min, EB dyeing；
(2) when the different DNA fragmentation of molecular weight is separated clearly, in 2000 gel imaging system of BIO-RAD Gel Doc Imaging；
(3) SNP polymorphism is analyzed according to agarose gel electrophoresis results:
With 2000 gel imaging system PHOTOGRAPHIC ANALYSIS of BIO-RAD Gel Doc, the polymorphism of SNP: microRNA- is judged When mutation by C to T occurs for the 320a-1 gene site (rs518926539:C > T), due to introducing base mispairing, PCR amplification The sequence of microRNA-320a-1 gene product isForm restriction enzyme PvuII (CAG^CTG) restriction enzyme site；When there is no being mutated constantly, the sequence of PCR amplification microRNA-320a-1 gene product isRestriction enzyme PvuII cannot identify, thus can to site SNP polymorphism into Row detection.The agarose gel electrophoresis knot of Qinchuan Cattle microRNA-320a-1 gene (rs518926539:C > T) loci polymorphism Fruit are as follows: CC genotypic expression is 188bp band, and CT genotypic expression is 188bp, 168bp, 20bp band, TT genotypic expression For 168bp and 20bp band.Since 20bp is smaller, therefore be not easy to observe in agarose electrophoretic analysis, but by 188bp and 168bp band is still able to accurately identify CC genotype, CT genotype and TT genotype.Such as Fig. 2,168bp band is not included For CC genotype individuals, not including 188bp band is TT genotype individuals, while include 188bp and 168bp band being CT gene Type individual.
5, the sequence verification of different genotype individual PCR product
Positive and negative bidirectional sequencing is carried out respectively to different genotype individual PCR product using 3730 sequenator of ABI；Meanwhile into Row SNP position analysis, is as a result shown in Fig. 3.The sequencing result of heterozygote CT genotype individuals comprising 188bp and 168bp band It is really C or T, and CC genotype, TT genotype are respectively C, T.
6, the frequency statistics analysis of Qinchuan Cattle population microRNA-320a-1 gene SNP site
(1) genotype frequency
Genotype frequency refers to that certain genotype individuals number in a group accounts for the ratio of total individual number.
PCC=NCC/N
PTT=NTT/N
Wherein PCC、PTTRepresent CC, TT genotype frequency in a certain site；NCC、NTTIndicate that there is CC, TT gene in group The number of individuals of type；N is the total quantity for detecting group.
(2) gene frequency
Gene frequency refers to that a certain gene number is to the relative ratios of its allele sum in a group.The formula of calculating It can be write as:
PT=(2NTT+NTT1+NTT2+NTT3+NTT4+……+NTTn)/2N
In formula, PTIndicate allele T frequency, NTTIndicate the individual amount in group with TT genotype, NTTiIndicate group There are TTi genotype individuals quantity, the n mutually different multiple alleles that a1-an is allele T in body.
Allele involved in this research is C and T, so specific gene frequency calculation formula are as follows:
PC=(2NCC+NGC)/2N
PT=(2NTT+NGC)/2N
In formula, PC, PTRespectively indicate the frequency of allele C and T allele, NCC、NCTAnd NTTRespectively indicate CC, CT and The individual amount of TT genotype, N indicate total group number.C allele in Qinchuan Cattle microRNA-320a-1 gene SNP Frequency and T gene frequency are as shown in table 3.
Table 3. Qinchuan Cattle microRNA-320a-1 gene (rs518926539:C > T) locus gene frequency distribution table
7, the correlation analysis of microRNA-320a-1 gene SNP site and Qinchuan Cattle growth traits
(1) data are analyzed
The character of measurement includes: weight, and body is high, and body is long, and bust, chest breadth, chest depth, point of the buttocks is wide, hip width, hip cross Height and buttocks are long.
The model of association analysis general linear: 17.0 software general linear model GLM (General of SPSS is used LinearModels Procedure) influence to each genotype to growth traits carries out significance test.According to this experiment Its body situation establishes following statistical model:
Yijk=μ+Ai+Gj+Eijk,
Wherein: Yijk: individual phenotypic record；μ: population mean；Ai: age effect；Gj: genotype effects；Eijk: with chance error Difference.
(2) association analysis result
It the results are shown in Table 4.Adult Qinchuan Cattle individual with TT genotype is noticeably greater than CT type in hip cross height, weight Body (P < 0.05).The above result shows that the site Qinchuan Cattle microRNA-320a-1 gene (rs518926539:C > T) difference base Because type phenotypic difference is dramatically different, if tending to the selection individual that physique is tall and big, weight is big, TT type should be selected.As a result, in the present In breeding work afterwards, it can refer to result above and chosen seeds and eliminated, to accelerate that there is Quality and economy character Qin Chuan ox kind The foundation of group.
4. Qinchuan Cattle microRNA-320a-1 gene (rs518926539:C > T) loci polymorphism of table and growth traits Association analysis
Note: numerical value indicates mean value ± standard error in table.Superscript have same letter indicate difference it is not significant (P > 0.05), letter is different indicates significant difference (P < 0.05).
Nucleotides sequence list
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Claims (6)

1. a kind of detection method of Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism, which is characterized in that including with Lower step: using Qinchuan Cattle complete genome DNA to be measured as template, using primer pair P2 as primer, PCR amplification Qinchuan Cattle microRNA- 320a-1 genetic fragment；The segment obtained with restriction enzyme PvuII digestion PCR amplification, then to the amplified fragments after digestion Carry out agarose gel electrophoresis；Monokaryon on Qinchuan Cattle microRNA-320a-1 gene is identified according to agarose gel electrophoresis results The genotype in nucleotide polymorphism site；
The primer pair P2 are as follows:
Upstream primer: 5`-AACTCCCACGTTGCGTCCAGC-3`；
Downstream primer: 5`-GCTCCCCTCCGCCTTCTCTT-3`；
The agarose gel electrophoresis results of the Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism are as follows: CC gene Type shows as mono- band of 188bp, and CT genotypic expression is tri- band of 188bp, 168bp and 20bp, and TT genotypic expression is 168bp With two band of 20bp；Adult Qinchuan Cattle individual with TT genotype is noticeably greater than CT type individual in hip cross height, weight.
2. the detection method of Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism as described in claim 1, special Sign is, the response procedures of the PCR amplification are as follows: 94 DEG C of initial denaturation 2min；94 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C are prolonged Stretch 15s, 30~36 circulations；72 DEG C of extension 5min.
3. the detection method of Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism as described in claim 1, special Sign is, the agarose gel electrophoresis use mass concentration for 3% Ago-Gel.
4. the detection method of Qinchuan Cattle microRNA-320a-1 gene mononucleotide polymorphism as described in claim 1 is in the Qin Application in the ox molecular marker assisted selection breeding of river.
5. application as claimed in claim 4, which is characterized in that the microRNA-320a-1 gene mononucleotide polymorphism Detection be applied to the meat growth traits of Qinchuan Cattle marker assisted selection.
6. application as claimed in claim 4, which is characterized in that the Qinchuan Cattle individual with mutant homozygous genotype TT is selected, Quickly establish the excellent Qinchuan Cattle population of genetic resources.
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