Method for efficiently detecting fungal whole genome level histone modification sites and abundance

Abstract

The invention discloses a method for efficiently detecting a fungal whole genome level histone modification site and abundance. The method for detecting the distribution and abundance of the histone modification in the whole genome of the tested fungus comprises the following steps: taking hyphae of the fungus, preparing protoplast, and then extracting cell nucleuses; the cell nucleus is subjected to CUTamp for detecting histone modification as a purpose; and Tag. According to the method and the device, the CUAMP is firstly converted into the CUAMP; the Tag technology is applied to detection of the histone modification content and positioning of fungi, and an important means is provided for studying the relation between fungus histone modification and growth and pathogenicity of the fungus histone modification.

Description

Technical Field

[0001] The present invention relates to a method for efficiently detecting histone modification sites and abundance at the whole genome level of fungi. Background Art

[0002] Nucleosomes are the basic structural units of chromatin. Nucleosomes are composed of an octamer formed by two copies of each of the four histones H2B, H2A, H3, and H4, and about 146bp of DNA wrapped around the outside. Histones, as important components of nucleosomes, are one of the decisive factors that determine the degree of chromatin packaging. The amino acids at the N-terminal tail of histones can undergo a variety of modifications, including acetylation, methylation, and ubiquitination. These modifications affect the compactness and accessibility of chromatin by changing the charge of nucleosomes or recruiting epigenetic regulatory factors that affect chromatin structure, further affecting gene expression, thereby participating in the regulation of the physiological development process of eukaryotic organisms. Histone modification is one of the most important epigenetic regulatory methods for eukaryotic organisms to regulate gene expression. In recent years, as more and more histone modifications have been discovered and the specific mechanisms of histone modifications involved in regulation have been revealed, this epigenetic regulatory method has received more and more attention and has gradually become a hot topic in life science research.

[0003] Chromatin Immunopreciptation with Sequencing (ChIP-Seq) is a traditional and important tool for studying histone modifications and is widely used in epigenetic studies of plants and animals. Its basic principle is to fix histones and DNA with formaldehyde, then break the cells, break the DNA with ultrasound, precipitate the protein-DNA complex with specific antibodies, release the DNA by cross-linking, and then purify the DNA, build a library for sequencing, and obtain histone-specific modification sites. However, this technology has many limitations: it is time-consuming; the sample volume required is high; the signal-to-noise ratio is low; the repeatability is poor; and the enrichment level in fungi is low.

[0004] In order to solve the drawbacks of this technology, Henikoff published a new technology CUT&Tag in Nature Communication in 2019. This technology uses Concanavalin A-coated magnetic beads to adsorb living cells and uses digitonin to increase cell membrane permeability. The target protein-specific antibody (primary antibody) enters the cell and binds to the specifically modified histone, 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+Under activation, the DNA at both ends of the target protein is cut, and the linker is added at the same time. The library can be built through PCR. After sequencing, the binding sites of the target protein or the modification level of histones at the whole genome level can be detected. The relative binding amount of the protein at the target gene or the modification level of histones can also be detected through qPCR. This method has a short cycle, high signal-to-noise ratio, good repeatability, requires a small number of cells, and is expected to achieve single-cell level. This method can keep cells in a living cell state and can more realistically reflect the modification level in the cell.

[0005] Since its publication, this technology has been widely used in animal cells. Currently, it has also been realized in plants to detect histone modification levels and transcription factor binding sites through CUT&Tag. Due to the presence of cell walls and some secondary metabolites in fungi, the application of this technology in fungi is more challenging, and there are no related reports in fungi. Histone modification plays an important role in the growth, development and pathogenicity of fungi. Since the application of ChIP technology in fungi has obvious disadvantages, a new method that can replace this technology is urgently needed. Summary of the invention

[0006] The purpose of the present invention is to provide a method for efficiently detecting histone modification sites and abundance at the whole genome level of fungi.

[0007] The present invention provides a method for detecting the distribution and abundance of histone modifications in the whole genome of a test organism, characterized in that:

[0008] The organism is a fungus;

[0009] The method provided by the present invention for detecting the distribution and abundance of histone modifications in the whole genome of a test organism is fungal CUT&Tag; compared with traditional CUT&Tag, the difference of the fungal CUT&Tag is that the following steps are added before adsorbing the cell nucleus: fungal hyphae are taken to prepare protoplasts.

[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 histone modifications in the entire genome of the test organism are obtained based on the sequencing results.

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

[0020] (1) taking fungal mycelium and 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) Library amplification: obtaining a DNA library through library amplification;

[0028] (9) The DNA library is subjected to high-throughput sequencing, and the distribution and abundance of histone modifications in the entire genome of the test organism are obtained based on the sequencing results.

[0029] CUT&Tag: Cleavage Under Targets and Tagmentation.

[0030] Specifically, the adsorption of cell nuclei is to use ConA beads to adsorb cell nuclei.

[0031] Specifically, the ConA beads are ConA Beads Pro.

[0032] The preparation of protoplasts comprises the following steps: taking mycelium, using Pro was enzymatically hydrolyzed and then the protoplasts were collected.

[0033] Specifically, enzymolysis is performed using an enzymolysis solution; in the enzymolysis solution, The content of Pro is 2g / 100ml.

[0034] Specifically, the conditions for the enzymatic hydrolysis are: 33° C., 60 rpm shaking reaction for 3 hours.

[0035] Specifically, the conditions for the enzymatic hydrolysis are: 33° C., 60 rpm shaking reaction for 30 min.

[0036] Specifically, the preparation of protoplasts includes the following steps: taking mycelium, placing it in an enzyme hydrolyzate, and shaking it at 33°C and 60rpm for 30min-3h (specifically 30min or 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.

[0037] 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.

[0038] Specifically, the preparation of protoplasts includes the following steps: taking mycelium, placing it in an enzymatic hydrolysis solution, and shaking the reaction at 33°C and 60rpm for 30min-3h (specifically 30min or 3h); then filtering with a double-layer magic filter cloth, collecting the filtrate, centrifuging the filtrate at 3800rpm for 10min, discarding the supernatant, and washing with SCS solution.

[0039] Enzyme hydrolysate: Take 0.25g Pro was dissolved in 12.5 ml of SCS solution and dissolved by rotation for 20 min, then filtered through a 0.22 μm pore size filter membrane and the filtrate was collected.

[0040] Specifically, the primary antibody incubation is carried out using a primary antibody working solution.

[0041] Specifically, the primary antibody incubation time is 16 hours.

[0042] Specifically, the secondary antibody incubation is carried out using a secondary antibody working solution.

[0043] Specifically, the secondary antibody incubation time is 60 minutes.

[0044] The pA / G-Tn5 transposase incubation time may specifically be 60 minutes.

[0045] 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.

[0046] Activation of pA / G-Tn5 transposase was achieved by adding TTBL working solution.

[0047] TTBL working solution: Mix 40 μl Dig-300 Buffer and 10 μl 5×TTBL.

[0048] Specifically, the CUT&Tag uses the following kit: Hyperactive Universal CUT&Tag AssayKit for Illumina Pro.

[0049] The amount of cell nuclei adsorbed in the CUT&Tag is the nuclei of 250,000 cells.

[0050] Specifically, the method for extracting cell nuclei comprises the following steps:

[0051] Take 1×10 6 For each protoplast, add 100 μL NE buffer, lyse on ice for 10 min, then centrifuge at 2500 rpm for 5 min, discard the supernatant, and add 100 μL Wash Buffer to resuspend the precipitate to obtain the resuspension solution.

[0052] Mix 25 μl of the resuspension with 75 μl of Wash buffer to obtain 100 μl of cell nucleus suspension for the subsequent cell nucleus adsorption step.

[0053] Specifically, the histone modification is H3K9me3, H3K27me3, H3K4me3, H3K36me3 or H3K27ac.

[0054] Specifically, the primary antibody is an H3K9me3 antibody, an H3K27me3 antibody, an H3K4me3 antibody, an H3K36me3 antibody or an H3K27ac antibody.

[0055] Specifically, the secondary antibody is Goat anti Rabbit IgG.

[0056] Specifically, the fungus is a filamentous fungus.

[0057] Specifically, the fungus is Verticillium dahliae, Neurospora crassa, or Ustilago officinalis.

[0058] Fungi have cell walls and relatively rich secondary metabolites. The digitonin used in traditional CUT&Tag technology is difficult to directly permeate the cell membrane of fungi. The present invention successfully applies CUT&Tag-seq technology to fungi by adding a pretreatment step (i.e., preparing protoplasts first and then extracting cell nuclei), and can detect its various histone modification levels, providing an important means for the study of histone modification in fungi.

[0059] Compared with traditional ChIP technology, the present invention has the following advantages:

[0060] ① The cell requirement is small, only 250,000 cells;

[0061] ② Short time consumption, easy operation, and can be completed in just two days;

[0062] ③High signal-to-noise ratio and low background;

[0063] ④Good repeatability.

[0064] This invention realizes for the first time the application of CUT&Tag technology to detect the content and location of histone modifications in fungi, providing an important means for studying the relationship between fungal histone modifications and their growth and pathogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is the result of the length distribution of the DNA library in Example 3.

[0066] Figure 2 This is the sequencing result of the DNA library in Example 3.

[0067] Figure 3 This is the result of the length distribution of the DNA library in Example 5.

[0068] Figure 4 This is the sequencing result of the DNA library in Example 5.

[0069] Figure 5 This is the sequencing result of the DNA library in Example 6.

[0070] Figure 6 This is the sequencing result of the DNA library in Example 7.

[0071] Figure 7 This is the sequencing result of the DNA library in Example 8. DETAILED DESCRIPTION

[0072] 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.

[0073] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field 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 all set up for three repeated experiments, and the results are averaged. Miracloth: pore size 22-25μm, Merck millipore, item number 475855-1R. Verticillium dahliae V592, PGKO-HPT vector, and PGKO-NAT-AGO1 vector were all donated by Guo Huishan's research group at the Institute of Microbiology, Chinese Academy of Sciences. The recombinant bacterium Nc△dim5 was obtained from the research group of He Qun at China Agricultural University and donated by Liu Xiao, a teacher at the Institute of Microbiology, Chinese Academy of Sciences. Neurospora crassa 87-3 was donated by Liu Xiao's research group at the Chinese Academy of Sciences. Sugarcane whip smut fungus haploid JG35 and Sugarcane whip smut fungus haploid JG36 are physiological races isolated from Guangxi Academy of Agricultural Sciences and donated by Professor Lu Shan from Guangxi University.

[0074] 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 a tablet of protease inhibitor (Roche, 04693132001) and dissolve it in 1ml ddH2O. Binding Buffer: Mix 30μl 10×Binding Buffer with 270μld ddH2O. Wash Buffer: 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.

[0075] Example 1. Establishment of the method

[0076] 1. Preparation of protoplasts

[0077] 1. Take a 100 mL Erlenmeyer flask, add the mycelium of the test fungus and 10 ml of enzymatic hydrolysate, and shake at 33°C and 60 rpm for reaction.

[0078] Enzyme hydrolysate: Take 0.25g Pro was dissolved in 12.5 ml of 0.7 M NaCl aqueous solution, and the solution was dissolved by rotation for 20 min, and then filtered through a 0.22 μm pore size filter membrane, and the filtrate was collected. Pro (the product is in the form of solid powder): Novozyme.

[0079] 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 (use a hemocytometer to count protoplasts)

[0080] 2. Obtaining the Cell Nucleus

[0081] 1. Take 1×10 6 protoplasts, add 100 μL NE buffer, lyse on ice for 10 min, then centrifuge at 2500 rpm for 5 min, discard the supernatant, and the precipitate is the cell nucleus.

[0082] 2. After completing step 1, add 100 μl Wash Buffer to resuspend the precipitate to obtain the resuspension solution.

[0083] 3. Mix 25 μl of the resuspension obtained in step 2 with 75 μl of Wash buffer to obtain 100 μl of cell nucleus suspension.

[0084] 3. Preparation of DNA library using CUT&Tag technology

[0085] 1. ConA beads processing

[0086] 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.

[0087] 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.

[0088] ③After completing step ②, add 10 μl Binding Buffer to each tube to resuspend ConA Beads Pro.

[0089] 2. Incubation of cell nuclei with ConA beads

[0090] 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.

[0091] 3. Primary Antibody Incubation

[0092] 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.

[0093] 4. Secondary Antibody Incubation

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 5. pA / G-Tnp incubation

[0101] 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.

[0102] pA / G-Tnp Pro working solution: Mix 2μl pA / G-Tnp Pro and 98μl Dig-300 Buffer.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 6. Fragmentation

[0108] 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.

[0109] TTBL working solution: Mix 40 μl Dig-300 Buffer and 10 μl 5×TTBL.

[0110] 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 2.5 pg to each tube, and incubate at 55° C. for 10 min (invert and mix 2-3 times during this time).

[0111] 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.

[0112] 7. DNA Extract Beads Pro Processing

[0113] 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.

[0114] ②After completing step ①, remove the 1.5ml centrifuge tube from the magnetic stand, add 200μl 1×B&WBuffer, mix well, place on the magnetic stand, and discard the supernatant after the solution is clarified.

[0115] 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.

[0116] 8. DNA extraction

[0117] ① Take the 8-tube strip containing the supernatant obtained in step 6, add 50 μl of the DNA ExtractBeads Pro suspension obtained in step 7 to each tube, invert to mix, and incubate at room temperature for 20 minutes (invert 2-3 times during this period).

[0118] ②After completing step ①, centrifuge instantly and place the 8-tube strip on a magnetic rack. After the solution becomes clear, discard the supernatant.

[0119] ③After completing step ②, keep the 8-tube strip on the magnetic rack, add 200μl 1×B&WBuffer to each tube, incubate at room temperature for 30 seconds, and discard the supernatant.

[0120] ④After completing step ③, keep the 8-tube strip on the magnetic rack, add 200μl 1×B&WBuffer to each tube, incubate at room temperature for 30 seconds, and discard the supernatant.

[0121] 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.

[0122] 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.

[0123] 9. Library Amplification

[0124] Prepare the amplification system according to Table 1, and then perform amplification according to Table 2.

[0125] Table 1

[0126] volume The resuspension prepared in step 8 15μl 2×CAM 25μl N5XX 5μl N7XX 5μl Total volume 50μl

[0127] Table 2

[0128]

[0129] 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.

[0130] 10. Purification and recovery of DNA

[0131] ① After completing step 9, 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 4. Sequencing

[0136] 1. Take the DNA library solution obtained in step 3, use Qubit dsDNA HS Assay Kit double-stranded DNA fluorescence quantitative kit, perform quantification according to the instructions, and use Qseq 400 to detect the peak distribution.

[0137] 2. Take the DNA library solution obtained in step 4 and sequence it.

[0138] The library was sequenced using the Illumina Novaseq 6000 sequencing platform in paired-end 150 bp sequencing mode.

[0139] Example 2: Construction of recombinant bacteria Vd△kmt1

[0140] KMT1: Histone H3K9me3 methyltransferase KMT1.

[0141] The KMT1 gene in the genomic DNA of Verticillium dahliae V592 is shown in Sequence 1 of the Sequence Listing.

[0142] Verticillium dahliae V592 and pGKO-HPT vector are described in the following literature: Wang S, Xing H, Hua C, Guo HS, Zhang J. An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae. Phytopathology. 2016 Jun; 106(6): 645-52. doi: 10.1094 / PHYTO-10-15-0280-R. Epub 2016 Mar 30. PMID: 26780432.

[0143] 1. Using pGKO-HPT vector as the starting vector, inserting the upstream homology arm (as shown in positions 1-1037 in Sequence 2 of the sequence table) into the upstream PacI restriction site of the HPT gene, inserting the downstream homology arm (as shown in positions 3535-4537 in Sequence 2 of the sequence table) into the downstream PacI restriction site of the HPT gene, and obtaining a recombinant plasmid. Sequencing verification shows that the recombinant plasmid contains the DNA molecule shown in Sequence 2 of the sequence table. In Sequence 2 of the sequence table, positions 1-1037 constitute the upstream homology arm, positions 1445-2470 encode the hygromycin resistance protein (HPT), and positions 3535-4537 constitute the downstream homology arm.

[0144] 2. The recombinant plasmid obtained in step 1 is introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

[0145] 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 named as the recombinant bacterium Vd△kmt1. Compared with the genomic DNA of Verticillium dahliae V592, the genomic DNA of the recombinant bacterium Vd△kmt1 differed only in that the KMT1 gene was replaced with the DNA molecule shown in positions 1038-3534 in sequence 2 of the sequence table.

[0146] Example 3: Detection of the distribution and abundance of H3K9me3 in Verticillium dahliae V592 and recombinant strain Vd△kmt1

[0147] The tested fungi were Verticillium dahliae V592 (represented by WT) or the recombinant bacterium Vd△kmt1.

[0148] 1. Preparation of mycelium

[0149] 1. Strain activation: Inoculate the test fungus stored at -80℃ onto a PDA medium plate and culture it at 26℃ for 3-4 days.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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 0.7MNaCl aqueous solution.

[0155] 2. Detection of the distribution and abundance of H3K9me3 at the fungal genome level

[0156] Take the mycelium prepared in step 1 and perform detection according to the method of Example 1.

[0157] In step 1 of Example 1, the oscillation reaction time is 3 h.

[0158] The primary antibody working solution is H3K9me3 antibody working solution. H3K9me3 antibody working solution: dilute the H3K9me3 antibody to 50 times the volume with Antibody Buffer to obtain H3K9me3 antibody working solution. H3K9me3 antibody (HistoneH3K9me3 antibody) (rabbit source, liquid product): Active Motif, catalog number 39161.

[0159] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat antiRabbit IgG): Novozymes, catalog number AB207-01-AA.

[0160] The results of the length distribution of the DNA library are shown in Figure 1 The DNA library solutions all met the sequencing requirements.

[0161] The sequencing results of the DNA library are shown in Figure 2 In the method provided by the present invention, H3K9me3 is well enriched. After knocking out the KMT1 gene, the level of H3K9me3 decreases.

[0162] Example 4: Construction of recombinant bacteria VdΔezh2

[0163] EZH2: H3K27me3 methyltransferase EZH2.

[0164] The EZH2 gene in the genomic DNA of Verticillium dahliae V592 is shown in Sequence 3 of the Sequence Listing.

[0165] The PGKO-NAT-AGO1 vector is described in the following literature: Wen, HG., Zhao, JH., Zhang, BS. et al. Microbe-induced gene silencing boosts crop protection against soil-borne fungal pathogens. Nat. Plants 9, 1409–1418 (2023). https: / / doi.org / 10.1038 / s41477-023-01507-9.

[0166] 1. Using PGKO-NAT-AGO1 vector as the starting vector, inserting the upstream homology arm (as shown in the 1st to 1020th position in the sequence 4 of the sequence table) into the upstream PacI restriction site of the NAT gene, inserting the downstream homology arm (as shown in the 2364th to 3288th position in the sequence 4 of the sequence table) into the downstream PacI restriction site of the NAT gene, and obtaining a recombinant plasmid. After sequencing verification, the recombinant plasmid has the DNA molecule shown in the sequence 4 of the sequence table. In the sequence 4 of the sequence table, the 1st to 1020th positions constitute the upstream homology arm, the 1390th to 1959th positions encode N-acetyltransferase (NAT), and the 2364th to 3288th positions constitute the downstream homology arm.

[0167] 2. The recombinant plasmid obtained in step 1 is introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

[0168] 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 NAT screening, which was named as the recombinant bacterium Vd△ezh2. Compared with the genomic DNA of Verticillium dahliae V592, the genomic DNA of the recombinant bacterium Vd△ezh2 differed only in that the EZH2 gene was replaced with the DNA molecule shown in positions 1021-2363 in sequence 4 of the sequence table.

[0169] Example 5. Detection of the distribution and abundance of H3K27me3 in Verticillium dahliae V592 and recombinant bacteria VdΔezh2. The test fungi were Verticillium dahliae V592 (expressed as WT) or recombinant bacteria VdΔezh2.

[0170] 1. Preparation of mycelium

[0171] Same as step 1 of Example 3.

[0172] 2. Detection of the distribution and abundance of H3K27me3 at the fungal genome level

[0173] Take the mycelium prepared in step 1 and perform detection according to the method of Example 1.

[0174] In step 1 of Example 1, the oscillation reaction time is 3 h.

[0175] The primary antibody working solution is H3K27me3 antibody working solution. H3K27me3 antibody working solution: dilute the H3K27me3 antibody to 50 times the volume with Antibody Buffer to obtain H3K27me3 antibody working solution. H3K27me3 antibody (HistoneH3K27me3 antibody) (rabbit source, product form is liquid, specification is 1μg / μl): Active Motif Company, catalog number 39155.

[0176] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat antiRabbit IgG): Novozymes, catalog number AB207-01-AA.

[0177] The results of the length distribution of the DNA library are shown in Figure 3 The DNA library solutions all met the sequencing requirements.

[0178] The sequencing results of the DNA library are shown in Figure 4 In the method provided by the present invention, H3K27me3 is well enriched. After knocking out the EZH2 gene, the level of H3K27me3 decreases.

[0179] Example 6: Detection of the distribution and abundance of H3K4me3, H3K36me3 or H3K27ac in Verticillium dahliae The test fungus was Verticillium dahliae V592.

[0180] 1. Preparation of mycelium

[0181] Same as step 1 of Example 3.

[0182] 2. Detection of the distribution and abundance of H3K4me3, H3K36me3 or H3K27ac at the fungal genome level

[0183] Take the mycelium prepared in step 1 and perform detection according to the method of Example 1.

[0184] In step 1 of Example 1, the oscillation reaction time is 3 h.

[0185] The primary antibody working solutions were H3K4me3 antibody working solution, H3K36me3 antibody working solution, H3K27ac antibody working solution, or IgG antibody working solution (as a control).

[0186] H3K4me3 antibody working solution: dilute the H3K4me3 antibody to 50 times the volume with Antibody Buffer to obtain H3K4me3 antibody working solution. H3K4me3 antibody (Histone H3K4me3 antibody) (rabbit source, liquid form): Active Motif, catalog number 39060.

[0187] H3K36me3 antibody working solution: dilute the H3K36me3 antibody to 50 times the volume with Antibody Buffer to obtain H3K36me3 antibody working solution. H3K36me3 antibody (Histone H3K36me3 antibody) (rabbit source, liquid form, specification 1μg / μl): Active Motif, catalog number 61902.

[0188] H3K27ac antibody working solution: dilute the H3K27ac antibody to 50 times the volume with Antibody Buffer to obtain H3K27ac antibody working solution. H3K27ac antibody (Histone H3K27ac antibody) (rabbit source, liquid form, specification 1μg / μl: Active Motif, catalog number 39034.

[0189] IgG antibody working solution: dilute Rabbit IgG control Polyclonal antibody to 50 times the volume with AntibodyBuffer to obtain IgG antibody working solution. Rabbit IgG control Polyclonal antibody (product form is liquid): Proteintech, catalog number 30000-0-AP.

[0190] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat antiRabbit IgG): Novozymes, catalog number AB207-01-AA.

[0191] The sequencing results of the DNA library are shown in Figure 5 By using the method provided by the present invention, H3K4me3 / H3K36me3 / H3K27ac all achieved good enrichment effects.

[0192] Example 7: Detection of Neurospora crassa

[0193] Neurospora crassa is an important eukaryotic model organism and plays an important role in genetics and molecular biology research. Neurospora crassa 87-3 (i.e., the wild-type strain in the literature) and recombinant strain Nc△dim5 (i.e., dim-5KO in the literature) are described in the following literature: Zhao Y, Shen Y, Yang S, Wang J, Hu Q, Wang Y, He Q. Ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation in Neurosporacrassa. J Biol Chem. 2010 Feb 12; 285(7): 4355-65. doi: 10.1074 / jbc.M109.034710. Epub 2009 Nov 30. PMID: 19948733; PMCID: PMC2836040. Compared with Neurospora crassa 87-3, the recombinant strain Nc△dim5 knocked out the dim5 gene.

[0194] The tested fungi were Neurospora crassa 87-3 (represented by WT) or the recombinant fungus Nc△dim5.

[0195] Take the hyphae of the test fungus and perform detection according to the method of Example 1.

[0196] In step 1 of Example 1, the oscillation reaction time is 30 min.

[0197] The primary antibody working solution is H3K9me3 antibody working solution. H3K9me3 antibody working solution: dilute the H3K9me3 antibody to 50 times the volume with Antibody Buffer to obtain H3K9me3 antibody working solution. H3K9me3 antibody (HistoneH3K9me3 antibody) (rabbit source, liquid product): Active Motif, catalog number 39161.

[0198] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat antiRabbit IgG): Novozymes, catalog number AB207-01-AA.

[0199] The sequencing results of the DNA library are shown in Figure 6 H3K9me3 is well enriched in Neurospora crassa 87-3, and the H3K9me3 level in the recombinant bacterium Nc△dim5 is decreased. The method provided by the present invention is suitable for the detection of histone modification in Neurospora crassa.

[0200] Example 8: Detection of Ustilago canefolia

[0201] Ustilago officinalis is the pathogen of sugarcane smut and is one of the most important diseases affecting sugarcane production worldwide. Histone modification plays an important role in the pathogenicity of this fungus, so a simple and reliable method for detecting histone modification is urgently needed. Ustilago officinalis haploid JG35 and Ustilago officinalis haploid JG36 are recorded in the following literature: Lan Xianruan, Wang Yukun, Wu Haoming, Lu Shan, Qiu Jinfeng, Li Ru, Chen Baoshan. Early visual observation of sugarcane infection by Ustilago officinalis[J]. Acta Microbiologica Sinica, 2021, 61(3): 596-606.

[0202] The tested fungi were: Ustilago officinalis haploid JG35 or Ustilago officinalis haploid JG36.

[0203] 1. Preparation of protoplasts

[0204] 1. Pick the test fungus and place it in YEPS liquid medium, shake and culture it at 28℃ and 200rpm for 1 day, then transfer it to YEPS liquid medium (starting OD 600nm ≈0.05), 28°C, 200 rpm shaking culture until OD6 00nm ≈0.7, then centrifuge at 1000g for 5 min to collect the bacteria, wash once with SCS solution, and centrifuge at 1000g for 5 min at room temperature to collect the bacteria.

[0205] YEPS liquid culture medium (pH 6.3): 10 g yeast extract, 20 g peptone, 20 g sucrose, add deionized water to make up to 1 L.

[0206] 2. Take a 100 mL Erlenmeyer flask, add the bacteria obtained in step 1 and 10 ml of enzymatic hydrolysate, and shake at 33°C and 60 rpm for 30 min.

[0207] Enzyme hydrolysate: Take 0.25g Pro was dissolved in 12.5 ml of SCS solution and dissolved by rotation for 20 min, then filtered through a 0.22 μm pore size filter membrane and the filtrate was collected. Pro (the product is in the form of solid powder): Novozyme.

[0208] SCS solution (pH 5.8): contains 20 mmol / L trisodium citrate and 1 mol / L sorbitol, with the balance being water.

[0209] 3. After completing step 2, centrifuge at 1000g for 10 minutes, discard the supernatant, wash the precipitate with SCS solution, and then resuspend the precipitate with 5mL SCS solution to obtain the protoplast solution (use a hemocytometer to count the protoplasts)

[0210] The subsequent steps are the same as step 2, step 3 and step 4 of Example 1.

[0211] The primary antibody working solution is H3K9me3 antibody working solution or H3K27me3 antibody working solution or IgG antibody working solution (as a control).

[0212] H3K9me3 antibody working solution: dilute the H3K9me3 antibody to 50 times the volume with Antibody Buffer to obtain H3K9me3 antibody working solution. H3K9me3 antibody (Histone H3K9me3 antibody) (rabbit source, liquid form): Active Motif, catalog number 39161.

[0213] H3K27me3 antibody working solution: dilute the H3K27me3 antibody to 50 times the volume with Antibody Buffer to obtain H3K27me3 antibody working solution. H3K27me3 antibody (Histone H3K27me3 antibody) (rabbit source, liquid form, specification 1μg / μl): Active Motif, catalog number 39155.

[0214] IgG antibody working solution: dilute Rabbit IgG control Polyclonal antibody to 50 times the volume with AntibodyBuffer to obtain IgG antibody working solution. Rabbit IgG control Polyclonal antibody (product form is liquid): Proteintech, catalog number 30000-0-AP.

[0215] Secondary antibody working solution: Take the secondary antibody and dilute it 100 times with Dig-wash Buffer. Secondary antibody (Goat antiRabbit IgG): Novozymes, catalog number AB207-01-AA.

[0216] The sequencing results of the DNA library are shown in Figure 7 The results showed that both H3K9me3 and H3K27me3 were well enriched, indicating that the method provided by the present invention can be used to detect the histone modification level of sugarcane whip smut fungus, providing a new method for subsequent research on the effect of epigenetic modification on the pathogenicity of sugarcane whip smut fungus.

[0217] 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 histone modifications in the whole genome of a test organism, characterized in that: The organism is a fungus; The method provided by the present invention for detecting the distribution and abundance of histone modifications in the whole genome of a test organism is fungal CUT&Tag; compared with traditional CUT&Tag, the difference of the fungal CUT&Tag is that the following steps are added before adsorbing the cell nucleus: fungal hyphae are taken to prepare protoplasts.

2. The method according to claim 1, characterized in that: The fungus CUT&Tag comprises the following steps in sequence: (1) taking fungal mycelium and 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) Library amplification: obtaining a DNA library through library amplification; (9) The DNA library is subjected to high-throughput sequencing, and the distribution and abundance of histone modifications in the entire genome of the test organism are obtained based on the sequencing results.

3. The method according to claim 1 or 2, characterized in that: The preparation of protoplasts comprises the following steps: taking mycelium, using Pro was enzymatically hydrolyzed to obtain protoplasts.

4. The method according to any one of claims 1 to 3, characterized in that: The amount of cell nuclei adsorbed in the fungal CUT&Tag is the nuclei of 250,000 cells.

5. The method according to any one of claims 1 to 4, characterized in that: The fungus is a filamentous fungus.

6. The method according to any one of claims 1 to 4, characterized in that: The fungus is Verticillium dahliae, Neurospora crassa or Ustilago officinalis.

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