A method for rapid separation and purification of plant cell nuclei

Abstract

The present invention provides a method for rapidly separating and purifying plant cell nuclei. The present invention can be used to rapidly separate, purify and enrich high-quality and high-purity plant cell nuclei. The obtained cell nuclei can be used for high-throughput sequencing such as single-cell nucleus RNA-seq, single-cell nucleus ATAC-seq and single-cell nucleus DNA methylation sequencing, which is of great significance for plant molecular biology and cell biology research.

Description

Technical Field

[0001] The invention relates to the technical field of plant molecular cell biology, and in particular to a method for rapidly separating and purifying plant cell nuclei. Background Art

[0002] Although research strategies such as proteomics, transcriptomics, and mutant or population screening play an important role in studying plant signal perception, transduction, and downstream gene response mechanisms, another important issue is that different types of cells in higher plants have cell-specific perception and response to signals. Therefore, for plant tissues with multiple cell types, this research strategy often ignores the problem of cell heterogeneity among different types of cells in response to hormones or environmental signals, thereby masking the signal perception, transduction, and downstream regulatory genes specific to each cell type. This is also an important limiting factor in the current identification of plant functional genes and in-depth analysis of plant signal transduction pathways. Therefore, how to present plant cell type-specific signal transduction and downstream response mechanisms in a high-resolution manner has become a high-tech topic in plant biology and crop science research. In-depth analysis of plant cell type-specific signal transduction and downstream response mechanisms can bring plant science research into a new era of high precision, thereby providing precise theoretical guidance for crop molecular breeding.

[0003] In recent years, single-cell high-throughput sequencing technology has been widely used in the fields of medicine, model plants and animals, and agricultural science, greatly enhancing human understanding of many life development processes and diseases. Single-cell high-throughput sequencing technology avoids the masking of cell type-specific regulatory information by cell heterogeneity, and brings medical science and life science research into an era of precise targeted analysis.

[0004] The application of single-cell RNA sequencing technology (scRNA-seq) in plants has improved people's understanding of cellular heterogeneity in plant species such as Arabidopsis, rice, maize, cotton, peanut and poplar (Zhang TQ, Xu ZG, Shang GD, Wang JW. A Single-Cell RNA Sequencing Profiles the Developmental Landscape ofArabidopsis Root. Mol Plant, 2019, 12(5):648-660; Zhang TQ, Chen Y, Liu Y,Lin WH, Wang JW. Single-cell transcriptome atlas and chromatin accessibilitylandscape reveal differentiation trajectories in the rice root. Nat Commun,2021, 12(1):2053; Ortiz-Ramírez C, Guillotin B, Xu X, Rahni R, Zhang S, YanZ, Coqueiro Dias Araujo P, Demesa-Arevalo E, Lee L, Van Eck J, Gingeras TR,Jackson D, Gallagher KL, Birnbaum KD. Ground tissue circuitry regulates organcomplexity in maize and Setaria. Science, 2021, 374(6572):1247-1252; Qin Y,Sun M, Li W, Xu M, Shao L, Liu Y, Zhao G, Liu Z, Xu Z, You J, Ye Z, Xu J,Yang X, Wang M, Lindsey K, Zhang X, Tu L. Single-cell RNA-seq reveals fatedetermination control of an individual fiber cell initiation in cotton(Gossypium hirsutum).Plant Biotechnol J, 2022, 20(12):2372-2388; Liu H, HuD, Du P, Wang L, Liang X, Li H, Lu Q, Li S, Liu H, Chen X, Varshney RK, HongY. Single-cell RNA-seq describes the transcriptome landscape and identifiescritical transcription factors in the leaf blade of the allotetraploid peanut(. Arachis hypogaeaL.). Plant Biotechnol J, 2021, 19(11):2261-2276; [ PubMed ] Li H, DaiX, Huang X, Xu M, Wang Q, Yan X, Sederoff RR, Li Q. Single-cell RNAsequencing reveals a high-resolution cell atlas of xylem in Populus. J IntegrPlant Biol, 2021, 63(11):1906-1921), in the presence of a chemical scientist, a chemical engineer and a scientist in the field(Zhang TQ, Xu ZG, [ PubMed ] Shang GD, Wang JW. A Single-Cell RNA SequencingProfiles the Developmental Landscape of Arabidopsis Root. Plant Mol, 2019, 12(5):648-660; [ PubMed ] Zhang TQ, Chen Y, Liu Y, Lin WH, Wang JW. Single-cell transcriptome atlas and chromatin accessibility landscape reveal differentiation trajectories in the rice root. Nat Commun, 2021, 12(1):2053;Ortiz-Ramirez C, Guillotin B, Xu X, Rahni R, Zhang S, Yan Z, Coqueiro DiasAraujo P, Demesa-Arevalo E, Lee L, Van Eck J, Gingeras TR, Jackson D,Gallagher KL, Birnbaum KD. Ground tissue circuitry regulates organ complexityin maize and Setaria.Science, 2021, 374(6572):1247-1252). The transcriptome heterogeneity of rice and Arabidopsis roots can be compared using scRNA-seq technology. The results show that there are two special cell populations in rice, sclerenchyma and exodermis, which do not exist in Arabidopsis. In rice fields, sclerenchyma and exodermis can prevent radial oxygen loss in rice roots (Zhang TQ, Chen Y, Liu Y, Lin WH, Wang JW. Single-cell transcriptomeatlas and chromatin accessibility landscape reveal differentiationtrajectories in the rice root. Nat Commun, 2021, 12(1):2053). The root differentiation of monocotyledons is different from that of dicotyledons. The lateral root caps and epidermis of monocotyledons do not have common initiator cells like the dicotyledonous model species Arabidopsis (Zhang L, He C, Lai Y, Wang Y, Kang L, Liu A, Lan C, SuH, Gao Y, Li Z, Yang F, Li Q, Mao H, Chen D, Chen W, Kaufmann K, Yan W. Asymmetric gene expression and cell-type-specific regulatory networks in the root of bread wheat revealed by single-cell multiomics analysis. Genome Biol, 2023, 24(1): 65), single-cell transcriptome sequencing (scRNA-seq) can reconstruct cell differentiation trajectories and provide new perspectives for organ development research. scRNA-seq of rice radicle revealed the differentiation trajectories of the outer epidermis, sclerenchyma and cortex. The results showed that these ground tissues originated from the same meristem cell population (Zhang TQ, Chen Y, Liu Y, Lin WH, Wang JW. Single-cell transcriptome atlas and chromatin accessibility landscape reveal differentiation trajectories in the rice root.Nat Commun, 2021, 12(1):2053), scRNA-seq studies can also deepen our understanding of gene function. Ortiz-Ramírez et al. demonstrated that the migration of the transcription factor SHORT-ROOT is essential for the division of the corn cortex (Ortiz-Ramírez C, Guillotin B, Xu X, Rahni R, Zhang S, Yan Z, Coqueiro Dias Araujo P, Demesa-Arevalo E, Lee L, Van Eck J, Gingeras TR, Jackson D, Gallagher KL, BirnbaumKD. Ground tissue circuitry regulates organ complexity in maize and Setaria. Science, 2021, 374(6572):1247-1252). .

[0005] The main bottleneck in applying single-cell transcriptome RNA sequencing technology to crop science research is the presence of cell wall structures in plant cells, which makes it difficult to isolate plant protoplasts. Prolonged separation time will not only lead to an increase in the dead cell ratio in the obtained plant cell protoplasts, but also increase the delayed effect of transcriptional regulation time in plant cell protoplasts, and cannot accurately characterize the immediate signal transduction and transcriptional response of plant cells to external stimuli in real time. Some studies have begun to apply single-cell nuclear sequencing technology to single-cell transcriptome sequencing analysis of Arabidopsis root tips (Long Y, Liu Z, JiaJ, Mo W, Fang L, Lu D, Liu B, Zhang H, Chen W, Zhai J. FlsnRNA-seq: protoplasting-free full-length single-nucleus RNA profiling in plants. GenomeBiol, 2021, 22(1):66). Single-cell nuclear transcriptome sequencing analysis has been applied in animal and disease research (Ding J, Adiconis X, Simmons SK, Kowalczyk MS, Hession CC, Marjanovic ND, Hughes TK, Wadsworth MH, Burks T, Nguyen LT, Kwon JYH, Barak B, Ge W, Kedaigle AJ, Carroll S, Li S, Hacohen N, Rozenblatt-Rosen O, Shalek AK, Villani AC, Regev A, Levin JZ. Systematic comparison of single-cell and single-nucleus RNA-sequencing methods. Nat Biotechnol, 2020, 38(6):737-746). This is because the nucleus is more stable than the protoplast, and the RNA in the nucleus can more accurately reflect the immediate transcriptional regulatory events inside the cell, avoiding the delayed effect of post-transcriptional regulation caused by RNA splicing and RNA transport (Shaw R, Tian X, Xu J. Single-Cell Transcriptome Analysis in Plants:Advances and Challenges.Mol Plant, 2021, 14(1):115-126). More importantly, the nucleus can exist relatively stably in the fixative solution and frozen environment. Therefore, in the study of crop development and environmental response, the processed samples can be quickly frozen to fix the transcriptional regulatory events in the nucleus at a specific time point, further avoiding the delayed effect of transcriptional regulation caused by the digestion of plant tissue materials by enzymatic hydrolysis. A study has conducted single-cell nuclear transcriptome sequencing (snRNA-seq) in wheat roots. This study explored the asymmetric gene expression patterns between subgenomes at single-cell resolution and constructed a cell-type-specific gene expression regulatory network (Zhang L, He C, Lai Y, Wang Y, KangL, Liu A, Lan C, Su H, Gao Y, Li Z, Yang F, Li Q, Mao H, Chen D, Chen W,Kaufmann K, Yan W. Asymmetric gene expression and cell-type-specificregulatory networks in the root of bread wheat revealed by single-cellmultiomics analysis. Genome Biol, 2023, 24(1):65), and more single-cell high-throughput sequencing technologies are mainly based on the nucleus, including single-cell nuclear transcriptome sequencing (snRNA-seq), single-cell chromatin accessibility sequencing (scATAC-seq), single-cell whole-genome methylation sequencing (scWGBS), single-cell ChIP-seq (scChIP-seq) and single-cell CUT&Tag sequencing (scCUT&Tag) (Zhang L, He C, Lai Y, Wang Y, KangL, Liu A, Lan C, Su H, Gao Y, Li Z, Yang F, Li Q, Mao H, Chen D, Chen W,Kaufmann K, Yan W. Asymmetric gene expression and cell-type-specificregulatory networks in the root of bread wheat revealed by single-cellmultiomics analysis.Genome Biol, 2023, 24(1):65; Farlik M, Sheffield NC,Nuzzo A, Datlinger P, Schönegger A, Klughammer J, Bock C. Single-cell DNAmethylome sequencing and bioinformatic inference of epigenomic cell-statedynamics. Cell Rep, 2015, 10(8):1386-97; Raimundo F, Prompsy P, Vert JP,Vallot C. A benchmark of computational pipelines for single-cell histonemodification data. Genome Biol, 2023, 24(1):143; Wu SJ, Furlan SN, MihalasAB, Kaya-Okur HS, Feroze AH, Emerson SN, Zheng Y, Carson K, Cimino PJ, KeeneCD, Sarthy JF, Gottardo R, Ahmad K, Henikoff S, Patel AP. Single-cell CUT&Taganalysis of chromatin modifications in differentiation and tumor progression.Nat Biotechnol, 2021, 39(7):819-824)。.

[0006] Typically, single-cell high-throughput sequencing based on the nucleus first requires the separation and purification of the nucleus and the maintenance of the nucleus in a free and dispersed state. Currently, the more commonly used single-cell purification method mainly relies on flow cytometry sorting (Zhang L, He C, Lai Y, Wang Y, Kang L, Liu A, Lan C, Su H, Gao Y, Li Z, Yang F, Li Q, Mao H, Chen D, Chen W, Kaufmann K, Yan W. Asymmetric gene expression and cell-type-specific regulatory networks in the root of breadwheat revealed by single-cell multiomics analysis. Genome Biol, 2023, 24(1):65), a good and efficient flow cytometer costs at least 3-4 million yuan, which is expensive. In addition, the flow cytometer has complicated operation steps. Usually, the flow sorting process of one or two single-cell sequencing samples takes more than one hour, including the instrument startup, calibration, machine testing, sorting, cleaning and shutdown. The operation process of the flow cytometer is complicated, the maintenance cost is high, and the instrument operation technology level is high. It usually requires professional personnel to be responsible and guided. This greatly limits the preparation process and number of single-cell nuclear sequencing samples. In particular, the state of the enriched nuclei after flow sorting is unstable, which is easy to cause nuclear rupture and aggregation. The sorted and enriched nuclei need to complete the single-cell machine work as soon as possible, otherwise the experiment will fail. The above factors have greatly and directly restricted the development of related single-cell sequencing work in many laboratories and companies.

[0007] In the previous research on single cell nucleus separation and purification, we found that by using special reagents, a large number of nuclei can be enriched at a relatively low centrifugal force, and after 2-3 washes, mitochondria and chloroplasts and other cytoplasmic components can be removed. However, the purity of the nuclei obtained by centrifugation is low and still contains some cell wall fragments. We found that after filtering and centrifugal enrichment through several cell sieves with different pore sizes, a large number of high-purity, free-dispersed nuclei can be quickly enriched. The nuclei are intact in morphology without rupture and aggregation. In other words, the nuclei obtained by the improved nucleus separation and enrichment method are very stable and can be preserved for a relatively long time. The advantages of this method are: (1 ) It abandons the excessive reliance on expensive flow cytometers. The operation steps are fast, simple and easy to understand. No professional personnel are required to operate and maintain the instrument, saving time and labor costs in the process of cell nucleus preparation; (2) The machine hour cost of the flow cytometer is 300-400 yuan / hour, saving the machine hour cost in the process of cell nucleus preparation; (3) The cell nuclei obtained by low centrifugal force enrichment are relatively complete, and the free cell nuclei are in a very stable state and can be preserved for more than two hours. They are very suitable for high-throughput sequencing research based on single cell nuclei. However, the cell nuclei obtained after flow sorting are prone to cell nucleus rupture and aggregation, and the subsequent machine operation needs to be completed as soon as possible. The time is relatively tight and it is not suitable for preparing multiple samples. Summary of the invention

[0008] The technical problem to be solved by the present invention is to establish a rapid and efficient method for isolating and purifying high-purity plant cell nuclei, abandoning the reliance on flow cytometry in the previous process of isolating high-purity plant cell nuclei, thereby reducing the time cost, labor cost and expensive instrument use cost of isolating and purifying plant cell nuclei, while improving the integrity and stability of the isolated and enriched cell nuclei, maintaining the free state of the cell nuclei for a long time, so as to facilitate high-throughput sequencing research based on single cell nuclei.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] The first aspect of the present invention provides a plant cell nucleus isolation reagent R buffer, wherein the R buffer reagent comprises: 0.4M mannitol, 20mM sodium citrate (pH=7.0), 20mM potassium chloride, 40mM magnesium chloride, 0.2% polyethylene glycol octylphenyl ether, 3mM dithiothreitol, and 1% bovine serum albumin.

[0011] A second aspect of the present invention provides a use of a plant cell nucleus isolation reagent R buffer in crude separation of plant cell nuclei.

[0012] A third aspect of the present invention provides a method for crudely isolating plant cell nuclei using a plant cell nucleus isolation reagent R buffer, comprising the following steps:

[0013] a. Add 400 μl of R buffer to a 60 mm sterile plate pre-cooled on ice, then soak 1 g of plant leaves in R buffer, and cut the young plant leaves into a homogenate state in R buffer using a double-sided blade;

[0014] b. Add 400 μl of R buffer to the sterile plate again, and gently shake the plate horizontally for 1 minute to fully lyse the cell membrane, release the cell nucleus, and obtain a homogenate suspension of young plant leaves after lysis;

[0015] c. The plant leaf homogenate suspension after lysis in step b was filtered through 35 μm and 20 μm cell sieves to remove the residue in the homogenate, and the filtrate was collected using a 1.5 ml centrifuge tube;

[0016] d. Add 400 μl of R buffer to the plate again, rinse the plate, and transfer the collected filtrate to 35 μm and 20 μm cell sieves for filtration and collection.

[0017] The fourth aspect of the present invention provides a plant cell nucleus purification reagent RDT buffer, the RDT buffer reagent comprising: 0.4M mannitol, 20mM sodium citrate (pH=7.0), 20mM potassium chloride, 40mM magnesium chloride, 3mM dithiothreitol, and 1% bovine serum albumin.

[0018] A fifth aspect of the present invention provides a method for purifying plant cell nuclei using a plant cell nucleus purification reagent RDT buffer, comprising the following steps:

[0019] (1) Crudely isolate plant cell nuclei and collect the isolated cell nucleus suspension;

[0020] (2) Filter and purify plant cell nuclei using RDT buffer and an 8 μm filter mesh;

[0021] (3) Wash and purify plant cell nuclei using RDT buffer;

[0022] Wherein, in step (2), the specific steps of filtering and purifying plant cell nuclei using RDT buffer and 8 μm filter mesh include:

[0023] e. Centrifuge the cell nuclear suspension collected in step (1) at 100 g, 4°C, 6 min, then gently and slowly discard the supernatant and retain 30-40 μl of the supernatant containing the cell nuclear precipitate at the bottom of the tube;

[0024] f. Add 700 μl of RDT buffer to the supernatant containing the cell nuclear pellet, resuspend the cell nuclear pellet, and then transfer the resuspended cell nuclear suspension to an 8 μm filter mesh for filtration and collection on ice;

[0025] g. Centrifuge the cell nucleus suspension collected after filtration in step f at 100 g, 4 ° C, 6 min, then gently and slowly discard the supernatant and retain 30-40 μl of the supernatant containing the cell nucleus precipitate at the bottom of the tube;

[0026] In step (3), the specific steps of washing and purifying the plant cell nuclei using RDT buffer include:

[0027] h. Add 700 μl of RDT buffer to the supernatant containing the nuclear pellet retained at the bottom of the tube in step g, resuspend the nuclear pellet, centrifuge the resuspended nuclear suspension again, the centrifugation conditions are 4°C, 100g, 6min, then gently and slowly discard the supernatant, and retain 30-40 μl of the supernatant containing the nuclear pellet at the bottom of the tube;

[0028] i. Repeat step h once: add 700 μl of RDT buffer to the supernatant containing the cell nucleus pellet retained at the bottom of the tube in step h, resuspend the cell nucleus pellet, centrifuge the resuspended cell nucleus suspension again, the centrifugation conditions are 4°C, 100g, 6min, then gently and slowly discard the supernatant, and retain 30-40 μl of supernatant at the bottom of the tube;

[0029] j. Use the supernatant remaining at the bottom of the tube in step i to resuspend the cell nuclear pellet, add 2 μl of 2 mg / ml 4',6-diamidino-2-phenylindole stain solution, stain on ice in the dark for 5 minutes, and examine under a fluorescence microscope.

[0030] Typically, the centrifugal force used for centrifugal enrichment of cell nuclei is between 500-1000 g. In a specific embodiment, D buffer is used as a cell nucleus separation reagent, and a differential centrifugation method is used to precipitate and remove larger tissue fragments and impurities, in order to obtain higher purity cell nuclei in the subsequent centrifugal enrichment process. According to the results of centrifugal enrichment, it is found that most of the cell nuclei and tissue fragments can be precipitated from the cell nucleus suspension after resuspending the precipitate obtained by 100 g low-speed centrifugation. This is significantly different from the higher centrifugal force used in previous cell nucleus centrifugation enrichment. Therefore, the subsequent exploration will continue to explore how to enrich cell nuclei with higher purity under low-speed centrifugation conditions of about 100 g.

[0031] In view of the presence of a large amount of tissue fragments in the cell nucleus suspension obtained by centrifugal enrichment with D buffer, in another specific embodiment of the present invention, after using D buffer to separate rice cell nuclei, a cell sieve with a smaller pore size of 20 μm is used to filter and separate the obtained cell nuclei in order to remove more tissue fragments. The results showed that there were still a large amount of tissue fragments in the cell nucleus suspension obtained by low-speed centrifugation enrichment, and there was a certain degree of cell nucleus aggregation. For this reason, the formula of the cell nucleus separation buffer was subsequently replaced in order to reduce the cell nucleus aggregation phenomenon.

[0032] Buffer C is another buffer for nuclear separation used in Arabidopsis. In a specific embodiment, buffer C was used to enrich rice nuclei under a centrifugal force of 100 g. The results showed that the nuclear aggregation phenomenon caused by buffer C was significantly weaker than that caused by buffer D, but there were still some tissue fragments remaining.

[0033] In order to further reduce the phenomenon of cell nuclei aggregation, the present invention innovates a new cell nucleus separation reagent based on D buffer and C buffer, which is named R buffer. In a specific embodiment, R buffer is used to enrich rice cell nuclei under a centrifugal force of 100g. The results show that the cell nuclei separated by R buffer have no obvious aggregation phenomenon, and most of the cell nuclei are in a free mononuclear state. However, there are still some cell wall fragments remaining. It is necessary to further optimize the specific operation method of separating cell nuclei with R buffer to further remove cell wall fragments and impurities.

[0034] Since under the centrifugal force of 100g, the cell nuclei enriched by the various cell nucleus separation buffers mentioned above all contain certain tissue or cell wall fragments, and the cell nuclei are relatively small in diameter, therefore, in a specific embodiment, a cell sieve with a smaller pore size of 8μm is used to filter and remove larger tissue or cell wall fragments. The results show that the cell nuclei obtained by separation using R buffer can be filtered through an 8μm cell sieve to obtain very pure and free cell nuclei, and no cell nucleus aggregation is observed.

[0035] In view of the fact that the above-mentioned cell nucleus separation buffer contains a detergent polyethylene glycol octyl phenyl ether, polyethylene glycol octyl phenyl ether is a mild surfactant, which can rupture the cell membrane to release the cell nucleus during the cell nucleus separation process, and can separate the cell nucleus from the endoplasmic reticulum and other structures in the cytoplasm. Although polyethylene glycol octyl phenyl ether has a weak destructive effect on the integrity of the cell nucleus and nuclear membrane, the cell nucleus is stored in a buffer containing polyethylene glycol octyl phenyl ether for a long time, and there is also a risk of causing damage to the cell nucleus and nuclear membrane. In a specific embodiment, the polyethylene glycol octyl phenyl ether in the R buffer used in the early stage is removed, and the buffer used for the later cell nucleus purification and the polyethylene glycol octyl phenyl ether is removed is named RDT buffer, so as to further ensure the integrity of the cell nucleus and nuclear membrane in the later purification process, so that the cell nucleus can maintain a stable free state for a relatively long time, avoiding cell nucleus rupture and aggregation.

[0036] The beneficial effects of the present invention are as follows: the reagents used in the reagent formula suitable for separation and purification of plant cell nuclei provided by the present invention are conventional laboratory reagents, which can better maintain the integrity and free state of plant cell nuclei. The reagents can also be stored at low temperatures for a long time and do not need to be prepared on demand. The cell nucleus separation and purification method provided by the present invention does not need to rely on expensive instruments and equipment, and is fast and safe to operate. It only requires a low-temperature centrifuge, several cell sieves with different pore sizes and several commonly used consumables. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 For the initial separation of enriched nuclei using buffer D;

[0038] Figure 2 For cell nuclei isolated using buffer D;

[0039] Figure 3 For cell nuclei isolated using buffer C;

[0040] Figure 4 For cell nuclei isolated using R buffer;

[0041] Figure 5 The cell nuclei were purified after separation using R buffer and filtration through an 8 μm cell sieve;

[0042] Figure 6 These are cell nuclei that have been stable in RDT buffer for more than 2 hours after purification.

[0043] illustrate: Figure 1 to Figure 6 In the middle, the left picture shows the cell nuclear morphology observed under bright field conditions of fluorescence microscopy, and the right picture shows the cell nuclear morphology observed under ultraviolet excitation conditions of fluorescence microscopy. DETAILED DESCRIPTION

[0044] In order to facilitate the understanding of the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments.

[0045] Example 1 Isolation of rice leaflet nuclei using D buffer

[0046] (1) Add 400 μl of D buffer (Table 1) to a 60 mm sterile plate pre-cooled on ice, then soak 1 g of rice leaves in D buffer and cut the rice leaves into a homogenate in D buffer using a double-sided blade;

[0047] (2) Add 400 μl of D buffer to the sterile plate again, and gently shake the plate horizontally for 1 min to fully lyse the cell membrane, release the cell nucleus, and obtain a homogenate suspension of young plant leaves after lysis;

[0048] (3) Transfer the lysed rice leaf homogenate suspension to a 35 μm cell sieve for filtration to remove the residue in the homogenate, and collect the filtrate in a 1.5 ml centrifuge tube;

[0049] (4) Add 400 μl of D buffer to the plate again, rinse the plate, and transfer the collected filtrate to a 35 μm cell sieve for filtration and collection;

[0050] (5) Centrifuge the cell nucleus suspension collected in steps (3) and (4) at 100 g and 4 °C for 6 min, then transfer most of the supernatant to another new centrifuge tube, leaving 30-40 μl of supernatant at the bottom of the tube;

[0051] (6) Transfer the supernatant from step (5) to another new centrifuge tube and centrifuge again at 4°C, 500 g, 6 min, then discard the supernatant and retain 30-40 μl of supernatant at the bottom of the tube;

[0052] (7) Label the precipitates obtained from the two centrifugations as 100 g precipitate and 500 g precipitate, respectively, and use the supernatant remaining at the bottom of the tube to resuspend the precipitates;

[0053] (8) Add 2 μl of 2 mg / ml 4',6-diamidino-2-phenylindole dye solution and stain on ice for 5 min in the dark. Examine under a fluorescence microscope.

[0054] Result analysis: Fluorescence microscopy results showed that a centrifugal force of 100 g could precipitate most of the cell nuclei and tissue fragments from the cell nucleus suspension ( Figure 1 ), while the pellet obtained at 500g centrifugal force mainly contains mitochondria and chloroplasts as well as smaller tissue fragments, and cell nuclei are almost undetectable.

[0055] Table 1 D buffer reagent formula

[0056]

[0057] Example 2 Purification of cell nucleus suspension by filtering with a 20 μm pore size cell sieve

[0058] In this embodiment, while using D buffer to separate rice cell nuclei, a cell sieve with a smaller pore size of 20 μm is used to filter the separated cell nuclei, and the cell nuclei obtained by centrifugal enrichment are washed multiple times with D buffer to remove more tissue fragments. The specific implementation steps are as follows:

[0059] (1) Add 400 μl of D buffer to a 60 mm sterile plate pre-cooled on ice, then soak 1 g of rice leaves in D buffer and cut the rice leaves into a homogenate in D buffer using a double-sided blade;

[0060] (2) Add 400 μl of D buffer to the sterile plate again, and gently shake the plate horizontally for 1 min to fully lyse the cell membrane, release the cell nucleus, and obtain a homogenate suspension of young plant leaves after lysis;

[0061] (3) Filter the lysed rice leaf homogenate suspension through 35 μm and 20 μm cell sieves in turn to remove the residue in the homogenate, and collect the filtrate using a 1.5 ml centrifuge tube;

[0062] (4) Add 400 μl of D buffer to the plate again, rinse the plate, and transfer the collected filtrate to 35 μm and 20 μm cell sieves for filtration and collection;

[0063] (5) Centrifuge the collected cell nucleus suspension at 100g and 4°C for 6 min, then use a pipette to gently and slowly discard the supernatant, leaving 30-40 μl of supernatant at the bottom of the tube;

[0064] (6) Resuspend the pellet with 700 μl of buffer D and centrifuge at 4°C, 100 g for 6 min. Then use a pipette to gently and slowly discard the supernatant, leaving 30-40 μl of supernatant at the bottom of the tube;

[0065] (7) Repeat step (6) twice;

[0066] (8) Use the supernatant remaining at the bottom of the tube to resuspend the pellet, add 2 μl of 2 mg / ml 4',6-diamidino-2-phenylindole dye solution, and stain on ice for 5 min in the dark. Examine under a fluorescence microscope.

[0067] Result analysis: Fluorescence microscopy results showed that although a large number of cell nuclei could be enriched by 100g low-speed centrifugation, there were still a lot of tissue fragments in the cell nucleus suspension, and there was a certain degree of cell nucleus aggregation ( Figure 2 ).

[0068] Example 3 Isolation of rice leaflet nuclei using buffer C

[0069] In this example, rice nuclei were centrifuged at a low centrifugal force of 100 g using C buffer. During the filtration and purification of the nucleus suspension, 35 μm and 20 μm cell sieves were used in series for filtration and multiple washing and purification steps. The specific implementation steps are as follows:

[0070] (1) Add 400 μl of C buffer (Table 2) to a 60 mm sterile plate pre-cooled on ice, then soak 1 g of rice leaves in C buffer and cut the rice leaves into a homogenate in C buffer using a double-sided blade;

[0071] (2) Add 400 μl of C buffer to the sterile plate again, and gently shake the plate horizontally for 1 min to fully lyse the cell membrane, release the cell nucleus, and obtain a homogenate suspension of young plant leaves after lysis;

[0072] (3) Filter the lysed rice leaf homogenate suspension through 35 μm and 20 μm cell sieves in turn to remove the residue in the homogenate, and collect the filtrate using a 1.5 ml centrifuge tube;

[0073] (4) Add 400 μl of C buffer to the plate again, rinse the plate, and transfer the collected filtrate to 35 μm and 20 μm cell sieves for filtration and collection;

[0074] (5) Centrifuge the collected cell nucleus suspension at 100g and 4°C for 6 min, then use a pipette to gently and slowly discard the supernatant, leaving 30-40 μl of supernatant at the bottom of the tube;

[0075] (6) Resuspend the pellet in 700 μl of buffer C and centrifuge at 4°C, 100 g for 6 min. Then use a pipette to gently and slowly discard the supernatant, leaving 30-40 μl of supernatant at the bottom of the tube;

[0076] (7) Repeat step (6) twice;

[0077] (8) Use the supernatant remaining at the bottom of the tube to resuspend the pellet. Add 2 μl of 2 mg / ml 4',6-diamidino-2-phenylindole dye solution, stain on ice in the dark for 5 min, and examine under a fluorescence microscope.

[0078] Result analysis: Fluorescence microscopy results showed that the nuclear aggregation caused by buffer C was significantly weaker than that caused by buffer D, but there were still some tissue fragments remaining ( Figure 3 ).

[0079] Table 2 C buffer reagent formula

[0080]

[0081] Example 4 Isolation of rice leaflet nuclei using R buffer

[0082] (1) Add 400 μl of R buffer to a 60 mm sterile plate pre-cooled on ice, then soak 1 g of rice leaves in R buffer and cut the rice leaves into a homogenate in R buffer using a double-sided blade;

[0083] (2) Add 400 μl of R buffer to the sterile plate again, and gently shake the plate horizontally for 1 min to fully lyse the cell membrane, release the cell nucleus, and obtain a homogenate suspension of young plant leaves after lysis;

[0084] (3) Filter the lysed rice leaf homogenate suspension through 35 μm and 20 μm cell sieves in turn to remove the residue in the homogenate, and collect the filtrate using a 1.5 ml centrifuge tube;

[0085] (4) Add 400 μl of R buffer to the plate again, rinse the plate, and transfer the collected filtrate to 35 μm and 20 μm cell sieves for filtration and collection;

[0086] (5) Centrifuge all the cell nuclear suspensions collected after filtration in steps (3) and (4) at 4°C, 100 g, for 6 min, then gently and slowly discard the supernatant using a pipette, leaving 30-40 μl of the supernatant containing the cell nuclear pellet at the bottom of the tube;

[0087] (6) Add 700 μl of R buffer to the 30-40 μl supernatant containing the cell nucleus pellet at the bottom of the tube in step (5), resuspend the pellet, and centrifuge at 4°C, 100g for 6 min. Then use a pipette to gently and slowly discard the supernatant, leaving 30-40 μl of supernatant at the bottom of the tube;

[0088] (7) Repeat step (6) twice;

[0089] (8) Use the supernatant remaining at the bottom of the tube to resuspend the pellet. Add 2 μl of 2 mg / ml 4',6-diamidino-2-phenylindole dye solution, stain on ice in the dark for 5 min, and examine under a fluorescence microscope.

[0090] Result analysis: Fluorescence microscopy results showed that the cell nuclei separated by R buffer were free mononuclear, and some cell wall fragments remained ( Figure 4 ).

[0091] Example 5 Removal of tissue fragments and impurities

[0092] In this embodiment, based on the use of R buffer to separate the cell nuclei, a smaller pore size of 8 μm centrifugal filter is further used to filter the cell nucleus suspension in order to completely remove larger tissue or cell wall fragments. The specific implementation steps are as follows:

[0093] (1) Add 400 μl of R buffer to a 60 mm sterile plate pre-cooled on ice, then soak 1 g of rice leaves in R buffer and cut the rice leaves into a homogenate in R buffer using a double-sided blade;

[0094] (2) Add 400 μl of R buffer to the sterile plate again, and gently shake the plate horizontally for 1 min to fully lyse the cell membrane, release the cell nucleus, and obtain a homogenate suspension of young plant leaves after lysis;

[0095] (3) Filter the lysed rice leaf homogenate suspension through 35 μm and 20 μm cell sieves in turn to remove the residue in the homogenate, and collect the filtrate using a 1.5 ml centrifuge tube;

[0096] (4) Add 400 μl of R buffer to the plate again, rinse the plate, and transfer the collected filtrate to 35 μm and 20 μm cell sieves for filtration and collection;

[0097] (5) Centrifuge all the cell nucleus suspensions collected after filtration in step (3) and step (4) at 4°C, 100 g, for 6 min, then gently and slowly discard the supernatant and retain 30-40 μl of the supernatant containing the cell nucleus pellet at the bottom of the tube;

[0098] (6) Add 700 μl of R buffer to the supernatant containing the cell nucleus precipitate retained at the bottom of the tube in step (5), resuspend the precipitate, and then transfer the resuspended cell nucleus suspension to an 8 μm filter screen for filtration on ice, and collect the filtrate;

[0099] (7) Centrifuge the collected filtrate at 4°C, 100 g, for 6 min, then gently and slowly discard the supernatant and retain 30-40 μl of the supernatant containing the cell nuclear pellet at the bottom of the tube;

[0100] (8) Add 700 μl of R buffer to the supernatant containing the cell nucleus precipitate at the bottom of the tube in step (7), resuspend the precipitate, and centrifuge at 4°C, 100 g for 6 min. Then, gently and slowly discard the supernatant, leaving 30-40 μl of supernatant at the bottom of the tube;

[0101] (9) Repeat step (8) once: add 700 μl of RDT buffer to the supernatant containing the cell nucleus precipitate at the bottom of the tube in step (8), resuspend the precipitate, and centrifuge at 4°C, 100 g for 6 min. Then, gently and slowly discard the supernatant, leaving 30-40 μl of supernatant at the bottom of the tube;

[0102] (10) Use the supernatant remaining at the bottom of the tube in step (9) to resuspend the precipitate. Add 2 μl of 2 mg / ml 4',6-diamidino-2-phenylindole dye solution, stain on ice in the dark for 5 min, and examine under a fluorescence microscope.

[0103] Result analysis: The results of fluorescence microscopy showed that the cell nuclei separated by R buffer were filtered through an 8μm cell sieve to obtain very pure and free cell nuclei, and no obvious cell nuclei aggregation was observed ( Figure 5 ).

[0104] Example 6 Isolation and purification of rice leaflet nuclei using R buffer and RDT buffer

[0105] (1) Add 400 μl of R buffer to a 60 mm sterile plate pre-cooled on ice, then soak 1 g of rice leaves in R buffer and cut the rice leaves into a homogenate in R buffer using a double-sided blade;

[0106] (2) Add 400 μl of R buffer to the sterile plate again, and gently shake the plate horizontally for 1 min to fully lyse the cell membrane, release the cell nucleus, and obtain a homogenate suspension of young plant leaves after lysis;

[0107] (3) Filter the lysed rice leaf homogenate suspension through 35 μm and 20 μm cell sieves in turn to remove the residue in the homogenate, and collect the filtrate using a 1.5 ml centrifuge tube;

[0108] (4) Add 400 μl of R buffer to the plate again, rinse the plate, and transfer the collected filtrate to 35 μm and 20 μm cell sieves for filtration and collection;

[0109] (5) Centrifuge the cell nucleus suspension collected after filtration in step (3) and step (4) at 100g, 4°C, for 6 min. Then, gently and slowly discard the supernatant, leaving 30-40 μl of the supernatant containing the cell nucleus pellet at the bottom of the tube;

[0110] (6) Add 700 μl of RDT buffer to the supernatant containing the cell nuclear pellet retained at the bottom of the tube in step (5), resuspend the cell nuclear pellet, and then transfer the resuspended cell nuclear suspension to an 8 μm filter screen for filtration and collection on ice;

[0111] (7) Centrifuge the cell nucleus suspension collected after filtration in step (6) at 100g, 4°C, 6 min. Then gently and slowly discard the supernatant, leaving 30-40 μl of supernatant at the bottom of the tube;

[0112] (8) Add 700 μl of RDT buffer to the supernatant containing the cell nuclear pellet at the bottom of the tube in step (7), resuspend the cell nuclear pellet, and centrifuge the resuspended cell nuclear suspension again at 4°C, 100g, 6 min. Then, gently and slowly discard the supernatant, leaving 30-40 μl of supernatant at the bottom of the tube;

[0113] (9) Repeat step (8) once: add 700 μl of RDT buffer to the supernatant containing the cell nuclear pellet at the bottom of the tube in step (8), resuspend the cell nuclear pellet, and centrifuge the resuspended cell nuclear suspension again at 4°C, 100g, 6 min. Then, gently and slowly discard the supernatant, leaving 30-40 μl of supernatant at the bottom of the tube;

[0114] (10) Use the supernatant remaining at the bottom of the tube in step (9) to resuspend the pellet, add 2 μl of 2 mg / ml 4',6-diamidino-2-phenylindole dye solution, stain on ice in the dark for 5 min, and examine under a fluorescence microscope.

[0115] Result analysis: Fluorescence microscopy results showed that the purified cell nuclei could remain in the free single cell nucleus state in RDT buffer for more than 2 hours, and no obvious cell nucleus aggregation was observed after 2 hours ( Figure 6 ).

[0116] The present invention uses specific embodiments to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the scope of the specific implementation methods. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A plant cell nucleus isolation reagent R buffer, characterized in that The R buffer reagent is: 0.4M mannitol, 20mM sodium citrate with pH=7.0, 20mM potassium chloride, 40mM magnesium chloride, 0.2% polyethylene glycol octylphenyl ether, 3mM dithiothreitol, and 1% bovine serum albumin.

2. Use of the reagent as claimed in claim 1 in crude separation of plant cell nuclei.

3. A method for crudely isolating plant cell nuclei using the reagent according to claim 1, characterized in that: The following steps are involved: a. Add 400 μl of R buffer to a 60 mm sterile plate pre-cooled on ice, then soak 1 g of plant leaves in R buffer, and cut the young plant leaves into a homogenate state in R buffer using a double-sided blade; b. Add 400 μl of R buffer to the sterile plate again, and gently shake the plate horizontally for 1 minute to fully lyse the cell membrane, release the cell nucleus, and obtain a homogenate suspension of young plant leaves after lysis; c. The plant leaf homogenate suspension after lysis in step b was filtered through 35 μm and 20 μm cell sieves to remove the residue in the homogenate, and the filtrate was collected using a 1.5 ml centrifuge tube; d. Add 400 μl of R buffer to the plate again, rinse the plate, and transfer the collected filtrate to 35 μm and 20 μm cell sieves for filtration and collection.

4. A method for purifying plant cell nuclei, characterized in that: The following steps are involved: (1) crudely isolating plant cell nuclei according to the method of claim 3, and collecting the isolated cell nuclei suspension; (2) Filter and purify plant cell nuclei using RDT buffer and an 8 μm filter mesh; The RDT buffer reagent is: 0.4M mannitol, 20mM sodium citrate with pH=7.0, 20mM potassium chloride, 40mM magnesium chloride, 3mM dithiothreitol, and 1% bovine serum albumin; (3) Wash and purify plant cell nuclei using RDT buffer; Wherein, in step (2), the specific steps of filtering and purifying plant cell nuclei using RDT buffer and 8 μm filter mesh include: e. Centrifuge the cell nuclear suspension collected in step (1) at 100 g, 4°C, 6 min, then gently and slowly discard the supernatant and retain 30-40 μl of the supernatant containing the cell nuclear precipitate at the bottom of the tube; f. Add 700 μl of RDT buffer to the supernatant containing the cell nuclear pellet at the bottom of the tube, resuspend the cell nuclear pellet, and then transfer the resuspended cell nuclear suspension to an 8 μm filter mesh for filtration and collection on ice; g. Centrifuge the cell nucleus suspension collected after filtration in step f at 100 g, 4 ° C, 6 min, then gently and slowly discard the supernatant and retain 30-40 μl of the supernatant containing the cell nucleus precipitate at the bottom of the tube; In step (3), the specific steps of washing and purifying the plant cell nuclei using RDT buffer include: h. Add 700 μl of RDT buffer to the supernatant containing the nuclear pellet retained at the bottom of the tube in step g, resuspend the nuclear pellet, centrifuge the resuspended nuclear suspension again, the centrifugation conditions are 4°C, 100g, 6min, then gently and slowly discard the supernatant, and retain 30-40 μl of the supernatant containing the nuclear pellet at the bottom of the tube; i. Repeat step h once: add 700 μl of RDT buffer to the supernatant containing the nuclear pellet retained at the bottom of the tube in step h again, resuspend the nuclear pellet, centrifuge the resuspended nuclear suspension again, the centrifugation conditions are 4°C, 100g, 6min, then gently and slowly discard the supernatant, and retain 30-40 μl of supernatant at the bottom of the tube; j. Use the supernatant remaining at the bottom of the tube in step i to resuspend the cell nuclear pellet, add 2 μl of 2 mg / ml 4',6-diamidino-2-phenylindole stain solution, stain on ice in the dark for 5 minutes, and examine under a fluorescence microscope.

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