Construction method of sequencing library for detecting high-resolution chromatin interaction of plants

Abstract

The invention discloses a construction method of a sequencing library for detecting high-resolution chromatin interaction of plants. The method comprises the following steps: mixing a purified to-be-detected plant cell nucleus with a dendritic high-molecular compound, irradiating for 30 minutes under ultraviolet light, crosslinking to obtain a crosslinked compound, digesting the crosslinked compound by using protease K, digesting for 30-45 minutes by using DNaseI, and connecting an enzyme-digested product by using a bridge linker to obtain a connecting product containing a target DNA fragment, and finally, enriching and purifying the connected product to obtain the interacting chromatin DNA captured by the dendritic high-molecular compound. By using the method disclosed by the invention, experimental data of high-resolution chromatin interaction in the plant can be captured, and the method can be used for identifying the interaction between gene promoter regions in a whole genome range of the plant.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for constructing a sequencing library for detecting high-resolution chromatin interaction in plants. Background Art

[0002] Organisms need to face complex environmental changes during their growth and development. These environmental changes may either promote or stress the development of organisms. Cold stress, due to its relatively regular occurrence cycle and wider scope of action, especially affects plants. As sessile organisms, plants cannot move to cope with changes in environmental temperature. Therefore, how to cope with cold stress has become an important issue in plant growth and development. To solve this survival problem, plants have evolved a perfect regulatory mechanism. This includes the well-studied cold stress regulator ICE1 (Inducer of CBF Expression 1), which is a transcriptional regulator under cold stress conditions. Currently, the research on plant cold stress still remains at the phenotypic and gene transcription levels. The specific molecular mechanism of gene expression regulation under cold stress conditions is still unclear.

[0003] The genetic material DNA of an organism is wound and stacked with histones to form chromatin, which is stored in the nucleus. The arrangement of chromatin in the nucleus is not linear but has a certain three-dimensional structure. That is, different regions on chromatin will approach and interact with other regions in terms of spatial distance. This interaction is of great significance for biological processes occurring on chromosomes in organisms, such as transcription, replication, etc. Among them, transcription is the process by which an organism expresses the stored genetic information, and it is a key link for an organism to maintain life activities and interact with the external environment. Therefore, exploring the mechanism of transcriptional regulation in organisms is an important topic in life science research. When organisms cope with complex environments (stress environments, such as cold stress) and developmental resistances, gene transcription is often not independent, but two or more genes are co-transcribed. Corresponding to the co-transcription phenomenon, organisms have also evolved a co-regulation mechanism - a mechanism that regulates co-transcription between gene pairs. Currently, the main method used in the research on co-transcription is to use WGCNA combined with multi-omics analysis. Based on the existing transcriptome and phenome data, the expression levels between two or several genes are extracted and weighted, and further correlation analysis and clustering analysis are carried out. This method can only speculate on gene pairs that may be co-expressed from the levels of expression and phenotype. However, the molecular mechanism of co-regulation between genes still lacks research.

[0004] During transcription, transcription factors interact with the promoter region, thereby promoting transcription. Therefore, the activity of the promoter region is crucial for understanding the transcriptional regulatory mechanism. For example, from the perspective of chromatin interaction, when the promoter regions of two or more genes are spatially close, while a transcription factor regulates the transcription of one gene, it may affect the expression of another gene due to spatial accessibility. This provides a possible mechanism for the same transcription factor to regulate the transcription of these genes simultaneously. Therefore, how to obtain high-resolution chromatin interaction data is an important link in breaking through the molecular mechanism of chromatin interaction on gene expression regulation.

[0005] Currently, the main research methods for chromatin interaction include 3C, 4C, 5C, and Hi-C technologies, etc. They observe chromatin interaction from different levels, covering the interaction between single genes to the interaction between whole genome regions. Among them, the Hi-C (High-throughput Chromatin Conformation Capture) technology is a technology that uses high-throughput sequencing and bioinformatics analysis methods to study the three-dimensional structure of chromosomes and the spatial interaction patterns of the genome. However, due to the limitations of the Hi-C library construction method (depending on the interaction between proteins and proteins), the resolution of chromosome interaction captured by it is relatively low, and it cannot accurately provide interaction information at the single gene level. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to construct a fine chromatin three-dimensional conformation capture platform based on the CAP-C technology in plants and / or how to capture high-resolution plant chromatin interaction information and / or how to capture the interaction between plant promoters.

[0007] To solve the above technical problems, the present invention first provides a method for obtaining chromatin DNA with interaction in plants, and the method may include the following steps:

[0008] A1) Grinding of plant tissues and purification of cell nuclei: Freeze-grind the (cross-linked) plant tissues to be tested to obtain powder, and extract the purified cell nuclei of the plant tissues to be tested from the powder;

[0009] A2) Ultraviolet cross-linking: Mix the purified cell nuclei with a dendritic polymer compound and irradiate under ultraviolet light for 30 minutes to obtain an ultraviolet cross-linked product; the ultraviolet cross-linked product contains a cross-linked complex of chromatin, protein, and dendritic polymer.

[0010] A3) DNA digestion and ligation: Digest the cross-linked complex with proteinase K to obtain a DNA-dendrimer complex; digest the DNA-dendrimer complex with DNase I to obtain a complex containing DNA fragments; after subjecting the complex containing DNA fragments to a ligation reaction with a bridge linker, remove the excess linker and add a ligase for ligation to obtain a ligation product 3 containing DNA fragments;

[0011] A4) Obtaining of target chromatin fragments: Enrich and purify the DNA fragments in the ligation product 3 to obtain interacting chromatin DNA pairs linked by dendritic macromolecules.

[0012] In the above method, the plant tissue to be tested can be the cross-linked plant tissue to be tested fixed with formaldehyde. After fixation with formaldehyde, glycine solution can be added to terminate the cross-linking, and the concentration of the glycine solution can be 0.125 M.

[0013] The termination of formaldehyde cross-linking can be carried out under vacuum conditions.

[0014] In the above method, the DNA fragments in A3) can be 50 - 200 bp.

[0015] In the above method, the dosage of DNase I in A3) can be 8000 U Dnase I per 2.0 g of plant tissue to be tested, and the digestion time is 30 - 45 minutes.

[0016] In the above method, the extraction in A1) can be carried out in a buffer solution. The composition of the buffer solution can be 10 mM Tris-HCl at pH 8.0, 10 mM NaCl, 0.2% non-ionic surfactant Igepal CA630, 1× protease inhibitor mixture and water. The dosage of the buffer solution can be 1 mL.

[0017] In the above method, the plant can be Arabidopsis thaliana.

[0018] To solve the above technical problems, the present invention also provides a method for detecting the interaction of promoters in the whole genome of plants. The method can include constructing a sequencing library of plants using the method described above, sequencing the sequencing library to obtain sequencing data, and performing bioinformatics analysis on the sequencing data to obtain the interaction of promoters in the whole genome of the plant to be tested.

[0019] In the above method, the plant can be Arabidopsis thaliana.

[0020] The present invention applies the CAP-C technology to the model plant Arabidopsis thaliana for the first time. Based on the experimental data of capturing high-resolution chromatin interactions, the interactions between gene promoters across the genome are identified, thus providing direct chromatin interaction evidence for the study of the molecular regulatory mechanism of gene co-expression / transcription factories. In this invention, experimental materials from different cold stress treatment groups under different conditions are used to identify co-regulated promoter-promoter interactions under cold treatment conditions, providing a basis for studying the molecular countermeasures of plants against cold stress.

[0021] Advantages of the present invention:

[0022] Firstly, at the experimental level in the early stage, after extracting the cell nuclei, when using DNA enzymes to digest fragments, different enzyme digestion concentrations and treatment times will affect the enzyme digestion results. The enzyme digestion conditions are an important factor directly determining the experimental detection sensitivity of the present invention. Therefore, in the present invention, it is necessary to re-explore experimental conditions different from existing studies (such as different species, different stress conditions, different tissues and organs, etc.), and form an experimental system applicable to this species and experimental conditions.

[0023] Different from the traditional Hi-C experiment, the method used in this invention to ligate large plant chromosome molecules is not based on the action of chromosome-binding proteins, but uses a polymer, namely dendrimer. And dendrimer itself is not a molecule existing in plants. Therefore, how to introduce this macromolecule into the plant cell nucleus is an innovative discovery of the present invention. At the same time, in previous experiments of the same type, the materials selected were animal tissues or cell materials, which are quite different from the Arabidopsis thaliana plants selected in this invention. The latter has a cell wall, which brings resistance to the research work. Therefore, it is crucial to explore the way for dendrimer to enter the cell nucleus. Description of the Drawings

[0024] Figure 1 Compared with the Hi-C technology, CAP-C captures chromatin interaction information with higher resolution. The left figure shows the results of chromatin interaction intensities at different resolutions in a partial section of Arabidopsis thaliana chromosome 1 obtained by the HicExplorer software. The vertical axis represents the coordinate information of a partial section of Arabidopsis thaliana chromosome 1, and the coordinates increase from top to bottom. The horizontal axis represents the coordinates of the same chromatin interval as the vertical axis, and increases from left to right. The right figure shows the change graph of the relative interaction frequency with the increase of genetic distance. The vertical axis represents the interaction frequency, that is, the interaction intensity, and the horizontal axis represents the continuously increasing genetic distance from left to right. The two lines shown in the figure, where the dark line is the chromatin interaction frequency corresponding to different genetic distances captured by the CAP-C technology, and the light line is the chromatin interaction frequency corresponding to different genetic distances captured by the Hi-C technology.

[0025] Figure 2 PPI network and transcription factor enrichment site information. a shows the interaction strength between gene promoters in the whole genome landscape of Arabidopsis thaliana, where each point represents the chromatin interaction between a pair of gene promoter regions, and the five regions with higher point density represent the five chromosomes of Arabidopsis thaliana; b Figure 2 is an enlarged picture of a region with higher density in a, showing a more local promoter interaction situation; c is the statistics of the interaction strength between gene promoters related to transcription factors, with the vertical coordinate being the interaction strength between promoters and the horizontal coordinate being the distance from the position to the binding site of transcription factor (TF); d is the statistics of the occurrence of promoter-promoter interactions in the upstream and downstream regions of the binding sites of three transcription factor families, with the horizontal coordinate being the three different transcription factor families and the distance upstream and downstream of the binding site of each family.

[0026] Figure 3 is the curve of interaction strength and genomic genetic distance of CAP-C under different temperature conditions. The vertical coordinate is the interaction frequency, i.e., the interaction strength, and the horizontal coordinate is the increasing genetic distance from left to right.

[0027] Figure 4 shows that the promoter-promoter interactions of three groups of co-expressed gene pairs exist in chromatin loops. Detailed implementation manners

[0028] The present invention will be further described in detail below in combination with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0029] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0030] Example 1: Construction of Arabidopsis thaliana chromatin interaction detection library

[0031] 1. Material acquisition:

[0032] The seeds of Arabidopsis thaliana (Col-0) of the Columbia ecotype were disinfected with 70% ethanol for 10 min and then washed three times with distilled water. After disinfection, they were sown on half-strength MS (Murashige and Skoog) medium supplemented with 1% sucrose. On the fourth day, the medium was transferred to long-day conditions (16 h light / 8 h dark) at 4 °C and cultured for 10 days. Then, the Arabidopsis seedlings were harvested and stored for the construction of the CAP-C library.

[0033] 2. CAP-C Library Construction Method

[0034] 2.1 Collection of Experimental Materials

[0035] The Arabidopsis seedlings obtained in step 1 were collected in a tube and fixed by vacuum with 1% formaldehyde at a final concentration for 15 minutes. Then, they were vacuum-treated with 0.125 M glycine at a final concentration for 5 minutes to terminate the reaction.

[0036] The Arabidopsis seedling tissues after formaldehyde fixation were rinsed three times with sterile water, the moisture attached to the materials was blotted dry with a tissue paper, quickly frozen in liquid nitrogen, and placed in a -80 ultra-low temperature refrigerator for later use (experimental materials without formaldehyde cross-linking can also be used).

[0037] 2.2 Nucleus Extraction and Dendrimer Cross-Linking

[0038] 2.2.1 Grinding and Nucleus Isolation

[0039] The collected Arabidopsis materials were thoroughly ground in liquid nitrogen and 10 ml of pre-cooled nucleus extraction buffer (consisting of 20 mM HEPES, 250 mM sucrose, 1 mM MgCl2, 0.25% (V / V) Triton X-100, 40% (V / V) glycerol, 0.1 mM PMSF, 0.1% (V / V) β-mercaptoethanol, 1× protease inhibitor cocktail, and water) was added.

[0040] The lysed sample was filtered twice through a double-layer Miracloth filter.

[0041] The filtrate was centrifuged at 4 °C, 3000 g for 15 minutes, and the supernatant was discarded. The pellet was carefully resuspended in the nucleus extraction buffer.

[0042] Centrifugation was performed at 4 °C, 1900 g for 5 minutes, the supernatant was discarded, and centrifugation was repeated. Crude nuclei were obtained.

[0043] The pellet was resuspended in 1 mL of CAP-C buffer (consisting of 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630, and 1× protease inhibitor mixture) for 20 minutes for purification to obtain purified nuclei of Arabidopsis thaliana.

[0044] 2.2.2 Ultraviolet Cross-Linked Dendrimers and Chromatin

[0045] Mix the purified nuclei with the dendrimer compound (HS code: 2932209090, final concentration 100 μM), and then cross-link the dendrimers with chromatin by cross-linking under ultraviolet light at 365 nm for 30 minutes to obtain a cross-linked complex of chromatin, protein, and dendrimer.

[0046] After cross-linking, centrifuge the solution at 4°C and 2500 g for 5 minutes, and then wash it twice with CAP buffer. Resuspend the pellet in proteinase K buffer. Then digest the cross-linked complex with proteinase K overnight.

[0047] 2.3 Extraction, Purification, and Fragmentation of DNA-Dendrimer Compounds

[0048] Digest the proteins in the cross-linked complex to obtain a DNA-dendrimer complex. Purify and extract the DNA-dendrimer complex with phenol / chloroform. Take the supernatant and add 800 μL of 100% ethanol and 50 μL of 3 M sodium acetate to precipitate the DNA at -80°C.

[0049] Take out the precipitate from -80°C and centrifuge for 30 minutes. Wash it twice with 80% (V / V) ethanol and finally dissolve it in 50 μL of ultrapure water.

[0050] Fragment the DNA-dendrimer complex (corresponding to 2.0 g of Arabidopsis thaliana) into DNA fragments of 50 - 200 bp with DNase I (enzyme activity 8000 U, referring to the KAPAfragmentation Kit) at 37°C for 40 minutes.

[0051] The product is subjected to end repair and A-tailing, and then ethanol precipitated again at -80°C.

[0052] Dissolve the precipitate in 100 μL of double-distilled water. Mix and react the Dendrimer-DNA mixture with the double-labeled linker (IDT) at 37°C for 4 hours to form a linker-dendrimer-DNA complex.

[0053] Remove the unreacted linker with 1.2 volumes of Ampure magnetic beads.

[0054] Add T4 DNA ligase to the linker-dendrimer-DNA complex to ligate the DNA fragments with the dendrimer complex and the bridge linker, obtaining a ligation product of the DNA fragment with the dendrimer complex and the bridgelinker, and directly ligating the linker to the complex DNA fragment to form a chimeric fragment. The reaction is placed at 16°C and mixed at 900 rpm overnight.

[0055] The bridge linker is a nucleotide sequence with a known sequence. One strand of it is labeled with biotin for enrichment, and the other strand is labeled with a DBCO (dibenzocyclooctyne) modification group, which can react with the azide group on the dendrimer to form a strong covalent bond. After the enzymatic digestion reaction, the chemical group label on the bridge linker reacts with the Et3N branch of the dendrimer.

[0056] 2.4 Purify and enrich the target chromatin fragments

[0057] After using the dendrimer to capture a pair of interacting DNA fragments, since the bridge linker we used carries biotin, then through the biotin label on the bridge linker, the DNA chimeric fragments in the ligation product 3 obtained in step 2.3 are enriched using biotin antibody and magnetic beads (Dynabeads Myone Streptavidin C1 beads).

[0058] 2.5 Construction and sequencing of the sequencing library

[0059] Use the NEB library preparation kit to construct a sequencing library for the obtained DNA fragments. Then place the library on the MGI platform (BGI) for 150bp paired-end sequencing.

[0060] 2.6 Obtain chromatin interactions through sequencing data analysis

[0061] The FASTQ-formatted data files of 150-bp paired-end sequencing were aligned with the Arabidopsis thaliana TAIR10 reference genome using HiC-Pro (version 3.0.0) (related literature: Servant N, et al. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. Genome Biol 16, 259 (2015)). The linker sequences were trimmed by cutadapt (related website: https: / / github.com / marcelm / cutadapt / parameters: cutadapt -n 10 –mask-g / -a) to perform quality control on the sequencing data. R1 and R2 were merged according to the reported method (related literature: You Q, et al. Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution. Nat Biotechnol 39, 225-235 (2021)). For each sample, the alignment results were filtered using MAPQ >= 10. Then, the merged sorted files were converted into.hic-format files using Juicer (related literature: Durand NC, et al. Juicer Provides a One-Click System for Analyzing Loop-Resolution Hi-C Experiments. Cell Syst 3, 95-98 (2016)).All interaction matrices at 200bp resolution were visualized as interaction heatmaps using HiCExplorer (version 3.5.1) (related literature: Wolff J, et al. Galaxy HiCExplorer 3: a webserver for reproducible Hi-C, capture Hi-C and single-cell Hi-C data analysis, quality control and visualization. Nucleic Acids Res 48, W177-W184 (2020)) and Genomic-Interactive-Visualization-Engine (GIVE) (related literature: 8. Cao X, Yan Z, Wu Q, Zheng A, Zhong S. GIVE: portable genome browsers for personal websites. Genome Biol 19, 92 (2018)). The genome-wide interaction distance decay plot was displayed using hicPlotDistVsCount of HiCEexplore. Genome-wide chromatin loops were identified at 200bp resolution using Fit-Hi-C (version 2.0.7) (related literature: Ay F, Bailey TL, Noble WS. Statistical confidence estimation for Hi-C data reveals regulatory chromatin contacts. Genome Res 24, 999-1011 (2014). Kaul A, Bhattacharyya S, Ay F. Identifying statistically significant chromatin contacts from Hi-C data with FitHiC2. Nat Protoc 15, 991-1012 (2020)).

[0062] Promoter–promoter interaction network identification: According to the annotation file (containing gene chromosomal location information, naming information, and transcript information), the position 2 kb upstream of the gene was found as the gene promoter region. The overlapping region between the defined gene promoter region and the detected interaction data was found to define promoter–promoter interaction. After extracting all the identified promoter–promoter interaction position information, Cytoscape (version 3.9.1) (related literature: Shannon P, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13, 2498-2504 (2003)) was used to integrate the promoter–promoter interaction network into the whole-genome network, and the network was visualized by AllegroLayout. Gephi45 (version 0.10.0) (related literature: Bastian M, Heymann S, Jacomy M. Gephi: An Open Source Software for Exploring and Manipulating Networks. Proceedings of the International AAAI Conference on Web and Social Media 3, 361-362 (2009)) was used to perform more detailed visualization of the promoter–promoter interaction network.

[0063] Example 2. Comparison of Arabidopsis chromatin interactions obtained from the CAP-C library of the present invention with the detection results of Hi-C technology

[0064] 1. The CAP-C library constructed in Example 1 was used for sequencing, and the obtained sequencing data was subjected to bioinformatics analysis to obtain chromatin interactions in Arabidopsis. At the same time, the same Arabidopsis material in Example 1 was used for Hi-C library construction and sequencing analysis to obtain the chromatin interaction results of the same Arabidopsis material based on Hi-C technology.

[0065] The experimental procedure of the Hi-C technology in this example includes the following steps:

[0066] 1) Crosslinking cells: First, cells were treated with a crosslinking agent such as formaldehyde to fix the interaction between proteins and DNA on chromatin.

[0067] 2) Digest and label DNA ends: Then, use restriction enzymes (such as HindIII or EcoRI) to cut chromatin, generating DNA fragments with sticky ends. Next, fill these sticky ends with biotin-labeled nucleotides to make the DNA fragments easy to identify and separate in subsequent experiments.

[0068] 3) Ligation of adapters: Inside the cell, the cut DNA fragments randomly ligate to form new DNA ligation points. These ligation points reflect the DNA regions that were originally close to each other in three-dimensional space.

[0069] 4) Reverse crosslinking and purification: Remove the crosslinking by methods such as heating to release DNA. Then, use the biotin tag to extract and purify the adapter-ligated DNA fragments.

[0070] 5) Library construction and high-throughput sequencing: Next, construct a DNA library and perform high-throughput sequencing. The sequencing data reflects the proximity of different DNA regions in three-dimensional space.

[0071] 6) Data analysis: Finally, analyze the sequencing data by bioinformatics analysis methods to reconstruct the three-dimensional organizational structure of chromatin, thereby understanding biological processes such as gene expression regulation and chromosomal structure changes.

[0072] By comparing the chromatin interaction results obtained by sequencing the libraries constructed using the CAP-C library construction method of the present invention and the Hi-C sequencing technology for the same Arabidopsis thaliana material, the results show that CAP-C captures chromatin interactions with higher resolution.

[0073] It should be noted that at different resolutions (20 kb, 10 kb, and 2 kb), the comparison of the centromeric regions between CAP-C and Hi-C shows that ( Figure 1 in the left middle figure), compared with Hi-C, the chromatin interaction signals captured by the CAP-C library constructed using the present invention are stronger and finer ( Figure 1 in the left middle figure, the darker the color represents a stronger captured interaction), and the background noise is lower under high-resolution conditions (the color of each point in the figure represents the intensity of its interaction, so the darker the color of the point, the higher the interaction intensity, and vice versa. Many points with lighter colors and lower interaction intensities are considered non-biological significant interaction regions, but not all light-colored points are non-biological significant interactions. And this mixture of positive and non-positive points forces us to discard some positive data, so we call them background noise). Lower background noise means that all captured signals are more real and effective. As Figure 1 shown in the right middle figure, compared with the previously detected Hi-C data in Arabidopsis thaliana, the CAP-C data can capture more short-range (less than 103 bp) interactions, thus enabling the detection of more refined interaction structures. Identify the promoter-promoter interaction (PPI) network across the genome.

[0074] Example 3: Using CAP-C technology to capture chromatin conformational changes in Arabidopsis thaliana under cold stress conditions

[0075] 1. Using CAP-C technology to capture chromatin interactions and conformational changes in Arabidopsis thaliana under cold stress conditions

[0076] To study the molecular mechanism of gene expression regulation under cold stress conditions. The present invention focuses on the changes in chromatin structure in Arabidopsis thaliana under cold stress conditions.

[0077] Cold treatment method: The condition for cold treatment of Arabidopsis thaliana seedlings is to directly transfer 10-day-old seedlings from 22°C to 4°C, and the light condition is ~25 μE·m-2·s-1. Divide into two groups and treat at 4°C for 3 h and 4°C for 12 h respectively. The control treatment condition is that 10-day-old seedlings grow at 22°C.

[0078] Using the CAP-C library construction method and data analysis method in Example 1, the chromatin structure and chromatin interactions of Arabidopsis thaliana under cold stress conditions were obtained. The results showed that compared with the control (22°C) without cold stress treatment, cold stress treatments with different treatment times ( Figure 3 in CAP-C 4°C 3 h and CAP-C 4°C 12 h represent h) did not cause large-scale changes in the chromatin structure of plants. On the contrary, local small-scale changes in chromatin conformation occurred ( Figure 3 ). This indicates that local chromatin interactions are more sensitive to cold stress responses, and it also indicates that there is a certain correlation between local chromatin conformation and gene expression regulation.

[0079] 2. Analyze the interactions in the promoter region and gene expression regulation based on chromatin interactions

[0080] After that, to explore the relationship between chromatin conformation and gene expression regulation. Using the same method as in Example 1, the promoter regions of Arabidopsis thaliana were identified according to the gene positions recorded in the annotation file, and the interaction information of the promoter regions was obtained by comparing with the chromatin interaction data obtained in step 1. As Figure 2As shown in a and b, the genome-wide promoter-promoter interaction (PPI) map of Arabidopsis thaliana under three temperature conditions (the three temperature conditions include different treatment temperatures and different treatment times at different temperatures. Here, the three temperature conditions refer to the above: ① treatment at 4°C for 3 hours; ② treatment at 4°C for 12 hours; ③ growth at 22°C) was drawn using Cytoscape 3.9.1. The five regions marked by dashed lines in the figure respectively correspond to the main regions of five chromosomes of Arabidopsis thaliana. The two larger points in the network are two PPI hotspots on chromosomes 2 and 3 respectively. The five separate chromosomes are shown in different colors. The size of the points represents the number of PPIs.

[0081] Transcription factors may be enriched around the PPI region. To explore the co-regulation mechanism between genes, the present invention combined chromatin immunoprecipitation method to obtain chromatin regions enriched with transcription factors, and co-localized according to the information of this region and the chromatin interaction regions captured by the CAP-C experimental technique. The characteristics of the PPI region were analyzed, and it was found that the PPI region highly overlapped with the targeting regions of many transcription factor families. Experimental data showed that according to the histogram drawn by 422 transcription factors involved in the statistics, it was found that the chromatin interaction intensity in the transcription factor enrichment region was relatively high ( Figure 2 in c). At the same time, the heat map ( Figure 2 in d) shows the intensity of the PPI distribution in the regions adjacent to the binding sites of bHLH, NAC and WRKY transcription factor families.

[0082] To verify the correlation between PPI and co-expression, the present invention selected three groups of co-expressed genes related to cold stress under cold treatment conditions ( Figure 4 ). According to the experimental data of transcriptional activity measurement, it was found that these three pairs of genes (AT1G60220 and AT1G60230, AT1G78210 and AT1G78230, AT1G80940 and AT1G80950) showed co-expression phenomena of co-upregulation or co-downregulation with different cold stress treatments (plaNET-seq data showed the expression activity of genes).

[0083] The relevant literature on the experimental steps of plaNET-seq is: Kindgren P, Ivanov M, Marquardt S. Native elongation transcript sequencing reveals temperature dependent dynamics of nascent RNAPII transcription in Arabidopsis. Nucleic Acids Res 48, 2332-2347 (2020).

[0084] The Arabidopsis seedlings obtained in step 3g were quickly frozen in liquid nitrogen and ground into a fine powder in a mortar. The powder was transferred to a tube containing 15 ml of NUC1 buffer and thawed by rotation at 4°C. After centrifugation (5000 g, 20 minutes, 4°C), the pellet was dissolved in 1 ml of NUC2 buffer and centrifuged again (12 000 g, 10 minutes, 4°C). The resulting pellet was dissolved in 0.3 ml of NUC3 buffer, placed on top of 0.9 ml of clean NUC3 buffer and centrifuged (16 000 g, 60 minutes, 4°C). The purified nuclear fraction was dissolved and lysed in 1.5 ml of plaNET-seq lysis buffer.

[0085] Lysis was carried out by rotation (2000 rpm) at 4°C, first treated with DNaseI (Invitrogen), and then centrifuged (10000 g, 10 minutes, 4°C). The supernatant was transferred to a new tube and gently rotated and incubated with Dynabeads M-270 (Invitrogen) conjugated with anti-FLAG antibody (10 μg, Sigma-Aldrich F3165) at 4°C for 2 hours. After washing six times with 1 ml of wash buffer, the bound protein was eluted with 3xFLAG peptide (0.5 mg / ml, ApexBio). Elution was carried out twice for 20 minutes at 4°C with 0.1 ml of 3xFLAG peptide. According to the instructions, the RNA attached to the purified protein complex was isolated using the miRNeasy kit (Qiagen). The RNA was quantified using the RNA-Pico kit on a Bioanalyzer 2100 (Agilent). After obtaining the nascent RNA, a library was constructed using the NEXTflex Small RNA-seq kit v3 from Bioo Scientific. Different from the original protocol of the kit, the improved protocol incorporated an RNA fragmentation step to avoid underrepresentation of longer molecules of nascent RNA compared to shorter molecules.

[0086] Calculation of differentially expressed genes was performed using htseq-count and DESeq2 in Python. Htseq-count was run with default parameters and --strand=yes was added.

[0087] The results showed that AT1G60220 and AT1G60230 encode a stress-responsive SUMO protease and a SAM superfamily protein involved in chloroplast function, respectively. The present invention found that their expression levels were both downregulated after 3 hours of cold treatment ( Figure 4 represented by 4°C 3h in the lower left figure in Figure 44°C for 12 h in the lower left middle figure (representing) were up-regulated jointly after). AT1G78210 and AT1G78230 encode an α / β hydrolase for enhancing freezing tolerance and the outer arm dynein light chain 1 (ODALC1) protein that regulates intracellular trafficking, cell division, and organelle localization, respectively. The present invention found that these two genes were jointly up-regulated after 3 hours of cold treatment ( Figure 4 4°C for 3 h in the lower middle figure (representing) were jointly up-regulated and returned to the control level after 12 hours of cold treatment ( Figure 4 4°C for 12 h in the lower middle figure (representing)) Figure 4 22°C in the lower middle figure (representing). AT1G80940 and AT1G80950 encode an SNF1-related protein kinase and lysine diethanolamine involved in cell membrane metabolism, respectively, and the former regulates stomatal closure during stress. The expression levels of both genes were strongly inhibited after 3 hours of cold treatment ( Figure 4 4°C for 3 h in the lower right figure (representing)) and returned to normal levels after 12 hours of cold treatment ( Figure 4 4°C for 12 h in the lower right figure (representing). It is worth noting that the interaction regions of these three groups of genes ( Figure 4 Chromatin Loop in the figure) highly overlap with the target regulatory region of the cold stress-related transcription factor ICE1, which indicates that the co-expression of these three groups of gene pairs shows that the degree of overlap between the PPI region and the transcription factor action region is relatively high, further indicating that the interaction between gene promoters is of great significance for the regulation of co-transcription. In addition, the present invention mainly identified a series of gene promoter-promoter interaction sites, providing data support for studying the gene expression regulation mechanism.

[0088] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. A method for obtaining chromatin DNA in which plants interact, characterized in that: The method includes the following steps: A1) Grinding of plant tissue and purification of cell nuclei: Freeze-grind the plant tissue to be tested to obtain a powder, and extract the purified cell nuclei of the plant tissue to be tested from the powder; A2) UV crosslinking: Mix the purified cell nuclei with a dendritic polymer compound and irradiate under ultraviolet light for 30 minutes for crosslinking to obtain a UV crosslinked product; the UV crosslinked product contains a crosslinked complex of chromatin, protein, and dendritic polymer; A3) DNase digestion and ligation: Digest the crosslinked complex with proteinase K to obtain a DNA-dendrimer complex; digest the DNA-dendrimer complex with DnaseI to obtain a complex containing DNA fragments; after subjecting the complex containing DNA fragments to a ligation reaction using a bridge linker, remove the excess linker, and add a ligase for ligation to obtain a ligation product 3 containing DNA fragments; A4) Obtaining of target chromatin fragments: Enrich and purify the DNA fragments in the ligation product 3 to obtain chromatin DNA that interacts with proteins.

2. The method according to claim 1, wherein: The concentration of the glycine solution is 0.125 M; the termination of crosslinking is carried out under vacuum conditions.

3. The method according to claim 1 or 2, characterized in that: A3) The DNA fragments are 50 - 200 bp.

4. The method according to any one of claims 1 to 3, characterized in that: A3) The dosage of DnaseI is 8000 U DnaseI per 2.0 g of the plant tissue to be tested, and the digestion time is 30 - 45 minutes.

5. The method according to any one of claims 1-4, characterized in that: The extraction in A1) is carried out in a buffer solution, and the composition of the buffer solution is 10 mM Tris-HCl at pH 8.0, 10 mM NaCl, 0.2% non-ionic surfactant Igepal CA630, 1× protease inhibitor mixture, and water, and the dosage of the buffer solution is 1 mL.

6. The method according to any one of claims 1-5, characterized in that: The plant is Arabidopsis thaliana.

7. A method for detecting the interaction of promoters in the whole genome of plants, characterized in that: The method includes constructing a sequencing library of a plant using the method described in any one of claims 1 - 5, sequencing the sequencing library to obtain sequencing data, and performing bioinformatics analysis on the sequencing data to obtain the interaction of the whole genome promoter of the plant to be tested.

8. The method according to claim 7, wherein: The plant is Arabidopsis thaliana.

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