CN107419026B

CN107419026B - Identification method and functional application of UCP2mRNA m6A methylation unit site related to pig fat deposition

Info

Publication number: CN107419026B
Application number: CN201710760315.XA
Authority: CN
Inventors: 王新霞; 汪以真; 江芹; 吴睿帆; 姚永曦; 蔡旻; 刘卿
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang Qinglian Food Co Ltd
Priority date: 2017-08-30
Filing date: 2017-08-30
Publication date: 2020-12-22
Anticipated expiration: 2037-08-30
Also published as: CN107419026A

Abstract

UCP2mRNA m related to pig fat deposition⁶Method for identifying A methylated Unit sites according to m⁶The sequence of A-seq shows that m is present in about 100nt of the fragment⁶A methylation site characterized by: the single-site position of the UCP2 gene was confirmed by the sequence of RRACH (R ═ G, A; H ═ A, C, T) with highly conserved methylation. Through bioinformatics analysis, a specific site of m6A is identified according to the conserved sequence of m 6A; provides a primer sequence and a specific method for identifying the methylation level of UCP2mRNA m6A in pigs; the function of the effect of UCP2m 6A site on fat deposition was demonstrated by altering UCP2m 6A levels by single-site mutation of the gene.

Description

Identification method and functional application of UCP2mRNA m6A methylation unit site related to pig fat deposition

Technical Field

The invention belongs to the field of molecular biology, and relates to a single gene mRNA m⁶Identification, function and application of A methylation site, in particular to UCP2mRNA m⁶Identification, function and application of A methylation sites.

Background

The reasonable deposition and regulation of pig fat are always the focus of animal husbandry. Excessive deposition of pig fat not only reduces the nutrient utilization efficiency, but also directly affects the health of consumers due to excessive body fat deposition, including metabolic diseases such as diabetes, cardiovascular diseases and the like caused by obesity; and the fat deposition of the pig body causes a series of problems of poor pork quality, low immunity, susceptibility to diseases, reduction of reproductive performance and the like. Local pigs in China are mostly high-fat and are called fatty pigs; the lean meat percentage of the introduced pig species is high, and the pig is called a lean type pig. The genetic difference of the pig carcass fat composition relates to the gene diversity and the gene regulation complexity, and is one of the difficult problems in the research of the genetic breeding field.

In recent years, mRNA difference display methods, gene chip technologies, transcriptome high-throughput sequencing, DNA methylation high-throughput sequencing, and the like have been widely used in the field of screening of related functional genes (bloom et al, 2008; Weber et al, 2008; Mingzhou Li, et al, 2012). Compared with other gene screening methods, m⁶The A-seq technology carries out differential gene screening from the mRNA post-transcriptional modification level, is closer to the execution of macromolecules-proteins of biological functions, and can accurately position differential genes. RNA methylation modification was found in 1974 to be one of the most common post-transcriptional level modifications, accounting for approximately two-thirds of all RNA modifications. In eukaryotes, the most common post-transcriptional modification of mRNA is a methylation modification occurring at the N atom at position 6 of the base A, m⁶A is about 0.1% to 0.4% of the total adenosine content of cellular mRNA, i.e., each mRNA in a mammal contains on average 3-5 6-methyl modified adenosines. m is⁶A methylation sites occur mainly in the highly conserved RRACH (R ═ G, A; H ═ A, C, T) sequence, and may play an important role in epigenetic inheritance.

Recently, uncoupling protein UCP2 is found to have a certain regulation and control effect on fat deposition, and m of UCP2mRNA is found according to early sequencing⁶A methylation level is largely different between fat and lean pigs, so it is necessary to control m of UCP2mRNA⁶A methylation sites and functions are identified, and a new molecular marker is provided for improvement of the pig fat deposition hereditary character.

Disclosure of Invention

The invention aims to provide UCP2mRNA m related to pig fat deposition⁶A methylation site identification method and detection methodMeasurement of UCP2mRNA m⁶Method for relative difference of A methylation level, method for changing UCP2mRNA m at gene level⁶Method for A methylation level, UCP2mRNA m⁶A methylation site identification method for fat deposition function.

The purpose of the invention is realized by the following technical scheme:

UCP2mRNA m related to pig fat deposition⁶Method for identifying A methylated Unit sites according to m⁶The sequence of A-seq shows that m is present in about 100nt of the fragment⁶A methylation site characterized by: the single-site position of the UCP2 gene was confirmed by the sequence of RRACH (R ═ G, A; H ═ A, C, T) with highly conserved methylation.

Changing m in UCP2 gene without changing amino acid sequence⁶Mutation of the third base of the A triplet codon, C257, to T257, altered UCP2mRNA m⁶A methylation level; the principle is as follows: the A site is positioned at the second position of the codon, and in order to not change the amino acid sequence, only the third position of the codon can be mutated to realize synonymous mutation; and m⁶A of A is positioned in a conserved sequence GGACU, C behind A is important for the formation of methylation on A, and mutation of C can change the methylation efficiency of A, cause methylation reduction and further achieve the aim of changing the methylation level.

Method for detecting UCP2mRNA m⁶A method for the relative difference in methylation levels, characterized by:

(1) fragmenting RNA by transferring plasmids before mutation (UCP2-WT) and after mutation (UCP2-MUT) into cells for 24 hours, and extracting total RNA from the cells;

(2) immunoprecipitating an RNA fragment containing the m6A modification site using the m6A antibody;

(3) after the precipitated RNA is extracted, the relative levels of methylation before and after mutation of UCP2 can be obtained by reverse transcription and real-time fluorescence quantification techniques.

Respectively designing fluorescent quantitative primers according to 21 bases in front and back of a methylation site A256 of UCP2 and an unmethylated site region:

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 the detection of UCP2mRNA m⁶A method for relative difference of methylation level comprises performing UCP2 mRNAm⁶Identification of fat deposition function by A methylation site:

(1) by introducing pre-mutation (UCP2-WT) and post-mutation (UCP2-MUT) plasmids into porcine preadipocytes;

(2) after 48 hours, inducing and differentiating the pig precursor fat cells into mature fat cells;

(3) the influence of UCP2-WT and UCP2-MUT on fat deposition is proved by oil red O staining and a fluorescent quantitative PCR method, so that the promotion effect of UCP2mRNA m6A locus on fat deposition is accurately proved.

According to the detection of UCP2mRNA m⁶A methylation level relative difference method is carried out, and the influence of UCP2mRNA m6A site on UCP2 protein expression is carried out:

(2) after 48 hours, collecting cells, and extracting total protein in the cells;

(3) the expression difference of UCP2-WT and UCP2-MUT to UCP2 protein is verified by using a Western blot method, so that the m6A locus of UCP2mRNA has an inhibition effect on UCP2 protein expression.

According to the detection of UCP2mRNA m⁶A method for relative differences in methylation levels of UCP2mRNA m⁶Effect of a site on UCP2mRNA stability:

(1) transfecting UCP2-WT and UCP2-MUT into porcine preadipocytes respectively;

(2) after 24 hours, actinomycin D which inhibits mRNA transcription is added into a cell culture solution at 5 mu M for treating for 0, 3 and 6 hours, cell total RNA is extracted, and the gene expression quantity of UCP2 is detected by qRT-PCR (quantitative reverse transcription-polymerase chain reaction), so that the difference of the gene expression quantity of UCP2-WT and UCP2-MUT is compared, and the mRNA stability is calculated, thereby accurately proving that UCP2mRNA M6A locus has an inhibiting effect on the UCP2mRNA stability.

The invention has the beneficial effects that: 1. through bioinformatics analysis, a specific site of m6A is identified according to the conserved sequence of m 6A; 2. provides a primer sequence and a specific method for identifying the methylation level of UCP2mRNA m6A in pigs; 3. the function of the influence of the UCP2m 6A site on fat deposition was demonstrated by altering UCP2m 6A levels by single-site mutation of the gene; 4. the UCP2m 6A methylation site plays an important role in fat deposition, can be used as a new molecular marker and a drug target for treating obesity, and provides a beneficial gene resource for molecular breeding or transgenosis of pigs.

Description of the drawings:

FIG. 1: UCP2mRNA m of Changbai pig (lean pig) and Jinhua pig (fatty pig)⁶A difference in methylation site and methylation level;

FIG. 2: UCP2mRNA m of pig⁶The specific position of the A methylation site;

FIG. 3: altering UCP2mRNA m in pigs⁶Single site mutation of A methylation level

FIG. 4: post-point mutation UCP2mRNA m⁶Change in A methylation level

FIG. 5: effect of UCP2mRNA point mutation on fat deposition

FIG. 6: effect of UCP2mRNA point mutation on UCP2 protein expression

FIG. 7: effect of UCP2mRNA Point mutation on UCP2mRNA stability

Detailed Description

The following will explain in detail specific embodiments of the present invention with reference to examples. Numerous changes and modifications may be made to the invention as shown in the specific embodiments by those skilled in the art without departing from the spirit and scope of the invention as broadly described.

Example 1: UCP2mRNA m of Changbai pig and Jinhua pig⁶Difference in methylation level of A

The experimental RNA is derived from adipose tissues of Changbai pigs and Jinhua pigs, and the specific operation is as follows:

1. obtaining tissue total RNA

Taking a pig adipose tissue sample, and extracting total RNA by using a Trizol method (a conventional method), wherein the specific method comprises the following steps:

1) taking out the muscle tissue sample frozen in liquid nitrogen, taking 50-100mg of the muscle tissue sample, grinding the muscle tissue sample into powder in the liquid nitrogen, and placing the powder into a 1.5ml eppendorf tube of RNase-free;

2) adding 1ml of Trizol and shaking vigorously to obtain a uniform solution;

3) adding 200ul chloroform, shaking vigorously to obtain a uniform solution, and centrifuging at 12000g at 4C for 15 min;

4) taking the supernatant into another RNase-free 1.5ml eppendorf tube, adding isopropanol with the same volume, gently mixing, incubating at room temperature for 10min, and centrifuging at 4C and 12000g for 10 min;

5) removing supernatant, washing precipitate with 1ml 75% ethanol, centrifuging at 4C, 7500g for 5min

6) Dissolving with 20-50ul RNase-free H2O.

2. Obtaining mRNA

mRNA is extracted by using a Genelute mRNA miniprep kit (Sigma), and the specific method comprises the following steps:

1) expanding the volume of total RNA to 250ul, adding 250ul binding solution, and mixing uniformly;

2) adding 15ul beads, shaking to obtain uniform solution, incubating at 70 deg.C for 3min, and standing at room temperature for 10 min;

3) centrifuging at maximum speed for 2min, and discarding supernatant;

4) resuspend beads with 500ul wash buffer and transfer to spin filter;

5) centrifuging for 1-2min, and discarding flow through;

6) repetition 4)5)

7) Adding Elution Buffer 50ul pre-heated to 70C, and incubating at 70C for 2-5min

8) The centrifuged product is mRNA

9) The mRNA obtained was concentrated to 9ul in vacuo

3. Fragmenting mRNA

RNA fragmentation system

The temperature and incubation time were: adding EDTA to stop the reaction at 70 ℃ for 15min

^＊Fragmentation buffer (10 ×) was: 800 μ L of RNase-free water, 100 μ L of 1M Tris-HCl (pH 7.0), 100 μ L of 1M ZnCl₂And (3) solution.

4. Detection of fragmented mRNA

The fragmented samples were subjected to 1 × TAE electrophoresis on 2% agarose gel electrophoresis to determine the fragment size (FIG. 2), and the fragments were subjected to subsequent experiments after 100-200bp, appropriate size and concentration to 1ug/100 ul.

5. Immunoprecipitation

1) Reagent preparation

10％Igepal CA-630(Sigma-Aldrich,cat.no.I8896)

5*IP buffer

Preparing 1 IP buffer:

(#SUPERase·In^TMRNase Inhibitor(20U/μL)Thermo Fisher(AM2696))

m6A competitive eluent

(#N6-Methyladenosine,50-monophosphate sodium salt(m6A,Sigma-Aldrich,cat.no.M2780))

1) m6A immunological binding of mRNA

The RNA fragments obtained in 4 were subjected to the following tests:

mRNA fragmentation	100μl
			5×IP buffer	40μl
RNAase inhibitor	10μl
		M6A-antibody(0.5mg/ml)	5-8μl
RNAase free water	42-45μl
		Total	200μl

incubation at 4 ℃ for 2h

2) (ii) sealing beads: (

Protein A，Life technologies,10002D)

Place 40. mu.l of beads on a magnetic frame, discard the supernatant, wash three times with 1ml of 1 IP buffer, resuspend in 1 IP blocking buffer, incubate for 2h at 4 ℃.1 IP blocking buffer was prepared as follows:

5×IP buffer	200μl
		RNAase inhibitor	10μl
BSA	25μl
		RNAase free water	765μl
Total	1000μl

after 2h incubation, three washes with 1ml of 1 × IP buffer and resuspension with 100 μ l of 1 × IP buffer;

3) beads and mRNA-antibody immunoprecipitation

The samples obtained in 2) and 3) above were mixed and incubated at 4 ℃ for 2 h.

4) Elution I

The sample from 4) was placed on a magnetic rack, the supernatant was discarded, washed three times with 500. mu.l 1 × IP buffer, resuspended in the elution buffer, and incubated at 4 ℃ for 1 h. Elution buffer was prepared as follows:

5×IP buffer	90μl
		RNAase inhibitor	7μl
M6A	150μl
		RNAase free water	203μl
Total	450μl

5) elution II

Placing the sample obtained in the step 5) on a magnetic frame, collecting the supernatant, adding 50ul of elution buffer into the beads, and incubating at 4 ℃ for 30 min;

6) mixing 5) and 6) together to obtain 200ul eluent;

7) ethanol precipitation: 200ul of RNA eluate +20ul of NaoAc +500ul of ethanol (100%) +4ul of glycogen (5mg/ml), 80 ℃ overnight precipitation;

8) the next day, 8) was removed, centrifuged at 14000rpm for 30min at 4C, the supernatant was discarded, 1ml of 75% ethanol was added, centrifuged at 14000rpm for 30min at 4C, the supernatant was discarded, and after air-drying, 9ul of RNase-free water was added and the concentration was measured.

6. Building warehouse

Using a Library Kit of TruSeq Stranded mRNA Library Prep Kit from Illumina,

1) first strand synthesis:

taking 6-7ul RNA in 5-9), adding 10-11ul FPF mix to obtain 17ul mixture; immediately after heating at 94 ℃ for 10s, the plates were placed on ice.

The mixture was placed in a PCR apparatus at 25 ℃ for 10min,42 ℃ for 15min,70 ℃ for 15min, and 4 ℃ for the 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) Second strand synthesis:

mixing the products in the step 1) according to the following system, putting the mixture into a PCR instrument, and keeping the operation at 16 ℃ for 1h and 4 ℃:

3) by using

25μl product from 1.	25μl
		Second strand marking master Mix	20μl
Resuspension buffer	5μl
		Total	50μl

AMPure XP magnetic bead purified cDNA

Adding 90ul AMPure XPbeads into 50ul cDNA in the step 2), standing at room temperature for 15min, transferring to a magnetic frame, separating for 5min, removing 135ul supernatant, adding 200ul 80% ethanol, washing magnetic beads for 2 times, standing at room temperature for 5min, and waiting for air drying; adding 20ul of phase buffer, standing and incubating at room temperature for 2min, transferring to a magnetic frame, standing for 5min, and transferring 17.5ul of supernatant to a new tube;

4) 3' terminal of synthesized cDNA added with A base

Adding 12.5ulA-tailing mix into 17.5ul of cDNA solution in 3); 30ul of the mixture was reacted at 37 ℃ for 30min, and then at 70 ℃ for 5min, and kept at 4 ℃.

5) Connecting joint

After the following system was prepared, the reaction was carried out at 30 ℃ for 10min, and 5ul of stop dilution buffer was added to terminate the reaction.

dscDNA from 4)	30μl
		Ligation mix	2.5μl
RNA adapter index	2.5μl
		resuspension buffer	2.5ul
Total	37.5μl

6) First purifying the sample

Adding 42ul of AMPurea XP beads into the mixture obtained in the step 5), standing at room temperature for 15min, transferring to a magnetic frame, standing for 5min, removing 79.5ul of supernatant, adding 200ul of 80% ethanol, washing magnetic beads twice, and airing at room temperature for 5 min; adding 52.5ul of resuspension buffer, standing at room temperature for 2min, transferring to a magnetic frame for 5min, and transferring 50ul of supernatant to a new tube;

7) second purification of the sample

Adding 50ul AMPurea XP beads into the mixture obtained in the step 6), standing at room temperature for 15min, transferring to a magnetic frame, standing for 5min, removing 95ul of supernatant, adding 200ul of 80% ethanol, washing magnetic beads twice, and airing at room temperature for 5 min; adding 22.5ul of resuspension buffer, standing at room temperature for 2min, transferring to a magnetic frame for 5min, and transferring 20ul of supernatant to a new tube;

8) PCR amplification

The reaction system is prepared as follows and then put into a PCR instrument for 30s at 98 ℃; 98 ℃,10s,60 ℃,30s,72 ℃,30s,13-15 cycles; 72 ℃ for 5 min; hold at 4 ℃ set PCR program

20μl product from 7	20μl
		PCR primer cocktail	5μl
PCR master mix	25μl
		Total	50μl

9) Purification of PCR products

Adding 50ul AMPurea XP beads into 50ul PCR products in the step 8), standing at room temperature for 15min, transferring to a magnetic frame, standing for 5min, removing 95ul supernatant, adding 200ul 80% ethanol, washing magnetic beads twice, and drying at room temperature for 5 min; 32.5ul of resuspension buffer was added, left at room temperature for 2min and then transferred to a magnetic stand for 5min, 20ul of the supernatant was transferred to a new tube, and the concentration was measured with a Qubit.

7. Sequencing and bioinformatics analysis

1) Bioanalyzer assay.

2) High-throughput sequencing is carried out by utilizing Hiseq 4000 platform of illumina company, bioinformatics analysis is carried out, and m of UCP2mRNA of Changbai pigs and Jinhua pigs is obtained⁶There was a significant difference in A levels (FIG. 1)

3) Based on the gene sequence and the predicted website (http://www.cuilab.cn) M of UCP2mRNA was determined⁶A is located at the 256 th position (fig. 2)

Example 2: altering UCP2mRNA m in pigs⁶Single site mutation of A methylation level

(Note: because the A site is located at the second position of the codon, only the third position of the codon can be mutated to realize the synonymous mutation so as not to change the amino acid sequence; and m⁶A of A is positioned in a conserved sequence GGACU, C behind A is important for the formation of methylation on A, and mutation of C can change the methylation efficiency of A, cause methylation reduction and further achieve the aim of changing the methylation level. )

Cloning the sequence of the porcine UCP2 gene (NM-214289.1) and the mutant sequence of T257 from C257, adding a FLAG sequence (5'-GACTACAAGGACGATGATGACAAG-3') at the N end, cloning to the HindIII and BamHI sites of Pcdna3.1(+) expression plasmid to obtain UCP2-WT and UCP2-MUT plasmids (the cloning process belongs to the conventional operation and the specific steps are omitted).

Example 3: change in the methylation level of UCP2mRNA m6A after Point mutation

1. Designing a primer:

fluorescent quantitative PCR primers were designed upstream and downstream of the mutation site and synthesized by Shanghai Biotechnology Ltd as follows:

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’

2. isolation and culture of porcine preadipocytes

The acquisition of pig preadipocytes is slightly improved according to the methods of Ding et al (1999) and Zhanghua et al (2005): and (d) taking fat tissues of the piglets for 5-7 days under the aseptic condition, soaking and washing the fat tissues by using PBS (phosphate buffer solution) containing high-concentration double antibodies, and removing visible blood vessels and muscles. Shearing into tissue blocks with uniform size with scissors, placing in a sterile bottle, digesting with digestive juice in 37 deg.C water bath shaking pot for 1.0h, neutralizing with isovolumetric complete culture medium after digestion, passing the digest through 200 mesh and 300 mesh nylon sieves at one time, collecting filtrate, and centrifuging at 1500rpm/min for 10 min. And (3) discarding the supernatant, adding erythrocyte lysate, uniformly blowing, standing at room temperature for 10min, centrifuging at 1500rpm/min for 5min, discarding the supernatant, suspending the cells by using a complete culture medium, centrifuging for 1-2 times, finally adding a complete culture medium, and uniformly blowing to obtain the pig precursor adipocytes.

3. UCP2-WT and UCP2-MUT were transfected into porcine preadipocytes, respectively:

using Lipofectamine^TMThe UCP2-WT and UCP2-MUT plasmids and empty plasmids were introduced into porcine preadipocytes using 2000 reagent (Invitrogen). The transfection method was performed according to the Lipofectamine TM 2000 kit instructions. The method mainly comprises the following steps:

1) one day before transfection, cells are digested by pancreatin and counted, and are cultured in a complete culture medium without antibiotics, and transfection is carried out when the cell density is 70% -90%;

2) mu.g of DNA was diluted with 50. mu.l of OPTI-MEM I medium per well of cells;

3) diluting 10. mu.l LIPOFECTAMINE 2000 reagent with 50. mu.l OPTI-MEM I medium at room temperature

Standing for 5 minutes;

4) mixing the diluted DNA (from step 2) with the diluted LIPOFECTAMINE 2000 (from step 3)

Step), keeping the temperature at room temperature for 20 min;

5) directly adding the compound into cells in each well, shaking the culture plate, gently mixing uniformly, and repeating the treatment for three times;

6) the culture medium is replaced after the temperature is kept for 4 to 6 hours at 37 ℃ in 5 percent CO2 without antibiotics;

7) after 24h of adding the complex to the cells, the cells were collected.

4. Total RNA extraction, chemical cleavage, immunoprecipitation, reverse transcription (same procedure as above)

5. Real-time fluorescence relative quantitative PCR

The reaction system of the experiment is 1 mu l of sample cDNA diluted by 5 times, 5 mu l of SYBR Green Real-time PCR Master Mix, 3 mu l of sterile double distilled water, 0.5ul of each target gene upstream and downstream primer (the concentration is 1 mu M), and 10 mu l of the total reaction system. The PCR reaction conditions are pre-denaturation at 95 ℃ for 2min-95 ℃ for 20s, at 64 ℃ for 20s and at 72 ℃ for 30s, and 45 cycles are repeated. The selection of internal reference is ATCB, the expression quantity of gene in Input, and the result analysis adopts 2^-ΔΔctThe method of (1), wherein Δ Δ ct is calculated as follows: Δ Δ Ct ═ (Ct)_Target-Ct_Input)x-(Ct_Target-Ct_Input)_Control

The results are shown in FIG. 4, and UCP2mRNA m after point mutation was obtained⁶Change in A methylation level.

Example 4: UCP2mRNA m⁶Effect of A methylation level changes on fat deposition

1. Isolation and culture of porcine preadipocytes (supra)

2. UCP2-WT and UCP2-MUT were transfected into porcine preadipocytes (methods as above)

3. Induced differentiation of porcine preadipocytes

After the cells are completely fused for 48h, the induction is started, 3-isobutyl-1-methylxanthine (IBMX) with the final concentration of 0.5mmol/L, 1 mu mol/L Dexamethasone (DEX) and 1.7 mu mol/L Insulin (INS) are added, namely 5.60ml of 50mM IBMX, 0.56ml of DEX and 1.54ml of complete medium of 40IU/ml Insulin are added into 500ml of complete medium for culturing for 48h, the complete medium is replaced by the complete medium containing 10mg/L Insulin for culturing for 48h, and finally the complete medium is replaced for continuous culturing, and the solution is replaced for once for 2d until 90% of the cells generate lipid drops.

4. Oil red O staining identification

After induction maturation of pig precursor adipocytes, the complete medium was discarded and washed 3 times with PBS. The cells were fixed with 10% formaldehyde solution at room temperature for 1h, and the fixing solution was discarded. Washing with 60% isopropanol for 2 times, adding oil red O solution, dyeing at room temperature for 30min-1h, discarding the dyeing solution, washing off the uncolored dyeing solution with PBS, and observing under inverted microscope.

5. Fluorescent quantitative PCR (same method as above)

The results are shown in FIG. 5, and UCP2 mRNAm after point mutation⁶The A methylation level further inhibits the fat deposition, and the expression of fat deposition related genes PPAR gamma, FABP4 and C/EBP alpha is also inhibited.

Example 5: UCP2mRNA m⁶Effect of A methylation level changes on UCP2 protein expression

1. Isolation and culture of porcine preadipocytes (supra)

2. UCP2-WT and UCP2-MUT were transfected into porcine preadipocytes, respectively (supra)

Western blot detection

(1) Adding 200 μ L cell lysate into each well of 6-well plate, standing on ice for 30min, scraping cells, and placing into 1.5ml centrifuge tube; centrifuging at 12000rpm and 4 ℃ for 10min, taking the supernatant, and performing protein concentration determination by using a QIAGEN BCA protein content detection kit;

(2) SDS-PAGE electrophoresis: and (3) cleaning the glass plate, pouring glue and loading, concentrating the glue at a voltage of 80v for half an hour, and separating the glue for 110v 1.5 h.

(3) Film transfer: soaking filter paper, two sponge pads and a membrane into the membrane transferring liquid for wetting, placing the sponge pads and the filter paper according to a sandwich principle, wherein the sponge pads, the filter paper, the membrane, gel, the filter paper and the sponge pads are arranged from the white side of the clamp to the upper side in sequence, and a glass rod is used for slightly rolling on the sponge pad on the uppermost layer to drive away bubbles in the sandwich; the clips with the films and gels are carefully placed in the transfer tank to ensure that the black side of the clip faces the black side of the tank and the white side faces the red side of the tank. In addition, when the film is rotated, a large amount of heat is generated, and ice blocks need to be put into the film to reduce the temperature. The cross-flow transfer is generally carried out for 1.5h with 300A.

(4) Immune reaction: sealing the membrane with 5% skimmed milk (5% skimmed milk powder and TBST), about 7-8ml on a shaker for 1 h; pouring out the skimmed milk in the box, adding the skimmed milk containing primary antibody again, shaking up by a shaking table, and standing overnight in a refrigerator at 4 ℃; the next day, discarding primary anti-diluent, washing with TBST for 3 times, each time for 5 min; the overnight primary antibody membrane was incubated with the secondary antibody dilution at room temperature for about 1 hour, the secondary antibody dilution was discarded, and the membrane was washed 3 times 5min each with TBST. The TBST was discarded, and a chemiluminescent solution was added and photographed.

The results are shown in FIG. 6, and UCP2mRNA m after point mutation⁶Promoting the expression of UCP2 protein after A methylation level.

Example 6: UCP2mRNA m⁶Effect of A methylation level changes on UCP2mRNA stability

1. Isolation and culture of porcine preadipocytes (supra)

Detection of mRNA stability

And (3) transfecting for 24h, adding 5 mu M actinomycin D for inhibiting mRNA transcription into a culture solution of the cells, treating for 0, 3 and 6 hours, extracting total RNA of the cells, and detecting the gene expression level of UCP2 by using qRT-PCR (quantitative reverse transcription-polymerase chain reaction), thereby comparing the difference of the UCP2-WT gene expression level and the UCP2-MUT gene expression level and calculating the mRNA stability.

The results are shown in FIG. 7, and UCP2mRNA m after point mutation⁶Increased UCP2mRNA stability following a methylation level. Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited toMany variations of the above embodiments are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

SEQUENCE LISTING

<110> Zhejiang university

<120> identification method and functional application of UCP2mRNA m6A methylation unit site related to pig fat deposition

<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

1.UCP2 mRNA m⁶The application of the A methylation single site as a molecular marker for predicting pig fat deposition is characterized in that: the sequence number of the UCP2mRNA in GenBank is nm _214289.1, and the UCP2mRNA m⁶A is located at position 256 of the nm _214289.1 sequence.

2. Reducing UCP2mRNA m⁶A methylation level method, characterized by: the sequence number of the UCP2mRNA in GenBank is nm _214289.1, and the UCP2mRNA m⁶A is at the 256 th position of the nm _214289.1 sequence, and C at the 257 th position of the nm _214289.1 sequence is changed into T.