High-throughput method for detecting transcription factor recognition motif based on protoplast CUTTag

Abstract

The invention discloses an application of a protoplast CUAmp; the invention discloses a high-throughput method for detecting a transcription factor recognition motif by using a Tag technology. The method comprises the following steps: 1) cutting up plant leaves, soaking the cut plant leaves in enzymatic hydrolysate, and separating cells, so that rice powder is completely soaked in the enzymatic hydrolysate; 2) after enzymolysis, wrapping filter residues with gauze, and extruding the filter residues to collect protoplast cells; (3) adding the protoplast cells into a centrifugal tube containing plasmids, instantaneously transferring the plasmids into the cells, and expressing genes transferred by the plasmids; (4) adding a GFP antibody, a secondary antibody and a Tn5 transposon, recovering the DNA combined with the GFP antibody, and carrying out subsequent NGS library building; 5) performing high-throughput sequencing to obtain a transcription factor sequence, and performing biological information analysis to obtain a transcription factor combined motif; and 6) verifying the interaction of the transcription factor and the recognition motif by using an EMSA method.

Description

Technical Field

[0001] The invention relates to the fields of molecular biology and bioinformatics, and in particular to a plant cell dissociation method, a transcription factor binding DNA separation method and a bioinformatics analysis method. Background Art

[0002] Transcription factors are key regulatory factors in life activities, regulating the expression of target genes by binding to specific regulatory regions (promoters or enhancers). Transcription factors generally recognize specific conserved sequences, which are called motifs. Determining the motif is the first step in studying the function of transcription factor genes, and only then can the regulatory network of transcription factors be further determined.

[0003] The identification methods of transcription factor binding motifs are divided into two categories: in vivo methods and in vitro methods. The advantage of in vivo methods is that they can be associated with cell types, stress conditions, and developmental stages, reflecting the real binding events of organisms. In vitro methods reflect the DNA binding characteristics of pure single transcription factors. The most typical in vivo method is the ChIP-seq (chromatin immunoprecipitation sequencing detection) method, which requires two basic conditions: either a stable transgenic strain is obtained through transgenic means, and then the binding DNA of the target transcription factor is obtained through immunoprecipitation with a label antibody, or the antibody of the target transcription factor is customized, and then the binding DNA of the target transcription factor is obtained through immunoprecipitation with a customized antibody. Both methods are time-consuming and costly. In vitro methods include the DAP-seq method and the TF-Centered Y1H method. The DAP-seq method is to express the transcription factor protein in vitro, and then obtain the transcription factor binding DNA fragment by affinity purification. The disadvantages of this method are high cost and long experimental cycle. The TF-Centered Y1H method constructs a saturated 7-base length random prey DNA library, then uses the transcription factor bait to retrieve the bound prey DNA, and finally obtains the transcription factor-bound DNA fragments through high-throughput sequencing. This method requires the synthesis of a large amount of prey DNA, which is costly and cannot obtain the specific binding sites of transcription factors on the genome. Summary of the invention

[0004] In order to solve the above technical problems, a high-throughput method based on protoplast CUT&Tag to detect transcription factor recognition motifs is provided. Through transient transfection of transcription factor vector plasmids in protoplasts, an exogenous transcription factor expression system is quickly obtained, and then the binding DNA of the exogenous transcription factor is obtained through CUT&tag technology. Finally, the motif recognized by the target transcription factor is obtained through high-throughput sequencing. It is specifically achieved through the following technical solutions:

[0005] A high-throughput method for detecting transcription factor recognition motifs based on protoplast CUT&Tag comprises the following steps: 1) chopping plant leaves and soaking them in a lysis solution to separate cells, and using a vacuum pump to draw a vacuum so that the rice pieces are completely immersed in the enzymatic solution; 2) wrapping the filter residue with gauze after enzymatic hydrolysis, and squeezing the filter residue to collect protoplast cells; 3) then adding the protoplast cells to a centrifuge tube containing a plasmid (containing a transcription factor gene), transiently transferring the plasmid into the cells, and expressing the gene transferred into the plasmid; 4) finally adding a GFP antibody, a secondary antibody and a Tn5 transposome, recovering the DNA bound by the GFP antibody and subsequently building a NGS library; 5) obtaining tens of millions of DNA sequences bound by transcription factors after high-throughput sequencing by Illumina Hiseq6000, and obtaining the motifs bound by transcription factors after bioinformatics analysis; 6) verifying the interaction between the transcription factor and the recognition motif by using an EMSA method.

[0006] The commonly used technical method ChIP-seq currently requires transgenic or antibody customization, which takes about 3 months to a year, while our technical process only takes about 2 days, which greatly improves the progress of research. The second commonly used technology is DAP-seq, which requires protein expression in advance, has a high experimental cost, and because it is an in vitro experiment, it eliminates the influence of chromatin structure and avoids the interaction of other transcription factors, completely losing the influence of the special cell environment in the body, and its authenticity is questionable. This method not only retains the advantages of the ChIP-seq method's cellular authenticity environment, but also ensures the convenience of implementation, and can quickly obtain the binding motifs of transcription factors.

[0007] The present invention combines plant cell dissociation methods, transcription factor binding DNA separation methods and bioinformatics analysis methods, and finally obtains the motifs to which the transcription factors bind, laying the foundation for the final study of the function of the target transcription factor. The method has the advantages of low cost, high speed, high efficiency and high utilization value, and can be widely used to detect the binding motifs of plant transcription factors. With the motifs, the gene function of the transcription factor can be further studied.

[0008] Furthermore, the protoplast preparation method comprises the following steps: soaking plant seeds and then culturing them in the dark; crushing yellowed plant seedlings and adding enzymatic solution to perform enzymatic hydrolysis at 20-30 degrees Celsius for 2-4 hours.

[0009] Specifically, the protoplast preparation method comprises the following steps:

[0010] A1) Cultivation of etiolated rice seedlings: After soaking rice seeds at 37 degrees Celsius for 24 hours, the seeds were placed in a moistened filter paper culture dish for dark culture at an ambient temperature of 25 degrees Celsius for 7 days.

[0011] A2) Chop 5-10g of yellowed seedlings with a blade, add enzymatic solution, and place in a conical flask. Then, vacuumize to allow the enzymatic solution to fully penetrate into the leaf tissue to facilitate full cell lysis.

[0012] A3) After 3 hours of enzymatic hydrolysis at 24 degrees Celsius, the enzymatic hydrolysis is complete. Gently shake the enzymatic solution and it turns green. Then use a microscope to observe the protoplast cells.

[0013] Furthermore, the step of transient transformation of protoplasts includes adding plasmid and then performing water bath.

[0014] Specifically, the protoplast transient transformation method;

[0015] B1) A transient expression vector was constructed using the PAN580 vector backbone, and a GFP peptide tag was added to the end of the target AP2 gene.

[0016] B2) Take 100ul of protoplast cell suspension, add PEG4000 solution, mix gently and place in a 23°C water bath for 15 minutes.

[0017] B3) Detect transformation efficiency by microscopic examination.

[0018] Furthermore, the protoplast CUT&Tag method includes the following steps: DNA cutting, activating the transposome, and cutting the DNA flanking region to which the target protein is bound.

[0019] Specifically, the protoplast CUT&Tag method comprises the following steps:

[0020] C1) Anchoring the target protein. The target antibody is co-incubated with the target protein to couple the target antibody with the target protein.

[0021] C2) Capture of target protein-DNA complex: Add pA-Tn5 transposome to obtain a coupling product of pA-Tn5 transposome-target antibody-target protein.

[0022] C3) DNA cleavage: Activate the pA-Tn5 transposome to cleave the DNA flanking region bound by the target protein.

[0023] C4) NGS library construction: The DNA obtained in step C3 was dissociated and then PCR amplified to construct the library.

[0024] C5) Sequencing and analysis: The library obtained in C4 was sequenced and analyzed on the Illumina Nova6000 platform to obtain the DNA interaction site information of the target protein.

[0025] Furthermore, the above method was verified in species such as Arabidopsis, tobacco, wheat, corn, soybean, cabbage, rapeseed, and tomato, and significantly enriched motifs were obtained.

[0026] The application of the above method in the study of plant gene regulatory networks includes the following steps:

[0027] 1) Find the binding motifs and binding sites of transcription factors;

[0028] 2) Design probes based on binding sites and conduct EMSA experiments for verification;

[0029] 3) The transcription factor sequence and the promoter sequence of the binding site were used to construct AD vector and BD vector respectively, and yeast one-hybrid verification was performed;

[0030] 4) Place the transcription factor sequence and the promoter sequence of the binding site into the effector vector and reporter expression vector respectively for luciferase verification.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. Low cost. Unlike ChIP-seq technology, which requires high cost of customized antibodies, which costs at least several thousand yuan, the present invention only requires the transcription factor sequence to carry the tag GFP sequence, and then uses the tag antibody to capture the DNA bound by the target transcription factor. The DAP-seq method requires in vitro protein expression, which increases a lot of costs. The TF-Centered Y1H method requires the synthesis of a large number of random DNA fragments for the construction of the prey library, which is costly.

[0033] 2. High efficiency. The ChIP-seq technology experiment process takes 4-5 days to complete the entire experimental process. The entire experiment of the present invention only takes two days, eliminating the previous cross-linking and ultrasound processes, and the sample requirement is low, only more than 100 cells are needed.

[0034] 3. High utilization value. DAP-seq technology and TF-Centered Y1H are both in vitro methods and cannot reflect the true binding state of transcription factors in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The transient transformation effect of protoplasts

[0036] Plant cells with green fluorescence are successfully transformed.

[0037] Figure 2 NGS library quality inspection chart

[0038] The captured DNA fragments were mainly distributed in the 300-500 bp region.

[0039] Figure 3 Reads enrichment distribution map

[0040] Reads are mainly enriched near TSS.

[0041] Figure 4 Motif display picture

[0042] Binding site motif display of transcription factors. DETAILED DESCRIPTION

[0043] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Routine experimental operations usually follow the conditions described in "Molecular Cloning" or the conditions recommended by the reagent manufacturers. The reagents used can be purchased at biochemical reagent stores unless otherwise specified, and the software used is free software.

[0045] 1. Select a rice variety 9311 for dark culture, obtain yellow flower seedlings, and take 3 grams of sample;

[0046] 2. Cut the leaves obtained in step 1 into pieces with a scalpel and place them in a lysis buffer for enzymatic hydrolysis;

[0047] 3. Filter the enzymatic hydrolysis product obtained in step 2 using a filter and collect the filtrate;

[0048] 4. Examine the filtrate obtained in step 3 under a microscope until complete spherical cells are observed. Then add the plasmid and place in a water bath to promote transformation;

[0049] 5. Centrifuge the protoplast suspension transformed in step 4, remove the supernatant and add nuclear extraction buffer to extract the cell nucleus;

[0050] 6. The cell nuclei obtained in step 5 are incubated with antibodies in the next step to enrich the target transcription factor binding DNA;

[0051] 7. Use the enriched DNA obtained in step 6 to construct an NGS library and perform quality inspection;

[0052] 8. Perform next-generation sequencing on the library that has passed the quality inspection in step 7;

[0053] 9. Perform high-throughput sequencing on the sequencing results obtained in step 8, and analyze the sequencing results;

[0054] 10. Analyze the motifs and binding sites recognized by the transcription factor, verify the binding sites by EMSA, and finally obtain the regulatory network of the transcription factor.

[0055] The above method has been verified in species such as Arabidopsis, tobacco, wheat, corn, soybean, cabbage, rapeseed, and tomato, and all of them can obtain significantly enriched motifs.

[0056] The specific implementation methods are as follows:

[0057] Embodiment 1:

[0058] A high-throughput method for detecting transcription factor recognition motifs based on protoplast CUT&Tag, the steps of which are:

[0059] 1. Protoplast preparation

[0060] A. Cut the rice leaves grown for about 7 days into pieces of about 0.5 mm with scissors, put them into a conical flask, and perform enzymatic hydrolysis at 30°C and 50 rpm for about 30 minutes in a dark environment;

[0061] B. Filter with a 150-mesh filter to remove the residue, collect the filtrate in a 50 ml centrifuge tube, and centrifuge at 1000 rpm for 10 minutes to remove the supernatant;

[0062] C. Add 2 ml of W5 to the precipitate, shake gently and mix, take 20 ul for microscopic examination, and judge the quality of the protoplasts by the roundness (complete sphere).

[0063] 2. Transformation of protoplasts

[0064] A. Add 5 plasmids: 5 protoplasts: 20 PEG to the corresponding centrifuge tube, mix gently, then add 1 ml PEG along the wall of the centrifuge tube and mix gently.

[0065] B. Immediately place the material with protoplasts in a water bath of appropriate temperature at 25°C for 45 minutes. Add 3 times the volume of W5 to terminate the reaction.

[0066] C. Rice was incubated at 1000 rpm for 10 minutes, and the supernatant was removed. The rice was resuspended in W5 and cultured in the dark for 24-48 hours for laser confocal microscopy.

[0067] 3. Cell nuclear extraction

[0068] A. Centrifuge the protoplast suspension at 1000×g for 5 minutes, remove the supernatant, resuspend the pellet with 1ml nuclear extract buffer, centrifuge at 1000×g for 10 minutes, remove the supernatant, and keep the pellet. Resuspend the pellet with 1ml nuclear extract buffer, let stand on ice for 30 minutes, centrifuge at 500×g for 10 minutes, remove the supernatant, repeat this step once, and resuspend the pellet with 0.5ml nuclear extract buffer.

[0069] B. Take a new 1.5ml EP tube and add 0.5ml nuclear extract buffer. Slowly add the suspension in step 2A along the tube wall. Centrifuge at 500×g for 60min, remove the supernatant and keep the precipitate. Resuspend with nuclear extract buffer, centrifuge at 2000×g for 30min, remove the supernatant, and repeat this step once.

[0070] C. Resuspend the precipitate in 100 μL of nuclear extraction buffer. Draw 5 μL of cells and stain with trypan blue. Count the cells with a cell counter. Draw 100,000 successfully transformed nuclei suspension for subsequent experimental operations based on the cell nucleus concentration and transformation efficiency.

[0071] 4. Binding of magnetic beads to cell nuclei

[0072] A. Take 20 μL of Con A magnetic beads and pipette to mix them in a PCR tube that has been pre-added with 40 μL Con A bind buffer. Place it on a magnetic rack until the beads adhere to the wall, and remove all the solution in the tube by aspiration. Remove the PCR tube, add 50 μL Con A bind buffer, pipette to mix, and place it on a magnetic rack until the beads adhere to the wall. Remove all the solution in the tube by aspiration, remove the PCR tube, add 20 μL Con A bind buffer, and pipette to mix.

[0073] B. Gently add the bead slurry resuspended in ConA bind buffer to the cell nucleus suspension, invert 5 times to mix, and place the PCR tube on a rotator to mix for 30 minutes.

[0074] C. Remove the magnetic beads that have bound to the cells, centrifuge them briefly (<100×g), place them on a magnetic rack until the beads adhere to the wall (no more than 2 minutes), and aspirate the solution in the tube. Add 50μL BF4+ / BF4-, and mix them by gently blowing or tapping the bottom with a pipette.

[0075] D. Rotate and incubate at room temperature for 2-4 hours or at 4°C overnight to ensure that the liquid is at the bottom of the tube. Tap the bottom to mix every 30 minutes. Remove the magnetic beads that have bound to the primary antibody, centrifuge them (<100×g), and place them on a magnetic rack until the beads adhere to the wall (no more than 2 minutes), and then aspirate the solution in the tube.

[0076] E. After adding 49.5 μL BF2, add 0.5 μL secondary antibody and gently vortex or tap the bottom to mix. Incubate at room temperature for 3 hours to ensure that the liquid is at the bottom of the tube. Every 30 minutes, tap the bottom or pipette gently to prevent the magnetic beads from agglomerating and drying.

[0077] F. Centrifuge the magnetic beads (<100×g) and place them on a magnetic rack until they adhere to the wall (no more than 2 minutes). Aspirate the solution in the tube. Add 49μL BF3 and then 1μL pA / G-Transposome Mix. Use a pipette to blow or flick the bottom to mix.

[0078] 5. Transposase activates Tn5 digestion

[0079] A. Centrifuge the magnetic beads (<500×g) and place them on a magnetic rack until the beads adhere to the wall (no more than 2 minutes). Aspirate the solution in the tube, add 30μL BF3, and then add 1μL 30× Activating buffer. Gently pipette or tap the bottom to mix.

[0080] B. Incubate at 37℃ with rotation for 3 hours to ensure that the liquid is at the bottom of the tube. Tap the bottom every 30 minutes to mix well and prevent the beads from agglomerating and drying.

[0081] 6. Proteinase K digestion and genomic DNA recovery

[0082] A. Add 2 μL 15×Terminate Solution and 1 μL 30×Protease K to each sample. Vortex the sample at full speed for about 20 minutes, centrifuge after mixing, and place the sample in a PCR instrument and digest at 55°C for 30 minutes or at 37°C overnight.

[0083] B. Briefly centrifuge (<100×g), place the PCR tube on a magnetic stand and let it stand for 10 minutes, and take 20μL of supernatant. Add 20μL of DNA Selection Beads equilibrated at room temperature, vortex to mix or pipette 10 times to mix, and incubate at room temperature for 15 minutes. Briefly centrifuge (<100×g), place the PCR tube on a magnetic stand and let it stand for 10 minutes, and aspirate the solution in the tube after the magnetic beads are completely attached to the wall.

[0084] C. Keep the PCR tube on the magnetic rack, add 200μL 80% ethanol from the other side to avoid directly washing the magnetic beads, let it stand for 30-60 seconds, and aspirate the solution in the tube. Keep the PCR tube on the magnetic rack, add 200μL 80% ethanol again, let it stand for 30-60 seconds, and aspirate the solution in the tube.

[0085] D. Keep the PCR tube on the magnetic rack at all times, open the lid and air dry until the magnetic beads just begin to crack (no more than 5 minutes). Remove the PCR tube from the magnetic rack, add 21μL ddH2O, vortex thoroughly, and let stand at room temperature for 10 minutes. Centrifuge the PCR tube briefly, place it on the magnetic rack to separate the magnetic beads and liquid, and after the solution is clarified (about 5 minutes), carefully transfer 20μL of the supernatant to a new EP tube and store at -20℃.

[0086] 7. Library Amplification

[0087] The amplification procedure is as follows:

[0088]

[0089] The reaction system is as follows:

[0090]

[0091] 8. Library purification

[0092] A. Mix the DNA Selection Beads magnetic beads balanced to room temperature by oscillation or inversion. Pipette 60μL (1.2×) DNA Selection Beads into the PCR reaction product in step 7, vortex to mix or pipette to mix 10 times, and incubate at room temperature for 10 minutes. Centrifuge briefly (<100×g), place the centrifuge tube on the magnetic stand and let it stand for 3 minutes. After the magnetic beads are completely attached to the wall, aspirate the solution in the PCR tube.

[0093] B. Keep the PCR tube on the magnetic rack, add 200μL 80% ethanol from the other side to avoid directly washing the magnetic beads, let it stand for 50-90 seconds, and aspirate the solution in the PCR tube. Keep the PCR tube on the magnetic rack, add 200μL 80% ethanol again, let it stand for 50-90 seconds, and aspirate the solution in the PCR tube.

[0094] C. Keep the PCR tube in the magnetic rack, open the lid and air dry until the magnetic beads just begin to crack (no more than 3 minutes). Remove the PCR tube from the magnetic rack, add 21 μL ddH2O, vortex thoroughly, and let it stand at room temperature for 10 minutes.

[0095] D. Centrifuge the PCR tube briefly and place it on a magnetic stand to separate the magnetic beads and liquid. After the solution is clear (about 5 minutes), carefully transfer 20 μL of the supernatant to a new EP tube and store it at -20°C. The library size can be detected using Qsep 1 or gel electrophoresis.

[0096] 9. NGS sequencing and data analysis

[0097] A. Place the library on the Illumina NovaSeq6000 sequencing platform for next-generation sequencing;

[0098] B. Use Trimmomatic software to filter data, and use fastQC software to perform data quality control on the filtering results;

[0099] C. Use bowtie2 software to align the clean data to the reference genome;

[0100] D. Use MACS2 software to analyze peaks;

[0101] E. Use meme-chip software to analyze motifs.

[0102] 10. Result Analysis

[0103] like Figure 1 As shown, the transient transfection effect meets the requirements and the next step of cell nucleus extraction can be carried out; Figure 2 As shown, the NGS library construction was qualified, and the target fragments were mainly distributed in the 300-500 main area; Figure 3 As shown in Figure 2, reads are mainly enriched near TSS, indicating that the captured target DNA fragments are mainly located near the gene transcription start site; Figure 4 As shown, the conserved motif was obtained after software analysis.

[0104] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A high-throughput method for detecting transcription factor recognition motifs based on protoplast CUT&Tag, characterized in that: The method comprises the following steps: 1) protoplast preparation: crushing the plant and soaking it in an enzymatic solution for enzymatic hydrolysis, and filtering after enzymatic hydrolysis to obtain protoplast cells; 3) protoplast transient transformation: adding plasmids to the protoplast cells and mixing them for transformation; 4) protoplast CUT&Tag: adding GFP antibody, secondary antibody and transposome, recovering DNA bound by GFP antibody and subsequent NGS library construction; 5) Sequencing and analysis.

2. The high-throughput method according to claim 1, characterized in that The protoplast preparation method comprises the following steps: soaking plant seeds and then culturing them in the dark; crushing yellowed plant seedlings, adding enzymatic solution and performing enzymatic hydrolysis at 20-30 degrees Celsius for 2-4 hours.

3. The high-throughput method according to claim 1, characterized in that The step of transient transformation of protoplasts includes adding plasmid and then performing water bath.

4. The high-throughput method according to claim 1, characterized in that The protoplast CUT&Tag method comprises the following steps: DNA cutting, activating the transposome, and cutting the DNA flanking region bound by the target protein.

5. The high-throughput method according to claim 4, characterized in that The plant comprises one of rice, Arabidopsis, tobacco, wheat, corn, soybean, cabbage, rapeseed and tomato.

6. An application of the high-throughput method according to any one of claims 1 to 5 in the study of plant gene regulatory networks.

7. The use according to claim 6, characterized in that The steps include: 1) Find the binding motifs and binding sites of transcription factors; 2) Design probes based on binding sites and conduct EMSA experiments for verification; 3) The transcription factor sequence and the promoter sequence of the binding site are used to construct AD vector and BD vector respectively, Conduct yeast one-hybrid validation; 4) Place the transcription factor sequence and the promoter sequence of the binding site into the effector vector and reporter expression vector respectively for luciferase verification.

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