A method for efficiently detecting distribution and abundance of DNA binding proteins at whole genome level of fungi

Abstract

The application discloses a method for efficiently detecting the distribution and abundance of DNA binding proteins at the whole genome level of fungi. The application provides a method for detecting the distribution and abundance of target proteins in the whole genome of a test organism, characterized in that the organism is fungi, and the target protein is a DNA binding protein in the test fungi; the method for detecting the distribution and abundance of target proteins in the whole genome of a test organism provided by the application is fungal CUT&Tag; compared with traditional CUT&Tag, the difference of the fungal CUT&Tag lies in that the following step is added before adsorbing the cell nucleus: taking fungal hyphae, performing formaldehyde cross-linking, and then preparing protoplasts; the following step is added between fragmentation and library amplification: removing formaldehyde cross-linking. The application first realizes the application of the CUT&Tag technology to the detection of the distribution and abundance of DNA binding proteins of filamentous fungi at the genome level, and provides an important means for the research on fungal DNA binding proteins.

Description

TECHNICAL FIELD

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

[0002] The CUT&Tag technology was published by the Henikoff team in Nature Communication in 2019. This technology can be used for research on histone modification sites and DNA binding protein target sites at the whole genome level. CUT&Tag technology uses Concanavalin A-coated magnetic beads to adsorb living cells, and uses digitonin to increase cell membrane permeability. The cells remain in a living state during the experiment, which can more truly reflect the state of histone modification and DNA binding protein in the cells. The target protein specific antibody (primary antibody) enters the cell to recognize and bind to the specific modified histone / DNA binding protein, the secondary antibody recognizes the primary antibody to amplify the signal, the pA / G-Tn5 transposase is combined with the primary antibody and the secondary antibody, and the DNA at both ends of the target protein is cut by the activation of Mg 2+ The sequencing adapter is added at the same time. Through PCR, the library can be constructed, and after sequencing, the binding sites of the target protein or the modification level of the histone in the whole genome range can be detected. Through qPCR, the relative binding amount of the protein or the modification level of the histone at the target gene can also be detected. Compared with the traditional ChIP technology, this method requires less starting material, can realize single-cell level, does not require ultrasonic crushing and other steps with high requirements for instruments and technology, 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 a large number of existing histone modifications, the relative content of DNA binding proteins (including transcription factors) is low, or the number of target genes is small, and the binding is unstable, which can all lead to the failure to enrich the target genes of the protein through the traditional CUT&Tag experiment. At present, only a few cases of detecting transcription factors through CUT&Tag experiment in animal and plant cells have been successful. Due to the existence of cell walls and some secondary metabolites in fungi, the application of this technology in fungi is more challenging, and there is no related report in filamentous fungi. SUMMARY

[0004] The purpose of the present application 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 application provides a method for detecting the distribution and abundance of target proteins 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 test fungus.

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

[0008] Before adsorbing the cell nucleus, the following steps are added: taking fungal hyphae, performing formaldehyde cross-linking, and then preparing protoplasts;

[0009] Between fragmentation and library amplification, the following steps are added: removing formaldehyde cross-linking.

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

[0011] (1) extracting the cell nucleus;

[0012] (2) adsorbing the cell nucleus;

[0013] (3) primary antibody incubation;

[0014] (4) secondary antibody incubation;

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

[0016] (6) fragmentation: activating pA / G-Tn5 transposase;

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

[0018] (8) performing high-throughput sequencing on the DNA library, and obtaining the distribution and abundance of target proteins in the whole genome of a test organism according to the sequencing results.

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

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

[0021] (2) extracting the cell nucleus;

[0022] (3) adsorbing the cell nucleus;

[0023] (4) primary antibody incubation;

[0024] (5) secondary antibody incubation;

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

[0026] (7) fragmentation: activating pA / G-Tn5 transposase;

[0027] (8) removing formaldehyde cross-linking;

[0028] (9) Library amplification: DNA library is obtained by library amplification;

[0029] (10) High-throughput sequencing of the DNA library, and obtaining the distribution and abundance of the target protein in the whole genome of the test organism according to the sequencing results.

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

[0031] Specifically, the adsorbed nuclei are adsorbed by ConA beads.

[0032] The formaldehyde crosslinking includes the following steps: taking mycelium, treating with a formaldehyde solution, and then adding glycine or a glycine solution to terminate the reaction.

[0033] The formaldehyde crosslinking includes the following steps: taking mycelium, treating with a formaldehyde solution, and then adding glycine or a glycine solution to terminate the reaction.

[0034] Specifically, the formaldehyde crosslinking includes the following steps: taking mycelium, reacting in a formaldehyde solution for 8 min; then adding glycine or a glycine solution, and incubating for 5 min.

[0035] Specifically, the formaldehyde crosslinking includes the following steps: taking mycelium, reacting in a formaldehyde solution for 8 min; then adding glycine or a glycine solution, and incubating for 5 min.

[0036] Specifically, the formaldehyde solution is a formaldehyde solution with a formaldehyde volume percentage content 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 content 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 the protoplasts comprises the following steps: after the cross-linking by formaldehyde is completed, the mycelium is taken and subjected to enzymatic hydrolysis using Pro, and then the protoplasts are collected.

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

[0045] Specifically, the enzymatic hydrolysis is performed at 33℃ with 60 rpm oscillation for 3 h.

[0046] Specifically, the preparation of the protoplasts comprises the following steps: after the cross-linking by formaldehyde is completed, the mycelium is taken and placed in an enzymatic hydrolysis solution, and subjected to enzymatic hydrolysis at 33℃ with 60 rpm oscillation for 3 h; then, the protoplasts are collected by filtering through double-layer magic filter cloth, and the filtrate is centrifuged at 3800 rpm for 10 min, the supernatant is discarded, and the filtrate is washed with 0.7M NaCl aqueous solution.

[0047] The enzymatic hydrolysis solution: 0.25 g of VinoTaste Pro is dissolved in 12.5 ml of 0.7M NaCl aqueous solution, and the solution is rotated for 20 min, and then filtered through a filter membrane with a pore size of 0.22 μm, and the filtrate is collected.

[0048] The removal of the formaldehyde cross-linking comprises the following steps: after the fragmentation is completed, the DNA fragments are taken, and the following operations are sequentially performed: NaCl or NaCl solution is added and reacted, then RNase or RNase solution is added and reacted, then proteinase K or proteinase K solution is added and reacted, and then protease inhibitor or protease inhibitor solution is added and reacted.

[0049] The removal of the formaldehyde cross-linking comprises the following steps: after the fragmentation is completed, the liquid phase system containing the DNA fragments is taken, and the following operations are sequentially performed: 5M NaCl aqueous solution is added and reacted at 65℃ for 18 h; then, RNase is added and reacted at 37℃ for 30 min; then, proteinase K solution is added and reacted at 37℃ for 1 h; and then, protease inhibitor is added and reacted for 5 min.

[0050] The removal of the formaldehyde cross-linking comprises the following steps: after the fragmentation is completed, 50 μl of the liquid phase system containing the DNA fragments is taken, and the following operations are sequentially performed: 3 μl of 5M NaCl aqueous solution is added and reacted at 65℃ for 18 h; then, 0.25 μl of RNase is added and reacted at 37℃ for 30 min; then, 1 μl of proteinase K solution is added and reacted at 37℃ for 1 h; and then, 1 μl of 50× protease inhibitor is added and reacted for 5 min.

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

[0052] Protease K (powder form): Merck, product number 539480. Protease K solution: Take Protease K, dissolve it with buffer (containing 50 mM Tris-HCl, 2 mM calcium acetate, the rest is water, pH 8.0) to make its concentration 5 mg / ml.

[0053] As a 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 fungus; the recombinant fungus is obtained by homologous recombination of an exogenous DNA molecule into the genomic DNA of the starting fungus; 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, and expresses the target protein with the 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. For example, 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 with the stop codon removed, and the tag gene. The exogenous DNA molecule also has a resistance screening gene. Specifically, the tag gene is shown in SEQ ID NO: 2 at positions 1088-1156. Specifically, the fusion gene is composed of, from upstream to downstream, the target gene with the stop codon removed, and a DNA molecule shown in SEQ ID NO: 2 at positions 1049-1156. Specifically, the resistance screening gene can be a hygromycin resistance gene. Specifically, the hygromycin resistance gene is shown in SEQ ID NO: 2 at positions 1531-2556. Specifically, the exogenous DNA molecule is shown in SEQ ID NO: 2 at positions 1049-3620.

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

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

[0057] Specifically, the time of the primary antibody incubation can be 16 hours.

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

[0059] Specifically, the time of the secondary antibody incubation can be 60 minutes.

[0060] As an example, the secondary antibody is Goat Anti mouse IgG. Specifically, the secondary antibody incubation is incubation with secondary antibody working solution. The secondary antibody is diluted with 50 μl Dig-wash Buffer to 100 times volume to obtain secondary antibody working solution.

[0061] Specifically, the time of the pA / G-Tn5 transposase incubation can be 60 min.

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

[0063] The activation of the pA / G-Tn5 transposase is achieved by adding TTBL working solution.

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

[0065] Specifically, in the fungal CUT&Tag, the amount of adsorbed nuclei is 4000000 cell nuclei.

[0066] Specifically, the fungus is filamentous fungus.

[0067] Specifically, the fungus is Lachancea ligniera.

[0068] Due to the relatively low abundance of DNA binding proteins such as transcription factors in cells, the target genes bound by the DNA binding proteins are also less, and the binding of the DNA binding proteins to DNA is unstable, so it is difficult to study the abundance and binding sites of the transcription factors by the traditional CUT&Tag technology, and the cell wall and rich secondary metabolites of fungi further increase the difficulty of the experiment.

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

[0070] ①The cell demand is small, only 4000000 cells;

[0071] ②The time consumption is short, and the operation is simple;

[0072] ③The signal-to-noise ratio is high, and the background is low;

[0073] ④The repeatability is good.

[0074] The present application first realizes the application of the CUT&Tag technology to the detection of the distribution and abundance of DNA binding proteins (including transcription factors) of filamentous fungi at the genomic level, and provides an important means for the research of the DNA binding proteins of fungi. BRIEF DESCRIPTION OF DRAWINGS

[0075] Figure 1 It is an element schematic diagram of the pGKO-Flag-HPT vector.

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

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

[0078] Figure 4 It is a result of length distribution of the DNA library prepared by the L. rosyum V592.

[0079] Figure 5 It is a sequencing result of the DNA library prepared by the L. rosyum V592 and the recombinant bacteria HP1-3Flag (using Flag antibody working solution).

[0080] Figure 6 It is a sequencing result of the DNA library prepared by the L. rosyum V592 and the recombinant bacteria HP1-3Flag (using Mouse IgG working solution). DETAILED DESCRIPTION

[0081] The application will be further described in conjunction with the specific embodiments, the examples given are only for illustrating the application, but not for limiting the scope of the application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application.

[0082] The experimental methods in the following examples are all routine methods, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified. The quantitative tests in the following examples are all set in triplicate, and the results are averaged, unless otherwise specified. Miracloth: pore size 22-25 μm, Merck millipore, item number 475855-1R. L. maculata V592 was kindly provided by Professor Hui-shan Guo's group in the Institute of Microbiology, Chinese Academy of Sciences.

[0083] Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro: Novagen, Cat. No. TD904-01. The kit provides the following components: NE buffer, 10x Wash Buffer, 10x Binding Buffer, ConA Beads Pro, Antibody Buffer(-), pA / G-Tnp Pro (2 mM), 10x Dig-300 Buffer, 5x TTBL, DNA Spike-in (5 ng / µl), DNA Extract Beads Pro, 2x B&W Buffer, 2x CAM, 10% SDS, 5% Digitonin. 50x Protease Inhibitor: Take one piece of Protease Inhibitor (Roche, 04693132001), dissolve in 1 ml ddH2O. Binding Buffer: Mix 30 µl 10x Binding Buffer with 270 µl ddH2O. Wash Buffer: Mix 150 µl 10x Wash Buffer, 30 µl 50x Protease Inhibitor with 1320 µl ddH2O. Antibody Buffer: Take 50 µl Antibody Buffer(-), add 0.5 µl 5% Digitonin, mix and pre-cool on ice. Dig-wash Buffer: Mix 792 µl Wash Buffer with 8 µl 5% Digitonin. Dig-300 Buffer: Take 100 µl 10x Dig-300 Buffer, add 2 µl 5% Digitonin and 20 µl 50x Protease Inhibitor, add 878 µl ddH2O, mix. 1x B&W Buffer: Mix 500 µl 2x B&W Buffer with 500 µl ddH2O.

[0084] Example 1, Establishment of the method

[0085] I. Preparation of mycelium

[0086] 1. Strain activation: The test fungus stored at -80°C was inoculated into PDA medium plate and incubated at 26°C for 3-4 days.

[0087] 2. Spore culture: After completing step 1, the mycelial block was inoculated into Czapek's liquid medium and incubated at 26°C with 220 rpm shaking for 24 h to obtain spore culture.

[0088] Chase 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 with ddH2O and make up to 1 L.

[0089] 3. Mycelium culture: inoculate 5 mL spore culture solution prepared in step 2 into 500 mL liquid YEPD medium, and cultivate at 26°C with 220 rpm shaking for 18 h.

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

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

[0092] II. Formaldehyde crosslinking

[0093] 1. Put the mycelium obtained in step I into a 50 mL centrifuge tube, add 9 mL 0.1% formaldehyde working solution, and react for 8 min.

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

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

[0096] 2M glycine solution: dissolve L-glycine in 0.7 M NaCl solution, and make the concentration of glycine 2 M.

[0097] 3. After step 2 is completed, wash the mycelium with 0.7 M NaCl solution for 3 times.

[0098] III. Preparation of protoplasts

[0099] 1. Take 100 mL triangular flask, add the mycelium obtained in step II and 10 ml enzyme solution, and react at 33°C with 60 rpm shaking for 3 h.

[0100] Enzyme solution: take 0.25 g Pro, dissolve in 12.5 ml 0.7 M NaCl solution, rotate for 20 min, then filter with 0.22 μm pore size filter membrane, and collect the filtrate. Pro (product form is solid powder): novozyme company.

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

[0102] Four, obtain the nucleus

[0103] 1、Take 4x10 6 μl of protoplast prepared in step three, add 100μl NE buffer, lyse on ice for 10min, then centrifuge at 2500rpm for 5min, discard the supernatant, and the precipitate is the nucleus.

[0104] 2、After step 1 is completed, add 100μl Wash Buffer to resuspend the precipitate, which is the nucleus suspension.

[0105] Five, prepare DNA library using CUT&Tag technology

[0106] 1、ConA beads treatment

[0107] ①Take an 8-tube tube, add 100μl Binding Buffer to each tube, then add 10μl ConA Beads Pro to each tube, mix well, then place the 8-tube tube on the magnetic stand, and after the solution is clarified, discard the supernatant.

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

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

[0110] 2、Nuclei and ConA beads incubation

[0111] Take the 8-tube tube completed in step 1, add 100μl of the nucleus suspension to each tube, mix well by inverting, then incubate at room temperature for 10min (invert and mix 2-3 times during the incubation), then centrifuge briefly and place the 8-tube tube on the magnetic stand, and after the liquid is clarified, discard the supernatant.

[0112] 3、Primary antibody incubation

[0113] Take the 8-tube tube completed in step 2, add 50μl of pre-cooled primary antibody working solution to each tube, mix well by inverting, then centrifuge briefly and place at 4℃ for 16h.

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

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

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

[0117] 4. Incubation of secondary antibody

[0118] ①Take the 8-tube pipe completed in step 3, centrifuge for a moment, then place it on the magnetic stand, and after the liquid is clarified, discard the supernatant.

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

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

[0121] ③Take the 8-tube pipe completed in step ②, centrifuge for a moment, then place the 8-tube pipe on the magnetic stand, and after the liquid is clarified, discard the supernatant.

[0122] ④Take the 8-tube pipe completed in step ③, add 200 μl of Dig-wash Buffer to each tube, invert and mix, centrifuge for a moment, place the 8-tube pipe on the magnetic stand, and after the liquid is clarified, discard the supernatant.

[0123] ⑤Take the 8-tube pipe completed in step ④, add 200 μl of Dig-wash Buffer to each tube, invert and mix, centrifuge for a moment, place the 8-tube pipe on the magnetic stand, and after the liquid is clarified, discard the supernatant.

[0124] ​Take the 8-tube from step 5, add 200 μl Dig-wash Buffer to each tube, mix well by inverting, centrifuge briefly, and place the 8-tube on the magnetic stand until the liquid is clear, then discard the supernatant.

[0125] 5. Incubate pA / G-Tnp

[0126] Take the 8-tube from step 4, add 100 μl pA / G-Tnp Pro working solution to each tube, mix well by inverting, and incubate at room temperature for 60 min.

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

[0128] Take the 8-tube from step 1, centrifuge briefly, and place the 8-tube on the magnetic stand until the liquid is clear, then discard the supernatant.

[0129] Take the 8-tube from step 2, add 200 μl Dig-300 Buffer to each tube, mix well by inverting, centrifuge briefly, and place the 8-tube on the magnetic stand until the liquid is clear, then discard the supernatant.

[0130] Take the 8-tube from step 3, add 200 μl Dig-300 Buffer to each tube, mix well by inverting, centrifuge briefly, and place the 8-tube on the magnetic stand until the liquid is clear, then discard the supernatant.

[0131] Take the 8-tube from step 4, add 200 μl Dig-300 Buffer to each tube, mix well by inverting, centrifuge briefly, and place the 8-tube on the magnetic stand until the liquid is clear, then discard the supernatant.

[0132] 6. Fragmentation

[0133] Take the 8-tube from step 5, add 50 μl TTBL working solution to each tube, mix well by inverting, and incubate at 37°C for 60 min.

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

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

[0136] After step 2, centrifuge briefly, place the 8-tube on the magnetic stand, and let it stand for about 2-3 min, then carefully transfer the supernatant to a new 8-tube.

[0137] 7. De-crosslinking

[0138] ① Take the 8-tube 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] ② After completion of step ①, add 0.25 μl of RNase to each tube, and react at 37°C for 30 min.

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

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

[0142] Protease K (product form: powder): Merck, product code 539480. Protease K solution: Take protease K, dissolve it in a buffer (containing 50 mM Tris-HCl, 2 mM calcium acetate, and the balance being water, pH 8.0) so as to have a concentration of 5 mg / ml.

[0143] ④ After completion of step ③, add 1 μl of 50x protease inhibitor to each tube, and react for 5 min.

[0144] 8. DNA Extract Beads Pro treatment

[0145] ① Take 25 μl of DNA Extract Beads Pro into a 1.5-ml centrifuge tube, add 200 μl of 1x B&W Buffer, mix well, place on a magnetic stand, and after the solution has clarified, discard the supernatant.

[0146] ② After completion of step ①, remove the 1.5-ml centrifuge tube from the magnetic stand, add 200 μl of 1x B&W Buffer, mix well, place on a magnetic stand, and after the solution has clarified, discard the supernatant.

[0147] ③ After completion of step ②, add 50 μl of 2x B&W Buffer to the 1.5-ml centrifuge tube to resuspend the DNA Extract Beads Pro, thereby obtaining a DNA Extract Beads Pro suspension.

[0148] 9. DNA extraction

[0149] ① Take the 8-tube obtained in step 7, add 50 μl of the DNA Extract Beads Pro suspension obtained in step 8 to each tube, mix well by inversion, and incubate at room temperature for 20 min (inverting 2-3 times during the incubation).

[0150] 2. After step 1, centrifuge the tubes briefly, place the 8-tube on the magnetic stand, and discard the supernatant after the solution is clear.

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

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

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

[0154] 6. After step 5, remove the 8-tube from the magnetic stand, add 15 μl of ddH2O to each tube to resuspend the DNA Extract 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 Resuspension prepared in Step 9 15 μl 2 x CAM 25 μl N5XX 5 μl N7XX 5 μl Total volume 50 μl

[0159] Table 2

[0160]

[0161] TruePrep Index Kit V2 for Illumina: Vazyme, item number TD202. TruePrep Index Kit V2 for Illumina provides 8 N5XX (N501-N508) and 12 N7XX (N701-N712), and any one of N5XX and N7XX can be selected from Table 1.

[0162] 11. Purification and recovery of DNA

[0163] 1. After step 10, add 100 μl of 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 the magnetic stand after the solution is clear. Carefully remove the supernatant.

[0164] ② After step ① is completed, keep the reaction tube on the magnetic stand, add 200 μl of 80% ethanol aqueous solution to each tube, incubate at room temperature for 30 sec, carefully remove the supernatant.

[0165] ③ After step ② is completed, keep the reaction tube on the magnetic stand, add 200 μl of 80% ethanol aqueous solution to each tube, incubate at room temperature for 30 sec, carefully remove the supernatant, and dry for 3-5 min with the cap open.

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

[0167] Six, sequencing

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

[0169] 2. Take the DNA library solution obtained in step five, and perform sequencing.

[0170] Use the double-end 150bp sequencing mode of Illumina Novaseq 6000 sequencing platform to sequence the library.

[0171] Example 2, construction of recombinant bacteria HP1-3Flag

[0172] Heterochromatin Protein 1 (HP1) is 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 L. maculans V592 is shown as sequence 1 of the sequence table.

[0173] Both Verticillium dahliae strain V592 and pGKO-Flag-HPT vector are described in 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. The schematic diagram of elements of pGKO-Flag-HPT vector is shown in FIG. 1. Figure 1 .

[0174] 1. Using pGKO-Flag-HPT vector as the starting vector, an upstream homologous arm (as shown in SEQ ID NO: 2, positions 1-1048) is inserted at the upstream PacI restriction site of the Flag-HPT box, and a downstream homologous arm (as shown in SEQ ID NO: 2, positions 3620-4623) is inserted at the downstream PacI restriction site of the Flag-HPT box to obtain a recombinant plasmid. Sequencing verification shows that the recombinant plasmid has the DNA molecule shown in SEQ ID NO: 2. In SEQ ID NO: 2, positions 1-1048 constitute the upstream homologous arm, positions 1088-1153 encode the 3Flag tag, positions 1154-1156 constitute the stop codon, positions 1531-2556 encode the hygromycin resistance protein, and positions 3621-4623 constitute the downstream homologous arm.

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

[0176] 3. The recombinant Agrobacterium obtained in step 2 is co-cultured with Verticillium dahliae V592 spores, and then recombinant Verticillium dahliae is obtained through hygromycin screening, which is named as recombinant bacteria HP1-3Flag. Compared with the genomic DNA of Verticillium dahliae V592, the difference of the genomic DNA of recombinant bacteria HP1-3Flag is that the stop codon TAA (the last 3 nucleotides in SEQ ID NO: 1) of HP1 gene shown in SEQ ID NO: 1 is replaced by the DNA molecule shown in positions 1046-3619 of SEQ ID NO: 2. In the genomic DNA of recombinant bacteria HP1-3Flag, the 3Flag gene is fused downstream of the HP1 gene without the stop codon, and the fusion gene is shown in SEQ ID NO: 3.

[0177] Example 3, verification of the effect of the method

[0178] The test fungi were: P. dahliae V592 or the recombinant bacteria HP1-3Flag constructed in Example 2.

[0179] The operation was performed according to the method of Example 1.

[0180] The results of the length distribution of the DNA library prepared by the above steps (using Flag antibody working solution) for the recombinant bacteria HP1-3Flag are shown in Figure 2 (the results of two repetitions). The results of the length distribution of the DNA library prepared by the above steps (using Mouse IgG working solution) for the recombinant bacteria HP1-3Flag are shown in Figure 3 (the results of two repetitions). The results of the length distribution of the DNA library prepared by the above steps (using Flag antibody working solution) for P. dahliae V592 are shown in Figure 4 (the left graph). The results of the length distribution of the DNA library prepared by the above steps (using Mouse IgG working solution) for P. dahliae V592 are shown in Figure 4 (the right graph). The DNA libraries all met the sequencing requirements.

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

[0182] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A method for detecting the distribution and abundance of a target protein across the whole genome of a test organism, characterized by: The organism is a fungus, and the target protein is a DNA binding protein in the fungus; The method comprises the following steps in sequence: (1) taking fungal mycelium, cross-linking with formaldehyde, and then preparing protoplasts; the formaldehyde cross-linking comprises the following steps: taking the mycelium, treating with a formaldehyde solution, and then adding a glycine solution to terminate the reaction; the preparation of protoplasts comprises the following steps: after the formaldehyde cross-linking is completed, the mycelium is taken, enzymolysis is performed by using VinoTaste® Pro, and protoplasts are obtained; (2) extracting cell nuclei; (3) adsorbing cell nuclei; (4) primary antibody incubation; (5) secondary antibody incubation; (6) pA / G-Tn5 transposase incubation; (7) fragmentation: activating pA / G-Tn5 transposase; (8) removing formaldehyde cross-linking; the removing of formaldehyde cross-linking comprises the following steps: after the fragmentation is completed, the DNA fragments are taken, and the following operations are performed in sequence: adding a NaCl solution and reacting, then adding an RNase solution and reacting, then adding a proteinase K solution and reacting, and then adding a protease inhibitor solution; (9) library amplification: obtaining a DNA library through library amplification; (10) performing high-throughput sequencing on the DNA library, and obtaining the distribution and abundance of the target protein in the whole genome of the test organism according to the sequencing results; The fungus is a filamentous fungus.

2. The method of claim 1, wherein: The primary antibody used in the primary antibody incubation is a primary antibody against the target protein.

3. The method of claim 1, 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 a 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, so that the target protein with the protein tag is expressed; The primary antibody used in the primary antibody incubation is an antibody against the protein tag.

4. The method of claim 1, wherein: The amount of adsorbed cell nuclei in the method is 4000000 cell nuclei.

5. The method of any one of claims 1 to 4, wherein: The fungus is Lecanicillium lecanii.

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