A CUT&Tag method applied to plant pollen

Abstract

The application discloses a CUT&Tag method applied to plant pollen. The CUT&Tag method of plant pollen protected by the application contains Percoll in a nuclear extraction solution, so as to remove starch in pollen cells. After the starch is removed, the method further comprises a step of removing polysaccharides and polyphenols in the pollen cells by using a buffer containing 1,6-hexanediol and glycerol. The concentration of the Percoll in the nuclear extraction solution can be 60%. Experiments prove that the CUT&Tag method of plant pollen established in the application can effectively remove polysaccharides, polyphenols, high starch and other impurities rich in the pollen, and can effectively identify the interaction between DNA and proteins in the plant pollen. The method of the application can also be popularized to the research on the interaction between DNA and proteins in other tissues of corn or other plants.

Description

TECHNICAL FIELD

[0001] The present application relates to a nucleic acid containing assay method in the field of biotechnology, in particular to a CUT&Tag method applied to plant pollen. BACKGROUND

[0002] CUT&Tag (Cleavage Under Targets and Tagmentation) is a new method for studying protein-DNA interactions. Compared with traditional ChIP-Seq, it has the following advantages: time-saving and efficient, less sample required, low background signal, good repeatability, etc. Even single-cell sequencing can be used. CUT&Tag is expected to change the study of protein-DNA interactions into a PCR-like routine operation, which has a revolutionary significance for the study of gene regulation and epigenetics.

[0003] In biological research, the interaction between DNA and protein (DNA-Protein Interaction, DPI) is crucial. Gene expression, regulation, replication, recombination and repair, RNA transport, translation and regulation all rely on DPI. Almost all life activities involve DPI.

[0004] The basic principle of CUT&Tag technology (reference: Kaya-Okur, H.S. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 10 (2019)) is as follows: under the mediation of a certain antibody, pA-Tn5 fusion protein only fragments the target DNA in the local part of the target group protein modification marker, transcription factor or chromatin regulatory protein binding chromatin, while adding a sequencing adapter. Since the pA-Tn5 transposome only binds and cuts the DNA in its adjacent space, the signal-to-noise ratio of the entire experiment is greatly improved, and the experimental steps are simplified. This method can be used for one-pot high-throughput application and can also be applied to single-cell sequencing.

[0005] The CUT&Tag method comprises the following steps:

[0006] A1) extracting the nucleus to obtain a nucleus extract; the target protein in the nucleus extract binds the target DNA to be detected;

[0007] A2) adding an antibody that specifically binds to the target protein to the nucleus extract obtained in A1) to bind the target protein; in the subsequent step of extracting the nucleus, a protease inhibitor and / or a cell membrane permeation treatment solution are added to the nucleus extract obtained in A1).

[0008] A3) adding pA-Tn5 transposome to the antibody; the Tn5 transposase in the pA-Tn5 transposome can specifically cut the DNA fragment near the target protein, fragmenting the target DNA;

[0009] A4) activating the pA-Tn5 transposome to fragment the target DNA, obtaining a mixed solution containing the fragmented target DNA; the protease inhibitor and / or the cell membrane permeation treatment solution are added during the fragmentation of the target DNA;

[0010] A5) extracting the fragmented target DNA, and sequencing to obtain the nucleotide sequence of the target DNA.

[0011] Due to the advantages of CUT&Tag technology, such as no need for cross-linking, less starting cell amount, high signal-to-noise ratio, etc., there is currently a trend to replace ChIP-seq technology, and its own application has also expanded to single-cell CUT&Tag and Multi-CUT&Tag for detecting multiple DPIs in the same tissue. However, there is still room for further improvement. For plant pollen, it is difficult to purify chromatin and identify DPI due to the presence of impurities such as polysaccharides, polyphenols, starch, etc. So far, there is no report on the application of CUT&Tag to plant pollen in the world. SUMMARY

[0012] The technical problem to be solved by the present application is how to perform CUT&Tag on plant pollen and / or how to identify the interaction between DNA and protein in plant pollen.

[0013] To solve the above technical problems, the present application first provides a method for CUT&Tag of plant pollen.

[0014] The method comprises extracting nuclei from plant pollen with an extraction solution containing Percoll to obtain a starch-removed nuclei extraction solution from pollen cells, and the nuclei extraction solution contains target proteins combined with DNA.

[0015] The Percoll is used to remove starch from pollen cells.

[0016] The method for CUT&Tag of plant pollen can be a method for obtaining DNA combined with target proteins or a method for obtaining DNA interacting with target proteins.

[0017] The above method can comprise the step of removing polysaccharides and polyphenols in the starch-removed nuclei extraction solution from pollen cells with an extraction solution containing 1,6-hexanediol and glycerol to obtain a starch-removed, polysaccharide-removed and polyphenol-removed nuclei extraction solution from pollen cells.

[0018] The method can comprise the following steps:

[0019] A1) precipitating the target protein: mixing the target antibody with the cell nucleus extract solution for removing starch, polysaccharide and polyphenol in the pollen cell, so that the target antibody specifically binds to the target protein;

[0020] A2) fragmenting the target DNA: adding pA-Tn5 transposome to obtain a coupling of the pA-Tn5 transposome with the target antibody and the target protein; the coupling contains DNA bound to the target protein;

[0021] A3) activating the pA-Tn5 transposome: activating the pA-Tn5 transposome to fragment the DNA to obtain a mixed solution containing the fragmented DNA.

[0022] In the method, the Percoll can have a volume content of 60% in the extract solution containing Percoll. In the extract solution containing 1,6-hexanediol and glycerol, the concentration of 1,6-hexanediol is 1M and the volume concentration of glycerol is 20%.

[0023] The composition of the extract solution containing Percoll can be: 60% (volume content, v / v) Percoll, 10mM Tris pH 8.0, 400mM sucrose, 10mM sodium butyrate, 0.1mM PMSF, 1× protease inhibitor, 5mM β-mercaptoethanol, and the rest is water. The composition of the extract solution containing 1,6-hexanediol and glycerol can be: 10mM Tris pH 8.0, 400mM sucrose, 10mM sodium butyrate, 1M 1,6-hexanediol, 20% v / v glycerol, 0.1mM PMSF, 1× protease inhibitor, 5mM β-mercaptoethanol, and the rest is water.

[0024] In the method, no protease inhibitor and / or cell membrane permeabilization solution can be added during the DNA fragmentation process.

[0025] In the method, the target protein can be histone or a histone variant. The histone can be H3K27ac; the histone variant can be centromeric histone CENH3. The target protein can also be a transcription factor or other protein. The target antibody can be an H3K27ac antibody; it can also be a CENH3 antibody rabbit anti-CENH3 antibody.

[0026] In the method, the pA-Tn5 transposome can be a fusion protein complex of Protein A and Tn5 transposase Protein A-Tn5, or a fusion protein complex of Protein G and Tn5 transposase Protein G-Tn5.

[0027] In the above method, the amount of pollen cells used in the cell pretreatment can be 0.1-0.2g.

[0028] The application of any of the methods described above in detecting DNA interacting with the target protein in pollen cells also falls within the scope of protection of this invention.

[0029] The application of Percoll, 1,6-hexanediol, and glycerol in the CUT&Tag method for removing starch, polysaccharides, and polyphenols from plant pollen is also within the scope of protection of this invention.

[0030] In the above method, the plant can be corn.

[0031] To address the aforementioned technical problems, the present invention also provides a composition. This composition is used to remove starch, polysaccharides, and polyphenols from plant pollen in a CUT&Tag method. The composition may consist of Percoll, 1,6-hexanediol, and glycerol.

[0032] In the above composition, Percoll, 1,6-hexanediol, and glycerol can be packaged separately and used in different steps of the CUT & Tag method for plant pollen.

[0033] In the embodiments of this invention, 60% Percoll was added to the nuclear extract of plant pollen using the CUT&Tag method, which effectively removed starch from pollen cells. After starch removal, a buffer solution containing 1,6-hexanediol and glycerol was used to remove polysaccharides and polyphenols from the pollen cells. Results showed that the plant pollen CUT&Tag method established in this invention can effectively remove impurities such as abundant polysaccharides, polyphenols, and high starch content in pollen. It can also effectively identify the interaction between DNA and proteins in plant pollen. The H3K27ac antibody was used to identify DNA in maize pollen that interacts with histone H3K27ac. The method of this invention can also be extended to the study of DNA-protein interactions in other tissues of maize or other plants.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] This invention is the first to propose a CUT & Tag method that can be used for pollen.

[0036] In this invention, protease inhibitors and digitalis saponins only need to be added once during the extraction of cell nuclei, instead of the multiple additions required in different versions of CUT&Tag published in plants, thus further saving costs.

[0037] This invention establishes a mature and stable CUT & Tag method for corn pollen; this method can also be extended to other tissues of corn or other plants.

[0038] This method is applicable to both cross-linked and non-cross-linked plant tissues. Attached Figure Description

[0039] Figure 1 This is a PCR electrophoresis image of H3K27ac antibody-mediated CUT&Tag method in maize pollen. The left lane shows the capillary electrophoresis results of the purified DNA fragments, and the right lane shows the DNA markers.

[0040] Figure 2 Comparison of peak plots obtained by H3K27ac antibody-mediated ChIP-seq and CUT&Tag methods. AD represents a comparison of the peak positions of the four genes. The top of the plot shows the start and end positions of the gene containing the peak or its flanking genes on the chromosome, with the gene name above the corresponding gene position. The upper half of each plot shows the H3K27ac antibody-mediated ChIP-seq results in B73 seedlings, and the lower half shows the H3K27ac antibody-mediated CUT&Tag results in B73 pollen.

[0041] Figure 3 Comparison of peak plots for the CENH3 antibody-mediated CUT&Tag method in two biological replicates. A and B compare the peak positions of the two genes. The top of the plot shows the start and end positions of the gene containing the peak or its flanking genes on the chromosome, with the gene name above the corresponding gene position. The upper peak plot of each figure represents the result of the first biological replicate of the CENH3 antibody-mediated CUT&Tag method in B73 pollen, and the lower peak plot represents the result of the second biological replicate of the CENH3 antibody-mediated CUT&Tag method in B73 pollen. Detailed Implementation

[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] The reagents and consumables in this embodiment of the invention are sourced from the following sources:

[0045] Tris pH 8.0: Thermo, catalog number AM9858;

[0046] Sucrose: Sigma, product number V900116-500G;

[0047] Sodium butyrate (Na-butyrate): Sigma, catalog number V900464-25g;

[0048] Benzyl sulfonyl fluoride (PMSF): Sigma, catalog number P7626-5G;

[0049] 1×Proteinase Inhibitors: Roche, Catalog No. 04693159001;

[0050] β-Mercaptoethanol: Sigma, catalog number M6250-250ML;

[0051] Percoll: Merck, product number P4937-500ML;

[0052] 1,6-Hexanediol solution: Sigma, catalog number 88571-100ML-F;

[0053] Glycerol (Sigma), product number G9012-100ML;

[0054] EDTA: Invitrogen, part number AM9260G;

[0055] Triton X-100: Sigma, product number T8787-100ML;

[0056] Sodium chloride (NaCl): Sigma, product number S5150-1L;

[0057] Bovine serum albumin (BSA): Sigma, catalog number V900933-100G;

[0058] Digitolin: Invitrogen, catalog number BN2006;

[0059] H3K27ac antibody: Abcam, catalog number ab4729.

[0060] Spermidine: Sigma, catalog number S2501-1G;

[0061] Tween-40: Sigma, product number P1504-500ML;

[0062] Magnesium chloride hexahydrate: Sigma, product number M2670-100G;

[0063] EDTA: Invitrogen, part number AM9260G;

[0064] SDS: Merck, Part No. 75746-1KG;

[0065] Proteinase K: Thermo, catalog number EO6491;

[0066] GlycoBlue: Ambion, item number AM9740;

[0067] Sodium acetate (pH 5.2): Sigma, catalog number S7899-100ML;

[0068] Hyperactive pA-Tn5 Transposase for CUT&Tag (hereinafter referred to as pA-Tn5 in the experimental procedure): Vazyme, catalog number S603-02;

[0069] Phase Lock Gel Heavy: Tiangen, part number WM5-2302830;

[0070] TruePrep DNA Library Prep Kit V2 for Illumina: Vazyme, Cat. No. TD501-01.

[0071] Example 1: CUT & Tag Method for Plant Pollen (Applied to Histone Modification Antibodies)

[0072] 1. CUT & Tag Methods for Plant Pollen

[0073] We performed cuts and tags on plant pollen and analyzed the DNA that interacts with the target protein H3K27ac in the pollen.

[0074] 1.1 Pollen grinding

[0075] Collect fresh pollen from the maize inbred line Chang 7-2 and flash-freeze it in liquid nitrogen. Take 1.0g and grind it in liquid nitrogen for about 30 minutes to ensure that the powder is uniform.

[0076] 1.2 Pollen cell pretreatment (nucleus extraction)

[0077] Using nucleic acid extraction solution II (containing Percoll) and nucleic acid extraction solution III (containing 1,6-hexanediol and glycerol) to extract pollen cell nuclei can effectively remove the starch, polysaccharides and polyphenols abundant in pollen cells.

[0078] (1) Weigh 0.2g of ground corn pollen, dissolve it in 20mL of nucleic acid extraction buffer I, and vortex to mix to obtain a mixture.

[0079] The composition of nucleic acid extraction solution I is as follows: 10 mM Tris pH 8.0, 400 mM sucrose, 10 mM sodium butyrate, 0.1 mM MPMSF, 1× protease inhibitor, 5 mM β-mercaptoethanol, and the remainder is water.

[0080] (2) Place the mixture in ice and gently rotate it on a horizontal shaker at 100 rpm for 15 minutes to obtain a fully dispersed pollen cell nucleus extract mixture.

[0081] (3) Prepare Nucleic Acid Extraction Buffer II (containing 60% v / v Percoll) and vortex to mix.

[0082] The composition of nucleic acid extraction solution II is as follows: 60% (v / v) Percoll, 10mM Tris pH 8.0, 400mM sucrose, 10mM sodium butyrate, 0.1mM PMSF, 1× protease inhibitor, 5mM β-mercaptoethanol, and the remainder is water. The 60% Percoll is used to remove starch-rich pollen; the combined use of PMSF, protease inhibitor, and β-mercaptoethanol inhibits various proteases and other enzymes.

[0083] Add 20 mL of nucleic acid extraction solution II to a 50 mL BD centrifuge tube, and slowly pour the fully dispersed pollen cell nucleus extraction mixture from step (2) onto nucleic acid extraction solution II.

[0084] (4) Centrifuge at 2880g and minimum acceleration at 4℃ for 20min.

[0085] (5) Take another 50mL BD centrifuge tube, freshly prepare Nucleic Acid Extraction Buffer III (containing 1M 1,6-hexanediol and 20% glycerol), vortex to mix, and pre-cool. Transfer about 1mL of Nucleic Acid Extraction Buffer III to another sterile 1.5mL centrifuge tube for later use.

[0086] The composition of nucleic acid extraction solution III is as follows: 10 mM Tris pH 8.0, 400 mM sucrose, 10 mM sodium butyrate, 1 M 1,6-hexanediol, 20% v / v glycerol, 0.1 mM PMSF, 1× protease inhibitor, 5 mM β-mercaptoethanol, and the remainder is water. The 1,6-hexanediol and glycerol effectively remove polysaccharides and polyphenols from pollen cells; the combined use of PMSF, protease inhibitor, and β-mercaptoethanol inhibits various proteases and other enzymes.

[0087] (6) After centrifugation in step (4), the mixture in the tube will be layered. Take the middle layer and transfer it to a BD centrifuge tube containing 19 mL of nucleic acid extraction solution III, and gently mix them.

[0088] (7) Centrifuge at 2880g and minimum acceleration at 4℃ for 10min.

[0089] (8) Discard the supernatant, add 1 mL of the nucleic acid extraction solution III prepared in step (5), and gently pipette to mix. Transfer to a 1.5 mL Eppendorf centrifuge tube.

[0090] (9) Centrifuge at 1000g and minimum acceleration at 4℃ for 5min.

[0091] (10) Discard the supernatant, add 120 μL of pre-cooled dilution buffer, and gently disperse the cell nuclei to obtain pollen cell nucleus extract. This cell nucleus extract contains the target protein H3K27ac, which binds to the target DNA to be detected. Digitalis saponins are used for permeability of the cell nuclear membrane.

[0092] The dilution buffer consisted of: 50 mM Tris pH 8.0, 1 mM EDTA, 0.1% v / v Triton X-100, 150 mM sodium chloride, 10 μg / mL bovine serum albumin, and 1.5 μL 5% digitalis saponins, with the remainder being water.

[0093] 1.3 Add antibody to bind target protein

[0094] (1) Add 1.0 μg of H3K27ac antibody to the cell nuclear extract obtained in step 1.2, gently invert several times to mix; place on an IntelliMixer instrument and rotate at 4°C for 2 hours (or overnight).

[0095] (2) Centrifuge at 1000g for 5 min and carefully remove the supernatant with a 200μL pipette.

[0096] (3) Add 1 mL of pre-cooled wash buffer, gently blow and let stand at room temperature for 5 min.

[0097] The washing buffer consisted of: 10 mM Tris pH 8.0, 0.5 mM spermidine, 0.05% v / v Tween-40, 150 mM sodium chloride, and the remainder was water.

[0098] (4) Centrifuge at 1000g for 5 min and carefully remove the supernatant with a 200μl pipette.

[0099] (5) Repeat steps (3) to (4).

[0100] 1.4pA-Tn5 transposon coupling with antibody and target DNA

[0101] (1) Add 150 μL of transposase incubation buffer and gently blow on the cell nuclei to disperse them.

[0102] The transposase incubation buffer consisted of: 20 mM Tris pH 8.0, 300 mM sodium chloride, 0.5 mM spermidine, and the remainder being water.

[0103] (2) Add 1.4 μL of pA-Tn5 and gently invert several times to mix. Place on an IntelliMixer instrument and rotate at 4°C for 1 hour.

[0104] (3) Centrifuge at 1000g for 5 min and carefully remove the supernatant with a 200μL pipette.

[0105] (4) Add 1 mL of pre-cooled wash buffer, gently blow and let stand at room temperature for 5 min.

[0106] (5) Centrifuge at 1000g for 5 min and carefully remove the supernatant with a 200μl pipette.

[0107] (6) Repeat steps (4) to (5).

[0108] 1.5 Activate the pA-Tn5 transposon to fragment the target DNA that binds to the target protein.

[0109] (1) Add 200 μL of magnesium-containing tagmentation buffer and gently blow on the cell nuclei to disperse them.

[0110] The fragmentation buffer consisted of: 20 mM Tris pH 8.0, 10 mM magnesium chloride, 300 mM sodium chloride, 0.5 mM spermidine, and the remainder being water.

[0111] (2) React in a metal bath at 37°C for 1-2 hours. A mixture containing fragmented target DNA (DNA bound to the target protein H3K27ac) is obtained.

[0112] 1.6 Target DNA extraction, PCR amplification, and product purification

[0113] (1) Add 6 μL of 0.5M EDTA and 2 μL of 10% SDS to the mixture containing fragmented target DNA obtained in step 1.4, and mix by inverting the mixture.

[0114] (2) Add 200 μL of elution buffer, then add 8 μL of 20 mg / mL proteinase K, invert and vortex to mix.

[0115] The elution buffer consisted of 10 mM Tris pH 8.0, 1 mM EDTA, 1% SDS, and the remainder was water.

[0116] (3) Place in a 55°C water bath and react for at least 1 hour.

[0117] (4) Add 400 μL of phenol:chloroform:isopropanol mixture at room temperature (volume percentage: 25:24:1), vortex vigorously for at least 10 s, and transfer to a 2 mL Phase Lock Gel Heavy centrifuge tube.

[0118] (5) Centrifuge at 13000 rpm for 5 min at room temperature.

[0119] (6) Take the supernatant and transfer it to a new 1.5 mL Eppendorf centrifuge tube. Add 2 μL of 20 mg / mL LGlycoBlue, 40 μL of sodium acetate (pH 5.5), and 400 μL of isopropanol. Vortex vigorously for at least 10 seconds to obtain a mixture. Quick freeze with liquid nitrogen for at least 30 seconds.

[0120] (7) Remove the mixture from the liquid nitrogen, wait for the liquid in the tube to melt slightly, and centrifuge at 4°C and 13000 rpm for 30 min.

[0121] (8) Wash twice with pre-cooled 80% ethanol and air dry.

[0122] (9) Add 50 μL ddH2O and dissolve it completely at 37℃.

[0123] (10) Take 24 μL as a template and perform PCR for 18 cycles using the TruePrep DNA Library Prep Kit V2 for Illumina. The PCR conditions are as follows:

[0124]

[0125] (11) Take 5 μl of PCR product and observe its size range on 1.5% agarose gel.

[0126] (12) The target DNA fragments of 300–700 bp in size were recovered using XP magnetic beads, and the PCR products were enriched between 300–500 bp. Figure 1 ).

[0127] 1.7. Target DNA Sequencing

[0128] On the NovaSeq sequencer, the purified target DNA fragment obtained in step 1.5 was sequenced at PE150 for 10G to obtain sequencing data containing sequencing reads.

[0129] 2. Analysis of pollen cut & tag method results

[0130] 2.1 Sequencing data were aligned to the reference genome using bowtie2 (related literature: Langmead, B. & Salzberg, SL Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357-359 (2012)). Reads were aligned to the maize B73 v4 reference genome (related literature: Jiao, Y. et al. Improved maize reference genome with single-molecule technologies. Nature 546, 524-527 (2017)).

[0131] 2.2 Peak Finding

[0132] Peak calling was performed using samtools (see reference: Li, H. et al. The Sequence Alignment / Map format and SAMtools. Bioinformatics 25, 2078-2079 (2009)) and macs2 (see reference: Zhang, Y. et al. Model-based Analysis of ChIP-Seq (MACS). Genome Biology 9, R137 (2008)). Each peak represents information about the DNA that binds to H3K27ac. The resulting DNA sequences interacting with H3K27ac and their locations on the genome were then obtained.

[0133] Using the method established in this invention, 30,446 peaks were identified in pollen from the maize B73 inbred line using the H3K27ac antibody. Compared with the published results obtained by ChIP-seq from 14-day seedling tissues of the same maize inbred line and the same antibody (related literature: Li, E. et al. Long-range interactions between proximal and distal regulatory regions in maize. Nat. Commun. 10, 2633 (2019)), the two methods share 26,221 common peaks.

[0134] Figure 2 A comparison of the peak patterns obtained by H3K27ac antibody-mediated ChIP-seq and CUT&Tag methods shows that, except for a very small number of peaks that may be due to tissue specificity, the peak patterns obtained by the two methods are highly similar. Furthermore, the peak values ​​of the latter are all higher than those of the former.

[0135] Example 2: Application of the CUT & Tag method for plant pollen (applied to centromere histone CENH3 antibody)

[0136] In this embodiment, the CUT&Tag method for plant pollen established in Example 1 was used, and the DNA bound to centromere histone CENH3 was analyzed using a centromere histone CENH3 antibody.

[0137] The experimental materials, CUT & Tag methods, and result analysis methods were the same as in Example 1. The target protein was centromere histone CENH3, and the target protein antibody was rabbit anti-CENH3 antibody (a generous gift from Professor Jin Weiwei's laboratory at China Agricultural University, which is available to the public from the applicant and is used only for replicating this invention. Related literature: Han, Y. et al. Divergence incentromere structure distinguishes related genomes in Coix lacryma-jobi and its wild relative. Chromosoma 119, 89-98 (2010)).

[0138] The analysis results showed that 16,672 peaks were identified in the pollen of the maize B73 inbred line using the CENH3 antibody, and the DNA sequence interacting with CENH3 and its location on the genome were finally obtained. Figure 3The results show that the peaks identified in the two biological replicates by the CENH3 antibody-mediated CUT&Tag method are highly consistent in their positions on the chromosomes.

[0139] In summary, the plant pollen CUT&Tag method established in this invention effectively removes impurities such as polysaccharides, polyphenols, and high starch content in pollen, and can effectively identify the interaction between DNA and proteins in plant pollen. For example, it can be applied to the identification of various DNAs that interact with histones or transcription factors; it can also be extended to the study of DNA-protein interactions in other tissues of maize or other plants.

[0140] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for cutting and tagging plant pollen, characterized in that: The method includes the following steps: A1) Precipitation of the target protein: Cell nuclei are extracted from plant pollen using an extract containing Percoll to obtain a nuclear extract in which starch in the pollen cells has been removed. Polysaccharides and polyphenols in the nuclear extract in which starch in the pollen cells has been removed are removed using an extract containing 1,6-hexanediol and glycerol to obtain a nuclear extract in which starch, polysaccharides, and polyphenols in the pollen cells have been removed. The nuclear extract contains the target protein bound to DNA. The target antibody is mixed with the nuclear extract in which starch, polysaccharides, and polyphenols in the pollen cells have been removed to allow the target antibody to specifically bind to the target protein. A2) DNA fragmentation: pA-Tn5 transposons are added to obtain a coupling product of pA-Tn5 transposons with the target antibody and the target protein; The coupling compound contains DNA that binds to the target protein; No protease inhibitors or cell membrane permeation treatment solutions are added during the DNA fragmentation process. A3) Activate the pA-Tn5 transposon: Activate the pA-Tn5 transposon to fragment the DNA, resulting in a mixture containing fragmented DNA.

2. The method according to claim 1, characterized in that: The Percoll content in the Percoll-containing extract is 60% by volume.

3. The method according to claim 1 or 2, characterized in that: In the extract containing 1,6-hexanediol and glycerol, the concentration of 1,6-hexanediol is 1M and the volume concentration of glycerol is 20%.

4. The use of the method according to any one of claims 1-3 in detecting DNA in pollen cells that interacts with the target protein.

5. Application of Percoll, 1,6-hexanediol and glycerol in the CUT&Tag method for removing starch, polysaccharides and polyphenols from plant pollen.

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