CN116536406A - Method for identifying plant open chromatin locus at whole genome level - Google Patents

Method for identifying plant open chromatin locus at whole genome level Download PDF

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CN116536406A
CN116536406A CN202310061337.2A CN202310061337A CN116536406A CN 116536406 A CN116536406 A CN 116536406A CN 202310061337 A CN202310061337 A CN 202310061337A CN 116536406 A CN116536406 A CN 116536406A
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beads
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张文利
史依宁
李兆国
韩琪
杨滢
李梦琪
何泽学
高志成
程雪姣
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Nanjing Agricultural University
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Abstract

The invention discloses a method for identifying plant open chromatin sites at the whole genome level (in situ DNase-seq). The method comprises the following steps: (1) cross-linking the immobilized plant material; (2) extracting and purifying the nuclei of the plant material; (3) Adding DNaseI, DNA polymerase I and biotin-dATP and biotin-dNTP, and performing enzyme digestion and in-situ marking of open chromatin sites; (4) recovering the DNA; (5) fragmenting the DNA; (6) DNA fragments marked by anti-streptavidins-beads binding biotin; (7) Library construction and Illumina sequencing, bioinformatic analysis, identified open chromatin sites at the whole genome level. The whole method has simple flow, short time consumption and strong DNA fragmentation visualization effect, and is theoretically suitable for various plants.

Description

Method for identifying plant open chromatin locus at whole genome level
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for identifying plant open chromatin loci at a whole genome level in a physiological state.
Background
Open chromatin, also known as accessibility chromatin (accessible chromatin), refers to the degree of physical compression of chromatin. In eukaryotic nuclei, the primary structure of chromatin is mainly a beaded linear structure composed of DNA together with histone octamers and other functional proteins such as transcription factors and chromatin remodeling complexes (Kumasaka N et al 2019,Nature Genetics,51 (1)). Chromatin can be divided into two major classes, open chromatin (open chromatin) and closed chromatin (closed chromatin), depending on the degree of physical compression of the chromatin, and can be used to indicate the physical "accessibility" and "inaccessibility" of DNA and regulatory proteins in the corresponding region, both chromatin states usually being associated with the relevant gene expression (Zhu B et al, 2015,Plant Cell,27 (9): 2415-2426).
Nucleosomes are the basic building blocks of eukaryotic chromatin, and due to histone protection, double-stranded DNA, which is often entangled in nucleosome octamers, is difficult to digest by nucleases such as deoxyribonuclease I (DNase I) and micrococcus nuclease (MNase). In contrast, the region of the nucleosome that is highly susceptible to nuclease digestion in the genome is referred to as the open chromatin site, since the DNA is in an naked state. The results of the related studies indicate that open chromatin sites usually contain cis-regulatory elements such as gene promoters and enhancers in the genome of animals and plants (Thurman R E et al 2012, nature,489 (7414); yue F et al 2014, nature,515 (7527); sheffield N C et al 2012, genes,3 (4)), and thus have a very important role in the regulation of gene expression.
Currently, methods for detecting open chromatin sites at the whole genome level are mainly: DNase I digestion sequencing (DNase-seq), transposase Tn5 based sequencing (ATAC-seq), MNase digestion sequencing (MH-seq), and formaldehyde assisted isolation regulatory element sequencing (faie-seq), and the like. Among them, DNase-seq is one of the experimental methods that was used at the earliest to identify open chromatin sites throughout the genome. DNaseI preferentially cleaves exposed double-stranded DNA in open chromatin, resulting in double-stranded DNA breaks (Hesselberth J R et al.,2009,Nature Methods,6 (4): 283-289;Boyle AP et al.,2008, cell,132 (2): 311-322). The basic principle of DNase-seq is: the purified cell nucleus is digested by DNaseI with a certain enzyme concentration unit, differential cutting is generated on double-strand DNA in different chromatin states of the cell nucleus, double-strand DNA of an open chromatin region is cut preferentially, and finally small-fragment or large-fragment DNA digested by DNaseI is collected and purified for single-ended or double-ended library construction sequencing, and a genome region enriched in enzyme cutting sites is identified at the whole genome level through bioinformatic analysis, which is called DNaseI hypersensitive site (DNaseIhypersensitivesites, DHSs). DHSs are generally associated with activation of gene expression (Wu C et al, 1979, cell,16 (4); wu C et al, 1980, nature,286 (5776)), and therefore, identification of DHSs is of great biological significance.
Currently, the DNase-seq method has been successfully applied to identify open chromatin sites in a variety of plants at the whole genome level (Zhang et al 2012,Plant Cell,24 (7): 2719-2731). The method mainly comprises the following steps: extracting and purifying cell nuclei, performing enzyme digestion on the purified cell nuclei by using DNase I with different concentrations, detecting enzyme digestion effects of DNase I with different concentrations by using pulse field electrophoresis or common agarose gel current, selecting a proper cell nucleus subjected to enzyme digestion, and extracting small-fragment or large-fragment DNA for library construction and sequencing. However, this method requires a large initial amount of cells and requires attempts at various cleavage concentrations.
Disclosure of Invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide a novel method for identifying plant open chromatin sites at the whole genome level; the method can identify whole genome open chromatin under plant physiological state; the method has been used at present to identify open chromatin sites at the whole genome level in normal growth of plants such as rice and wheat and under stress conditions.
The aim of the invention can be achieved by the following technical scheme:
in a first aspect, the present invention provides a method for identifying plant open chromatin sites at the whole genome level (abbreviated as in situ DH method), wherein a proper amount of deoxyriboendonuclease I (Deoxyribonuclease I, DNase I) is used for digesting cell nuclei, and simultaneously, DNA polymerase I (DNA polymerase I) is combined for timely repairing the cleaved in situ DNase-seq method.
Specifically, a single-stranded incision is generated on the double-stranded DNA of the coreless small body region by utilizing the non-specific deoxyriboendonuclease I, and simultaneously, under the action of the DNA polymerase I, the single-stranded incision is repaired in a base pairing mode by taking another complete DNA single strand as a template, so that the repaired double DNA contains biotin-marked dATP and dCTP nucleotides, and the DNA fragments marked by biotin are captured and enriched for library building and sequencing, and bioinformatics analysis is combined, so that the open chromatin locus is identified at the whole gene level.
The method of the invention provides a new technology and method for in-situ identification of plant whole genome open chromatin sites under physiological state and analysis of plant material regulatory elements. In addition, some key DH sites identified by the method are expected to be used for crop molecular breeding.
As a preferred embodiment of the present application, the method for identifying plant open chromatin sites at the whole genome level mainly comprises the following steps:
(1) Crosslinking and fixing plant materials;
(2) Extracting and purifying the nuclei of the plant material;
(3) Adding DNase I, DNA polymerase I and biotin-dATP and biotin-dCTP into cell nucleus, and performing enzyme digestion and in-situ marking of open chromatin sites;
(4) Digesting the RNA with RNAse A, and performing uncrosslinking at 65 ℃ overnight to recover a DNA fragment;
(5) Subjecting the recovered DNA fragment to a fragmentation treatment and detecting the fragmentation effect, for example, detecting the fragmentation effect by agarose gel electrophoresis;
(6) DNA fragments labeled with anti-streptavidins-beads conjugated with biotin (biotin);
(7) The beads, to which the biotin-labeled DNA fragment of interest was bound, were used for library construction and Illumina sequencing, and bioinformatic analysis was performed to identify open chromatin sites at the whole genome level.
As a preferred embodiment, the process of crosslinking the immobilized plant material in step (1) is: cutting fresh plant material (width about 0.2 cm), immersing in the cross-linking liquid, cross-linking at 4 deg.C for 10min under vacuum condition, adding 125mM glycine, vacuum treating at 4 deg.C for 5min, sterilizing, washing with sterilized water for 3 times, absorbing water on the surface of leaf with absorbent paper, quick freezing in liquid nitrogen, and storing at-80 deg.C.
Preferably, the formula of the crosslinking liquid is as follows: 400mM sucrose, 10mM Tris-HCl pH8.0,1mM EDTA (pH 8.0), 1% formaldehyde.
As a preferred technical solution, the process of extracting and purifying the nuclei of the plant material in step (2) is as follows: fully grinding plant materials into powder by liquid nitrogen, adding an equal volume of nuclear extraction buffer solution into the powder, fully stirring into homogenate, and then placing on ice; filtering, centrifuging, and discarding supernatant to obtain cell nucleus precipitate; adding a cell nucleus cleaning solution to resuspend cell nucleus precipitate, centrifuging again, discarding supernatant, repeating the cell nucleus cleaning process until the cell nucleus is white or faint yellow, re-suspending the purified cell nucleus by using an RSB buffer solution, centrifuging, and discarding the supernatant; finally, the pellet, i.e., purified nuclei, was retained and used directly in downstream experiments.
Preferably, the core extraction buffer (H1B) is formulated as follows: 20mM Tris-HCl,50mM EDTA,5mMSpermidine,0.15mMspermine,40%Glycerol,0.1% Mercap ethanol.
Preferably, the formula of the cell nucleus cleaning liquid (H1 BW) is as follows: 20mM Tris-HCl,50mM EDTA,5mMSpermidine,0.15mMspermine,40% glychol 0.1%Mercaptoethanol,0.5%Triton X-100.
Preferably, the RSB buffer is formulated as follows: 10mM Tris-HCl,10mMNaCl,3mM MgCl 2 。
As a preferred technical scheme, the process of enzyme digestion and in-situ labeling of open chromatin sites in the step (3) comprises the following steps: fully re-suspending the cell nuclei purified in the step (2) in 1x NEBuffer2, respectively adding dTTP, dGTP, dCTP, dATP and biotin (biotin) marked dCTP (orb 64049, biorbyt), adding the biotin marked dATP (orb 533181, biorbyt), adding DNA ploymerase I and DNase I, and reacting for 2 hours at 25 ℃, and gently mixing every half hour; finally, EDTA was added to terminate the reaction at a final concentration of 50 mM.
Preferably, the final concentration of dTTP, dGTP, dCTP, dATP and biotin-labeled dCTP and dATP is 0.05mM,0.025mM,0.035mM,0.025mM,0.015mM.
As a preferred embodiment, the method for recovering DNA fragments in the step (4) comprises the steps of: adding RNase A into the reaction solution, incubating for 0.5-1 h in a water bath at 37 ℃, then adding protease K and 20% SDS (w/v), and incubating overnight in a water bath at 65 ℃; the next day, the supernatant was retained after centrifugation with an equal volume of phenol-based replica (1:1), 1/10 of 3M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold absolute ethanol were added, and after mixing, the mixture was left at-20℃for 0.5 to 1.5 hours, and after centrifugation, the DNA precipitate was recovered, washed with 75% ethanol and dried, and then dissolved in 50. Mu. lEB buffer.
Preferably, the final concentration of SDS is 2%;
preferably, the EB buffer is formulated as follows: 10mM Tris-HCl, pH8.0.
As a preferable technical scheme, the fragmentation processing and the detection of the fragmentation effect in the step (5) are as follows: the DNA dissolved in the EB buffer was treated with a non-contact sonicator (parameters of the sonicator pre-chilled at 4℃were set to High energy, 20s was on, 40s was off for 1 cycle) for 3 to 15 cycles, and the fragmentation effect was detected by agarose gel electrophoresis.
If the fragmented DNA fragments are uniformly distributed in 100-250bp, extracting with an equal volume of phenol imitation (1:1), centrifuging, retaining supernatant, adding 1/10 volume of 3M sodium acetate (pH 5.2) and 2.5 times of ice-cold absolute ethyl alcohol, mixing uniformly, standing at-20 ℃ for 0.5-1.5 h, centrifuging, recovering DNA precipitate, washing and drying the recovered DNA precipitate with 75% alcohol, and dissolving with 20 mu lEB buffer solution.
As a preferred technical scheme, the process of recovering the biotin-labeled DNA fragment by using the anti-streptavidin-beads in the step (6) is as follows: fully re-suspending the anti-streptavidine-beads stored in a refrigerator at the temperature of 4 ℃ and subpackaging the beads into a new centrifuge tube; re-suspending the Beads with 1xTWB Buffer pre-cooled at 4 ℃, standing for 1min, recovering the Beads with a magnetic plate, and discarding the supernatant; repeating for 3 times, removing the supernatant for 1 time as much as possible, adding 1xTWB Buffer to resuspend the beads, uniformly mixing the DNA compound containing the biotin-label with the equivalent 2xBB Buffer, adding the mixture into the washed beads, and rotating for 0.5h at the temperature of 25-30 ℃ and the speed of 10rpm on a uniformly mixing instrument; the beads were collected by magnetic plate, the reaction mixture was discarded, and DNA fragments which could not bind to biotin were isolated.
Preferably, the formula of the 1xTWB Buffer is as follows: 0.5mM EDTA,5mM Tris-HCl, pH 7.5,1M NaCl,0.05% Tween 20.
The formula of the 2xBB buffer is as follows: 1mM EDTA,10mM Tris-HCl, pH 7.5,2M NaCl.
As a preferred technical scheme, the specific process of the step (7) is as follows: directly using the beads combined with the target DNA fragment in the step (6) to construct an Illumina sequencing library, and separating and purifying the 200-350bp DNA fragment to be used for 2×150 PE sequencing by using an IlluminaNovaSeq sequencing platform.
A further preferred detailed procedure of step (7) is: according to the specific instructions of the database construction kit, EB buffer solution with corresponding volume is added to resuspend the beads, and then the reagents used for database construction are directly added. After blunt end repair and linker ligation were completed according to kit instructions, beads were collected with a magnetic plate and the reaction solution was discarded. Suspending the beads with 1xTWB Buffer, standing for 1min, recovering the beads with a magnetic plate, and discarding the supernatant; repeating for 2 times, removing supernatant as much as possible for the last 1 time, re-suspending the beads with EB buffer solution, standing for 1min, recovering the beads with a magnetic plate, and removing supernatant as much as possible. Appropriate amount of EB buffer was added to resuspend the beads and reagents used for library amplification were added according to kit instructions. After the library amplification was completed, the beads were separated by magnetic plate, the supernatant was retained, and the beads were discarded. The supernatant was purified to give library DNA for sequencing, and 2×150 PE sequencing was performed on an IlluminaNovaSeq sequencing platform.
In a second aspect, the invention also provides the use of a method for identifying plant open chromatin loci at the whole genome level as described hereinbefore in crop molecular breeding
Preferably, the plant is rice, wheat, maize, arabidopsis, and the like.
Advantageous effects
Compared with the existing method, the method has the following advantages:
(1) The whole method has simpler flow. The method can be used for experiment and library establishment only in three days. Whereas the DNase-seq method reported requires running the gel to recover DNA, at least 4 days.
(2) The method of the invention requires a small initial amount of cells. The total amount of plant material required is only 1/5-1/7 of the DNase-seq reported.
Drawings
FIG. 1 is a flow chart of an in situ DNase-seq system established in the experiment;
FIG. 2 shows DNA subjected to cleavage and Biotin labeling; wherein CK is DNA which is not subjected to enzyme digestion and biotin labeling, and is treated into DNA subjected to enzyme digestion and biotin labeling;
FIG. 3 is a DNA fragment after ultrasonic treatment;
FIG. 4 is a schematic diagram of DNA fragments used to construct an Illumina sequencing library;
FIG. 5 is a graph showing the effect of visualization of open chromatin sites.
Fig. 2 to 5 are diagrams made under the condition of normal growth for rice Japanese leaf material.
Detailed Description
The invention is further illustrated, but not limited, by the following specific examples. The reagents or instrumentation used are not manufacturer specific and are considered to be commercially available conventional products.
The invention relates to a method for identifying plant open chromatin loci at the whole genome level, which mainly comprises the following steps: crosslinking and fixing plant materials, extracting and purifying cell nuclei, adding a proper amount of DNaseI, DNA polymelase I, biotin-dATP and biotin-dCTP, performing enzyme digestion and in-situ marking of open chromatin sites at the same time, extracting genome DNA marked by biotin, fragmenting the genome DNA into fragments of 100-250bp by utilizing ultrasonic waves, extracting fragmented genome DNA, combining the fragmented genome DNA with anti-streptavidins (65001,Life Technologies) and DNA fragments containing the biotin marks for library building and sequencing, and identifying open chromatin sites at the whole genome level by bioinformatics analysis.
Example 1
(1) Crosslinking and fixing plant materials:
selecting 2-5g of experimental material (wheat, corn, rice or arabidopsis leaves) to be tested, cutting the experimental material (width is about 0.2 cm) into pieces, immersing the pieces in a crosslinking liquid, crosslinking the pieces at the temperature of 4 ℃ for 10 minutes under vacuum, adding glycine with the final concentration of 125mM, continuing to carry out vacuum treatment at the temperature of 4 ℃ for 5 minutes, stopping crosslinking, washing the pieces with sterilized water for 3 times, fully absorbing water on the surfaces of the leaves by using water absorbing paper, quick-freezing the pieces in liquid nitrogen, and storing the pieces at the temperature of-80 ℃ for later use.
The formula of the crosslinking liquid is as follows: 400mM sucrose, 10mM Tris-HCl, pH8.0,1mM EDTA,1% formaldehyde.
(2) Extracting and purifying the nuclei of the plant material:
leaves stored in a-80℃refrigerator were thoroughly ground with liquid nitrogen into powder, and 2ml of the powder was used for extracting nuclei. An equal volume of nuclear extraction buffer (H1B) was added to the powder, stirred to homogenize, and the centrifuge tube was placed flat on ice at 100rpm and shaken for 6min.
The homogenate was filtered through 2 layers of microcloth gauze into 1 new 50ml centrifuge tubes and centrifuged at 3,000rpm at 4℃for 12min at an ascending and descending rate of 8. Discarding the supernatant to obtain a cell nucleus precipitate, adding 2ml H1B Washing Buffer (HIBW)), lightly resuspending the cell nucleus with the brush head, flushing the cell nucleus remained on the brush head with 3ml of HIBW, collecting the cell nucleus in the same 1 centrifuge tube, lightly reversing and mixing the cell nucleus with the same centrifuge tube for 3 to 5 times, centrifuging the cell nucleus for 12min at a speed of 8 rpm for 3,000rpm, discarding the supernatant, repeating the steps for 3 times until the cell nucleus is white or light yellow in color, discarding the supernatant as much as possible, and precipitating the cell nucleus into a purified cell nucleus.
Purified nuclei were resuspended in 5ml of RSB buffer, centrifuged at 8 at 3,000rpm for 12min at 4℃and the supernatant discarded and the pellet (nuclei) retained. The nuclei were resuspended in 400 μl of RSB buffer and transferred to 1 new 1.5ml centrifuge tubes; the supernatant was discarded and the pellet (nucleus) was retained for downstream experiments at 4℃and 3,000rpm for 8 centrifugation for 12min.
The formula of the nuclear extraction buffer (H1B) is as follows: 20mM Tris-HCl (pH=8.0), 50mM EDTA,5mMSpermidine,0.15mMspermine,40% (v/v) Glycerol,0.1% (v/v) Mercapetone.
The formula of the cell nucleus cleaning liquid (H1 BW) is as follows: 20mM Tris-HCl (pH=8.0), 50mM EDTA,5mMSpermidine,0.15mMspermine,40% (v/v) Glycerol,0.1% (v/v) Mercaptoethanol,0.5% (v/v) Triton X-100.
The formula of the RSB buffer solution is as follows: 10mM Tris-HCl (pH=7.4), 10mMNaCl,3mM MgCl 2 。
(3) Cleavage and in situ labelling of open chromatin sites:
the purified nuclei were fully resuspended in 162.8. Mu. l H 2 To O, 20. Mu.l of 10 XNEBuffer 2, 1. Mu.l of 10mM dTTP, 1. Mu.l of 10mM dGTP, 0.5. Mu.l of 10mM dCTP, 0.7. Mu.l of 1mM biotin-dCTP, 3. Mu.l of 1mM biotin-dATP, 1. Mu.l of DNA ploymerase I (10U/. Mu.l) and 5. Mu.l of DNase I (0.01U/. Mu.l) were added, and the mixture was reacted at 25℃for 2 hours, with gentle mixing every half hour. Finally, 20. Mu.l of 0.5M EDTA was added to terminate the reaction.
(4) Recovering the DNA fragment:
to the reaction solution, 5. Mu.l of RNase A (10. Mu.g/. Mu.l) was added, followed by incubation in a water bath at 37℃for 0.5 to 1 hour, and then 20. Mu.l of Proteinase K (10. Mu.g/. Mu.l) and 20. Mu.l of 20% SDS (w/v) were added, followed by incubation in a water bath at 65℃overnight; the next day was extracted with an equal volume of phenol imitation (1:1), after centrifugation at 12,000rpm at 4℃for 10min, the supernatant was retained in 1 new 1.5ml centrifuge tube, 1/10 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold absolute ethanol were added, after mixing well, after standing at-20℃for 0.5 to 1.5h, the DNA precipitate was recovered, washed 2 times with 75% alcohol, dried in air for 5min, and the DNA was dissolved with 50. Mu.l of EB (10 mM Tris-HCl, pH 8.0) buffer and the concentration was measured; the next reaction was immediately carried out or the DNA was kept in a refrigerator at-20℃until use.
(5) Fragmenting the recovered DNA fragment and detecting the fragmentation effect:
the DNA dissolved in the EB buffer was treated with a non-contact sonicator (parameters of the sonicator pre-chilled at 4 ℃ C. Were set to High energy, 20s was on, 40s was stopped at 1 cycle) for 3 to 15 cycles, and the fragmentation effect was detected by 1.5% agarose gel electrophoresis. Finally, the fragmented DNA fragments are evenly distributed in 100-250bp, an equal volume of phenol imitation (1:1) is added, after being vigorously mixed, the mixture is centrifuged for 10min at 12,000rpm and 4 ℃, the supernatant is reserved in 1 new 1.5ml centrifuge tube, 1/10 volume of 3M sodium acetate (pH 5.2) and 2.5 times volume of ice-cold absolute ethyl alcohol are added, after being uniformly mixed, the mixture is placed at-20 ℃ for 0.5-1.5 h, DNA sediment is recovered, the recovered DNA sediment is washed by 75% alcohol for 2 times, the mixture is dried in the air for 5min, and 20 mu l of EB buffer solution is used for dissolving the DNA and measuring the concentration; the next reaction was immediately carried out or the DNA was kept in a-20℃refrigerator for use.
(6) Recovery of the biotin-labeled DNA complexes with anti-streptavidine-beads:
fully re-suspending the anti-streptavidine-beads stored in a refrigerator at the temperature of 4 ℃ and subpackaging the beads into a new centrifuge tube; re-suspending the Beads with 1xTWB Buffer pre-cooled at 4 ℃, standing for 1min, recovering the Beads with a magnetic plate, and discarding the supernatant; repeating for 3 times, removing the supernatant for 1 time as much as possible, adding 1xTWB Buffer to resuspend the beads, uniformly mixing the DNA compound containing the biotin-label with the equivalent 2xBB Buffer, adding the mixture into the washed beads, and rotating for 0.5h at the temperature of 25-30 ℃ and the speed of 10rpm on a uniformly mixing instrument; the beads were collected by magnetic plate, the reaction mixture was discarded, and DNA fragments which could not bind to biotin were isolated.
The formula of the 1xTWB Buffer is as follows: 0.5mM EDTA,5mM Tris-HCl, pH 7.5,1M NaCl,0.05% Tween 20.
The formula of the 2xBB buffer is as follows: 1mM EDTA,10mM Tris-HCl, pH 7.5,2M NaCl.
(7) DNA labeled with Biotin was used to construct Illumina sequencing libraries and sequenced:
according to the specific instructions of the database construction kit, EB buffer solution with corresponding volume is added to resuspend the beads, and then the reagents used for database construction are directly added. After blunt end repair and linker ligation were completed according to kit instructions, beads were collected with a magnetic plate and the reaction solution was discarded. The beads were resuspended with 200. Mu.l of 1xTWB Buffer, and after 1min of standing, the beads were recovered with a magnetic plate and the supernatant was discarded; repeating for 2 times, removing supernatant as much as possible for the last 1 time, re-suspending the beads with 50 μl of EB buffer, standing for 1min, recovering the beads with a magnetic plate, and removing supernatant as much as possible. And adding a proper amount of EB buffer solution to resuspend the beads, adding reagents for library amplification according to the instruction of the kit, and the like. After the library amplification was completed, the beads were separated by magnetic plate, the supernatant was retained, and the beads were discarded. The supernatant is purified to obtain sequencing library DNA, and the sequencing library DNA is used for 2×150 PE sequencing by an IlluminaNovaSeq sequencing platform.
The experimental results show that:
(1) The flow of the in-situ DNase-seq system established in the experiment is shown in figure 1;
(2) The DNA subjected to enzyme digestion and Biotin labeling is shown in FIG. 2;
(3) The DNA fragments after ultrasonic treatment are uniformly distributed within the range of 100-250bp, as shown in FIG. 3.
(4) An appropriate amount of DNA was used to construct an Illumina sequencing library, and 200-350bp size DNA fragments were recovered and purified for sequencing, and the library construction assay is shown in FIG. 4.
(5) By bioinformatic analysis, open chromatin sites were identified, and the visual effect of which is shown in figure 5.
FIGS. 2-5 are graphs made under conditions of normal growth for rice Japanese leaf material for 14 days, and the applicant has also performed the method of the present invention for in situ identification of whole genome open chromatin sites in plant material such as wheat, maize or Arabidopsis under normal growth and stress conditions.
Noun interpretation of related art terms
Crosslinking fixing solution: a buffer solution for immobilizing proteins and DNA on chromatin.
Nuclear extraction buffer (HIB): buffer solution for extracting nuclei.
Nuclear cleaning solution (H1 BW): buffer solution for purification of nuclei.
DNA polymerase I: and DNA polymerase I, an enzyme which takes parent DNA as a template and catalyzes the polymerization of substrate dNTP molecules to form child DNA.
Biotin: biotin
DNaseI: deoxyribonuclease I, an endonuclease that digests single-stranded or double-stranded DNA to produce a single-stranded or double-stranded oligodeoxynucleotide.
RNaseA: ribonuclease for degrading RNA.
Proteinase K: proteinase K, which mainly degrades proteins.
TWB: buffer for washing beads.
BB: buffers for dilution of DNA.
Tris-HCl: tris (hydroxymethyl) aminomethane. .
EDTA: ethylenediamine tetraacetic acid.
Illumina sequencing library: a DNA library established based on a second generation DNA sequencing technique.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.

Claims (20)

1. A method for identifying plant open chromatin locus at whole genome level is characterized in that single-stranded nicks are generated on double-stranded DNA of a coreless body region by utilizing deoxyribonuclease I, meanwhile, under the action of DNA polymerase I, single-stranded nicks are repaired in a base pairing mode by taking another complete DNA single strand as a template, so that the repaired double-stranded DNA contains biotin-marked dATP and dCTP nucleotides, and the open chromatin locus is identified at whole genome level by capturing and enriching biotin-marked DNA fragments, constructing a library and sequencing and combining with bioinformatics analysis.
2. A novel method for identifying plant open chromatin loci at the whole genome level according to claim 1, comprising the steps of:
(1) Crosslinking and fixing plant materials;
(2) Extracting and purifying the nuclei of the plant material;
(3) Adding DNase I, DNA polymerase eI and biotin-dNTP into the cell nucleus, and performing enzyme digestion and in-situ marking of open chromatin sites;
(4) Digesting the RNA with RNAse A, uncrosslinking overnight, and recovering the DNA fragment;
(5) Fragmenting the recovered DNA fragments and detecting the fragmentation effect;
(6) DNA fragments marked by anti-streptavidins-beads binding biotin;
(7) Using the beads combined with the target DNA fragment for library construction and Illumina sequencing; open chromatin sites are identified at the whole genome level by bioinformatic analysis.
3. The method of claim 2, wherein the process of cross-linking the immobilized plant material in step (1) is: cutting plant materials, immersing the cut plant materials into crosslinking liquid, crosslinking under vacuum condition, adding glycine with the final concentration of 125mM, continuing vacuum treatment, stopping crosslinking, cleaning with sterilized water, absorbing water on the surface of the material, quick-freezing in liquid nitrogen, and storing at-80 ℃ for later use.
4. A method according to claim 3, wherein the cross-linking liquid is formulated as follows: 400mM sucrose, 10mM Tris-HCl pH8.0,1mM EDTA,1% formaldehyde.
5. The method of claim 2, wherein the process of extracting and purifying the nuclei of the plant material in step (2) is: fully grinding the plant material crosslinked in the step (1) into powder by liquid nitrogen, adding an equal volume of nuclear extraction buffer solution into the powder, fully stirring to be homogenate, and then placing on ice; filtering, centrifuging, discarding supernatant to obtain cell nucleus precipitate, adding cell nucleus cleaning solution to resuspend cell nucleus precipitate, centrifuging again, discarding supernatant, and repeating cell nucleus cleaning process until cell nucleus is white or yellowish; and re-suspending the purified cell nucleus by using RSB buffer solution, centrifuging, discarding the supernatant, and finally, reserving the precipitate to obtain the purified cell nucleus.
6. The method of claim 5, wherein the core extraction buffer is formulated as follows: 20mM Tris-HCl,50mM EDTA,5mMSpermidine,0.15mMspermine,40%Glycerol,0.1%Mercaptoethanol.
7. The method of claim 5 or 6, wherein the formulation of the nuclear wash is: 20mM Tris-HCl,50mM EDTA,5mMSpermidine,0.15mMspermine,40%Glycerol,0.1%Mercaptoethanol,0.5%Triton X-100.
8. The method of claim 5 or 6, wherein the RSB buffer is formulated as follows: 10mM Tris-HCl,10mMNaCl,3mM MgCl 2 。
9. The method of claim 2, wherein the step (3) of cleaving and in situ labeling the DNA comprises: fully re-suspending the cell nucleus purified in the step (2) in 1x NEBuffer2, respectively adding dTTP, dGTP, dCTP, dATP and biotin marked dCTP and dATP, adding DNA ploymerase I and DNase I, and reacting for 2 hours at 25 ℃; finally, EDTA was added to a final concentration of 50mM to terminate the reaction.
10. The method of claim 9, wherein the final concentration of said dTTP, dGTP, dCTP, dATP and biotin-labeled dCTP and dATP is 0.05mm,0.025mm,0.035mm,0.025mm,0.015mm, respectively.
11. The method according to claim 2, wherein the method for recovering the DNA fragments in step (4) is: adding RNase A into the reaction solution, incubating for 0.5-1 h in a water bath at 37 ℃, then adding protease K and 20% SDS (W/V), and incubating overnight in a water bath at 65 ℃; the next day, the supernatant was retained after centrifugation with an equal volume of phenol, 1/10 of 3M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold absolute ethanol were added, and after mixing, the mixture was left at-20℃for 0.5 to 1.5h, and after centrifugation to recover the DNA pellet, the recovered DNA pellet was washed with 75% alcohol, dried and then dissolved in EB buffer.
12. The method of claim 11, wherein the EB buffer is formulated as follows: 10mM Tris-HCl, pH8.0.
13. The method according to claim 2, wherein the fragmenting treatment and detection of the fragmentation effect in step (5) is: treating DNA dissolved in an EB buffer solution for 3-15 cycles by using a non-contact ultrasonic breaker, and detecting fragmentation effect by using agarose gel electrophoresis; the DNA fragments after fragmentation treatment are uniformly distributed in 100-250bp, the DNA fragments are subjected to phenol imitation extraction with equal volume, supernatant is reserved after centrifugation, 1/10 volume of 3M sodium acetate and 2.5 times volume of ice-cold absolute ethyl alcohol are added, after uniform mixing, the DNA fragments are placed at-20 ℃ for 0.5-1.5 h, DNA precipitation is recovered after centrifugation, recovered DNA precipitation is washed by 75% alcohol, and the DNA precipitation is dissolved by EB buffer solution after drying.
14. The method according to claim 2, wherein the recovery of the biotin-labeled DNA fragment using anti-strepavidin-beads in step (6) is carried out by: fully re-suspending the anti-streptavidine-beads stored in a refrigerator at the temperature of 4 ℃ and subpackaging the beads into a new centrifuge tube; re-suspending the Beads with 1xTWB Buffer pre-cooled at 4 ℃, standing for 1min, recovering the Beads with a magnetic plate, and discarding the supernatant; repeating for 3 times, removing the supernatant for 1 time as much as possible, adding 1xTWB Buffer to resuspend the beads, uniformly mixing the DNA compound containing the biotin-label with the equivalent 2xBB Buffer, adding the mixture into the washed beads, and rotating for 0.5h at the temperature of 25-30 ℃ and the speed of 10rpm on a uniformly mixing instrument; the beads were collected by magnetic plate, the reaction mixture was discarded, and DNA fragments which could not bind to biotin were isolated.
15. The method of claim 14, wherein the 1xTWB Buffer is formulated as follows: 0.5mM EDTA,5mM Tris-HCl, pH 7.5,1M NaCl,0.05%Tween 20.
16. The method of claim 14 or 15, wherein the 2xBB buffer is formulated as follows: 1mM EDTA,10mM Tris-HCl, pH 7.5,2M NaCl.
17. The method according to claim 2, wherein the specific process of step (7) is: directly using the beads combined with the target DNA fragment in the step (6) to construct an Illumina sequencing library, and separating and purifying the 200-350bp DNA fragment to be used for 2×150 PE sequencing by using an IlluminaNovaSeq sequencing platform.
18. The method of claim 17, wherein the detailed process of step (7) is: according to the specification of a specific library construction kit, adding EB buffer solution with corresponding volume to resuspend the beads in the step (6), and directly adding reagents used for library construction; after finishing flat end repair and joint connection according to the kit specification, collecting beads by using a magnetic plate, and discarding a reaction solution; suspending the beads with 1xTWB Buffer, standing for 1min, recovering the beads with a magnetic plate, and discarding the supernatant; repeating for 2 times, removing the supernatant as much as possible for the last 1 time, re-suspending the beads with EB buffer solution, standing for 1min, recovering the beads with a magnetic plate, and removing the supernatant as much as possible; adding a proper amount of EB buffer solution to resuspend the beads, and adding reagents for library amplification according to the instruction of the kit; after the amplification of the library is finished, separating the beads by using a magnetic plate, reserving supernatant, and discarding the beads; the supernatant was purified to obtain library DNA for sequencing, which was used for 2×150 PE sequencing on an IlluminaNovaSeq sequencing platform.
19. Use of a method according to any one of claims 1-18 for identifying plant open chromatin sites at the whole genome level in molecular breeding of crops.
20. The use according to claim 19, wherein the plant is one or more of rice, wheat, maize or arabidopsis.
CN202310061337.2A 2023-01-16 2023-01-16 Method for identifying plant open chromatin locus at whole genome level Pending CN116536406A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117431294A (en) * 2023-10-23 2024-01-23 华中农业大学 ChIP-seq chromatin co-immunoprecipitation method aiming at important economic organs of plants

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117431294A (en) * 2023-10-23 2024-01-23 华中农业大学 ChIP-seq chromatin co-immunoprecipitation method aiming at important economic organs of plants

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