Method for efficiently detecting distribution and abundance of fungal whole genome level DNA binding protein

Abstract

The invention discloses a method for efficiently detecting distribution and abundance of fungal whole genome level DNA (Deoxyribose Nucleic Acid) binding protein. The invention provides a method for detecting the distribution and abundance of a target protein in a whole genome of a tested organism, which is characterized in that the organism is a fungus, and the target protein is a DNA (Deoxyribose Nucleic Acid) binding protein in the tested fungus; the method for detecting the distribution and abundance of the target protein in the whole genome of the tested organism provided by the invention is a fungus CUTamp; a Tag; the method is compared with a conventional CUTamp; in comparison with Tag, the fungi CUTamp; tag is characterized in that the following steps are added before cell nucleus adsorption: fungal hyphae are taken for formaldehyde crosslinking, and then protoplast is prepared; a step of removing formaldehyde crosslinking is added between fragmentation and library amplification. According to the method and the device, the CUAMP is firstly converted into the CUAMP; the Tag technology is applied to detection of distribution and abundance of the DNA binding protein of the filamentous fungi in the genome level, and an important means is provided for related research of the DNA binding protein of the fungi.

Description

Technical Field

[0001] The present invention relates to a method for efficiently detecting the distribution and abundance of DNA binding proteins at the whole genome level of fungi. Background Art

[0002] The CUT&Tag technology was publicly released by the Henikoff team in Nature Communication in 2019. This technology can be used to study histone modification sites and DNA binding protein target sites at the whole genome level. The CUT&Tag technology uses Concanavalin A-coated magnetic beads to adsorb living cells and uses digitonin to increase cell membrane permeability. The cells always remain in a living cell state during the experiment, which can more realistically reflect the state of histone modifications and DNA binding proteins in the cells. The target protein-specific antibody (primary antibody) enters the cell to recognize and bind to the specifically modified histone / DNA binding protein, the secondary antibody recognizes the primary antibody and amplifies the signal, and the pA / G-Tn5 transposase binds to the primary and secondary antibodies. 2+ The activation of the target protein cuts the DNA at both ends, and sequencing adapters are added at the same time. The library can be built by PCR. After sequencing, the binding sites of the target protein or the modification level of histones in the whole genome can be detected. The relative binding amount of the protein at the target gene or the modification level of histones can also be detected by qPCR. Compared with the traditional ChIP technology, this method requires less starting materials and can achieve single-cell level. It does not require ultrasonic disruption and other steps that require high instrumentation and technology. It has a short cycle, and the experimental operation and library construction process are relatively simple. The data signal-to-noise ratio is high and the repeatability is good.

[0003] Compared with the large number of histone modifications, the relative low content of DNA binding proteins (including transcription factors), or the small number of their target genes, as well as unstable binding, will result in the inability to enrich the target genes of the protein through traditional CUT&Tag experiments. Currently, there are only a few successful cases of detecting transcription factors through CUT&Tag experiments in animal and plant cells. Since fungi have cell walls and some secondary metabolites, the application of this technology in fungi is more challenging, and there have been no related reports in filamentous fungi. Summary of the invention

[0004] The purpose of the present invention is to provide a method for efficiently detecting the distribution and abundance of DNA binding proteins at the whole genome level of fungi.

[0005] The present invention provides a method for detecting the distribution and abundance of a target protein in the whole genome of a test organism, characterized in that:

[0006] The organism is a fungus, and the target protein is a DNA binding protein in the tested fungus;

[0007] The method provided by the present invention for detecting the distribution and abundance of a target protein in the whole genome of a test organism is fungal CUT&Tag; compared with the traditional CUT&Tag, the difference of the fungal CUT&Tag is that:

[0008] The following steps are added before adsorbing the nucleus: fungal hyphae are taken, cross-linked with formaldehyde, and then protoplasts are prepared;

[0009] Add the following step between fragmentation and library amplification: Remove formaldehyde cross-links.

[0010] The traditional CUT&Tag includes the following steps in sequence:

[0011] (1) Extracting cell nuclei;

[0012] (2) Adsorption of cell nuclei;

[0013] (3) Primary antibody incubation;

[0014] (4) Secondary antibody incubation;

[0015] (5) pA / G-Tn5 transposase incubation;

[0016] (6) Fragmentation: Activation of pA / G-Tn5 transposase;

[0017] (7) Library amplification: obtaining a DNA library through library amplification;

[0018] (8) The DNA library is subjected to high-throughput sequencing, and the distribution and abundance of the target protein in the entire genome of the test organism is obtained based on the sequencing results.

[0019] The fungus CUT&Tag comprises the following steps in sequence:

[0020] (1) taking fungal mycelium, performing formaldehyde cross-linking, and then preparing protoplasts;

[0021] (2) Extracting cell nuclei;

[0022] (3) Adsorption of cell nuclei;

[0023] (4) Primary antibody incubation;

[0024] (5) Secondary antibody incubation;

[0025] (6) pA / G-Tn5 transposase incubation;

[0026] (7) Fragmentation: Activation of pA / G-Tn5 transposase;

[0027] (8) Removal of formaldehyde cross-linking;

[0028] (9) Library amplification: obtaining a DNA library through library amplification;

[0029] (10) The DNA library is subjected to high-throughput sequencing, and the distribution and abundance of the target protein in the entire genome of the test organism is obtained based on the sequencing results.

[0030] CUT&Tag: Cleavage Under Targets and Tagmentation.

[0031] Specifically, the adsorption of cell nuclei is to use ConA beads to adsorb cell nuclei.

[0032] The formaldehyde cross-linking comprises the following steps: taking mycelium, treating it with formaldehyde solution, and then adding glycine or glycine solution to terminate the reaction.

[0033] The formaldehyde cross-linking comprises the following steps: taking mycelium, treating it with a formaldehyde solution, then adding glycine or a glycine solution to terminate the reaction, and then washing it.

[0034] Specifically, the formaldehyde cross-linking comprises the following steps: taking mycelium and placing it in a formaldehyde solution for reaction for 8 minutes; then adding glycine or a glycine solution and incubating for 5 minutes.

[0035] Specifically, the formaldehyde cross-linking comprises the following steps: taking mycelium and placing it in a formaldehyde solution for reaction for 8 minutes; then adding glycine or a glycine solution and incubating for 5 minutes; and then washing with a NaCl solution.

[0036] Specifically, the formaldehyde solution is a formaldehyde solution with a formaldehyde volume percentage of 0.1%.

[0037] Specifically, the preparation method of the formaldehyde solution is: dissolving formaldehyde in a 0.7M NaCl aqueous solution, and making the volume percentage of formaldehyde 0.1%.

[0038] Specifically, the glycine solution is a 2M glycine solution.

[0039] Specifically, the preparation method of the glycine solution is: dissolving L-glycine in a 0.7M NaCl aqueous solution, and making the concentration of glycine 2M.

[0040] Specifically, the NaCl solution is a 0.7M NaCl solution.

[0041] Specifically, the NaCl solution is a 0.7M NaCl aqueous solution.

[0042] Specifically, the working concentration of glycine is 0.2M.

[0043] The preparation of protoplasts comprises the following steps: after the formaldehyde cross-linking is completed, the mycelium is taken, and Pro was enzymatically hydrolyzed and then the protoplasts were collected.

[0044] Specifically, enzymolysis is performed using an enzymolysis solution; in the enzymolysis solution, The content of Pro is 2g / 100ml.

[0045] Specifically, the conditions for the enzymatic hydrolysis are: 33° C., 60 rpm shaking reaction for 3 hours.

[0046] Specifically, the preparation of protoplasts includes the following steps: after completing formaldehyde cross-linking, taking mycelium, placing it in an enzyme hydrolyzate, and shaking it at 33°C and 60rpm for 3h; then, filtering it with a double-layer magic filter cloth, collecting the filtrate, centrifuging the filtrate at 3800rpm for 10min, discarding the supernatant, and washing it with a 0.7M NaCl aqueous solution.

[0047] Enzyme hydrolysate: Dissolve 0.25 g of VinoTaste Pro in 12.5 ml of 0.7 M NaCl aqueous solution, rotate and dissolve for 20 min, then filter with a 0.22 μm pore size filter membrane and collect the filtrate.

[0048] The method for removing formaldehyde crosslinks comprises the following steps: after fragmentation, taking DNA fragments, and sequentially performing the following operations: adding NaCl or NaCl solution and reacting, then adding RNase or RNase solution and reacting, then adding proteinase K or proteinase K solution and reacting, and then adding protease inhibitor or protease inhibitor solution and reacting.

[0049] The method for removing formaldehyde cross-linking comprises the following steps: after fragmentation is completed, a liquid phase system containing DNA fragments is taken, and the following operations are performed in sequence: a 5M NaCl aqueous solution is added, and the reaction is carried out at 65°C for 18 hours; then, RNAse is added, and the reaction is carried out at 37°C for 30 minutes; then, a proteinase K solution is added, and the reaction is carried out at 37°C for 1 hour; then, a protease inhibitor is added, and the reaction is carried out for 5 minutes.

[0050] The method for removing formaldehyde cross-linking comprises the following steps: after fragmentation is completed, 50 μl of a liquid phase system containing DNA fragments is taken, and the following operations are performed in sequence: 3 μl of a 5M NaCl aqueous solution is added, and the reaction is carried out at 65° C. for 18 hours; then, 0.25 μl of RNAse is added, and the reaction is carried out at 37° C. for 30 minutes; then, 1 μl of a proteinase K solution is added, and the reaction is carried out at 37° C. for 1 hour; then, 1 μl of a 50× proteinase inhibitor is added, and the reaction is carried out for 5 minutes.

[0051] RNase A (Ribonuclease A) (product form is liquid, specification is 10 mg / ml): Takara Biomedical Technology, product number is 2158.

[0052] Proteinase K (Powder): Merck, Catalog No. 539480. Proteinase K solution: Dissolve proteinase K in a buffer solution (containing 50 mM Tris-HCl, 2 mM calcium acetate, the remainder is water, pH 8.0) to a concentration of 5 mg / ml.

[0053] In one form, the primary antibody used in the primary antibody incubation is a primary antibody against the target protein.

[0054] As another form, a protein tag can be added to the target protein, and the primary antibody used in the primary antibody incubation is a primary antibody against the protein tag. For this form, the fungus is a recombinant bacterium; the recombinant bacterium is obtained by integrating an exogenous DNA molecule into the genomic DNA of the starting fungus through homologous recombination; the exogenous DNA molecule has a gene encoding a protein tag; the gene encoding the target protein in the genomic DNA of the starting fungus forms a fusion gene with the gene encoding the protein tag to express the target protein with a protein tag. The integration of the exogenous DNA molecule into the genomic DNA of the test fungus is specifically: replacing the stop codon of the target gene in the genomic DNA of the test fungus with the exogenous DNA molecule. Exemplarily, the protein tag is a Flag tag, and any other protein tag in the prior art can also be used. The fusion gene includes, from upstream to downstream, the target gene and the tag gene without the stop codon. The exogenous DNA molecule also has a resistance screening gene. Specifically, the tag gene is shown in positions 1088-1156 in sequence 2 of the sequence table. Specifically, the fusion gene is composed of the following elements from upstream to downstream: the target gene with the termination codon removed, and the DNA molecule shown at positions 1049-1156 in Sequence 2 of the sequence table. Specifically, the resistance screening gene can be a hygromycin resistance gene. Specifically, the hygromycin resistance gene is shown at positions 1531-2556 in Sequence 2 of the sequence table. Specifically, the exogenous DNA molecule is shown at positions 1049-3620 in Sequence 2 of the sequence table.

[0055] Specifically, the CUT&Tag uses the following kit: Hyperactive Universal CUT&Tag AssayKit for Illumina Pro.

[0056] Specifically, the ConA beads are ConA Beads Pro.

[0057] The primary antibody incubation time may specifically be 16 hours.

[0058] As an example, the primary antibody is a Flag antibody. Specifically, the primary antibody incubation is incubation with a Flag antibody working solution. The Flag antibody is diluted with Antibody Buffer to obtain a Flag antibody working solution; the antibody content in each 50 μl Flag antibody working solution is 1 μg. Flag antibody (monoclonal anti- M2 mouse antibody, product form is liquid, specification is 1mg / mL): Sigma, product number is F1804.

[0059] The secondary antibody incubation time may specifically be 60 minutes.

[0060] As an example, the secondary antibody is Goat Anti mouse IgG. Specifically, the secondary antibody incubation is incubation with a secondary antibody working solution. Take the secondary antibody and dilute it to 100 times the volume with 50 μl Dig-wash Buffer, which is the secondary antibody working solution.

[0061] The pA / G-Tn5 transposase incubation time may specifically be 60 minutes.

[0062] The pA / G-Tn5 transposase is composed of a pA / G-Tn5 fusion protein and a linker. The pA / G-Tn5 fusion protein is a fusion protein of Tn5 transposase and Protein A / G. The linker is bound to the Tn5 transposase. Specifically, the pA / G-Tn5 transposase is pA / G-TnpPro. Specifically, the pA / G-Tn5 transposase incubation is incubation with a pA / G-Tn5 transposase working solution. pA / G-Tnp Pro working solution: Mix 2μl pA / G-Tnp Pro and 98μl Dig-300 Buffer.

[0063] Activation of pA / G-Tn5 transposase was achieved by adding TTBL working solution.

[0064] TTBL working solution: Mix 40 μl Dig-300 Buffer and 10 μl 5×TTBL.

[0065] Specifically, in the fungal CUT&Tag, the amount of adsorbed cell nuclei is 4,000,000 cell nuclei.

[0066] Specifically, the fungus is a filamentous fungus.

[0067] Specifically, the fungus is Verticillium dahliae.

[0068] Since the abundance of DNA-binding proteins such as transcription factors in cells is relatively low, the number of target genes they bind to is relatively small, and their binding to DNA is unstable, it is difficult to study the abundance and binding sites of transcription factors using traditional CUT&Tag technology. In addition, fungi have cell walls and rich secondary metabolites, which further increases the difficulty of the experiment.

[0069] Compared with traditional ChIP technology, the present invention has the following advantages:

[0070] ① The cell requirement is small, only 4,000,000 cells;

[0071] ② Short time consumption and easy operation;

[0072] ③High signal-to-noise ratio and low background;

[0073] ④Good repeatability.

[0074] The present invention realizes for the first time the application of CUT&Tag technology to detect the distribution and abundance of DNA-binding proteins (including transcription factors) of filamentous fungi at the genome level, providing an important means for the research related to fungal DNA-binding proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Schematic diagram of the elements of the pGKO-Flag-HPT vector.

[0076] Figure 2 This is the length distribution result of the DNA library prepared by the recombinant bacterium HP1-3Flag (using Flag antibody working solution).

[0077] Figure 3 This is the length distribution result of the DNA library prepared by the recombinant bacterium HP1-3Flag (using Mouse IgG working solution).

[0078] Figure 4 Results of the length distribution of the DNA library prepared for Verticillium dahliae V592.

[0079] Figure 5 The sequencing results of the DNA libraries prepared for Verticillium dahliae V592 and the recombinant bacterium HP1-3Flag (using Flag antibody working solution).

[0080] Figure 6 The sequencing results of the DNA libraries prepared for Verticillium dahliae V592 and the recombinant bacterium HP1-3Flag (using Mouse IgG working solution). DETAILED DESCRIPTION

[0081] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0082] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are repeated three times, and the results are averaged. Miracloth: pore size 22-25 μm, Merck millipore, item number 475855-1R. Verticillium dahliae V592 was kindly donated by Guo Huishan's research group at the Institute of Microbiology, Chinese Academy of Sciences.

[0083] Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro: Novozymes, catalog number TD904-01. The kit provides the following components: NE buffer, 10×Wash Buffer, 10×BindingBuffer, ConA Beads Pro, Antibody Buffer(-), pA / G-Tnp Pro(2μM), 10×Dig-300Buffer, 5×TTBL, DNA Spike-in(5ng / μl), DNA Extract Beads Pro, 2×B&W Buffer, 2×CAM, 10%SDS, 5%Digitonin. 50×Protease Inhibitor: Take one tablet of protease inhibitor (Roche, 04693132001) and dissolve it in 1ml ddH2O. Binding Buffer: Mix 30μl 10×Binding Buffer with 270μl ddH2O. WashBuffer: Mix 150μl 10×Wash Buffer, 30μl 50×Protease Inhibitor and 1320μl ddH2O. Antibody Buffer: Take 50μl Antibody Buffer(-), add 0.5μl 5% Digitonin, mix well and place on ice for precooling. Dig-wash Buffer: Mix 792μl Wash Buffer with 8μl 5% Digitonin. Dig-300Buffer: Take 100μl 10×Dig-300 Buffer, add 2μl 5% Digitonin and 20μl 50×Protease Inhibitor, add 878μl ddH2O and mix well. 1×B&W Buffer: Mix 500μl 2×B&W Buffer with 500μl ddH2O.

[0084] Example 1. Establishment of the method

[0085] 1. Preparation of mycelium

[0086] 1. Strain activation: Inoculate the test fungus stored at -80℃ onto a PDA medium plate and culture it at 26℃ for 3-4 days.

[0087] 2. Spore culture: After completing step 1, pick the bacterial blocks from the plate and inoculate them into Czapek culture medium, and culture them at 26°C and 220 rpm for 24 hours to obtain spore culture medium.

[0088] Czapek medium (pH 7.2): Take 30 g sucrose, 3 g NaNO3, 0.5 g MgSO4·7H2O, 0.5 g KCl, 100 mg FeSO4·7H2O and 1 g K2HPO4, dissolve them with ddH2O and make up to 1 L.

[0089] 3. Mycelial culture: inoculate 5 mL of the spore culture solution prepared in step 2 into 500 mL of liquid YEPD medium and culture at 26°C and 220 rpm for 18 h.

[0090] Liquid YEPD medium: Take 3 g yeast extract, 10 g peptone, and 20 g glucose, dissolve them with ddH2O and make up to 1 L.

[0091] 4. After completing step 3, filter and collect the mycelium with magic filter cloth, wash it 3 times with ddH2O, and then wash it 3 times with 0.7M NaCl aqueous solution.

[0092] 2. Formaldehyde cross-linking

[0093] 1. Place the mycelium obtained in step 1 in a 50 mL centrifuge tube, add 9 mL of 0.1% formaldehyde working solution, and react for 8 minutes.

[0094] 0.1% formaldehyde working solution: Dissolve formaldehyde in 0.7M NaCl aqueous solution and make the volume percentage of formaldehyde be 0.1%.

[0095] 2. After completing step 1, add 1 mL of 2 M glycine solution and incubate for 5 min.

[0096] 2M glycine solution: Dissolve L-glycine in 0.7M NaCl aqueous solution to obtain a glycine concentration of 2M.

[0097] 3. After completing step 2, wash the mycelium three times with 0.7M NaCl aqueous solution.

[0098] 3. Preparation of Protoplasts

[0099] 1. Take a 100 mL Erlenmeyer flask, add the mycelium obtained in step 2 and 10 ml of enzymatic hydrolysate, and shake at 33°C and 60 rpm for 3 h.

[0100] Enzyme hydrolysate: Take 0.25g Pro was dissolved in 12.5 ml of 0.7 M NaCl aqueous solution, rotated to dissolve for 20 min, and then filtered through a 0.22 μm pore size filter membrane to collect the filtrate. Pro (the product is in the form of solid powder): Novozyme.

[0101] 2. After completing step 1, filter with a double-layer magic filter cloth, collect the filtrate, centrifuge the filtrate at 3800rpm for 10min, discard the supernatant, wash the precipitate with 0.7M NaCl aqueous solution, and then resuspend the precipitate with 10ml 0.7M NaCl aqueous solution to obtain the protoplast solution (count the protoplasts with a hemocytometer).

[0102] 4. Obtaining the Cell Nucleus

[0103] 1. Take 4×10 6 Add 100 μL NE buffer to the protoplasts prepared in step 3, lyse on ice for 10 min, then centrifuge at 2500 rpm for 5 min, discard the supernatant, and the precipitate is the cell nucleus.

[0104] 2. After completing step 1, add 100 μl Wash Buffer to resuspend the pellet to obtain the cell nucleus suspension.

[0105] 5. Preparation of DNA library using CUT&Tag technology

[0106] 1. ConA beads processing

[0107] ① Take an 8-tube strip, add 100μl Binding Buffer to each tube, then add 10μl ConABeads Pro to each tube, mix well, and then place the 8-tube strip on a magnetic rack. After the solution is clarified, discard the supernatant.

[0108] ②After completing step ①, remove the 8-tube strip from the magnetic rack, add 100μl Binding Buffer to each tube, mix well, and then place the 8-tube strip on the magnetic rack. After the liquid is clarified, discard the supernatant.

[0109] ③After completing step ②, add 10 μl Binding Buffer to each tube to resuspend ConA Beads Pro.

[0110] 2. Incubation of cell nuclei with ConA beads

[0111] Take the 8-tube strips from step 1, add 100 μl of cell nucleus suspension to each tube, invert to mix, and then incubate at room temperature for 10 minutes (invert 2-3 times to mix), place the 8-tube strips on a magnetic rack after instant centrifugation, and discard the supernatant after the liquid becomes clear.

[0112] 3. Primary Antibody Incubation

[0113] Take the 8-tube strip obtained in step 2, add 50 μl of pre-cooled primary antibody working solution to each tube, invert to mix, centrifuge briefly, and let stand at 4°C for 16 h.

[0114] The primary antibody working solution was Flag antibody working solution or Mouse IgG working solution. Mouse IgG working solution was used as a control.

[0115] Flag antibody working solution: Dilute the Flag antibody with Antibody Buffer to obtain Flag antibody working solution; the antibody content in each 50μl Flag antibody working solution is 1μg. Flag antibody (monoclonal anti- M2 mouse antibody, product form is liquid, specification is 1mg / mL): Sigma, product number is F1804.

[0116] Mouse IgG working solution: Dilute Mouse IgG with Antibody Buffer to obtain Mouse IgG working solution; the antibody content in each 50μl Mouse IgG working solution is 1μg. Mouse IgG (product form is liquid, specification is 1mg / mL): Proteintech, catalog number is B900620.

[0117] 4. Secondary Antibody Incubation

[0118] ① Take the 8-tube strips from step 3, centrifuge them momentarily, and then place them on a magnetic rack. After the liquid becomes clear, discard the supernatant.

[0119] ②Take the 8-tube strips prepared in step ①, add 50 μl of secondary antibody working solution to each tube, invert to mix, and incubate at room temperature with rotation for 60 min.

[0120] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat Anti mouse IgG): Novozymes, catalog number Ab208-01-AA.

[0121] ③ Take the 8-tube strips from step ②, centrifuge them momentarily, then place the 8-tube strips on a magnetic rack, wait for the liquid to become clear, and discard the supernatant.

[0122] ④ Take the 8-tube strips prepared in step ③, add 200 μl Dig-wash Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strips on a magnetic rack, wait for the liquid to become clear, and discard the supernatant.

[0123] ⑤ Take the 8-tube strips obtained in step ④, add 200 μl Dig-wash Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strips on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0124] ⑥Take the 8-tube strips obtained in step ⑤, add 200 μl Dig-wash Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strips on a magnetic rack, wait for the liquid to become clear, and discard the supernatant.

[0125] 5. pA / G-Tnp incubation

[0126] ① Take the 8-tube strip obtained in step 4, add 100 μl pA / G-Tnp Pro working solution to each tube, invert to mix, and incubate at room temperature with rotation for 60 min.

[0127] pA / G-Tnp Pro working solution: Mix 2μl pA / G-Tnp Pro and 98μl Dig-300 Buffer.

[0128] ② Take the 8-tube strips obtained in step ①, centrifuge them momentarily, place them on a magnetic rack, wait for the liquid to become clear, and discard the supernatant.

[0129] ③Take the 8-tube strips obtained in step ②, add 200 μl Dig-300 Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strips on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0130] ④ Take the 8-tube strips prepared in step ③, add 200 μl Dig-300 Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strips on a magnetic rack, wait for the liquid to become clear, and discard the supernatant.

[0131] ⑤ Take the 8-tube strips obtained in step ④, add 200 μl Dig-300 Buffer to each tube, invert to mix, centrifuge briefly, place the 8-tube strips on a magnetic rack, wait for the liquid to clarify, and discard the supernatant.

[0132] 6. Fragmentation

[0133] ① Take the 8-tube strips prepared in step 5, add 50 μl of TTBL working solution to each tube, invert to mix, and incubate at 37°C for 60 min.

[0134] TTBL working solution: Mix 40 μl Dig-300 Buffer and 10 μl 5×TTBL.

[0135] ② Take the 8-tube strips prepared in step ①, centrifuge briefly, add 2 μl of 10% SDS and DNA Spike-in with a DNA content of 10 pg to each tube, and incubate at 55°C for 10 min (invert and mix 2-3 times during this time).

[0136] ③After completing step ②, centrifuge instantly, place the 8-tube strip on the magnetic rack, let it stand for about 2-3 minutes, and carefully transfer the supernatant to a new 8-tube strip.

[0137] 7. De-crosslinking

[0138] ① Take the 8-tube strip containing the supernatant obtained in step 6, add 3 μl of 5M NaCl aqueous solution to each tube, and react at 65°C for 18 h.

[0139] ②After completing step ①, add 0.25 μl RNase to each tube and react at 37°C for 30 min.

[0140] RNase A (Ribonuclease A) (product form is liquid, specification is 10 mg / ml): Takara Biomedical Technology, product number is 2158.

[0141] ③After completing step ②, add 1 μl of proteinase K solution to each tube and react at 37°C for 1 hour.

[0142] Protease K (product form: powder): Merck, catalog number 539480. Protease K solution: Take proteinase K and dissolve it in a buffer solution (containing 50 mM Tris-HCl, 2 mM calcium acetate, the remainder is water, pH 8.0) to a concentration of 5 mg / ml.

[0143] ④After completing step ③, add 1 μl of 50× protease inhibitor to each tube and react for 5 minutes.

[0144] 8. DNA Extract Beads Pro Processing

[0145] ① Take 25μl DNA Extract Beads Pro to a 1.5ml centrifuge tube, add 200μl 1×B&W Buffer, mix well, place on a magnetic rack, and discard the supernatant after the solution is clarified.

[0146] ②After completing step ①, remove the 1.5ml centrifuge tube from the magnetic stand, add 200μl 1×B&W Buffer, mix well, place on the magnetic stand, and discard the supernatant after the solution is clarified.

[0147] ③After completing step ②, add 50μl 2×B&W Buffer to a 1.5ml centrifuge tube to resuspend DNA Extract Beads Pro to obtain DNA Extract Beads Pro suspension.

[0148] 9. DNA extraction

[0149] ① Take the 8-tube strip obtained in step 7, add 50 μl of the DNA Extract Beads Pro suspension obtained in step 8 to each tube, invert to mix, and incubate at room temperature for 20 minutes (invert 2-3 times during this period).

[0150] ②After completing step ①, centrifuge instantly and place the 8-tube strip on a magnetic rack. After the solution becomes clear, discard the supernatant.

[0151] ③After completing step ②, keep the 8-tube strip on the magnetic rack, add 200μl 1×B&W Buffer to each tube, incubate at room temperature for 30 seconds, and discard the supernatant.

[0152] ④After completing step ③, keep the 8-tube strip on the magnetic rack, add 200μl 1×B&W Buffer to each tube, incubate at room temperature for 30 seconds, and discard the supernatant.

[0153] ⑤After completing step ④, open the lid and let it stand at room temperature for 2-5 minutes until there is no liquid left in the tube and the surface of the magnetic beads is not reflective.

[0154] ⑥After completing step ⑤, remove the 8-tube strip from the magnetic stand and add 15μl ddH2O to each tube to resuspend the DNAExtract Beads Pro.

[0155] 10. Library Amplification

[0156] Prepare the amplification system according to Table 1, and then perform amplification according to Table 2.

[0157] Table 1

[0158] volume The resuspension prepared in step 9 15μl 2×CAM 25μl N5XX 5μl N7XX 5μl Total volume 50μl

[0159] Table 2

[0160]

[0161] TruePrep Index Kit V2 for Illumina: Novozymes, catalog number TD202. TruePrep Index Kit V2 for Illumina provides 8 N5XX (N501-N508) and 12 N7XX (N701-N712). You can choose any one of the N5XX and one of the N7XX in Table 1.

[0162] 11. Purification and recovery of DNA

[0163] ① After completing step 10, add 100 μl VAHTS DNA Clean Beads (Vazyme#N411) to each reaction tube, mix well, incubate at room temperature for 5 minutes, centrifuge briefly, and then place the reaction tube on a magnetic rack. After the solution is clarified, carefully remove the supernatant.

[0164] ②After completing step ①, keep the reaction tubes on the magnetic rack, add 200 μl of 80% ethanol aqueous solution to each tube, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0165] ③After completing step ②, keep the reaction tubes on the magnetic rack, add 200 μl of 80% ethanol aqueous solution to each tube, incubate at room temperature for 30 seconds, carefully remove the supernatant, open the lid and dry for 3-5 minutes.

[0166] ④After completing step ③, remove the reaction tube from the magnetic rack, add 22μl ddH2O to each tube for elution, mix well, incubate at room temperature for 5min, centrifuge briefly, and then place the reaction tube on the magnetic rack. After the solution is clarified, carefully pipette 20μl of supernatant into a new EP tube, which is the DNA library solution.

[0167] 6. Sequencing

[0168] 1. Take the DNA library solution obtained in step 5, use Qubit dsDNA HS Assay Kit double-stranded DNA fluorescence quantitative kit, follow the instructions for quantification, and use Qseq 400 to detect the peak distribution.

[0169] 2. Take the DNA library solution obtained in step 5 and sequence it.

[0170] The library was sequenced using the Illumina Novaseq 6000 sequencing platform in paired-end 150 bp sequencing mode.

[0171] Example 2: Construction of recombinant HP1-3Flag

[0172] Heterochromatin Protein 1 (HP1) was used as the target protein. HP1 can recognize histone H3K9me3 modification, participate in the formation and maintenance of heterochromatin, and is an important DNA binding protein. The HP1 gene in the genome of Verticillium dahliae V592 is shown in Sequence 1 of the sequence table.

[0173] Verticillium dahliae strain V592 and pGKO-Flag-HPT vector are both described in the following literature: Yang, J.; Liu, M.; Jiao, Y.; Guo, H.-S.; Shan, C.-M.; Wang, H. An Efficient Homologous Recombination-Based In Situ Protein-Labeling Method in Verticillium dahliae. Biology 2024, 13, 81. https: / / doi.org / 10.3390 / biology13020081. Schematic diagram of the elements of the pGKO-Flag-HPT vector is shown in Figure 1 .

[0174] 1. Using pGKO-Flag-HPT vector as the starting vector, insert the upstream homology arm (as shown in the 1st to 1048th position in the sequence 2 of the sequence table) at the upstream PacI restriction site of the Flag-HPT box, and insert the downstream homology arm (as shown in the 3620th to 4623rd position in the sequence 2 of the sequence table) at the downstream PacI restriction site of the Flag-HPT box to obtain a recombinant plasmid. After sequencing verification, the recombinant plasmid has the DNA molecule shown in the sequence 2 of the sequence table. In the sequence 2 of the sequence table, the 1st to 1048th positions constitute the upstream homology arm, the 1088th to 1153rd positions encode the 3Flag tag, the 1154th to 1156th positions constitute the stop codon, the 1531st to 2556th positions encode the hygromycin resistance protein, and the 3621st to 4623rd positions constitute the downstream homology arm.

[0175] 2. The recombinant plasmid obtained in step 1 is introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

[0176] 3. The recombinant Agrobacterium obtained in step 2 was co-cultured with spores of Verticillium dahliae V592, and then the recombinant Verticillium dahliae was obtained by hygromycin screening, and the recombinant bacterium HP1-3Flag was named. Compared with the genomic DNA of Verticillium dahliae V592, the difference in the genomic DNA of the recombinant bacterium HP1-3Flag is that the termination codon TAA (the last 3 nucleotides in sequence 1) of the HP1 gene shown in sequence 1 of the sequence table is replaced with the DNA molecule shown in positions 1046-3619 in sequence 2 of the sequence table. In the genomic DNA of the recombinant bacterium HP1-3Flag, the 3Flag gene is fused downstream of the HP1 gene with the termination codon removed, and the fusion gene is shown in sequence 3 of the sequence table.

[0177] Example 3: Verification of the effect of the method

[0178] The tested fungi were Verticillium dahliae V592 or the recombinant HP1-3Flag constructed in Example 2.

[0179] The procedure was carried out according to Example 1.

[0180] The length distribution of the DNA library prepared by the recombinant strain HP1-3Flag in the above steps (using Flag antibody working solution) is shown in Figure 2 (Results of two replicates). The length distribution of the DNA library prepared by the recombinant strain HP1-3Flag in the above steps (using Mouse IgG working solution) is shown in Figure 3 (Results of two replicates). The length distribution of the DNA library prepared by the above steps (using Flag antibody working solution) for Verticillium dahliae V592 is shown in Figure 4 The length distribution of the DNA library prepared by the above steps (using Mouse IgG working solution) of Verticillium dahliae V592 is shown in Figure 4 Right figure. All DNA libraries meet the sequencing requirements.

[0181] The sequencing results of the DNA library prepared by the above steps (using Flag antibody working solution) of Verticillium dahliae V592 and recombinant strain HP1-3Flag are shown in Figure 5 . Figure 5 In the table, WT represents Verticillium dahliae V592, and HP1-3Flag represents recombinant HP1-3Flag (two replicates). The sequencing results of the DNA library prepared by the above steps (using Mouse IgG working solution) of Verticillium dahliae V592 and recombinant HP1-3Flag are shown in Figure 6 . Figure 6 In the table, WT represents Verticillium dahliae V592, and HP1-3Flag represents recombinant HP1-3Flag (two replicates). The results show that the use of the Flag antibody working solution according to the method provided by the present invention has a good enrichment effect on the target gene bound by the target protein (HP1), and the method has good repeatability. The results show that the method provided by the present invention can effectively detect the distribution and abundance of the target protein in the whole fungal genome.

[0182] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for detecting the distribution and abundance of a target protein in the whole genome of a test organism, characterized in that: The organism is a fungus, and the target protein is a DNA binding protein in the tested fungus; The method provided by the present invention for detecting the distribution and abundance of a target protein in the whole genome of a test organism is fungal CUT&Tag; compared with the traditional CUT&Tag, the difference of the fungal CUT&Tag is that: The following steps are added before adsorbing the nucleus: fungal hyphae are taken, cross-linked with formaldehyde, and then protoplasts are prepared; Add the following step between fragmentation and library amplification: Remove formaldehyde cross-links.

2. The method according to claim 1, characterized in that: The fungus CUT&Tag comprises the following steps in sequence: (1) taking fungal mycelium, performing formaldehyde cross-linking, and then preparing protoplasts; (2) Extracting cell nuclei; (3) Adsorption of cell nuclei; (4) Primary antibody incubation; (5) Secondary antibody incubation; (6) pA / G-Tn5 transposase incubation; (7) Fragmentation: Activation of pA / G-Tn5 transposase; (8) Removal of formaldehyde cross-linking; (9) Library amplification: obtaining a DNA library through library amplification; (10) The DNA library is subjected to high-throughput sequencing, and the distribution and abundance of the target protein in the entire genome of the test organism is obtained based on the sequencing results.

3. The method according to claim 1 or 2, characterized in that: The formaldehyde cross-linking comprises the following steps: taking mycelium, treating it with formaldehyde solution, and then adding glycine or glycine solution to terminate the reaction.

4. The method according to any one of claims 1 to 3, characterized in that: The preparation of protoplasts comprises the following steps: after the formaldehyde cross-linking is completed, the mycelium is taken, and Pro was enzymatically hydrolyzed to obtain protoplasts.

5. The method according to any one of claims 1 to 4, characterized in that: The method for removing formaldehyde crosslinks comprises the following steps: after fragmentation, taking DNA fragments, and sequentially performing the following operations: adding NaCl or NaCl solution and reacting, then adding RNase or RNase solution and reacting, then adding proteinase K or proteinase K solution and reacting, and then adding protease inhibitor or protease inhibitor solution.

6. The method according to any one of claims 1 to 5, characterized in that: The primary antibody used in the primary antibody incubation is a primary antibody against the target protein.

7. The method according to any one of claims 1 to 5, characterized in that: The fungus is a recombinant fungus; the recombinant fungus is obtained by integrating an exogenous DNA molecule into the genomic DNA of the starting fungus through homologous recombination; the exogenous DNA molecule has a gene encoding a protein tag; the gene encoding the target protein in the genomic DNA of the starting fungus forms a fusion gene with the gene encoding the protein tag to express the target protein with the protein tag; The primary antibody used in the primary antibody incubation is an antibody against a protein tag.

8. The method according to any one of claims 1 to 7, characterized in that: The amount of cell nuclei adsorbed in the fungal CUT&Tag is 4,000,000 cell nuclei.

9. The method according to any one of claims 1 to 8, characterized in that: The fungus is a filamentous fungus.

10. The method according to any one of claims 1 to 8, characterized in that: The fungus is Verticillium dahliae.

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