Method for detecting methylation level of heterozygous heterologous DNA in saccharomyces cerevisiae

Abstract

The invention discloses a method for detecting methylation level of heterozygous heterologous DNA (deoxyribonucleic acid) in saccharomyces cerevisiae. Comprising the following steps: preparing a saccharomyces cerevisiae genome, carrying out enzymolysis on the saccharomyces cerevisiae genome, and detecting the methylation level of heterozygous heterologous DNA in saccharomyces cerevisiae by using an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer. According to the method, saccharomyces cerevisiae cell walls can be better removed, the use time of protease K is adjusted, protein combined with DNA molecules is better removed, saccharomyces cerevisiae genomes are extracted with high quality, and impurities are greatly reduced; the PMSF protease inhibitor is added in the saccharomyces cerevisiae genome extraction process, so that the activity of protease K can be effectively inhibited, the residual protease K is prevented from degrading nuclease and other key enzymes in the subsequent DNA enzymolysis process, and the DNA enzymolysis efficiency is improved. The method can be directly applied to DNA methylation detection of saccharomyces cerevisiae.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for detecting the methylation level of heterozygous DNA in brewer's yeast. Background Art

[0002] DNA methylation modification is the most common epigenetic modification in organisms. It can regulate gene expression without changing the genome sequence and is an important regulatory mode involved in life processes. DNA methylation exists in most organisms, including prokaryotes and eukaryotes. Eukaryotic DNA methylation is mainly mediated by DNA methyltransferases, and uses S-adenosylmethionine (SAM) as a methyl donor to produce three types of DNA methylation modifications at genomic CPG sites, with 5-methylcytosine (5mC) being the main one. DNA methylation can be divided into two processes: establishment and maintenance. Methylation of two unmethylated DNA chains is called de novo methylation. During DNA replication, the process in which the newly synthesized chain is remethylated is called maintenance methylation. The principle of detecting the overall level of DNA methylation is to detect the ratio of 5mC to the total amount of 5mC and C, which is expressed by a formula: overall DNA methylation level = 5mC / (5mC+C).

[0003] Since DNA methylation does not exist in Saccharomyces cerevisiae, traditional DNA methylation detection methods do not involve detecting DNA methylation in Saccharomyces cerevisiae.

[0004] At present, the main method for detecting the overall level of DNA methylation is to use liquid chromatography to detect the 5mC content in the sample to determine the overall DNA methylation level. The traditional overall level of DNA methylation detection is to extract the genome of animal and plant cells according to the genome extraction method in the "Molecular Cloning Experiment Guide", and use enzymes to break down the complete genome into nucleoside monomers for detection. Although this method can also be applied to brewer's yeast, the genome obtained by this method may be mixed with a large amount of protein or other impurities, which will affect the test results. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for detecting the methylation level of heterozygous DNA in Saccharomyces cerevisiae.

[0006] The technical solution of the present invention is summarized as follows:

[0007] A method for detecting the methylation level of heterozygous DNA in Saccharomyces cerevisiae comprises the following steps:

[0008] 1) Preparation of Saccharomyces cerevisiae genome:

[0009] (1) Add 1 ml of YPD liquid medium to a 10 ml disposable sterile test tube, add a single colony of Saccharomyces cerevisiae cells, and culture to obtain primary seeds;

[0010] (2) Measure the OD600 of the primary seeds and transfer them to 5 ml of SC liquid medium at an OD600 of 0.2 to obtain secondary seeds with an OD600 between 4 and 5. Centrifuge at 4°C, 5000 rpm for 5 min and discard the medium.

[0011] (3) Resuspend the precipitate with 5 ml of sterile water, centrifuge at 5000 rpm at 4°C for 5 min, and discard the supernatant;

[0012] (4) Repeat step (3) once;

[0013] (5) resuspending the precipitate obtained in step (4) in 5 ml of 1 M sorbitol aqueous solution precooled at 4°C, refrigerating at 4°C; centrifuging at 4°C, 3500 rpm for 5 min, and removing the supernatant;

[0014] (6) Resuspend the pellet with 1 ml of SPE solution, add 5 μl of snail enzyme 20T, 5 μL of mercaptoethanol, incubate at 30 °C, 75 rpm, and incubate for 1 h;

[0015] (7) Centrifuge at 500 G for 10 min at 4°C and remove the supernatant;

[0016] (8) Resuspend the pellet with 250 μL buffer A;

[0017] (9) Add 6 μL of 10 mg / ml RNase A solution, shake for 15 seconds, and leave at room temperature for 5 minutes;

[0018] (10) Add 10 μL of 10 mg / ml proteinase K aqueous solution, mix by inversion, add 250 μL of buffer B, shake for 15 seconds, and place in a 50 °C water bath for more than 24 hours;

[0019] (11) Add 5 μL of 100 mM PMSF, shake for 15 s, centrifuge at 12,000 rpm for 1 min at 4°C, transfer the supernatant to another new sterilized 1.5 mL EP tube, add 250 μL of anhydrous ethanol to the EP tube, shake for 15 s, centrifuge, and collect the water droplets on the inner wall;

[0020] (12) placing the DNA adsorption column into a collection tube, placing the liquid obtained in step (11) into the DNA adsorption column, adsorbing on ice for 3 min, and centrifuging at 4°C, 12,000 rpm, for 1 min;

[0021] (13) Place the liquid in the collection tube back into the DNA adsorption column; adsorb on ice for 3 min, then centrifuge at 4°C, 12,000 rpm, for 1 min;

[0022] (14) Repeat step (13) twice; discard the waste liquid in the collection tube, add 500 μL buffer C to the DNA adsorption column, remove water from the DNA molecules, centrifuge at 4°C, 12000 rpm, and centrifuge for 1 min; discard the waste liquid in the collection tube,

[0023] (15) Add 700 μL of rinse solution W2 to the DNA adsorption column to wash away impurities, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; then add 500 μL of rinse solution W2 to the DNA adsorption column to wash away impurities, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; then centrifuge the DNA adsorption column at 12000 rpm for 1 min, and discard the waste liquid;

[0024] (16) placing the DNA adsorption column obtained in step (15) in a 65°C dry bath for 5 min to dry the rinse solution W2;

[0025] (17) Transfer the DNA adsorption column obtained in step (16) to a clean EP tube, drip 200 μL of 65°C sterilized pure water into the middle of the DNA adsorption column, leave it at room temperature for 2-5 minutes, and centrifuge it at 12000 rpm for 1 minute; add the liquid in the EP tube back to the middle of the DNA adsorption column, leave it at room temperature for 2-5 minutes, and centrifuge it at 12000 rpm for 1 minute; discard the DNA adsorption column, and the liquid in the EP tube is the Saccharomyces cerevisiae genome;

[0026] 2) Enzymatic digestion of the Saccharomyces cerevisiae genome

[0027] (1) placing the Saccharomyces cerevisiae genome in a 100° C. dry bath for 30 min and placing it in an ice bath for 1 min;

[0028] (2) Add 1 μL nuclease P1 and 2 μL nuclease P1 buffer and incubate in a 37°C water bath for 12 h;

[0029] (3) Add 4 μL of 0.001 unit / μL phosphodiesterase aqueous solution and incubate in a 37°C water bath for 2 h;

[0030] (4) Add 1 μL Quick CIP and 2 μL resmart buffer and place in a 37°C water bath for 2 h;

[0031] (5) Add the liquid obtained in the previous step to a 10KDA centrifugal ultrafiltration tube, centrifuge at 12000 rpm for 30 min, add 50 μL of purified water, and centrifuge at 12000 rpm for 10 min to obtain the liquid that is the enzymatic hydrolysis product of the Saccharomyces cerevisiae genome;

[0032] 3) Detection of heterozygous DNA methylation levels in Saccharomyces cerevisiae using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry:

[0033] (1) Use ultra-high performance liquid chromatography-triple quadrupole mass spectrometry;

[0034] (2) preparing a 0.1% formic acid aqueous solution, filtering it through a 0.22 μm water-based microporous filter membrane, and ultrasonically degassing it for 30 min to obtain mobile phase A1; preparing a 0.1% formic acid methanol solution, filtering it through a 0.22 μm organic-based microporous filter membrane, and ultrasonically degassing it for 30 min to obtain mobile phase B1;

[0035] (3) Set the flow rate of the mobile phase to 0.3 ml / min, and the total gradient time of the mobile phase to 10 min. The specific settings are:

[0036] Initially 95% mobile phase A1, 5% mobile phase B1;

[0037] At 0.5 min, it was 95% mobile phase A1, 5% mobile phase B1;

[0038] At 3 min, it was set to 76% mobile phase A1 and 24% mobile phase B1;

[0039] At 4.5 min, it was set to 5% mobile phase A1 and 95% mobile phase B1;

[0040] At 7 min, it was 5% mobile phase A1 and 95% mobile phase B1;

[0041] At 7.01 min, it was set to 95% mobile phase A1 and 5% mobile phase B1;

[0042] At 10 min, it was set to 95% mobile phase A1 and 5% mobile phase B1;

[0043] The mass spectrometer ionization mode was set to ESI+;

[0044] (4) Prepare mixed standard solution with purified water:

[0045] Mixed standard solution 1: 5-methylcytosine at a final concentration of 1 ng / ml and cytosine at a final concentration of 300 ng / ml;

[0046] Mixed standard solution 2: 5-methylcytosine at a final concentration of 2 ng / ml and cytosine at a final concentration of 400 ng / ml;

[0047] Mixed standard solution 3: 5-methylcytosine at a final concentration of 5 ng / ml and cytosine at a final concentration of 500 ng / ml;

[0048] Mixed standard solution 4: 5-methylcytosine at a final concentration of 10 ng / ml and cytosine at a final concentration of 600 ng / ml;

[0049] Mixed standard solution 5: 5-methylcytosine at a final concentration of 15 ng / ml and cytosine at a final concentration of 700 ng / ml;

[0050] Mixed standard solution 6: 5-methylcytosine at a final concentration of 20 ng / ml and cytosine at a final concentration of 800 ng / ml;

[0051] The six mixed standard solutions were placed in six liquid phase insert tubes respectively, and then placed in an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for detection to obtain a 5mC standard curve and a C standard curve;

[0052] (5) adding the enzymatic hydrolyzate sample of the Saccharomyces cerevisiae genome obtained in step 2) into a liquid phase insert tube; then placing the sample into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for detection to obtain the concentration of 5mC and the concentration of C;

[0053] Divide the concentration of 5mC by the relative molecular mass of 5mC, 241.2, to get the molar concentration of 5mC;

[0054] Divide the concentration of C by the relative molecular mass of C, 227.2, to obtain the molar concentration of C;

[0055] Heterozygous DNA methylation level in Saccharomyces cerevisiae = 5mC molar concentration / (5mC molar concentration + C molar concentration).

[0056] Advantages of the present invention:

[0057] The present invention applies the protoplast preparation method to the extraction of the yeast genome, which can better remove the yeast cell wall, and adjusts the use time of proteinase K to better remove the protein bound to the DNA molecule, so that the yeast genome can be extracted with high quality and impurities can be greatly reduced;

[0058] PMSF protease inhibitor was added during the extraction of Saccharomyces cerevisiae genome, which can effectively inhibit the activity of proteinase K, prevent residual proteinase K from degrading key enzymes such as nucleases during subsequent DNA hydrolysis, and improve the efficiency of DNA hydrolysis.

[0059] The present invention uses ultra-high performance liquid chromatography-triple quadrupole mass spectrometry instead of high performance liquid chromatography, which can more accurately detect the content of 5-methylcytosine in the sample.

[0060] The method of the present invention can be directly applied to the detection of DNA methylation in Saccharomyces cerevisiae;

[0061] The present invention optimizes the method for extracting the genome of Saccharomyces cerevisiae and can prepare the genome with high quality;

[0062] The detection result response value of liquid chromatography used in the prior art is relatively low, making it difficult to detect DNA methylation. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It is the standard curve of ultra-high performance liquid chromatography, where A is the standard curve of cytosine and B is the standard curve of 5-methylcytosine.

[0064] Figure 2 This is a comparison chart of the methylation levels of the control bacteria BY4741 and the experimental bacteria yZZY0814. DETAILED DESCRIPTION

[0065] Buffer A (tris, SDS), buffer B (tris, guanidine salt), collection tube, buffer C (guanidine salt, ethanol) and rinse solution W2 are from the Yeast Genomic DNA Kit (ZP302) of Beijing Zhuangmeng International Biogene Technology Co., Ltd.

[0066] Preparation of SPE solution: Take 182g sorbitol, 2.04g Na2HPO4·7H2O, 0.32g NaH2PO4·H2O, 20mL 0.5M PH=7.5EDTA, and add purified water to 1L.

[0067] PMSF (purchased from Beijing Solebow Technology Co., Ltd.)

[0068] DNA adsorption column (purchased from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd.)

[0069] Nuclease P1, Nuclease P1 buffer (purchased from New England Biolabs)

[0070] Phosphodiesterase (purchased from MedChemexpress)

[0071] Quick CIP (purchased from New England Biolabs)

[0072] Resmart buffer (purchased from New England Biolabs)

[0073] 5-methylcytosine, abbreviated as 5mC; cytosine, abbreviated as C.

[0074] The above reagents are disclosed for a better understanding of the present invention, but are not intended to limit the present invention.

[0075] The present invention will be further described below in conjunction with specific embodiments.

[0076] Example 1

[0077] A method for detecting the methylation level of heterozygous DNA in Saccharomyces cerevisiae comprises the following steps:

[0078] 1) Preparation of Saccharomyces cerevisiae genome:

[0079] (1) Add 1 ml of YPD liquid medium to a 10 ml disposable sterile test tube, add a single colony of Saccharomyces cerevisiae yZZY0814 (prepared in Example 2) with a heterologous DNA methylation system, and culture for 16 hours to obtain primary seeds;

[0080] (2) Measure the OD600 of the primary seeds and transfer them to 5 ml SC liquid culture medium at an OD600 of 0.2 and culture for 48 h to obtain secondary seeds with an OD600 between 4 and 5 to maintain high cell activity and a greater degree of DNA methylation; centrifuge at 4°C, 5000 rpm for 5 min and discard the culture medium;

[0081] (3) Resuspend the precipitate with 5 ml of sterile water, centrifuge at 5000 rpm at 4°C for 5 min, and discard the supernatant;

[0082] (4) Repeat step (3) once;

[0083] (5) resuspending the precipitate obtained in step (4) in 5 ml of 1 M sorbitol aqueous solution precooled at 4°C, refrigerating at 4°C for 12 h; centrifuging at 4°C, 3500 rpm for 5 min, and removing the supernatant;

[0084] (6) Use 1 ml of SPE solution to resuspend the precipitate, add 5 ul of snail enzyme 20T, 5 μL of mercaptoethanol, incubate at 30°C, rotate at 75 rpm, and incubate for 1 h to enzymatically hydrolyze the cell wall of Saccharomyces cerevisiae and make the yeast cells into protoplasts;

[0085] (7) Centrifuge at 500 G for 10 min at 4°C and remove the supernatant;

[0086] (8) Resuspend the pellet with 250 μL buffer A. The SDS in buffer A destroys the cell membrane and nuclear membrane structure, releasing cytoplasm and DNA. The tris in buffer A stabilizes the DNA molecules to prevent degradation.

[0087] (9) Add 6 μL of 10 mg / ml RNase A solution, shake for 15 seconds, and leave at room temperature for 5 minutes to enzymatically hydrolyze RNA;

[0088] (10) Add 10 μL of 10 mg / ml proteinase K aqueous solution, invert to mix, add 250 μL of buffer B. The guanidine salt in buffer B inactivates nucleases, releases nucleic acids, and protects nucleic acid molecules. Oscillate for 15 seconds and place in a 50°C water bath for more than 24 hours to remove proteins.

[0089] (11) Add 5 μL of 100 mM PMSF and shake for 15 seconds to inhibit proteinase K activity; centrifuge at 12,000 rpm for 1 minute at 4°C, transfer the supernatant to another new sterilized 1.5 mL EP tube, add 250 μL of anhydrous ethanol to the EP tube, shake for 15 seconds to precipitate DNA, centrifuge, and collect water droplets on the inner wall;

[0090] (12) placing the DNA adsorption column into a collection tube, placing the liquid obtained in step (11) into the DNA adsorption column, adsorbing on ice for 3 min, and centrifuging at 4°C, 12,000 rpm, for 1 min;

[0091] (13) Place the liquid in the collection tube back into the DNA adsorption column; adsorb on ice for 3 min, then centrifuge at 4°C, 12,000 rpm, for 1 min;

[0092] (14) Repeat step (13) twice; discard the waste liquid in the collection tube, add 500 μL buffer C to the DNA adsorption column to stabilize the nucleic acid molecules and remove water from the DNA molecules to better adsorb the DNA on the DNA adsorption column; centrifuge at 4°C, 12000 rpm for 1 min; discard the waste liquid in the collection tube,

[0093] (15) Add 700 μL of rinse solution W2 to the DNA adsorption column to wash away impurities, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; then add 500 μL of rinse solution W2 to the DNA adsorption column to wash away impurities, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; then centrifuge the DNA adsorption column at 12000 rpm for 1 min, and discard the waste liquid;

[0094] (16) placing the DNA adsorption column obtained in step (15) in a 65° C. dry bath for 5 min to dry the rinse solution W2, which contains ethanol. Drying the DNA adsorption column is used to remove the ethanol to avoid affecting the elution effect;

[0095] (17) Transfer the DNA adsorption column obtained in step (16) to a clean EP tube, drip 200 μL of sterilized pure water at 65°C into the middle part of the DNA adsorption column, let it stand at room temperature for 3 min (it can also be any value of 2-5 min, such as 2 min, 2.5 min, 4 min or 5 min), centrifuge at 12000 rpm for 1 min, wash the DNA off, and transfer it into water; add the liquid in the EP tube back to the middle part of the DNA adsorption column, let it stand at room temperature for 3 min (it can also be any value of 2-5 min, such as 2 min, 2.5 min, 4 min or 5 min), centrifuge at 12000 rpm for 1 min; discard the DNA adsorption column, and the liquid in the EP tube is the Saccharomyces cerevisiae genome;

[0096] 2) Enzymatic digestion of the Saccharomyces cerevisiae genome

[0097] (1) Place the Saccharomyces cerevisiae genome obtained in the previous step in a 100°C dry bath for 30 min and place in an ice bath for 1 min;

[0098] (2) Add 1 μL of nuclease P1 and 2 μL of nuclease P1 buffer and place in a 37°C water bath for 12 h to allow the nuclease to fully digest the DNA molecules;

[0099] (3) Add 4 μL of 0.001 unit / μL phosphodiesterase aqueous solution and incubate in a 37°C water bath for 2 h;

[0100] (4) Add 1 μL Quick CIP and 2 μL resmart buffer and place in a 37°C water bath for 2 h;

[0101] (5) Add the liquid obtained in the previous step to a 10KDA centrifugal ultrafiltration tube, centrifuge at 12000 rpm for 30 min, add 50 μL of purified water, and centrifuge at 12000 rpm for 10 min to obtain the liquid that is the enzymatic hydrolysis product of the Saccharomyces cerevisiae genome;

[0102] 3) Detection of heterozygous DNA methylation levels in Saccharomyces cerevisiae using ultra performance liquid chromatography-triple quadrupole mass spectrometry:

[0103] (1) Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (XEVO-TQ-XS, ACQUITY column) BEH C18 1.7μm;)

[0104] (2) preparing a 0.1% formic acid aqueous solution, filtering it through a 0.22 μm water-based microporous filter membrane, and ultrasonically degassing it for 30 min to obtain mobile phase A1; preparing a 0.1% formic acid methanol solution, filtering it through a 0.22 μm organic-based microporous filter membrane, and ultrasonically degassing it for 30 min to obtain mobile phase B1;

[0105] (3) Set the flow rate of the mobile phase to 0.3 ml / min, and the total gradient time of the mobile phase to 10 min. The specific settings are:

[0106] Initially 95% mobile phase A1, 5% mobile phase B1,

[0107] At 0.5 min, it was 95% mobile phase A1, 5% mobile phase B1;

[0108] At 3 min, it was set to 76% mobile phase A1 and 24% mobile phase B1;

[0109] At 4.5 min, it was set to 5% mobile phase A1 and 95% mobile phase B1;

[0110] At 7 min, it was 5% mobile phase A1 and 95% mobile phase B1;

[0111] At 7.01 min, it was set to 95% mobile phase A1 and 5% mobile phase B1;

[0112] At 10 min, it was set to 95% mobile phase A1 and 5% mobile phase B1;

[0113] The mass spectrometer ionization mode was set to ESI+;

[0114] (4) Prepare mixed standard solution with purified water:

[0115] Mixed standard solution 1: 5-methylcytosine at a final concentration of 1 ng / ml and cytosine at a final concentration of 300 ng / ml;

[0116] Mixed standard solution 2: 5-methylcytosine at a final concentration of 2 ng / ml and cytosine at a final concentration of 400 ng / ml;

[0117] Mixed standard solution 3: 5-methylcytosine at a final concentration of 5 ng / ml and cytosine at a final concentration of 500 ng / ml;

[0118] Mixed standard solution 4: 5-methylcytosine at a final concentration of 10 ng / ml and cytosine at a final concentration of 600 ng / ml;

[0119] Mixed standard solution 5: 5-methylcytosine at a final concentration of 15 ng / ml and cytosine at a final concentration of 700 ng / ml;

[0120] Mixed standard solution 6: 5-methylcytosine at a final concentration of 20 ng / ml and cytosine at a final concentration of 800 ng / ml;

[0121] The six mixed standard solutions were placed in six liquid phase insert tubes respectively, and then placed in the above-mentioned ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for detection to obtain a 5mC standard curve and a C standard curve; see Figure 1

[0122] (5) adding the enzymatic hydrolyzate sample of the Saccharomyces cerevisiae genome obtained in step 2) into a liquid phase insert tube; then placing the sample into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for detection, and obtaining a 5mC concentration of 38.14 ng / ml and a C concentration of 3218.06 ng / ml;

[0123] Divide the concentration of 5mC by the relative molecular mass of 5mC, 241.2, and you get the molar concentration of 5mC to be 0.158126036

[0124] nmol / ml);

[0125] Divide the concentration of C by the relative molecular mass of C, 227.2, to obtain the molar concentration of C (14.164 nmol / ml);

[0126] Heterozygous DNA methylation level in Saccharomyces cerevisiae = 5mC molar concentration / (5mC molar concentration + C molar concentration) = (1.10%)

[0127] Comparison of methylation levels between control strain BY4741 and experimental strain yZZY0814 is shown in Figure 2 .

[0128] Example 2

[0129] The construction of a heterologous DNA methylation system of Saccharomyces cerevisiae yZZY0814 comprises the following steps:

[0130] 1) Construction of Dnmt plasmid:

[0131] (1) Using a plasmid containing the human Dnmt1 gene as a template, the sequence shown in SEQ ID NO.1 as an upstream primer, and the sequence shown in SEQ ID NO.2 as a downstream primer, PCR amplification was performed to obtain fragment 1; the 3' end of this fragment contained a 40 bp homology arm of the terminator CYC1t;

[0132] Using the plasmid containing the human Dnmt3A gene as a template, the sequence shown in SEQ ID NO.3 as an upstream primer, and the sequence shown in SEQ ID NO.4 as a downstream primer, PCR amplification was performed to obtain fragment 2; the 3' end of this fragment contains a 40 bp homology arm of the terminator CYC1t;

[0133] Using the plasmid containing the human Dnmt3B gene as a template, the sequence shown in SEQ ID NO.5 as an upstream primer, and the sequence shown in SEQ ID NO.6 as a downstream primer, PCR amplification was performed to obtain fragment 3; the 3' end of this fragment contains a 40 bp homology arm of the terminator CYC1t;

[0134] Using the plasmid containing the human Dnmt3L gene as a template, the sequence shown in SEQ ID NO.7 as an upstream primer, and the sequence shown in SEQ ID NO.8 as a downstream primer, PCR amplification was performed to obtain fragment 4; the 3' end of this fragment contains a 40 bp homology arm of the terminator CYC1t; among them: Dnmt1: NM_001130823.3, Dnmt3B: NM_006892.4, Dnmt3A: NM_022552.5, Dnmt3L: NM_175867.3

[0135] (2) Using the genome of Saccharomyces cerevisiae BY4741 as a template, using the high-fidelity enzyme KOD one, using the sequence shown in SEQ ID NO.9 as an upstream primer and the sequence shown in SEQ ID NO.10 as a downstream primer, PCR amplification was performed to obtain the endogenous promoter TEF1p of Saccharomyces cerevisiae BY4741 as fragment 5, the 3' end of which contained a 40 bp homology arm of the Dnmt1 gene;

[0136] Using the genome of Saccharomyces cerevisiae BY4741 as a template, using the high-fidelity enzyme KOD one, using the sequence shown in SEQ ID NO.11 as an upstream primer, and using the sequence shown in SEQ ID NO.12 as a downstream primer, PCR amplification was performed to obtain the endogenous promoter ADH1p of Saccharomyces cerevisiae BY4741 as fragment 6, and the 3' end of the fragment 6 contained a 40 bp homology arm of the Dnmt3B gene;

[0137] Using the genome of Saccharomyces cerevisiae BY4741 as a template, using the high-fidelity enzyme KOD one, using the sequence shown in SEQ ID NO.13 as an upstream primer, and using the sequence shown in SEQ ID NO.14 as a downstream primer, PCR amplification was performed to obtain the endogenous promoter TEF2p of Saccharomyces cerevisiae BY4741 as fragment 7, and the 3' end of the fragment 7 contained a 40 bp homology arm of the Dnmt3L gene;

[0138] Using the genome of Saccharomyces cerevisiae BY4741 as a template, using the high-fidelity enzyme KOD one, using the sequence shown in SEQ ID NO.15 as an upstream primer, and using the sequence shown in SEQ ID NO.16 as a downstream primer, PCR amplification was performed to obtain the endogenous promoter TEF1p of Saccharomyces cerevisiae BY4741 as fragment 8, and the 3' end of the fragment 8 contained a 40 bp homology arm of the Dnmt3A gene;

[0139] Using the genome of Saccharomyces cerevisiae BY4741 as a template, the sequence shown in SEQ ID NO.17 as an upstream primer, and the sequence shown in SEQ ID NO.18 as a downstream primer, PCR amplification was performed to obtain the endogenous terminator CYC1t of Saccharomyces cerevisiae BY4741 as fragment 9, the 5' end of the fragment 9 contained two NLS nuclear localization sequences, and the nuclear localization sequence was shown in SEQ ID NO.27;

[0140] (3) Using fragment 1, fragment 5 and fragment 9 as templates, the sequence shown in SEQ ID NO. 9 as the upstream primer, and the sequence shown in SEQ ID NO. 18 as the downstream primer, PCR amplification was performed to obtain the TEF1p+Dnmt1+CYC1t combined fragment;

[0141] Using fragment 2, fragment 8, and fragment 9 as templates, the sequence shown in SEQ ID NO.15 as an upstream primer, and the sequence shown in SEQ ID NO.18 as a downstream primer, PCR amplification was performed to obtain a TEF1p+Dnmt3A+CYC1t combined fragment;

[0142] Using fragment 3, fragment 6, and fragment 9 as templates, the sequence shown in SEQ ID NO.11 as an upstream primer, and the sequence shown in SEQ ID NO.18 as a downstream primer, PCR amplification was performed to obtain the ADH1p+Dnmt3B+CYC1t combined fragment;

[0143] Using fragment 4, fragment 7, and fragment 9 as templates, the sequence shown in SEQ ID NO.13 as an upstream primer, and the sequence shown in SEQ ID NO.18 as a downstream primer, PCR amplification was performed to obtain a TEF2p+Dnmt3L+CYC1t combined fragment;

[0144] (4) using KOD one high-fidelity enzyme and pRS413 plasmid as template, the sequence shown in SEQ ID NO.19 as upstream primer, and the sequence shown in SEQ ID NO.20 as downstream primer, PCR amplification was performed to obtain pRS413 vector as vector 1, wherein the 3' end of the vector 1 contains a 20 bp homology arm of the promoter ADH1p; and the 5' end contains a 20 bp homology arm of the CYC1t terminator;

[0145] Using KOD one high-fidelity enzyme and pRS415 plasmid as template, the sequence shown in SEQ ID NO.21 as upstream primer, and the sequence shown in SEQ ID NO.22 as downstream primer, PCR amplification was performed to obtain pRS415 vector as vector 2, wherein the 3' end of the vector 2 contains a 20 bp homology arm of the promoter TEF2p; and the 5' end contains a 20 bp homology arm of the CYC1t terminator;

[0146] Using KOD one high-fidelity enzyme and pRS416 plasmid as template, the sequence shown in SEQ ID NO.23 as upstream primer, and the sequence shown in SEQ ID NO.24 as downstream primer, PCR amplification was performed to obtain the pRS416 vector as vector 3, wherein the 3' end of the vector 3 contains a 20 bp homology arm of the promoter TEF1p; and the 5' end contains a 20 bp homology arm of the CYC1t terminator;

[0147] Using KOD one high-fidelity enzyme and pRS413 plasmid as template, the sequence shown in SEQ ID NO.25 as upstream primer, and the sequence shown in SEQ ID NO.26 as downstream primer, PCR amplification was performed to obtain pRS413 vector as vector 4, wherein the 3' end of the vector 4 contains a 20 bp homology arm of the promoter TEF1p; and the 5' end contains a 20 bp homology arm of the CYC1t terminator;

[0148] (5) TEF1p+Dnmt1+CYC1t combined fragment and vector 3 were added to a 300 μL EP tube at a molar ratio of 2:1, and an equal volume of Gibson seamless cloning enzyme was added;

[0149] The TEF1p+Dnmt3A+CYC1t combined fragment and vector 4 were added to a 300 μL EP tube at a molar ratio of 2:1, and an equal volume of Gibson seamless cloning enzyme was added;

[0150] The TEF2p+Dnmt3L+CYC1t combined fragment and vector 2 were added to a 300 μL EP tube at a molar ratio of 2:1, and an equal volume of Gibson seamless cloning enzyme was added;

[0151] The ADH1p+Dnmt3B+CYC1t combined fragment and vector 1 were added to a 300 μL EP tube at a molar ratio of 2:1, and an equal volume of Gibson seamless cloning enzyme was added;

[0152] The mixture was assembled at 50°C for 1 hour to obtain Dnmt1 recombinant plasmid, Dnmt3A recombinant plasmid, Dnmt3L recombinant plasmid, and Dnmt3B recombinant plasmid in sequence;

[0153] 2) Chemical transformation of E. coli:

[0154] The Dnmt1 recombinant plasmid, Dnmt3A recombinant plasmid, Dnmt3L recombinant plasmid, and Dnmt3B recombinant plasmid were transformed into competent Escherichia coli GB05 respectively; cultured at 37°C for 12-16 hours; selected transformants with good growth, verified the upper and lower connection ports by PCR, and selected transformants with both upper and lower interfaces for gene sequencing; the correctly sequenced transformants were inoculated into LB liquid culture medium containing a final concentration of 100 mg / L ampicillin, and cultured for 12-16 hours; extracted and obtained: Dnmt1 recombinant plasmid, Dnmt3A recombinant plasmid, Dnmt3L recombinant plasmid, and Dnmt3B recombinant plasmid;

[0155] 3) Plasmid yeast transformation:

[0156] The Dnmt1 recombinant plasmid, Dnmt3A recombinant plasmid, Dnmt3L recombinant plasmid and Dnmt3B recombinant plasmid were simultaneously transferred into Saccharomyces cerevisiae (Saccharomyces cerevisiae BY4741 is included in the instruction manual) by the lithium acetate transformation method, and cultured at 30°C for 2-3 days; transformants with better growth were selected for verification; the correct transformant yZZY0814 was obtained, that is, Saccharomyces cerevisiae yZZY0814 with a heterologous DNA methylation system was obtained.

Claims

1. A method for detecting the methylation level of heterozygous DNA in Saccharomyces cerevisiae, characterized in that The steps include: 1) Preparation of Saccharomyces cerevisiae genome: (1) Add 1 ml of YPD liquid medium to a 10 ml disposable sterile test tube, add a single colony of Saccharomyces cerevisiae cells, and culture to obtain primary seeds; (2) Measure the OD600 of the primary seeds and transfer them to 5 ml of SC liquid medium at an OD600 of 0.2 to obtain secondary seeds with an OD600 between 4 and 5. Centrifuge at 4°C, 5000 rpm for 5 min and discard the medium. (3) Resuspend the precipitate with 5 ml of sterile water, centrifuge at 5000 rpm at 4°C for 5 min, and discard the supernatant; (4) Repeat step (3) once; (5) resuspending the precipitate obtained in step (4) in 5 ml of 1 M sorbitol aqueous solution precooled at 4°C, refrigerating at 4°C; centrifuging at 4°C, 3500 rpm for 5 min, and removing the supernatant; (6) Resuspend the pellet with 1 ml of SPE solution, add 5 μl of snail enzyme 20T, 5 μL of mercaptoethanol, incubate at 30 °C, 75 rpm, and incubate for 1 h; (7) Centrifuge at 500 G for 10 min at 4°C and remove the supernatant; (8) Resuspend the pellet with 250 μL buffer A; (9) Add 6 μL of 10 mg / ml RNase A solution, shake for 15 seconds, and leave at room temperature for 5 minutes; (10) Add 10 μL of 10 mg / ml proteinase K aqueous solution, mix by inversion, add 250 μL of buffer B, shake for 15 seconds, and place in a 50 °C water bath for more than 24 hours; (11) Add 5 μL of 100 mM PMSF, shake for 15 s, centrifuge at 12,000 rpm for 1 min at 4°C, transfer the supernatant to another new sterilized 1.5 mL EP tube, add 250 μL of anhydrous ethanol to the EP tube, shake for 15 s, centrifuge, and collect the water droplets on the inner wall; (12) placing the DNA adsorption column into a collection tube, placing the liquid obtained in step (11) into the DNA adsorption column, adsorbing on ice for 3 min, and centrifuging at 4°C, 12,000 rpm, for 1 min; (13) Place the liquid in the collection tube back into the DNA adsorption column; adsorb on ice for 3 min, then centrifuge at 4°C, 12,000 rpm, for 1 min; (14) Repeat step (13) twice; discard the waste liquid in the collection tube, add 500 μL buffer C to the DNA adsorption column, remove water from the DNA molecules, centrifuge at 4°C, 12000 rpm, and centrifuge for 1 min; discard the waste liquid in the collection tube, (15) Add 700 μL of rinse solution W2 to the DNA adsorption column to wash away impurities, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; then add 500 μL of rinse solution W2 to the DNA adsorption column to wash away impurities, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; then centrifuge the DNA adsorption column at 12000 rpm for 1 min, and discard the waste liquid; (16) placing the DNA adsorption column obtained in step (15) in a 65°C dry bath for 5 min to dry the rinse solution W2; (17) Transfer the DNA adsorption column obtained in step (16) to a clean EP tube, drip 200 μL of 65°C sterilized pure water into the middle of the DNA adsorption column, leave it at room temperature for 2-5 minutes, and centrifuge it at 12000 rpm for 1 minute; add the liquid in the EP tube back to the middle of the DNA adsorption column, leave it at room temperature for 2-5 minutes, and centrifuge it at 12000 rpm for 1 minute; discard the DNA adsorption column, and the liquid in the EP tube is the Saccharomyces cerevisiae genome; 2) Enzymatic digestion of the Saccharomyces cerevisiae genome (1) placing the Saccharomyces cerevisiae genome in a 100° C. dry bath for 30 min and placing it in an ice bath for 1 min; (2) Add 1 μL nuclease P1 and 2 μL nuclease P1 buffer and incubate in a 37°C water bath for 12 h; (3) Add 4 μL of 0.001 unit / μL phosphodiesterase aqueous solution and incubate in a 37°C water bath for 2 h; (4) Add 1 μL Quick CIP and 2 μL resmart buffer and place in a 37°C water bath for 2 h; (5) Add the liquid obtained in the previous step to a 10KDA centrifugal ultrafiltration tube, centrifuge at 12000 rpm for 30 min, add 50 μL of purified water, and centrifuge at 12000 rpm for 10 min to obtain the liquid that is the enzymatic hydrolysis product of the Saccharomyces cerevisiae genome; 3) Detection of heterozygous DNA methylation levels in Saccharomyces cerevisiae using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry: (1) Use ultra-high performance liquid chromatography-triple quadrupole mass spectrometry; (2) preparing a 0.1% formic acid aqueous solution, filtering it through a 0.22 μm water-based microporous filter membrane, and ultrasonically degassing it for 30 min to obtain mobile phase A1; preparing a 0.1% formic acid methanol solution, filtering it through a 0.22 μm organic-based microporous filter membrane, and ultrasonically degassing it for 30 min to obtain mobile phase B1; (3) Set the flow rate of the mobile phase to 0.3 ml / min, and the total gradient time of the mobile phase to 10 min. The specific settings are: Initially 95% mobile phase A1, 5% mobile phase B1; At 0.5 min, it was 95% mobile phase A1, 5% mobile phase B1; At 3 min, it was set to 76% mobile phase A1 and 24% mobile phase B1; At 4.5 min, it was set to 5% mobile phase A1 and 95% mobile phase B1; At 7 min, it was 5% mobile phase A1 and 95% mobile phase B1; At 7.01 min, it was set to 95% mobile phase A1 and 5% mobile phase B1; At 10 min, it was set to 95% mobile phase A1 and 5% mobile phase B1; The mass spectrometer ionization mode was set to ESI+; (4) Prepare mixed standard solution with purified water: Mixed standard solution 1: 5-methylcytosine at a final concentration of 1 ng / ml and cytosine at a final concentration of 300 ng / ml; Mixed standard solution 2: 5-methylcytosine at a final concentration of 2 ng / ml and cytosine at a final concentration of 400 ng / ml; Mixed standard solution 3: 5-methylcytosine at a final concentration of 5 ng / ml and cytosine at a final concentration of 500 ng / ml; Mixed standard solution 4: 5-methylcytosine at a final concentration of 10 ng / ml and cytosine at a final concentration of 600 ng / ml; Mixed standard solution 5: 5-methylcytosine at a final concentration of 15 ng / ml and cytosine at a final concentration of 700 ng / ml; Mixed standard solution 6: 5-methylcytosine at a final concentration of 20 ng / ml and cytosine at a final concentration of 800 ng / ml; The six mixed standard solutions were placed in six liquid phase insert tubes respectively, and then placed in an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for detection to obtain a 5mC standard curve and a C standard curve; (5) adding the enzymatic hydrolyzate sample of the Saccharomyces cerevisiae genome obtained in step 2) into a liquid phase insert tube; then placing the sample into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer for detection to obtain the concentration of 5mC and the concentration of C; Divide the concentration of 5mC by the relative molecular mass of 5mC, 241.2, to get the molar concentration of 5mC; Divide the concentration of C by the relative molecular mass of C, 227.2, to obtain the molar concentration of C; Heterozygous DNA methylation level in Saccharomyces cerevisiae = 5mC molar concentration / (5mC molar concentration + C molar concentration).

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