CN107419026A - Label of pig fat deposition description correlation UCP2 mRNA m6A methylate the authentication method and application of function of unit point - Google Patents

Label of pig fat deposition description correlation UCP2 mRNA m6A methylate the authentication method and application of function of unit point Download PDF

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CN107419026A
CN107419026A CN201710760315.XA CN201710760315A CN107419026A CN 107419026 A CN107419026 A CN 107419026A CN 201710760315 A CN201710760315 A CN 201710760315A CN 107419026 A CN107419026 A CN 107419026A
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王新霞
汪以真
江芹
吴睿帆
姚永曦
蔡旻
刘卿
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Zhejiang Qinglian Food Co Ltd
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Abstract

Label of pig fat deposition description correlation UCP2 mRNA m6A methylates the authentication method of unit point, according to m6What A seq were sequenced to obtain be 100nt or so fragment in m be present6A methylation sites, it is characterised in that：According to RRACH (R=G, the A for methylating highly conserved；H=A, C, T) sequence carry out UCP2 genes unit point position confirmation.By bioinformatic analysis, according to m6A conserved sequence, m6A specific site is identified；Provide the primer sequence and specific method that UCP2 mRNA m6A methylation levels are identified in pig；The function of influence of the UCP2 m6A sites to fat deposition is demonstrated by Gene unit point mutation change UCP2 m6A levels.

Description

Label of pig fat deposition description correlation UCP2 mRNA m6A methylate unit point authentication method and Application of function
Technical field
The invention belongs to biology field, is related to a kind of individual gene mRNA m6The identification of A methylation sites, function And application, and in particular to UCP2 mRNA m6The identification of A methylation sites, function and application.
Background technology
Rationally deposition and regulation and control are always animal husbandry focus of attention problem to pork fat.Pork fat over-deposit not only reduces Nutrientuse efficiency, and too high Body fat retention can directly affect the health of consumer, including easily trigger the fat sugar brought The metabolic disease such as urine disease and angiocarpy；And the very few deposition of pig body fat then brings poor meat quality, hypoimmunity, easy infection disease A series of problems, such as disease and reproductive performance decline.Chinese native pig breed is mostly higher fatty acid, referred to as lard type；Introduce the thin of pig kind Meat rate is high, referred to as bacon hogs.It is various that the hereditary difference of pig kind carcass lipid composition is related to gene, and gene regulation is complicated, always It is one of problem of genetic breeding area research.
In recent years, mRNA differential displays, biochip technology, transcript profile high-flux sequence, DNA methylation high pass measure Sequence etc. be widely used to correlation function gene screening field (Bulow et al., 2008；Weber et al.,2008； Mingzhou Li,et al.,2012).Compared with other genetic screening methodologies, m6A-seq technologies are from mRNA posttranscriptional modification water It is flat to carry out differential gene screening, perform macromolecular-albumen more further with biological function, more can precise positioning differential gene. The RNA modifications that methylate are found in 1974, are a kind of most common post-transcriptional level modifications, are constituted about the three of whole RNA modifications / bis-.In eucaryote, most common mRNA posttranscriptional modifications are that methylating on the 6th N atom of base A occurs to repair Decorations, m6A constitutes about the 0.1%-0.4% of cell mRNA whole adenosine content, i.e., average each mRNA contains in mammal The adenosine of 3-5 6- methyl modification.m6A methylation sites occur mainly in highly conserved RRACH (R=G, A；H=A, C, T) In sequence, it may be played an important role in epigenetic.
A kind of UCPS UCP2 of discovered in recent years has certain regulating and controlling effect to fat deposition, while is surveyed according to early stage Sequence finds UCP2 mRNA m6There is larger difference in A methylation levels, it is therefore necessary to right in lard type and bacon hogs UCP2 mRNA m6A methylation sites, function identified, for label of pig fat deposition description inhereditary feature improvement provide it is a kind of new Molecular labeling.
The content of the invention
It is an object of the present invention to provide a kind of UCP2 mRNA m related to label of pig fat deposition description6A methylation sites authentication method, One kind detection UCP2 mRNA m6Method, one kind of A methylation level relative differents change UCP2 mRNA m in gene level6A The method of methylation level, UCP2 mRNA m6A methylation sites are to fat deposition Function Identification method.
The purpose of the present invention is achieved through the following technical solutions：
Label of pig fat deposition description correlation UCP2 mRNA m6A methylates the authentication method of unit point, according to m6A-seq is sequenced to obtain Be 100nt or so fragment in m be present6A methylation sites, it is characterised in that：According to the RRACH (R for methylating highly conserved =G, A；H=A, C, T) sequence carry out UCP2 genes unit point position confirmation.
On the premise of amino acid sequence is not changed, change in UCP2 genes and contain m63rd base of A codeword triplets, I.e. C257 is mutated into T257, so as to change UCP2 mRNA m6A methylation levels；Its principle is：A sites are positioned at codon Second, in order to not change amino acid sequence, can only Mutated codons the 3rd realize same sense mutation；And m6A A is position In conserved sequence GGACU, the formation that the C behind A methylates above for A is critically important, and C mutation can change methylating for A Efficiency, the reduction that methylates is caused, so as to reach the purpose for changing methylation level.
One kind detection UCP2 mRNA m6The method of A methylation level relative differents, it is characterised in that：
(1) after by (UCP2-WT) before being mutated and after being mutated, the plasmid of (UCP2-MUT) is transferred to cell 24 hours, from thin Total RNA are extracted in born of the same parents, carry out RNA fragmentation；
(2) m6A antibody is utilized, by the RNA fragment immunoprecipitations containing m6A decorating sites；
(3) by after the RNA extractions of precipitation, reverse transcription and real time fluorescent quantitative technology are carried out, it is possible to obtain UCP2 mutation The front and rear relative level to methylate.
Fluorescence is separately designed according to totally 21 bases before and after UCP2 methylation sites A256, and non-methylation sites region Quantitative primer：
pUCP2-m6A-peak-F、5’-CTGCCACTGTGAAGTTCCT-3’；
pUCP2-m6A-peak-R、5’-GATCTGCAGCCGGACTTTA-3’；
pUCP2-m6A-nonpeak-F、5’-TCTACAAAGGGTTCACGCCC-3’；
pUCP2-m6A-nonpeak-R、5’-CTCAAAAGGGAGCCTCTCGG-3’。
According to detection UCP2 mRNA m6The method of A methylation level relative differents, has carried out UCP2 mRNAm6A methylates Identification of the site to fat deposition function：
(1) porcine preadipocyte is transferred to by (UCP2-MUT) plasmid after (UCP2-WT) before being mutated and mutation；
After (2) 48 hours, carry out porcine preadipocyte is induced to differentiate into mature fat cell；
(3) shadows of the UCP2-WT and UCP2-MUT to fat deposition is proved using oil red O stain and fluorescence quantifying PCR method Ring, there is facilitation to fat deposition so as to accurately demonstrate UCP2 mRNA m6A sites.
According to detection UCP2 mRNA m6The method of A methylation level relative differents, UCP2 mRNA m6A sites are carried out Influence to UCP2 protein expressions：
(1) porcine preadipocyte is transferred to by (UCP2-MUT) plasmid after (UCP2-WT) before being mutated and mutation；
After (2) 48 hours, cell is collected, extracts total protein in cell；
(3) differential expression using Western blot method validation UCP2-WT and UCP2-MUT to UCP2 albumen, so as to It is inhibited to UCP2 protein expressions accurately to demonstrate UCP2 mRNA m6A sites.
According to detection UCP2 mRNA m6The method of A methylation level relative differents, UCP2 mRNA m are carried out6A sites Influence to UCP2 mRNA stabilities：
(1) UCP2-WT and UCP2-MUT are transfected into porcine preadipocyte respectively；
After (2) 24 hours, 5 μM of actinomycin D treatments 0 for suppressing mRNA transcriptions are added in the nutrient solution of cell, 3,6 is small When after extract cell total rna, with qRT-PCR detect UCP2 gene expression amount, so as to compare UCP2-WT and UCP2-MUT genes The difference of expression quantity, mRNA stability is calculated, it is stable to UCP2 mRNA so as to accurately demonstrate UCP2 mRNA m6A sites Property is inhibited.
The beneficial effects of the present invention are：1st, by bioinformatic analysis, according to m6A conserved sequence, m6A is identified Specific site；2nd, the primer sequence and specific method that UCP2 mRNA m6A methylation levels are identified in pig are provided；3rd, lead to Cross Gene unit point mutation and change the function that UCP2 m6A levels demonstrate influence of the UCP2 m6A sites to fat deposition；4th, table Bright UCP2 m6A methylation sites play an important role in fat deposition, can be as the new molecular labeling for the treatment of obesity And drug target, molecular breeding or transgenosis simultaneously for pig are provided with the genetic resources of benefit.
Brief description of the drawings：
Fig. 1：Landrace (bacon hogs) and Jinhua Pigs (lard type) UCP2 mRNA m6A methylation sites and methylate
Horizontal difference；
Fig. 2：The UCP2 mRNA m of pig6The particular location of A methylation sites；
Fig. 3：Change the UCP2 mRNA m of pig6The single-site mutant of A methylation levels
Fig. 4：UCP2 mRNA m after point mutation6The change of A methylation levels
Fig. 5：Influence after UCP2 mRNA point mutation to fat deposition
Fig. 6：UCP2 protein expressions are influenceed after UCP2 mRNA point mutation
Fig. 7：Influence after UCP2 mRNA point mutation to UCP2 mRNA stabilities
Embodiment
With reference to embodiments, the embodiment of the present invention is described in further detail.People in the art Member can make a variety of change and modifications without departing from this hair when being broadly described to the present invention shown in specific embodiment Bright spirit and scope.
Embodiment 1：Landrace and Jinhua Pigs UCP2 mRNA m6The difference of A methylation levels
Experiment is as follows with adipose tissues of the RNA from Landrace and Jinhua Pigs, concrete operations：
1st, total tissue RNA is obtained
Adipose Tissue sample is taken, extracts total serum IgE (conventional method) using Trizol methods, specific method is：
1) muscle tissue sample frozen in liquid nitrogen is taken out, takes 50-100mg grind into powders in liquid nitrogen, be placed in In RNase-free 1.5ml eppendorf pipes；
2) add 1mlTrizol and jiggle greatly to uniform solution；
3) after adding 200ul chloroforms, uniform solution, 4C, 12000g centrifugation 15min are jiggled greatly to；
4) supernatant is taken into another RNase-free 1.5ml eppendorf pipes, adds same volume isopropanol, gently It is light to mix, it is incubated at room temperature 10min, 4C, 12000g centrifugation 10min；
5) remove supernatant, precipitation, 4C, 7500g centrifugation 5min are washed with the ethanol of 1ml 75%
6) dissolved with 20-50ul RNase-free H2O.
2nd, mRNA is obtained
MRNA is extracted using Genelute mRNA miniprep Kits (Sigma), specific method is：
1) total RNA volumes are expanded to 250ul, add 250ul binding solution, mixed；
2) 15ul beads are added, jiggle greatly to uniform solution, after being placed in 70C incubations 3min, are placed in room temperature 10min；
3) maximum velocity centrifugation 2min, supernatant is discarded；
4) beads is resuspended with 500ul wash buffer, and is transferred to spin filter；
5) 1-2min is centrifuged, discards flow through；
6) repeat 4) 5)
7) Elution Buffer 50ul, the 70C incubations 2-5min for being previously heated to 70C is added
8) it is mRNA to centrifuge product
9) gained mRNA is utilized and is concentrated in vacuo to 9ul
3rd, fragmentation mRNA
RNA fragmentation systems
Temperature, incubation time are：70 DEG C, 15min, add EDTA terminating reactions
*Fragmentation buffer (10X) is：800 μ L RNase-free water, 100 μ L 1M Tris-HCl (pH=7.0), 100μL 1M ZnCl2Solution.
4th, fragmentation mRNA detection
Sample after fragmentation is subjected to 1*TAE electrophoresis detections clip size (Fig. 2), piece in 2% agarose gel electrophoresis Section properly and after being concentrated into 1ug/100ul carries out follow-up test in 100-200bp, size.
5th, immunoprecipitation
1) reagent prepares
10%Igepal CA-630 (Sigma-Aldrich, cat.no.I8896)
5*IP buffer
Fresh 1*IP buffer：
(#SUPERase·InTMRNase Inhibitor(20U/μL)Thermo Fisher(AM2696))
M6A competitive elution liquid
(#N6-Methyladenosine,50-monophosphate sodium salt(m6A,Sigma-Aldrich, cat.no.M2780))
1) mRNA m6A is immune combines
By the RNA fragments of gained in 4, tests below is carried out：
mRNA fragmentation 100μl
5×IP buffer 40μl
RNAase inhibitor 10μl
M6A-antibody(0.5mg/ml) 5-8μl
RNAase free water 42-45μl
Total 200μl
2h is incubated at 4 DEG C
2) close beads (Protein A, Life technologies, 10002D)
Take 40 μ l beads to be placed on magnetic frame, supernatant discarding, washed three times with 1ml 1*IP buffer, be resuspended in 1*IP Close in buffer, 2h is incubated in 4 DEG C.1*IP closings buffer is formulated as follows：
5×IP buffer 200μl
RNAase inhibitor 10μl
BSA 25μl
RNAase free water 765μl
Total 1000μl
After being incubated 2h, washed three times with 1ml 1*IP buffer, be resuspended with 100 μ l 1*IP buffer；
3) beads precipitates with mRNA- antibody mediated immunities
2) and 3) the above-mentioned sample obtained is mixed, 4 DEG C of incubation 2h.
4) I is eluted
4) gained sample is placed on magnetic frame, supernatant is abandoned, is washed three times with 500 μ l 1*IP buffer, be resuspended in elution Buffer, it is placed in 4 DEG C of incubation 1h.Elution buffer is formulated as follows：
5) II is eluted
5) gained sample is placed on magnetic frame, collects supernatant, 50ul elution buffer are then added in beads, It is placed in 4 DEG C of incubation 30min；
6) it will 5), 6) be merged together, obtain 200ul eluents；
7) ethanol precipitation：By 200ul RNA eluent+20ul NaoAc+500ul ethanol (100%)+4ul glycogen (5mg/ml), -80 DEG C, overnight precipitation；
8) it second day, will 8) take out, and in 4C, 14000rpm centrifugation 30min, abandon supernatant, and add 1ml75% ethanol in 4C, 14000rpm centrifuges 30min, abandons supernatant, after air-drying, adds 9ul RNase-free water, and determine concentration.
6th, Jian Ku
Storehouse kit is built using Illumina TruSeq Stranded mRNA Library Prep Kit,
1) the first chain synthesizes：
Take 5-9) in 6-7ul RNA, add 10-11ul FPF mix obtain 17ul mixtures；Stood after 94 DEG C are heated 10s It is placed on ice.
By PCR instrument is put into after following mixing, by 25 DEG C of 10min, 42 DEG C of 15min, 70 DEG C of 15min, 4 DEG C of holdings are tried Test.
17μl mixture 17μl
First strand synthesis Act D mix(FSA) 7.2μl
Superscript II reverse transcriptase 0.8μl
Total 25μl
(Superscript II reverse transcriptase invitrogen 18064-014)
2) the second chain synthesizes：
1) PCR instrument is put into after the product in is mixed by following system, by 16 DEG C of 1h, 4 DEG C remain on：
3) purified using magnetic bead to 2) AMPure XP and add 90ul AMPure XP in cDNA in 50ul cDNA It is transferred to after beads, room temperature placement 15min on magnetic frame and separates 5min, 135ul supernatants is abandoned in suction, add 200ul 80% second After alcohol washes magnetic bead 2 times, room temperature places 5min, and wait is dried；Add 20ul elution buffer stationary incubations at room temperature 2min, it is transferred on magnetic frame and places 5min, 17.5ul supernatants is transferred in new pipe；
4) ends of cDNA 3 ' of synthesis plus A bases
12.5ul A-tailing mix are added in 3) in 17.5ul cDNA solution；30ul mixtures is anti-at 37 DEG C After answering 30min, 70 DEG C of reaction 5min, 4 DEG C are maintained at.
5) jointing
It is formulated as follows after system and reacts 10min at 30 DEG C, adds 5ul stop ligation buffer terminating reactions.
dscDNA from 4) 30μl
Ligation mix 2.5μl
RNA adapter index 2.5μl
resuspension buffer 2.5ul
Total 37.5μl
6) first time purification of samples
42ul AMPurea XP beads are added in 5), room temperature places 15min, is transferred to magnetic frame and places 5min, inhales 79.5ul supernatants are abandoned, after the addition ethanol of 200ul 80% washes magnetic bead twice, room temperature dries 5min；Add 52.5ul Resuspension buffer, room temperature are transferred to magnetic frame 5min after placing 2min, take 50ul supernatants to be transferred in new pipe；
7) second of purification of samples
50ul AMPurea XP beads are added to 6) middle, room temperature places 15min, is transferred to magnetic frame and places 5min, inhales 95ul supernatants are abandoned, after the addition ethanol of 200ul 80% washes magnetic bead twice, room temperature dries 5min；Add 22.5ul Resuspension buffer, room temperature are transferred to magnetic frame 5min after placing 2min, take 20ul supernatants to be transferred in new pipe；
8) PCR is expanded
Reaction system is put into PCR instrument after being formulated as follows, by 98 DEG C of 30s；98℃,10s,60℃,30s,72℃,30s, 13-15cycles；72℃,5min；4 DEG C of setting PCR programs of Hold at
20μl product from 7 20μl
PCR primer cocktail 5μl
PCR master mix 25μl
Total 50μl
9) purified pcr product
50ul AMPurea XP beads are added into 50ul PCR primers 8), room temperature places 15min, is transferred to magnetic Power frame places 5min, and 95ul supernatants are abandoned in suction, and after the addition ethanol of 200ul 80% washes magnetic bead twice, room temperature dries 5min；Add 32.5ul resuspension buffer, room temperature are transferred to magnetic frame 5min after placing 2min, take 20ul supernatants to be transferred to newly Guan Zhong, concentration is determined with Qubit.
7th, sequencing and bioinformatic analysis
1) Bioanalyzer is tested and analyzed.
2) high-flux sequence is carried out using the platforms of illumina companies Hiseq 4000, and has carried out biological information credit Analysis, has obtained the UCP2 mRNA of Landrace and Jinhua Pigs m6Significant difference (Fig. 1) be present in A levels
3) according to gene order and prediction website (http://www.cuilab.cn) UCP2 mRNA m is determined6A positions In the 256th (Fig. 2)
Embodiment 2：Change the UCP2 mRNA m of pig6The single-site mutant of A methylation levels
(explanation：Because A sites are the seconds positioned at codon, in order to not change amino acid sequence, password can only be mutated Son the 3rd realizes same sense mutation；And m6A A is located in conserved sequence GGACU, and the C behind A is for methyl above A The formation of change is critically important, and C mutation can change the A efficiency that methylates, and cause the reduction that methylates, and is methylated water so as to reach change Flat purpose.)
Clone pig UCP2 genes (NM_214289.1) sequence and C257 sport T257 mutant nucleotide sequence, are added in N-terminal FLAG sequences (5 '-GACTACAAGGACGATGATGACAAG-3 '), it is cloned into the HindIII of Pcdna3.1 (+) expression plasmid And BamHI sites, obtain UCP2-WT and UCP2-MUT plasmids (cloning procedure belongs to routine operation, and specific steps are omited).
Embodiment 3：The change of UCP2 mRNA m6A methylation levels after point mutation
1. design of primers：
Upstream and downstream in mutational site designs fluorescence quantification PCR primer, and giving birth to work biology Co., Ltd by Shanghai synthesizes, such as Under：
2. the separation and culture of porcine preadipocyte
The acquisition of porcine preadipocyte slightly improves with reference to the method for (1999) and Cheong Kuoc Va etc. (2005) such as Ding： Aseptically, 5~7 days (d) piglet adipose tissues are taken, with the dual anti-PBS immersions containing high concentration, rinses, removes visible Blood vessel and muscle.Tissue block of uniform size is cut into scissors, is placed in aseptic bottle, is vibrated with digestive juice in 37 DEG C of water-baths Digest 1.0h in pot, digestion is used after terminating in isometric complete medium and digestive juice, digest once by 200 mesh with 300 mesh nylon mesh, collect filtered fluid, 1500rpm/min centrifugations 10min.Supernatant is abandoned, adds erythrocyte cracked liquid, piping and druming is equal It is stored at room temperature 10min after even, 1500rpm/min centrifugation 5min abandon supernatant, then are resuspended cell with complete medium, centrifugation 1~ 2 times, complete medium is eventually adding, piping and druming is uniform, produces porcine preadipocyte.
3. UCP2-WT and UCP2-MUT are transfected into porcine preadipocyte respectively：
Use LipofectamineTM2000 reagents (Invitrogen companies) by UCP2-WT and UCP2-MUT plasmids and Empty plasmid is mediated into porcine preadipocyte.Transfection method is carried out with reference to the kit specifications of LipofectamineTM 2000. Key step is as follows：
1) day before transfection, trypsin digestion cell simultaneously count, antibiotic-free complete medium culture, when cell density is 70%-90% is transfected；
2) per hole cell, 3 μ g DNA are diluted with the culture mediums of 50 μ l OPTI-MEM I；
3) reagents of 10 μ l LIPOFECTAMINE 2000 are diluted with the culture mediums of 50 μ l OPTI-MEM I, room temperature places 5 points Clock；
4) DNA (by the 2nd step) of the mixed diluting and LIPOFECTAMINE 2000 (by the 3rd step) of dilution, is protected in room temperature Warm 20min；
5) directly compound is added in every hole cell, shakes culture plate, gently mix, often handle in triplicate；
6) complete medium without antibiotic is changed after 4-6h is incubated in 37 DEG C, 5% CO2；
7) after adding compound 24h in cell, cell is collected.
4. Total RNAs extraction, chemical disruption, immunoprecipitation, reverse transcription (step is same as above)
5. real-time fluorescence relative quantification PCR
This experiment reaction system is sample cDNA1 μ l, the SYBR Green Real-time PCR Master of 5 times of dilutions The μ l of Mix 5, aseptic double-distilled water 3 μ l, each 0.5ul of target gene upstream and downstream primer (concentration is 1 μM), overall reaction system is 10 μ l. PCR reaction conditions are 95 DEG C of pre-degenerations 2min-95 DEG C of 20s, 64 DEG C of 20s, 72 DEG C of 30s, repeat 45 circulations.Internal reference selects ATCB, expression quantity of the gene in Input, interpretation of result use 2-△△ctMethod, wherein △ △ ct calculation formula is as follows： △ △ Ct=(CtTarget-CtInput)x-(CtTarget-CtInput)Control
As a result as shown in figure 4, obtaining UCP2 mRNA m after point mutation6The change of A methylation levels.
Embodiment 4：UCP2 mRNA m6Influence of the change of A methylation levels to fat deposition
1. the separation and culture (being same as above) of porcine preadipocyte
2. UCP2-WT and UCP2-MUT are transfected into porcine preadipocyte (method is same as above) respectively
3. the induction differentiation of porcine preadipocyte
Start to induce after cell merges 48h completely, it is fast to add final concentration of 0.5mmol/L 3- isobutyl groups -1- methyl yellows Purine (IBMX), 1 μm of ol/L dexamethasone (DEX), 1.7 μm of ol/L insulin (INS) are to be added in 500ml complete medium 5.60ml 50mM IBMX, 0.56ml DEX, 1.54ml 40IU/ml Insulin complete medium culture 48h, change with The complete medium of the insulin containing 10mg/L is further cultured for 48h, finally changes complete medium and continues to cultivate, and 2d changes liquid once, until There are fat drips in 90% cell.
4. oil red O stain is identified
After porcine preadipocyte induced maturation, complete medium is discarded, is washed 3 times with PBS.With 10% formalin room temperature Lower fixed cell 1h, discards fixer.60% isopropanol is washed 2 times, is added oil red O solution, is dyed 30min-1h at room temperature, discard Dyeing liquor, uncoloured dyeing liquor is washed away with PBS, is observed under inverted microscope.
5. quantitative fluorescent PCR (method is same as above)
As a result as shown in figure 5, UCP2 mRNA m after point mutation6The heavy of fat is further suppress after A methylation levels Product, while fat deposition related gene PPAR γ, FABP4 and C/EBP α expression are also inhibited.
Embodiment 5：UCP2 mRNA m6The change of A methylation levels influences on UCP2 protein expressions
1. the separation and culture (being same as above) of porcine preadipocyte
2. UCP2-WT and UCP2-MUT are transfected into porcine preadipocyte (are same as above) respectively
3.Western blot are detected
(1) 6 orifice plate per hole add 200 μ L cell pyrolysis liquids, place 30min on ice, cell scrapes, be put into 1.5ml from Heart pipe；12000rpm, centrifuges 10min, takes supernatant by 4 DEG C, and protein concentration is carried out using triumphant base BCA protein contents detection kit Measure；
(2) SDS-PAGE electrophoresis：Glass plate, encapsulating and loading are cleaned, voltage when concentrating glue, 80v half an hours, separation Glue 110v 1.5h.
(3) transferring film：Filter paper, two blocks of foam-rubber cushions and film are immersed in transferring film liquid and soaked, according to " sandwich " principle Foam-rubber cushion, filter paper are put, is up foam-rubber cushion, filter paper, film, gel, filter paper, foam-rubber cushion successively from clip white one side, uses Glass bar being rolled on the superiors' foam-rubber cushion gently, drives the bubble in " sandwich " away；The folder of film and gel will be installed Son is carefully placed into transfer groove, it is ensured that the black flour of one side face groove of clip black, the red face of white one side face groove.Separately Outside, during transferring film can a large amount of heat production, it is necessary to be put into ice block cooling.Typically flow over 300A and shift 1.5h.
(4) immune response：By film 5% skim milk (5% skimmed milk power and TBST configuration forms), about 7- 8ml closes 1h on shaking table；Skim milk in box is poured out and rejoins the skim milk containing primary antibody, after shaking table shakes up, 4 DEG C refrigerator overnight；Next day, primary antibody dilution is discarded, cleaned 3 times with TBST, each 5min；Primary antibody mistake is incubated with secondary antibody dilution The film at night, room temperature about 1 hour, secondary antibody dilution is discarded, TBST is cleaned 3 times, each 5min.TBST is discarded, adds chemiluminescence Liquid, observation are taken pictures.
As a result as shown in fig. 6, UCP2 mRNAm after point mutation6Promote the expression of UCP2 albumen after A methylation levels.
Embodiment 6：UCP2 mRNA m6Influence of the change of A methylation levels to UCP2 mRNA stabilities
1. the separation and culture (being same as above) of porcine preadipocyte
2. UCP2-WT and UCP2-MUT are transfected into porcine preadipocyte (are same as above) respectively
The detection of 3.mRNA stability
24h is transfected, 5 μM of actinomycin D treatments for suppressing mRNA transcriptions are added in the nutrient solution of cell after 0,3,6 hours Cell total rna is extracted, UCP2 gene expression amount is detected with qRT-PCR, so as to compare UCP2-WT and UCP2-MUT gene expressions The difference of amount, calculate mRNA stability.
As a result as shown in fig. 7, UCP2 mRNA m after point mutation6UCP2 mRNA stabilities are improved after A methylation levels. Finally, it is also necessary to it is noted that listed above is only several specific embodiments of the invention.It is clear that the invention is not restricted to Above example, there can also be many deformations.One of ordinary skill in the art can directly export from present disclosure Or all deformations associated, it is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Zhejiang University
<120>Label of pig fat deposition description correlation UCP2 mRNA m6A methylate the authentication method and application of function of unit point
<130> 2017.8.27
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 930
<212> DNA
<213> Sus scrofa
<400> 1
atggttggat tcaaggccac agaggtcccc ccgactgcca ctgtgaagtt cctgggggct 60
ggcacagctg cctgcatcgc agacctcatc acctttcccc tggacacggc taaagtccgg 120
ctgcagatcc agggagaaag gcgggggcca gtgcaggccg cggccagtgc ccagtaccgc 180
ggggtgctgg gcaccattct caccatggtg cgcaacgagg gcccccgcag cctctacaac 240
gggctggtgg ccggcctgca gcgccagatg agcttcgcct ccgtccgcat cggcctctac 300
gactccgtca agcatttcta caccaagggc tcagagcatg ctggcatcgg gagccgcctc 360
ctggcaggca gcaccacggg ggccttggct gtggccgtgg cccagccaac agacgtggta 420
aaggtccggt tccaagcgca ggcccgggcc ggcggaggcc ggcggtaccg gagcactgtc 480
gacgcctaca agaccatcgc ccgagaggag gggctgcggg gcctctggaa agggacctca 540
cccaatgtcg ctcgtaatgc cattgtcaac tgtgctgagc tggtgaccta tgacctcatc 600
aaggacacgc tcctgaaggc cgacctcatg acagatgacc ttccctgcca cttcacgtcc 660
gccttcgggg cgggcttctg caccaccgtc atcgcctctc ccgtggacgt ggtcaagacg 720
agatacatga actctgcccc gggccagtac agcagcgctg gccactgtgc cctcaccatg 780
ctccagaagg agggtccccg agccttctac aaagggttca cgccctcctt tctccgattg 840
gggtcctgga acgtggtgat gtttgtcacc tatgagcagc tgaagagggc cctcatggct 900
gcccgcgctt cccgggaggc tcccttttga 930

Claims (7)

1. label of pig fat deposition description correlation UCP2 mRNA m6A methylates the authentication method of unit point, according to m6A-seq is sequenced what is obtained M be present in the fragment for being 100nt or so6A methylation sites, it is characterised in that：According to the RRACH (R=for methylating highly conserved G,A；H=A, C, T) sequence carry out UCP2 genes unit point position confirmation.
2. label of pig fat deposition description correlation UCP2 mRNA m according to claim 16A methylates the authentication method of unit point, its It is characterised by：On the premise of amino acid sequence is not changed, change in UCP2 genes and contain m63rd alkali of A codeword triplets Base, i.e. C257 are mutated into T257, so as to change UCP2 mRNA m6A methylation levels；Its principle is：A sites are to be located at password The second of son, in order to not change amino acid sequence, can only Mutated codons the 3rd realize same sense mutation；And m6A A It is to be located in conserved sequence GGACU, the formation that the C behind A methylates above for A is critically important, and C mutation can change A first Base efficiency, the reduction that methylates is caused, so as to reach the purpose for changing methylation level.
3. one kind detection UCP2 mRNA m6The method of A methylation level relative differents, it is characterised in that：
(1) after being transferred to cell 24 hours by the plasmid of (UCP2-MUT) after (UCP2-WT) before being mutated and mutation, from cell Total RNA are extracted, carry out RNA fragmentation；
(2) m6A antibody is utilized, by the RNA fragment immunoprecipitations containing m6A decorating sites；
(3) by after the RNA of precipitation extraction, reverse transcription and real time fluorescent quantitative technology are carried out, it is possible to before and after obtaining UCP2 mutation The relative level to methylate.
4. detection UCP2 mRNA m according to claim 36The method of A methylation level relative differents, it is characterised in that Separately design fluorescent quantitation according to totally 21 bases before and after UCP2 methylation sites A256, and non-methylation sites region and draw Thing：
pUCP2-m6A-peak-F、5’-CTGCCACTGTGAAGTTCCT-3’；
pUCP2-m6A-peak-R、5’-GATCTGCAGCCGGACTTTA-3’；
pUCP2-m6A-nonpeak-F、5’-TCTACAAAGGGTTCACGCCC-3’；
pUCP2-m6A-nonpeak-R、5’-CTCAAAAGGGAGCCTCTCGG-3’。
5. detection UCP2 mRNA m according to claim 36The method of A methylation level relative differents, has carried out UCP2 mRNAm6Identification of the A methylation sites to fat deposition function：
(1) porcine preadipocyte is transferred to by (UCP2-MUT) plasmid after (UCP2-WT) before being mutated and mutation；
After (2) 48 hours, carry out porcine preadipocyte is induced to differentiate into mature fat cell；
(3) influences of the UCP2-WT and UCP2-MUT to fat deposition is proved using oil red O stain and fluorescence quantifying PCR method, from And accurately demonstrate UCP2 mRNA m6A sites has facilitation to fat deposition.
6. detection UCP2 mRNA m according to claim 36The method of A methylation level relative differents, has carried out UCP2 Influence of the mRNA m6A sites to UCP2 protein expressions：
(1) porcine preadipocyte is transferred to by (UCP2-MUT) plasmid after (UCP2-WT) before being mutated and mutation；
After (2) 48 hours, cell is collected, extracts total protein in cell；
(3) differential expression using Western blot method validation UCP2-WT and UCP2-MUT to UCP2 albumen, so as to accurate To demonstrate UCP2 mRNA m6A sites inhibited to UCP2 protein expressions.
7. detection UCP2 mRNA m according to claim 36The method of A methylation level relative differents, has carried out UCP2 mRNA m6Influence of the A sites to UCP2 mRNA stabilities：
(1) UCP2-WT and UCP2-MUT are transfected into porcine preadipocyte respectively；
After (2) 24 hours, 5 μM of actinomycin D treatments for suppressing mRNA transcriptions are added in the nutrient solution of cell after 0,3,6 hours Cell total rna is extracted, UCP2 gene expression amount is detected with qRT-PCR, so as to compare UCP2-WT and UCP2-MUT gene expressions The difference of amount, mRNA stability is calculated, had so as to accurately demonstrate UCP2 mRNA m6A sites to UCP2 mRNA stabilities There is inhibitory action.
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