CN110172516A - Detect the method and its application of ox lncFAM200B gene 5 ' flanking region single nucleotide polymorphism - Google Patents
Detect the method and its application of ox lncFAM200B gene 5 ' flanking region single nucleotide polymorphism Download PDFInfo
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- CN110172516A CN110172516A CN201910218571.5A CN201910218571A CN110172516A CN 110172516 A CN110172516 A CN 110172516A CN 201910218571 A CN201910218571 A CN 201910218571A CN 110172516 A CN110172516 A CN 110172516A
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- lncfam200b
- flanking region
- single nucleotide
- nucleotide polymorphism
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a kind of method and its application for detecting ox lncFAM200B gene 5' flanking region single nucleotide polymorphism, using native Chinese cattle complete genome DNA as template, PCR amplification ox lncFAM200B gene 5' flank region sequence, then agarose gel electrophoresis and DNA sequencing are carried out to pcr amplification product；2 single nucleotide variations of ox lncFAM200B gene 5' flanking region are identified according to sequencing result；Association analysis finds that this 2 mononucleotide sites of lncFAM200B gene 5' flanking region have important regulating and controlling to growth traits, can be used as molecular labeling.Detection method provided by the invention provides simple, quick approach to establish lncFAM200B gene SNP and growth traits relationship, and testing result can be used for the marker assisted selection of growth traits, the excellent ox population of Speed-up Establishment genetic resources.
Description
Technical field
The invention belongs to modern biotechnologies and cattle breeding field, relate to the use of PCR amplification and DNA sequencing technology detection
Long-chain non-coding RNA lncFAM200B (long noncoding RNA family with sequence
Similarity 200member B) 5 ' flanking region single nucleotide polymorphism genetic marker sites.
Background technique
With the rapid development of society, people's lives level is continuously improved, and increases sharply to the demand of livestock products, right
The requirement of livestock products quality continues to develop variation, improves so that breeding objective be pushed to continue to develop.Ancient human is starting to tame house
Starting breeding activity when poultry, they are selected and are met the individual of people's demand and reserve seed for planting by Phenotypic Observation, thus
The generations of production performance for improving domestic animal.Into after 20th century, it was recognized that the importance of gene pairs biological phenotype,
Then gradually start the production performance for considering how to improve domestic animal from hereditary angle.Slowly develop by hybridization etc. means into
Row breeding, to achieve the purpose that merge the excellent performance of different cultivars.In recent years, breeding process develops increasingly
System is not limited in domestic animal phenotype, also relates to economic benefit and environmental protection, in the hope of maximization of economic benefit,
In the case where not influencing environment, the production performance of domestic animal is improved to greatest extent.Meanwhile in the process of breeding, also no longer only
Only consider single production performance, but comprehensively consider the indexs such as growth, breeding, meat, is reasonably educated to make one
Kind scheme.With the continuous development of molecular cytobiology technology, more and more modern biotechnologies are applied to domestic animal and educate
In kind, such as molecular marker assisted selection (Marker-assisted selection, MAS) breeding, gene editing breeding, embryo
The technologies such as tire transplanting.The application of these technologies greatly improves breeding speed and efficiency, and can not influence preferably
On the basis of other characters, towards the direction directive breeding of people's needs.
Molecular marker assisted selection is the rapid development with modern molecular biology technique and generates most suitable current
The new breeding method of technology development.The technology can on a molecular scale fast and accurately select idiotype.Benefit
It the characteristics of with purpose character and molecular labeling close linkage, is marked and is screened by detection molecules, to reach selection purpose
The purpose of character has the characteristics that not interfered by external environmental condition quickly, accurately,.The implementation of this method is built upon known
On the basis of key variation, i.e., in practical applications, need first to find the quantitative trait locus (QTL) for controlling a certain crucial character
Or crucial hereditary variation is as genetic marker, such as copy number variation (Copy number variation, CNV), short-movie section
Insertion and deletion (Insertion/Deletion), single nucleotide polymorphism (Single nucleotide polymorphism,
SNP) etc., and then pass through the individual for selecting that there is in certain characters excellent performance type come indirect selections to genetic marker.
In these genetic markers, since SNP pertains only to conversion, transversion, insertion or the missing of single base,
In the heritable variation of all genomes, SNP is distributed most wide, occurrence frequency highest.In heritable variation of the mankind, SNP
80% or more of all known polymorphisms is accounted for, just has 1 SNP in average every 500~1000 base-pairs.In several changes of SNP
In different, the probability for converting generation is always apparently higher than other several variations, the especially mutation of C-T.In genomic DNA, SNP
Be likely to occur in any position of gene, may in intergenic region, gene internal, include sub-district, exon 1 and gene tune
Control area.The SNP of gene coding region can be further divided into missense mutation, nonsense mutation, same sense mutation.And gene control region SNP
The SNP of the different locations such as Enhancer district, 3' non-translational region, 5 ' non-translational regions, 3' flanking region, 5 ' flanking regions can be divided into again.Base
Because the missense mutation and nonsense mutation of code area will lead to the change of gene coding amino acid sequence, and then influence the structure of albumen
And function, same sense mutation may influence the production quantity of albumen by influencing the skewed popularity of amino acid in translation process, in turn
Influence the performance of protein function.The SNP of gene control region can be by influencing the interaction with trans-acting factor etc., Jin Erying
The transcription and translation for ringing gene is horizontal.The SNP of 5 ' flanking regions may regulate and control base by the combination of transformation promoter and transcription factor
The expression of cause.To sum up, the SNP of different location can influence the expression of gene by different approach, and then influence biology
Phenotypic character.
In recent years, sequencing technologies and bioinformatics are quickly grown, to whole-genome association (Genome occur
Wide association study, GWAS) etc. research hereditary variation and characters correlation method.GWAS is to multiple samples
The hereditary variation of full-length genome range carries out polymorphic detection, obtains genotype, and genotype and phenotypic character are counted
Analysis is found out to the significant relevant variation of purpose character and gene as genetic marker, is mentioned for molecular marker assisted selection breeding
For basic material.GWAS research have found it is many before not it has been found that key gene and chromosomal region, be that domestic animal is complicated
The breeding of character provides more clues.
In animal productiong practice, the detection and its application of SNP has great importance.Currently, the side of detection SNP variation
Method mainly has: (1) method for directly carrying out DNA sequencing to PCR product after PCR amplification, this method is the inspection of SNP the most accurate
Survey method.With popularizing for sequencing technologies, the price of DNA sequencing is lower and lower, and more and more sequencing companies appear in market
On, therefore, this method becomes more and more practical.(2) allele specific PCR, i.e. AS-PCR, also referred to as amplification Refracting Mutation body
It is PCR (amplification refractory mutation system, ARMS), this method can only be used to detect known
The genotype in mutational site, and new variation cannot be excavated.The principle of this method is to design mutating alkali yl at the end 3' of primer, by
It is exo-acting not have 3'-5's ' in Taq archaeal dna polymerase, so the primer of 3' mispairing is not easy to form phosphodiester bond, hinders to expand
Increase.It follows that enlarged fragment is obstructed between mutant primer and conventional mould, custom primer and mutagenesis template, when electrophoretic separation,
There are not bands of a spectrum, it is on the contrary then will appear particular bands.On the basis of the method, scientific research personnel has further invented four primers
The method of Refracting Mutation system PCR (Tetra-primer-ARMS-PCR) makes a variation to detect the SNP of single locus.Tetra-
Primer-ARMS-PCR designs the opposite inner primer comprising mutational site of two extending directions and two contrary outer are drawn
The amplified production of object, four primer combination of two, two Outside primers is positive control, and two inner primers separately include mutation
A kind of base in site theoretically only has an inner primer if a certain individual only has a kind of genotype in the site
PCR product can be amplified with opposite Outside primer.To bring the genotype of analysis individual according to the item of PCR product.But mesh
There is also some problems, some base mispairing types cannot prevent completely primer extend for preceding this method, specific in application
Analysis.(3) PCR-SSCP and DNA sequencing combined techniques, the testing cost of this method is relatively lower compared with DNA sequencing, but quasi-
True rate is without DNA sequencing height, and experimentation is comparatively laborious.(4)CAPs(cleaved amplification
Polymorphism sequence-tagged sites) technology is also known as restriction fragment length polymorphism polymerase chain reaction
Answer (PCR-RFLP) technology, a kind of common method when PCR-RFLP is detection specific site variation.This method is first with a pair
Primer amplification includes the region where mutational site, is then cut using specific restriction enzyme, this is restricted interior
Enzyme cutting can only be identified comprising a kind of sequence of genotype in mutational site, then carry out agarose or polyacrylate hydrogel electrophoresis point
Analysis, accurately identifies the genotype of SNP site according to the size of band after digestion.The accuracy of PCR-RFLP method ratio AS-PCR
Height, but it is lower than direct Sequencing accuracy, and also restriction enzyme price is higher, and test operation is cumbersome.Therefore, to sum up institute
It states, with the development of sequencing technologies and the reduction of sequencing cost, the method that DNA sequencing is directly carried out after PCR amplification is to reflect at present
Fixed and detection sample genotype most easy, most accurate method.
Currently, having there is many crucial hereditary variation sites for influencing animal economic characters to be revealed and apply.2017,
Li et al. utilizes the genotype of 61 Korea Spro ox breeding oxens of GAWS and chip analysis and 35968 SNP sites of 486 offsprings, and
6 growths and carcase quality character are determined, as a result, it has been found that 129 SNPs and these growth traits and the significant phase of carcase quality
It closes, wherein No. 14 chromosomes are the major chromosomals for influencing killing-out weight, No. 20 chromosomes are to influence weaning weight, one-year-old heavy, trunk
The major chromosomal (Li et al., 2017) of weight.2014, Doran etc. was in the genome of 5705 Irish Holstein cows
The genotype in 54001 sites SNPs is analyzed, and is constituted and butchered with genetic ability for carcass weight, content of fat in body, filial generation trunk
It is associated analysis again, it was found that 557 candidate genes (Doran et al., 2014).The discovery chromosome in 2014 such as Kelly
1, the region on 9,14,16,19,23,26,29 and X and fat composition and quantitative character are related (Kelly et al., 2014).
In addition to growth traits and carcass trait, the research in milk character also has many new discoveries.2008, Daetwyler etc.
It is detected using genotype of the GWAS to 484 holstein cow quantitative trait locus (QTL), it is found that 102 influences produce
The potential QTL of milk character, 144 with the milk production trait significantly associated site SNPs (Daetwyler et al., 2008).2011
Year Schopen etc. is analyzed using 50228 SNP sites of the GWAS to 1712 Dutch holstein cows, as a result, it has been found that with
The relevant genome area of milk protein fractions be predominantly located on the 5th, 6,11 and No. 14 chromosome (Schopen et al.,
2011).In addition to this, there are also many research and utilization GWAS to have excavated crucial variant sites relevant to the disease of domestic animals etc. or area
Domain.
Since GWAS is to excavate from the angle of full-length genome to crucial variation, before practical application, also
The reliability of GWAS result must be detected in animal population by the method for experimental verification.Many crucial variations existing at present exist
It is tested and analyzed, and is applied in cattle breeding in group.Myostatin gene Myostatin (MSTN,
1997) expressing quantity and muscle development are closely related, and the mutation of MSTN is the decisive gene for leading to double-muscling shape, and the world writes
The Belgian Blue ox of name forms termination codon in advance just because of the missing of 11 bases on the 3rd exon of MSTN gene
Son causes its " double flesh sterns " phenotype.Subsequent various countries researcher is utilized respectively the technologies such as gene knockout, transgenosis to MSTN
It is studied on the biology such as mouse, rat, sheep, ox, and has cultivated the livestock and poultry new varieties that meat is excellent, meat productivity is high,
New approach is opened for traditional breeding work, accelerates the speed of breeding.2017, the old Yulin of Xibei Univ. of Agricultural & Forest Science & Technology
Team has cultivated while having knocked out MSTN gene using technologies such as CRISPR/Cas9 system, hormone, embryo transfers, influenced
Beta carotene double deoxidation enzyme-hydro (β-carotene-9', 10'-dioxygenase, the BCO2) gene and shadow of fatty color
The Ningxia sheep of Agouti signal protein (Agouti Signal Protein, ASIP) gene of hair color is rung, is multiple gene editor
The application of breeding is laid a good foundation.By to " lean meat species " (the special blue pig of such as Large White, skin) and " fat meat type " pig (such as wild boar, plum
Mountain pig) qtl analysis discovery, the on insulin-like growth factor 2 (Insulin-like growth factor2, IGF2) the 3rd
There are the mutation of G to A for 3072nd bit base of three intrones, and the SNP site is to the speed of growth of pig, lean meat percentage, the thickness of backfat, back
The characters such as longissimus area have a significant impact.In proliferation, the farrowing of the SNP variation and sheep of many research discovery GDF9 genes
Number is closely related, by editing it, is expected to improve the reproductive performance of sheep.In addition to this, there are also much have important regulating and controlling function
Can hereditary variation have been found and identify, but be not applied in breeding practice also, but with the continuous development of biotechnology, this
A little crucial variations will be applied in breeding practice quickly.
LncFAM200B (long no-coding RNA FAM200B) gene is by will be in Qinchuan Cattle different times bone
A lncRNAs of differential expression is as seed sequence in bone flesh, with Bos indicus, Bison bison, Bos tauru base
Because group compare (up to 99%~100%, 100%) coverage reaches the consistency with protein coding gene FAM200B partial sequence,
And the long-chain non-coding RNA (lncRNA) determined after encoding histone capability analysis.Result of study shows that lnc FAM200B may
Ox muscle and adipose tissue development are influenced, but only finds the single nucleotide variations site (example in lncFAM200B genetic transcription region at present
Such as, 1 SNP mutation existing for the 1st introne of lncFAM200B gene) with it is several in the early development (6 monthly age) of Nanyang cattle
The significant correlation of the difference of a growth traits.
Currently, about lncFAM200B gene nontranslated region (for example, 5 ' flanking regions) single nucleotide polymorphism and China
The relevance of the middle and later periods growth and development Comprehensive Traits difference of Chinese native cattle breeds is there is not yet report.
Summary of the invention
It is an object of that present invention to provide a kind of detection ox lncFAM200B gene 5 ' flanking region single nucleotide polymorphism
Method and its application, to accelerate fine-variety breeding speed.
In order to achieve the above objectives, the invention adopts the following technical scheme:
A method of detection ox lncFAM200B gene 5 ' flanking region single nucleotide polymorphism, comprising the following steps:
Using ox complete genome DNA to be measured as template, pass through PCR amplification ox lncFAM200B gene 5 ' flanking region part
Segment, carries out electrophoresis to the amplified production of PCR, carries out DNA to electrophoretic band size and the matched PCR product of the Partial Fragment
Sequencing；The gene of two sites SNP1 and SNP2 in ox lncFAM200B gene 5 ' flanking region are identified according to sequencing result
Type, wherein SNP1 is the SNP site (rs519952828) for being positioned at reference sequences NC_037333.1:g.110851366,
SNP2 is the SNP site (rs380849013) for being positioned at reference sequences NC_037333.1:g.110851618.
Preferably, the amplimer that the PCR is used is to P1 are as follows:
Upstream primer: 5 '-AATCGGTGGACTGCTAACCT-3 '；
Downstream primer: 5 '-CGAGCGCCAGTGTACCTC-3 '.
Preferably, the ox lncFAM200B gene 5 ' flanking region Partial Fragment is positioned at reference sequences NC_
037333.1:g.110851260-110852103.
Preferably, the amplified reaction program of the PCR are as follows: 95.0 DEG C of initial denaturation 5min；95.0 DEG C of denaturation 30s, 60.0 DEG C
Renaturation 30s, 72.0 DEG C of extension 1.0min are recycled 40 times；72.0 DEG C of extension 10min.
Preferably, the electrophoresis use mass fraction for 1.0% Ago-Gel.
Preferably, the matched electrophoretic band size of the Partial Fragment is 844bp.
A kind of kit detecting ox lncFAM200B gene 5 ' flanking region single nucleotide polymorphism, including it is used for PCR
Expand the primer pair P1, the primer pair P1 of above-mentioned ox lncFAM200B gene 5 ' flanking region Partial Fragment are as follows:
Upstream primer: 5 '-AATCGGTGGACTGCTAACCT-3 '；
Downstream primer: 5 '-CGAGCGCCAGTGTACCTC-3 '.
The method of above-mentioned detection ox lncFAM200B gene 5 ' flanking region single nucleotide polymorphism is in ox molecular labeling
Application in assisted selection.
Preferably, the site SNP1 and SNP2 is molecular labeling relevant to ox individual growth character advantage.
Preferably, the growth traits is selected from 6~24 monthly age weight, body length, bust, hip width, hip cross height, average day
It is a variety of in weight gain, body length index.
Preferably, the genotype of the SNP1 is the ox individual growth character that the genotype of GG, GA and SNP2 are CC
It is more excellent.
Preferably, the ox is selected from Nanyang cattle or Qinchuan Cattle.
The beneficial effects of the present invention are embodied in:
The present invention by PCR amplification ox lncFAM200B gene 5 ' flanking region segment, and to PCR amplification target fragment into
Row DNA sequencing can simply, fast, accurately detect two hereditary variation sites of ox lncFAM200B gene 5 ' flanking region
Genotype.The present invention is not only the foundation of relationship between the hereditary variation and growth traits of lncFAM200B gene 5 ' flanking region
Effective means is provided, and testing result can be used for the marker assisted selection (MAS) of native Chinese cattle growth traits, fastly
The excellent ox population of genetic resources is stood in run-up.
Detailed description of the invention
Fig. 1 is the pcr amplification product electrophoretogram of ox lncFAM200B gene 5 ' flanking region.
Fig. 2 is the sequencer map of the 3 kinds of genotype in the site gene 5 ' flanking region SNP1 ox lncFAM200B: reference sequences are
The ox LOC100849050 gene order (NC_037333.1) announced on NCBI.
Fig. 3 is the sequencer map of the 2 kinds of genotype in the site gene 5 ' flanking region SNP2 ox lncFAM200B: reference sequences are
The ox LOC100849050 gene order (NC_037333.1) announced on NCBI.
Specific embodiment
It elaborating with reference to the accompanying drawings and examples to the present invention, the embodiment is explanation of the invention, and
Non- limiting the scope of the invention.
It is directed to long-chain non-coding RNA (lncRNA) gene lncFAM200B (long no-coding RNA
FAM200B), according to the full length sequence of the lncFAM200B of early-stage study clone, it is determined that the transcription of lncFAM200B gene rises
Beginning site.By the transcription initiation site of determining lncFAM200B gene, the position SNP that will have been announced in Ensembl database
Point carries out the matching analysis with lncFAM200B gene 5 ' flanking region.
Simultaneously as not being associated analysis to SNP and growth traits in database, the present invention is utilized by product for many years
DNA sample and growth data tired, that collect the different yellow cattle breeds (Nanyang cattle, Qinchuan Cattle) obtained, to lncFAM200B gene
The hereditary variation of partial sequence (Chromosome 6-NC_037333.1:110851260-110852103) in 5 ' flanking regions
Site has carried out screening and identification, and base is established in the screening of the SNP site to can be used as molecular labeling on lncFAM200B gene
Plinth.
Finally, the present invention is using PCR amplification, agarose gel electrophoresis detection and DNA sequencing technology, by 2 oxes
The genotype of the SNP site of the lncFAM200B gene 5 ' flanking region of kind carries out detection and gene frequency analysis, and to upper
State 2 SNP sites and Qinchuan Cattle and Nanyang cattle growth traits (such as: weight, body height, body length, chest breadth, bust, body length index)
It is associated analysis, the results showed that 2 SNP sites (SNP1, SNP2) of lncFAM200B gene 5 ' flanking region can be used as and mention
The molecular genetic marker of high ox growth traits.
(1) experimental drug and reagent
1. biochemical reagents and biological reagent: 1. PCR MasterMix (purchased from Chinese hundred Imtech)；2. Proteinase K
(being purchased from Huamei Bio-Engrg Co.)；3. Marker D2000 (is purchased from TIANGEN Biotech (Beijing) Co., Ltd.)；
2. general reagent: general reagent is bought from Huamei Bio-Engrg Co., dispenses product: citric acid, lemon for import
Sour sodium, glucose, Tris, EDTA, NaCl, NaOH, KCl, Na2HPO4、KH2PO4, it is Tris saturated phenol, chloroform, isoamyl alcohol, anhydrous
Ethyl alcohol, sodium acetate, dodecyl sodium sulfate (SDS), ethidium bromide (EB), bromophenol blue, dimethyl benzene cyanogen FF, acetic acid, sucrose, boron
Acid, agarose etc..
3. solution and buffer: all solution and buffer are all made of the preparation of deionization ultrapure water.Autoclave conditions are
15bf/in(1.034×105Pa), 25min.Preparation of reagents method refer to that Sambrook etc. writes " Molecular Cloning: A Laboratory refers to
South ".Specific solution and buffer are as follows:
(1) solution used in sample
Anti-coagulants ACD: citric acid 0.48g, sodium citrate 1.32g, glucose 1.47g, constant volume is in 100mL water, high pressure
Sterilizing.The ACD liquid of 1mL is added in every 6mL new blood.This anti-coagulants is better than EDTA, can be more preferable during blood storage
Ground saves high molecular DNA.It can be in 0 DEG C of preservation a couple of days or -70 DEG C of long-term preservations through its anticoagulant blood.
(2) blood sample genomic DNA separates solution used
1. PBS buffer solution: NaCl 8g, KCl 0.2g, Na2HPO41.44g KH2PO40.24g adds ultrapure water extremely
1000mL adjusts pH to 7.4, high pressure sterilization, 4 DEG C of preservations.
2. 10%SDS:10g SDS is dissolved in the ultrapure water of 90mL, 68 DEG C of water-bath dissolutions, fixed with HCl tune pH to 7.2
Hold to 100mL.
③0.5mol/L EDTANa2: EDTA Na2186.1g is dissolved in the ultrapure water of 800mL, extremely with NaOH tune pH
8.0, it is settled to 1000mL, high pressure sterilization, 4 DEG C of preservations.
4. 1mol/L TrisHCl:121.14g Tris, is dissolved in 800mL ultrapure water, HCl adjusts pH to 8.0, constant volume
To 1000mL.High pressure sterilization, 4 DEG C of preservations.
5. 5mol/L NaCl:NaCl 292.2g is dissolved in 1000mL ultrapure water.
6. DNA extraction buffer: taking 0.5mol/L EDTANa24mL, 1mol/L TrisHCl 10mL, 5mol/L
NaCl 4mL, 10%SDS 20mL are settled to 100mL, 20 μ g/mL of RNase.
7. NaAc buffer: NaAc3H2O 20.4g；Ultrapure water 40mL；Dilute HAc tune pH to 7.4；It is settled to 50mL.
8. TE buffer: TrisHCl buffer (pH 8.0) 10mmol/L, edta buffer liquid (pH 8.0) 0.1mmol/
L, high pressure sterilization, 4 DEG C of preservations.
9. Proteinase K: being made into 20mg/mL, -20 DEG C of preservations with ultrapure water.
(3) solution used in tissue sample DNA is extracted
Other than public solution when extracting genome DNA, following reagent is also prepared:
1. 2mol/L NaCl:11.688g is dissolved in water, it is settled to 100mL, high pressure sterilization.
2. tissue DNA extracting solution (100mL): l mol/L Tris HCl (pH 8.0) l mL, 0.5mol/L EDTA (pH
8.0) 20mL, 2mol/L NaCl 5mL, are settled to 100mL.
(4) agarose gel electrophoresis analyzes solution used
1 × tbe buffer liquid: 10 × TBE 50mL is taken to be settled to 1000mL.
(2) design of the PCR primer of ox lncFAM200B gene 5 ' flanking region SNP detection
The sequence of ox LOC100849050 gene is retrieved on NCBI, and can using NCBI Primer Blast design
PCR primer of the amplification comprising ox lncFAM200B gene 5 ' flanking region is to P1, and primer pair P1 sequence is following, and (design is completed in
In May, 2017):
Upstream primer: 5 '-AATCGGTGGACTGCTAACCT-3 '；
Downstream primer: 5 '-CGAGCGCCAGTGTACCTC-3 '；
With above-mentioned primer pair P1 to ox genome amplification, can expand comprising ox lncFAM200B gene 5 ' flanking region
Partial Fragment；Expand post-fragment electrophoresis detection as shown in Figure 1, Marker swimming lane be Marker D2000 (it is descending according to
Secondary is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp).1~3 swimming lane is the target fragment of different samples amplification
(844bp)。
After carrying out sequencing identification to the correct segment of amplification, the 110851366th alkali in NC_037333.1 sequence is found
Peak figure is sequenced as shown in Fig. 2, being ox lncFAM200B gene 5 ' flank by being analyzed to identify the site there are G > A mutation in base
One SNP (being denoted as SNP1, rs519952828) in area.110851618th alkali of the discovery in NC_037333.1 sequence simultaneously
Peak figure is sequenced as shown in figure 3, being ox lncFAM200B gene 5 ' flank by being analyzed to identify the site there are C > T mutation in base
Another SNP (being denoted as SNP2, rs380849013) in area.
(3) the lncFAM200B gene 5 ' flanking region segment of PCR amplification ox to be measured is carried out with primer pair P1
1, the acquisition of ox sample
Experiment animal used is that 2 yellow cattle breeds amount to 206 samples.Stochastical sampling mode is all made of when sampling to take
The blood sample of breeding station ox individual.Anticoagulant using ACD when taking blood sample, ice chest low temperature, which takes back laboratory and is placed on -80 DEG C, to be frozen.2
The sample message of a yellow cattle breed is as follows: (1) totally 139 parts of Qinchuan Cattle (QC) blood sample, using group's stochastical sampling mode in 2009
November in year collects in Baoji, Shaanxi province city, Qinchuan Cattle conservation field, Fufeng；(2) totally 67 parts of Nanyang cattle (NY) blood sample, in August, 2012
Pick up from Nanyang City, Henan Province Nanyang cattle conservation field.
2, separation, extraction, the purifying of blood sample genomic DNA
1) blood sample (predominantly haemocyte) thaw at RT is freezed, 500 μ L to 1.5mL Eppendorf centrifuge tubes is shifted, adds
Enter isometric PBS buffer solution, mix well, 12000r/min be centrifuged 10min (4 DEG C), discard supernatant liquid, repeat the above steps to
Supernatant is transparent, precipitating is in faint yellow；
2) 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；
3) plus 3 μ L (20mg/mL) Proteinase Ks into above-mentioned mixed liquor and mix, and 55 DEG C are incubated overnight to clarification；It is not yet clear
Clear person can add and continue digestion after 1 μ L Proteinase K mixes until clarification；
4) 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 centrifugation 10min, supernatant is transferred in another 1.5mL centrifuge tube, is repeated once；
5) chlorination imitates 500 μ L, mixes well 20min, and supernatant is transferred to another by 4 DEG C, 12000r/min centrifugation 10min
In 1.5mL centrifuge tube；
6) chlorination is imitative: 500 μ L of isoamyl alcohol mixed liquor (24:1), mixes well 20min, 4 DEG C, 12000r/min centrifugation
Supernatant is transferred in another 1.5mL centrifuge tube by 10min；
7) the NaAc buffer of 0.1 times of volume and the ice-cold dehydrated alcohol of 2 times of volumes, mixing is added to rotate centrifuge tube, directly
Flocculent deposit to white is precipitated, -20 DEG C of 30~60min of preservation；
8) 4 DEG C, 12000r/min centrifugation 10min, discard supernatant liquid, are precipitated 2 times with 70% ice cold ethanol rinsing DNA；
9) 4 DEG C, 12000r/min centrifugation 10min, discard supernatant liquid, make ethyl alcohol volatilization clean at room temperature；
10) DNA after drying is dissolved in the ultrapure water of 80~100 μ L, and 4 DEG C of preservations are completely dissolved up to DNA, 0.8% agar
Its quality of sugared detected through gel electrophoresis, -80 DEG C of preservations.
3, agarose gel electrophoresis detects DNA
1) it by the glue box of Ago-Gel processed and offset plate wash clean, dries, the bottom plate of the glue of wash clean is put into glue
In box, comb is plugged.
2) agarose for weighing 0.3g, is transferred in triangular flask, and 1 × TBE 30mL, which is added, makes its suspension, and microwave ingle adds
Heat is taken out for 2 times wait boil, the nucleic acid dye of final concentration of 0.5 μ g/mL is added when it is cooled to non-scald on hand.Gentle agitation is prevented
Only there is bubble.
3) after mixing (about 60 DEG C), agarose solution is poured into slot immediately.Such as there is bubble, is moved immediately with pipettor
Out.
4) completely after cooled and solidified (about 25~40min), comb is pulled out, gel is moved into electrophoresis tank.
5) 1 × tbe buffer liquid is added into electrophoresis tank, liquid level is made to be higher by 2~5mm of glue surface.
6) 2~4 μ L of DNA sample is taken, is mixed after adding 2 μ L sample-loading buffers, unified loading (notices that the sequence of pipette tips answers front and back
It is corresponding), and DNA Marker is added on one side.
7) 110V electrophoresis 40min.
8) it is observed on uv analyzer, if there is RNA, then needs to purify, if there is obvious degradation, phase need to be extracted again
Answer the DNA of sample.
4, the purifying of DNA
1) in the DNA solution of 500 μ L be added 10%SDS make its final concentration of 0.1%, be added Proteinase K to final concentration reach
To 100 μ g/mL；55 DEG C of heat preservation 10h or so.
2) with 1) the isometric phenol of solution: chloroform: isoamyl alcohol (25:24:1) and chloroform extract once respectively.
3) 12000r/min is centrifuged 5min split-phase, inhales upper strata aqueous phase into another centrifuge tube.
4) 1/10 volume 3mol/L sodium acetate is added and 2 times of volumes ice cold dehydrated alcohols precipitate DNA.
5) liquid is outwelled, 60 μ L sterilizing ultrapure water dissolution is added in airing after 70% ethanol washing, and 4 DEG C to be detected.
5, spectrophotometry DNA
With OD value of the ultraviolet light photometric determination DNA sample at 260nm, 280nm.Calculate DNA content and OD260/OD280
Ratio.Such as OD260/OD280Ratio illustrates then be purified in sample containing more protein or phenol less than 1.6；If
Ratio is greater than 1.8, then should consider to remove RNA purifying.Wherein, DNA concentration (ng/ μ L)=50 × OD260Value × extension rate.
DNA detect after, take out a certain amount be diluted to 50ng/ μ L, be stored in -20 DEG C it is spare, remaining deposits in -80 DEG C.
6, PCR amplification
PCR reaction system is using mixing sample-adding method, the quantity of various components according to needed for each reaction system and 1 time
The number of PCR reaction, calculates the total amount of various reactive components, is added in 1 1.5mL centrifuge tube needed for reaction, sufficiently mixed
Brief centrifugation after even, then be dispensed into each 0.2mL Eppendorf PCR pipe, after template DNA, then brief centrifugation is then added
Carry out PCR amplification；PCR reaction system include PCR MasterMix (including Taq archaeal dna polymerase, dNTPs and reaction buffer,
Concentration is 2 ×) 12.5 μ L；0.3 μ L of upstream primer；0.3 μ L of downstream primer (upstream and downstream primer concentration is 10pmol/ μ L)；Gene
Group DNA (concentration is 50ng/ μ L genomic DNA) 0.5 μ L；11.4 μ L of deionized water；Totally 25.0 μ L.
The program of 7.PCR reaction
95.0 DEG C of initial denaturation 5min；95.0 DEG C of denaturation 30s, 60.0 DEG C of renaturation 30s, 72.0 DEG C of extension 1.0min, 40 are followed
72.0 DEG C of extension 10min, 4.0 DEG C of preservation amplified productions after ring.
8. agarose gel electrophoresis detection and DNA sequencing
Agarose gel electrophoresis detection and DNA sequencing are divided into 4 steps: 1) making concentration is 1.0% Ago-Gel (addition core
Acid dye), 110V electrophoresis 20min；2) it is imaged in Bio-Rad gel imaging system, and expanding effect is judged according to image；
3) by stripe size, correctly (correct electrophoretic band size is 844bp), band is single, the higher PCR product of band brightness is sent
It is sequenced (each sample at least PCR product of 30 μ L)；4) sequencing result is compared with reference sequences NC_037333.1, is reflected
The genotype of other SNP site.
The heredity of ox lncFAM200B gene 5 ' flanking region is become by the method for direct Sequencing after agarose gel electrophoresis
Different polymorphism is detected, and analysis is then associated with growth traits.
(4) the frequency statistics analysis in ox lncFAM200B gene 5 ' flanking region hereditary variation site
1) gene and genotype frequency
2 SNP sites are found altogether in lncFAM200B gene 5 ' flanking region, are respectively designated as SNP1 to SNP2.In Nanyang
In ox and Qinchuan Cattle, two SNP sites show dimorphism, and gene frequency is all larger than 1%, and at least two genes
Type number of individuals is greater than 2, therefore can carry out data statistic analysis.Two yellow cattle breeds separately divide when being associated analysis
Analysis.
Genotype frequency refers to that certain genotype individuals number of a certain character in a group accounts for the ratio of total individual number.PII
=NII/ N, wherein PIIRepresent the II genotype frequency in a certain site；NIIIndicate the number of individuals in group with II genotype；N is
Detect the total quantity of group.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 can be write as: PI=(2NII+NIa1+NIa2+NIa3+NIa4+……+NIan)/2N, in formula, PIIndicate allele I
Frequency, NIIIndicate the individual amount in group with II genotype, NIaiIndicate that there is I in groupaiGenotype individuals quantity,
a1- anFor the n mutually different multiple alleles of allele I.
In Qinchuan Cattle and Nanyang cattle, the genotype frequency of 2 SNP sites of lncFAM200B gene 5 ' flanking region and wait
Position gene frequency is as shown in table 1.The site SNP1, there are three types of genotype, GG, GA and AA, Nanyang in Nanyang cattle and Qinchuan Cattle
The frequency of three kinds of genotype of ox is respectively 0.567,0.388 and 0.045, the frequency of three kinds of genotype of Qinchuan Cattle is respectively 0.655,
0.324 and 0.021；SNP2 is C > T mutation, and frequency of the CC genotype in Nanyang cattle and Qinchuan Cattle is respectively 0.925 He
0.892；Two sites, which are in Hardy's Weinberg equilibrium state, to be found to Hardy's Weinberg's imbalance analysis.
1. ox lncFAM200B gene 5 ' flanking region SNP mutant gene frequency distribution table of table
(5) association analysis of ox lncFAM200B gene 5 ' flank region SNP mutant gene effect
Genotype data: tri- kinds of genotype of SNP1:GG, GA and AA；Two kinds of genotype of SNP2:CC, CT；
Nanyang cattle growth data includes: birth weight, and 6 monthly ages, 12 monthly ages, 18 monthly ages and the weight at 24 monthly ages, body height, body are oblique
Length, bust, hip width, point of the buttocks be wide, daily gain, body body index, bust index, body length index, pipe enclose index；
Qinchuan Cattle growth data includes: the body height of Adult Bovine, hip cross height, body length, bust, chest breadth, chest depth, buttocks is wide, sits
Epiphysis is wide, hip width, weight, body body index, bust index, chest breadth index, the wide index of buttocks.
Relation analysis model: kind, the factors such as environment and gene loci and growth are analyzed using SPSS (20.0) software
Characters correlation.The statistical analysis descriptive to the data obtained is first had to, to determine whether there is outlier.Then according to number
According to characteristic, using variance analysis (when there are three types of in the presence of genotype, and different genotype individual be all larger than 3), multiple linear
(when only there are two types of in the presence of genotype, and different genotype number of individuals is all larger than 3) and then to analyze gene for model or t analysis
The effect of type.During data processing, the difference of the factor according to the index for influencing growth traits, it is contemplated that age, ring
The effect in border and the effect of genotype carry out correlation analysis using fixed model.In addition, being carried out according to physical condition
It accepts or rejects, complete model is as follows: Y=u+E+Age+ G+E, wherein Y: individual phenotypic record；U: population mean；E: Environmental Effect
It answers；Age: age effect；G: marker genetype effect；E: random error.
Referring to table 2, the results showed that the site ox lncFAM200B gene 5 ' flanking region SNP1 and SNP2 different genotype frequency
The distribution of rate and gene frequency has a significant impact the certain growth traits of ox.As can be seen from Table 2, the site SNP1 is in south
Different genotype and 6 monthly age weight, bust, hip width, average daily gain in positive ox, the hip width at 12 monthly ages averagely increase day by day
Weight, body length index, the bust at 18 monthly ages, the bust at 24 monthly ages is significant related (P < 0.05), wild-type genotype (GG genotype)
There is preferable growth traits with heterozygous (GA genotype) individual, and the phenotype of the individual of mutated homozygous (AA genotype) is equal
Significantly it is worse than heterozygous individual.Meanwhile the hip cross height of the Qinchuan Cattle individual of the site SNP1 GG genotype is also significantly greater than AA base
Because of type.These results of study explanation, G to the A in the site SNP1 sport detrimental mutation.The site SNP2 is in Nanyang cattle group, CC
6 monthly age weight, the body length, average daily gain, body length index of genotype individuals, the body at 18 and 24 monthly ages is long, the bust at 24 monthly ages,
Body length index is significantly better than CT genotype individuals (P < 0.05).The mutation for illustrating C to the T in the site SNP2 is also detrimental mutation,
The individual containing detrimental mutation should be rejected in production.
By further analyzing and test it can be concluded that, the SNP site SNP1 of ox lncFAM200B gene 5 ' flanking region
It is the molecular labeling of ox growth traits marker assisted selection with SNP2, there is the Huang of wild-type genotype by two sites of screening
Ox individual quickly establishes growth traits and develops stable, advantage significantly excellent ox population.
The influence that 2. ox lncFAM200B gene 5 ' flanking region SNP of table makes a variation to ox growth traits
Note: M: monthly age；Average value shoulder, which is put on, indicates that difference is not significant (P > 0.05) with same letter, and different letters indicate
Significant difference (P < 0.05), and the individual that a letter is indicated on shoulder is better than indicating the individual of b letter.
In conclusion the variation of lncFAM200B gene 5 ' flanking region generates important influence to the growth traits of ox,
Above 2 SNP sites have important regulating and controlling effect to Nanyang cattle, Qinchuan Cattle growth and development.Ox occupies in agro based economic development
Critical positions, therefore, the present invention for the hereditary variation of ox lncFAM200B gene 5 ' flanking region experiment and as a result, for
Effectively accelerate native Chinese cattle dominant population breeding process to have great theoretical and practical significance.
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>method and its application of ox lncFAM200B gene 5' flanking region single nucleotide polymorphism are detected
<160> 2
<210> 1
<211> 20
<212> DNA
<213>artificial synthesized
<400> 1
aatcggtgga ctgctaacct 20
<210> 2
<211> 18
<212> DNA
<213>artificial synthesized
<400> 2
cgagcgccag tgtacctc 18
Claims (9)
1. a kind of method for detecting ox lncFAM200B gene 5' flanking region single nucleotide polymorphism, it is characterised in that: including
Following steps:
Using ox complete genome DNA to be measured as template, pass through PCR amplification ox lncFAM200B gene 5' flanking region part piece
Section, carries out electrophoresis to the amplified production of PCR, carries out DNA survey to electrophoretic band size and the matched PCR product of the Partial Fragment
Sequence；The genotype of two sites SNP1 and SNP2 in ox lncFAM200B gene 5' flanking region are identified according to sequencing result,
Wherein, SNP1 is the SNP site for being positioned at reference sequences NC_037333.1:g.110851366, and SNP2 is to be positioned at reference to sequence
Arrange the SNP site of NC_037333.1:g.110851618.
2. a kind of side for detecting ox lncFAM200B gene 5' flanking region single nucleotide polymorphism according to claim 1
Method, it is characterised in that: the amplimer that the PCR is used is to P1 are as follows:
Upstream primer: 5 '-AATCGGTGGACTGCTAACCT-3 '；
Downstream primer: 5 '-CGAGCGCCAGTGTACCTC-3 '.
3. a kind of side for detecting ox lncFAM200B gene 5' flanking region single nucleotide polymorphism according to claim 1
Method, it is characterised in that: the ox lncFAM200B gene 5' flanking region Partial Fragment is positioned at reference sequences NC_
037333.1:g.110851260-110852103.
4. a kind of side for detecting ox lncFAM200B gene 5' flanking region single nucleotide polymorphism according to claim 1
Method, it is characterised in that: the amplified reaction program of the PCR are as follows: 95.0 DEG C of initial denaturation 5min；95.0 DEG C of denaturation 30s, 60.0 DEG C multiple
Property 30s, 72.0 DEG C of extension 1.0min, recycle 40 times；72.0 DEG C of extension 10min.
5. a kind of side for detecting ox lncFAM200B gene 5' flanking region single nucleotide polymorphism according to claim 1
Method, it is characterised in that: the electrophoresis use mass fraction for 1.0% Ago-Gel.
6. a kind of side for detecting ox lncFAM200B gene 5' flanking region single nucleotide polymorphism according to claim 1
Method, it is characterised in that: the matched electrophoretic band size of Partial Fragment is 844bp.
7. a kind of kit for detecting ox lncFAM200B gene 5' flanking region single nucleotide polymorphism, it is characterised in that: packet
Include the primer pair P1, the primer pair P1 for PCR amplification ox lncFAM200B gene 5' flanking region Partial Fragment are as follows:
Upstream primer: 5 '-AATCGGTGGACTGCTAACCT-3 '；
Downstream primer: 5 '-CGAGCGCCAGTGTACCTC-3 '.
8. a kind of method of detection ox lncFAM200B gene 5' flanking region single nucleotide polymorphism as described in claim 1
Application in ox molecular marker assisted selection breeding.
9. application according to claim 8, it is characterised in that: the site SNP1 and SNP2 be and ox individual growth
The relevant molecular labeling of shape advantage.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090117556A1 (en) * | 2007-11-07 | 2009-05-07 | Stephen Stewart Moore | Association of Single Nucleotide Polymorphisms in the CBFA2T1 and DECR1 Genes with Performance and Carcass Merit of Beef Cattle |
| CN102816836A (en) * | 2012-05-09 | 2012-12-12 | 西北农林科技大学 | Detection method of single nucleotide polymorphism and molecular markers of cattle FBXO32 genes |
| CN104878099A (en) * | 2015-05-15 | 2015-09-02 | 西北农林科技大学 | Method for detecting single-nucleotide polymorphism of goat ATBF1 gene and application of goat ATBF1 gene |
-
2019
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090117556A1 (en) * | 2007-11-07 | 2009-05-07 | Stephen Stewart Moore | Association of Single Nucleotide Polymorphisms in the CBFA2T1 and DECR1 Genes with Performance and Carcass Merit of Beef Cattle |
| CN102816836A (en) * | 2012-05-09 | 2012-12-12 | 西北农林科技大学 | Detection method of single nucleotide polymorphism and molecular markers of cattle FBXO32 genes |
| CN104878099A (en) * | 2015-05-15 | 2015-09-02 | 西北农林科技大学 | Method for detecting single-nucleotide polymorphism of goat ATBF1 gene and application of goat ATBF1 gene |
Non-Patent Citations (3)
| Title |
|---|
| JIN, YY等: "Identification of a Novel Polymorphism in Bovine lncRNA ADNCR Gene and Its Association with Growth Traits", 《ANIMAL BIOTECHNOLOGY》 * |
| 张思欢: "牛肌肉与脂肪发育相关lncRNA lncFAM200B的鉴定、启动子活性及遗传变异研究", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
| 徐子叶等: "调控肌内脂肪沉积的分子机制研究进展", 《中国畜牧杂志》 * |
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