Method for separating and purifying cell nucleuses from cucurbitaceae plant tissues

Abstract

The invention relates to a method for separating and purifying cell nucleuses from tissues of cucurbitaceae plants, which comprises the following steps of: optimizing a system by limiting the density gradient of conventional stem tissues and calluses at grafting parts of the cucurbitaceae plants; the technical bottleneck that high-purity and high-integrity cell nucleuses can be efficiently and stably obtained from conventional stem tissues of cucurbitaceae plants and special grafting part calluses is overcome, so that the strict requirements of downstream high-throughput molecular biology experiments such as single-cell multi-omics analysis on sample quality are met, dependence on an expensive flow cytometry sorter is avoided, and the method is suitable for industrial production. And the experiment cost and the technical threshold are reduced, so that related researches can be developed in wider laboratories.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant molecular biology and cell biology, and particularly relates to a method for isolating and purifying cell nuclei from Cucurbitaceae plant tissues. BACKGROUND

[0002] The nucleus is the main carrier of genetic information and the core site of gene expression regulation in eukaryotic cells. The integrity of its structure and function is crucial for life science research. Obtaining high-quality nucleus samples is a prerequisite and key step for downstream molecular biology analysis such as genomics, transcriptomics, epigenetics, and other molecular biology analysis (e.g. single-cell sequencing, ChIP-seq, ATAC-seq, etc.). Compared with animal cells, the preparation process of intact cells (protoplasts) is relatively complex due to the presence of tough cell walls in plant cells, and the enzymatic process may induce the expression of stress-related genes, interfering with the authenticity of experimental results. Therefore, directly isolating cell nuclei from plant tissues has become a more direct and widely used strategy.

[0003] However, the extraction of plant cell nuclei faces a series of challenges. First, during the mechanical disruption of tissues, the rupture of organelles (especially vacuoles) releases a large amount of hydrolytic enzymes (such as proteases, nucleases), phenolic compounds, pigments, and secondary metabolites, which easily lead to nucleic acid degradation, protein denaturation, damage to the structure of the nuclear membrane, or the formation of sticky precipitates that interfere with subsequent isolation and purification. Second, the size and shape of cell fragments formed after tissue disruption vary, increasing the difficulty of effectively isolating and purifying cell nuclei through conventional filtration and centrifugation techniques.

[0004] Currently, flow cytometry sorting technology (FACS) can be used for the purification of cell nuclei, but it is expensive, complex to operate, and has certain requirements for sample size. The high flow rate and pressure of the sheath fluid during the sorting process can cause damage to the membrane integrity of plant cell nuclei (especially some fragile types), while reducing the flow rate will prolong the processing time and increase the risk of RNA degradation.

[0005] Cucurbitaceae crops, such as cucumbers, pumpkins, watermelons, etc., play an important role in global agricultural production. Grafting is a commonly used technique in the production of Cucurbitaceae crops, which can effectively improve the resistance, yield, and quality of crops. The cell physiological state of the grafting site (the healing zone of the rootstock and scion) is complex, involving processes such as the coexistence of different genotypes, cell fusion, material exchange, signal communication, and the formation and differentiation of callus. Callus cells usually have characteristics such as rapid division, active metabolism, and possibly increased cell nucleus volume. In addition, stem tissues of Cucurbitaceae plants, especially grafted callus, often contain sticky polysaccharides, phenolic substances, lignin, and calcium oxalate crystals, which can easily form interfering substances during cell nucleus extraction, making it particularly difficult to obtain high-purity and high-integrity cell nuclei.

[0006] Therefore, it is of great practical significance and application value to develop a method for separating and purifying nuclei, which is simple to operate, low in cost, efficient and fast, does not depend on flow cytometry sorting, and can effectively overcome the inherent extraction difficulties of Cucurbitaceae plant tissues (especially conventional stem tissues and grafted callus tissues), for promoting the functional genomics of Cucurbitaceae plants, the analysis of grafted biology mechanisms, and related breeding improvement research. SUMMARY

[0007] To solve the above problems, the present application provides a method for separating and purifying nuclei of Cucurbitaceae plant tissues. This method optimizes the key steps in the extraction process, aiming to solve the technical bottleneck of efficiently and stably obtaining high-purity and high-integrity nuclei from Cucurbitaceae plant tissues, including conventional stem tissues and special grafted callus tissues, to meet the stringent requirements of downstream high-throughput molecular biology experiments, such as single-cell multi-omics analysis, for sample quality.

[0008] The method for separating and purifying nuclei of Cucurbitaceae plant tissues provided by the present application comprises the following steps:

[0009] S1, taking Cucurbitaceae plant tissues, and washing them thoroughly with pre-cooled nuclear extraction buffer;

[0010] S2, placing the thoroughly washed plant tissues in a pre-cooled grinding container, and cutting the tissues until fine and uniform tissue homogenate is formed;

[0011] S3, filtering the tissue homogenate obtained in S2 through two cell sieves with decreasing pore sizes in sequence, and collecting all the filtrate passing through the cell sieves;

[0012] S4, density gradient centrifugation:

[0013] S4-1, sample preparation: mixing the filtrate collected in S3 with a 50% iodixanol solution in a volume ratio of 1:1 to form a sample;

[0014] S4-2, gradient construction: constructing a three-layer discontinuous iodixanol density gradient in a pre-cooled 2 mL centrifuge tube, wherein:

[0015] For Cucurbitaceae stem tissues: first, take 600 μL of the sample into the centrifuge tube as the sample layer, then add 1000 μL of a 30% iodixanol solution as the middle layer, and then add 600 μL of a 40% iodixanol solution as the lower layer;

[0016] For the callus of the gourd family plant grafting site: first take 600 μL sample into a centrifuge tube as a sample layer, then add 1000 μL of 30% iodixanol solution as a middle layer, and then add 600 μL of 45% iodixanol solution as a lower layer;

[0017] S4-3, centrifugation: centrifuge the centrifuge tube with the density gradient at 4℃, 10000xg for 30min;

[0018] S4-5, targeted collection: after centrifugation, the milky white cloud-shaped strip at the interface or immediately adjacent to the interface of the middle layer and the lower layer of iodixanol solution is sucked, which is the nucleus enrichment liquid, and the volume is 100-150 μL;

[0019] S4-6, washing and resuspension: the nucleus enrichment liquid collected in S4-5 is washed, centrifuged and resuspended to obtain the preliminary purified nucleus suspension.

[0020] Further, the basic solution of the nucleus extraction buffer is from the CelLytic PN separation / extraction kit, and 1.0% Triton X-100, reducing agent, protease inhibitor and RNase inhibitor are additionally added.

[0021] Further, in S2, the well-washed plant tissue is placed in a pre-cooled grinding container, 1-3 mL of pre-cooled nucleus extraction buffer is added per gram of tissue, and the tissue is quickly and thoroughly cut using a sharp cutting tool for 8-12 minutes in an ice bath or 0-4℃ environment until a fine and uniform tissue homogenate is formed.

[0022] Further, in S3, the tissue homogenate obtained in S2 is filtered through two cell screens with decreasing pore sizes in turn, first through a first cell screen with a pore size of 30-50 μm to remove large cell clumps and unbroken tissue fragments, and then through a second cell screen with a pore size of 10-30 μm to further remove small cell fragments and part of the organelles, and the filtrate passing through the cell screens is collected.

[0023] Further, the S4-5 targeted collection step is: after centrifugation, the milky white cloud-shaped strip at the interface or immediately adjacent to the interface of the middle layer and the lower layer of iodixanol solution is sucked with a wide-bore pipette, which is the nucleus enrichment liquid, and the volume is 100-150 μL.

[0024] Further, the S4-6 washing and resuspension step is: transferring the S4-5 collected nucleus enrichment liquid to a new pre-cooled centrifuge tube, adding 3-5 times the volume of pre-cooled nucleus extraction buffer, mixing, and then centrifuging at low speed at 4°C for 5 minutes, discarding the supernatant, and resuspending the nucleus precipitate with the nucleus extraction buffer to obtain a preliminary purified nucleus suspension.

[0025] Further, the filter before S3 can be pre-wetted with a small amount of nucleus extraction buffer.

[0026] Further, the composition of the nucleus extraction buffer is: 1x base solution, 0.1 mM dithiothreitol, 0.4 U / μL RNase inhibitor, 1x cocktail protease inhibitor mixture, and 1.0% Triton X-100 with a final concentration.

[0027] The beneficial effects of the present application are as follows:

[0028] 1. The present application provides an optimized nucleus preparation technology that is not dependent on flow cytometry sorting technology and is suitable for various tissue types of Cucurbitaceae plants, especially for stem tissue (conventional stem tissue, i.e., stem tissue that has not been grafted) and grafted callus tissue with special physiological state, which completely avoids the dependence on expensive flow cytometry sorting instruments, reduces experimental costs and technical barriers, and enables more extensive laboratories to conduct related research.

[0029] 2. The method of the present application is optimized for the characteristics of conventional stem tissue and complex grafted callus tissue of Cucurbitaceae plants, with special attention to the difficulties of extracting grafted callus tissue nucleus of Cucurbitaceae. By adjusting the density gradient parameters, the recovery rate and quality of such special material nucleus are significantly improved, and the nucleus can be efficiently separated from conventional stem tissue and grafted callus tissue. The overall operation process is designed compactly and can be completed in a short time (e.g., total time controlled within 30-40 minutes), which minimizes the degradation of RNA and DNA in the nucleus by endogenous nucleases, and is beneficial for obtaining high-quality nucleic acid state for subsequent analysis.

[0030] 3. The method of the present application can effectively remove cell wall fragments, cytoplasmic components, chloroplasts, mitochondria, and other impurity particles through fine mechanical disruption, multi-stage filtration, and optimized multi-layer density gradient centrifugation (especially the application of iodixanol medium), to obtain a nucleus population with complete morphology, intact membrane structure, and high purity. The final nucleus suspension can be flexibly adjusted in composition according to different downstream applications (such as snRNA-seq, snATAC-seq, ChIP-seq, flow cytometry ploidy analysis, etc.), and has good experimental compatibility. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a flow chart of the method of the present application;

[0032] Figure 2 is a quality assessment chart of cucumber stem tissue cell nuclei extracted using the method of the present application;

[0033] Figure 3 is a display chart of part of the library quality assessment indicators after single cell nucleus RNA sequencing of the cucumber stem tissue cell nuclei extracted using the method of the present application;

[0034] Figure 4 is a comparison chart of the forward scatter light (FSC) and DAPI fluorescence signal intensity distribution of the callus tissue of the cucumber-pumpkin grafting site and the non-grafting cucumber stem tissue cell nuclei extracted using the method of the present application, detected by flow cytometry;

[0035] Figure 5 is a schematic diagram of the stratification state of the callus tissue cell nuclei of the cucumber-pumpkin grafting site extracted using the method of the present application in the centrifuge tube of Example 2, the left chart is the state before centrifugation after adding each layer of solution, and the right chart is the state of the cell nuclei enriched at the specific interface after centrifugation;

[0036] Figure 6 is a schematic diagram of the distribution state of the cell nuclei in the centrifuge tube or the purity and integrity under a microscope after the cell nuclei of cucumber stem tissue extracted using the Percoll density gradient centrifugation method, sucrose density gradient centrifugation method and the iodixanol density gradient centrifugation method described in the present application in Comparative Examples 1 and 2. DETAILED DESCRIPTION

[0037] The present application will be further described below in conjunction with examples.

[0038] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and unless otherwise specified, the reagents used in the present method are of analytical purity or above, and the percentages used, unless otherwise specified, generally refer to volume percentage (v / v).

[0039] Example 1, Isolation and Purification of Cucurbitaceae (Cucumber) Conventional Stem Tissue Cell Nuclei and Optimization of Key Parameters:

[0040] The present example aims to establish and optimize a method for isolating and purifying high-quality cell nuclei from conventional stem tissue of Cucurbitaceae representative plant cucumber, and to screen key operating parameters.

[0041] 1.1, Optimization of Triton X-100 Concentration in Cell Nucleus Extraction Buffer:

[0042] 1.1.1, Prepare the nuclear extraction buffer with the following composition: 1x NIB (base solution from CelLytic PN isolation / extraction kit), 0.1 mM dithiothreitol (DTT) as reducing agent, 0.4 U / μL RNase inhibitor, 1x Cocktail protease inhibitor mixture (Roche), and add 0.01%, 0.1%, 0.5%, 1.0% and 2.0% (v / v) Triton X-100 respectively to prepare a series of nuclear extraction buffers, all of which are pre-cooled on ice. The nuclear extraction buffer used in the following examples is the same as 1.1.1.

[0043] 1.1.2, Take the stem segments (about 1 g / sample) of cucumber seedlings grown for 7 days, rinse them with pre-cooled sterile deionized water, and dry the surface water, then place them in pre-cooled sterile glass culture dishes.

[0044] 1.1.3, Add 3 mL of pre-cooled nuclear extraction buffer corresponding to the concentration to each culture dish.

[0045] 1.1.4, Use a new sterile disposable scalpel blade (or double-sided blade) to quickly and carefully cut the tissue for about 10 minutes on an ice bath until a uniform and delicate tissue homogenate is formed.

[0046] 1.1.5, Pass the tissue homogenate through a 40-μm pore size cell strainer and a 20-μm pore size cell strainer (both purchased from pluriSelect company) pre-wetted with a small amount of the corresponding buffer in sequence, and collect the filtrate in a pre-cooled 15-mL centrifuge tube.

[0047] 1.1.6, Take a small amount of the filtrate, add DAPI staining solution (final concentration 10 μg / mL) dropwise, and incubate in the dark for 5 minutes, then observe under a fluorescence microscope and count the number of nuclei, and record the number of nuclei per unit volume. At the same time, observe the color of the suspension with the naked eye to evaluate the removal effect of chloroplasts (the depth of green represents the amount of chloroplasts remaining), and the results are shown in Table 1.

[0048] Table 1 Optimization screening experiment of Triton X-100 concentration in nuclear extraction buffer

[0049]

[0050] Comprehensive comparison of nuclear yield, purity (chloroplast removal effect) and nuclear morphological integrity, while considering the potential risk of damage to the nuclear membrane by high concentration of Triton X-100, therefore, the final concentration of 1.0% (v / v) Triton X-100 in the nuclear extraction buffer is selected as the optimal concentration for the extraction of cucumber stem tissue nuclei.

[0051] 1.2. Optimization of iodixanol density gradient centrifugation parameters:

[0052] The process of extracting plant cell nuclei using iodixanol density gradient centrifugation system is as follows: Figure 1 and Figure 2 As shown, however, the concentration setting of the iodixanol density gradient centrifugation system varies for different plant varieties and different positions of the same plant variety.

[0053] 1.2.1. Optimization of iodixanol concentration in the middle and lower layers:

[0054] 1.2.1.1. Prepare nuclear extraction buffer according to the optimal Triton X-100 concentration (1.0%) determined in 1.1. Treat cucumber stem tissue according to steps 1.1.2-1.1.5 above to obtain the filtrate.

[0055] 1.2.1.2. Gently mix the filtrate with 50% (v / v) iodixanol solution (prepared by diluting 60% (w / v) OptiPrep with CB) at a 1:1 volume ratio. This is the sample layer (final iodixanol concentration 25%).

[0056] 1.2.1.3. Construct a three-layer discontinuous iodixanol density gradient in a pre-chilled 2 mL centrifuge tube. The top layer is a 600 μL sample layer. Test different iodixanol concentration combinations in the middle and bottom layers, with the volumes of the middle and bottom layers being 1000 μL and 200 μL, respectively.

[0057] (a) Test series A (optimized middle layer): The lower layer was fixed at 40% (v / v) iodixanol, and the middle layer concentrations were set at 25%, 30%, 35%, and 40% (v / v).

[0058] (b) Test series B (optimized lower layer): the middle layer was fixed at 30% (v / v) iodixanol, and the concentrations of the lower layer were set at 30%, 35%, 40%, 45%, and 50% (v / v).

[0059] 1.2.1.4. Centrifuge the constructed gradient tube at 4°C, 10,000×g for 30 minutes.

[0060] 1.2.1.5. After centrifugation, use a wide-mouthed pipette to aspirate the milky white, cloudy strip located at or adjacent to the interface between the middle and lower iodixanol solutions. This is the nucleus enrichment solution, and the aspirated volume is 100-150 μL.

[0061] 1.2.1.6, Resuspend and wash the nuclei once (4°C, 800 x g, 5 min) with 400 μL of nuclei extraction buffer (1 x NIB, 0.1 mM DTT, 0.4 U / μL RNase inhibitor, 1 x cocktail protease inhibitor mixture).

[0062] 1.2.1.7, Count the nuclei after DAPI staining and observe the purity, and the results are shown in Table 2.

[0063] Table 2 Comparison of iodixanol density gradient optimization for cucumber stem tissue

[0064]

[0065] Based on the data in Table 2 and the results of microscopic examination of the nuclei suspension, it is considered that the higher the concentration of the lower iodixanol system, the stronger the retention effect on cell debris and other impurities. Therefore, in order to ensure a high yield and optimal purity, for cucumber stem tissue, a gradient combination of sample layer (25% iodixanol) / 30% iodixanol (middle layer) / 40% iodixanol (lower layer) can obtain a higher yield and purity of nuclei.

[0066] 1.2.2, Optimization of density gradient centrifugation time:

[0067] 1.2.2.1, Prepare the sample and construct the density gradient using the parameters optimized in 1.1 and 1.2.1 (Triton X-100 concentration 1.0%, iodixanol gradient sample layer 25% / middle layer 30% / lower layer 40%).

[0068] 1.2.2.2, Centrifuge at 4°C, 10000 x g for 5 min, 15 min, 30 min, and 45 min, respectively.

[0069] 1.2.2.3, Collect the target layer nuclei, process and count according to the steps of 1.2.1.6-1.2.1.7.

[0070] Table 3 Comparison of gradient centrifugation time optimization for cucumber stem tissue

[0071]

[0072] As can be seen from Table 3, the optimal recovery effect of nuclei for cucumber stem tissue gradient centrifugation time is 30 minutes.

[0073] 1.3, Comprehensive evaluation of the quality of cucumber stem tissue nuclei extracted by the method of the present application:

[0074] Cucumber stem nuclei were extracted using the above-optimized conditions (Triton X-100 1.0%, iodixanol gradient: sample layer 25% / middle layer 30% / lower layer 40%, 10000xg centrifugation for 30 minutes).

[0075] 1.3.1, Morphological observation: A small amount of the final nuclear suspension was taken, dyed with DAPI, and observed under a fluorescence microscope.

[0076] 1.3.2, Flow cytometry analysis:

[0077] (a) Purity detection: The nuclear suspension was dyed with DAPI and detected by flow cytometry (Beckman Coulter CytoFLEX). The appropriate gating strategy was set, and the particle proportion of DAPI positive signal was analyzed.

[0078] (b) Ploidy analysis and endopolyploidy: DAPI fluorescence intensity histogram was analyzed.

[0079] Figure 2 is a quality evaluation diagram of cucumber stem tissue nuclei extracted by the method of the present application, wherein, Figure 2 A is the signal distribution histogram of the nuclei before staining in the DAPI channel, and B is the signal distribution histogram of the nuclei after staining in the DAPI channel, from Figure 2 It can be seen that the cucumber stem tissue nuclei extracted by the method of the present application exhibit a typical endopolyploidy pattern, with clear nuclei peaks of different DNA contents, indicating that the extracted nuclei maintain the DNA content characteristics in their physiological state. From Figure 2 AB comparison of the present application, the nuclei extracted by the method maintain their physiological state of different DNA contents due to different cell cycles in the plant body; Figure 2 C is the scatter plot of the nuclei before staining in the side scatter light channel and the DAPI channel, and D is the scatter plot of the nuclei after staining in the side scatter light channel and the DAPI channel, from Figure 2 CD comparison of the present application, the negative and positive populations are clearly separated at a signal value of 10 4 , and the positive cell population is distributed densely, indicating that the nuclei extracted by the method have high integrity; Figure 2 AC and BD comparison of the present application, the purity of DAPI positive nuclei of cucumber stem tissue nuclei extracted by the method of the present application can reach more than 90%.

[0080] Figure 2 E is a morphological observation diagram of the nuclear suspension after DAPI staining under a fluorescence field, wherein the left arrow points to the nuclei and the right arrow points to the tissue debris; F is a morphological observation diagram of the nuclear suspension after DAPI staining under a bright field; from Figure 2As shown in E and F, the cucumber stem tissue nucleus suspension extracted by the method of the present invention has a low content of debris and has no biological significance and downstream research value; Figure 2 G is a morphological observation diagram of the complete cell nucleus under the bright field (left) and fluorescence (right) fields of view. As can be seen from G, the cell nuclei of the cucumber stem tissue extracted by the method of the present invention have regular morphology, clear edges, uniform DAPI staining, clean background, and very little cell debris and organelle contamination, indicating that the cell nuclei of the cucumber stem tissue extracted by the method of the present invention are of good quality.

[0081] 1.4. Cucumber stem tissue single-nucleus RNA sequencing (snRNA-seq) library construction and quality assessment:

[0082] 1.4.1. Extract cucumber stem nuclei strictly according to the optimized conditions described in 1.3. Perform the entire procedure in an RNase-free environment, and add RNase inhibitor (final concentration 0.4 U / μL) to all solutions. To further reduce environmental RNA contamination, increase the number of washes to 2-3 in the final nuclei wash step.

[0083] 1.4.2. Use a cell counter to accurately count the cell nuclei concentration and dilute the cell nuclei suspension to the target concentration (e.g., 250-400 cells / μL) according to the requirements of the downstream single-cell library construction platform (e.g., 10xGenomics or GEXSCOPE).

[0084] 1.4.3. Follow the standard operating instructions of the selected single-cell platform for loading the cell nucleus suspension, single-cell nucleus capture, reverse transcription, cDNA amplification, and sequencing library construction.

[0085] 1.4.4. The constructed library is analyzed for fragment size distribution using an Agilent Bioanalyzer or similar instrument, and accurately quantified using Qubit. High-throughput sequencing is then performed on an Illumina sequencing platform (NovaSeq 6000).

[0086] 1.4.5. Sequencing data were aligned, quantified (Cellranger), quality controlled (SoupX), and subjected to preliminary cluster analysis (Seurat) using standard bioinformatics pipelines.

[0087] The results are as follows Figure 2 As shown, Figure 3 This is a diagram showing some of the library quality assessment indicators after single-cell nuclear RNA sequencing (snRNA-seq) of cucumber stem tissue nuclei extracted using the method of the present invention, among which: Figure 3A is the density distribution plot of the number of genes detected in each nucleus (nFeature_RNA), showing the median and distribution of the number of genes detected in each nucleus (nFeature_RNA), Figure 3 A in FIG. 1 shows that the median gene number reaches 1000, and most of the nuclei (such as >80%) detect more than 500 genes; Figure 3 B in FIG. 1 is a cell tag UMI ordering plot, and the UMI number (nCount_RNA) is ordered by the number of nuclei (knee plot), which presents a clear knee point at 2400 barcodes, indicating that high-quality nuclei are effectively distinguished from background noise (empty droplets or low-quality nuclei); Figure 3 C in FIG. 1 is a cell subpopulation distribution plot obtained after dimensionality reduction clustering based on gene expression profiles, which shows that the cucumber stem tissue nuclei extracted by the method of the present application can be clustered into at least 7 biologically meaningful cell subpopulations after UMAP or t-SNE dimensionality reduction based on gene expression profiles, reflecting that the extracted cell nucleus population retains the original cell heterogeneity and transcriptome characteristics of the tissue.

[0088] Example 2, Isolation and purification of gourd (cucumber-pumpkin) grafting site callus nuclei:

[0089] This example applies the method established in Example 1 to the more challenging gourd grafting site callus and optimizes it according to its characteristics.

[0090] 2.1, Comparison and analysis of cucumber-pumpkin grafted callus and non-grafted stem tissue nuclei size:

[0091] 2.1.1, Material preparation: Take cucumber as scion and pumpkin as stock for grafting. After grafting, carefully peel off the callus formed at the grafting interface after 14 days of culture. At the same time, take the non-grafted stem tissue of the same batch of cucumber plants as a control.

[0092] 2.1.2, Nuclei extraction: Refer to the optimized conditions of Example 1.3 to extract nuclei from equal amounts (about 1 g) of cucumber-pumpkin grafted callus and non-grafted cucumber stem tissue. For grafted callus, you can initially try using the same iodixanol gradient as stem tissue (sample layer 25% / middle layer 30% / lower layer 40%).

[0093] 2.1.3, Flow cytometry analysis: After DAPI staining, the two groups of nuclei suspensions were detected by flow cytometry. Focus on the forward scatter light (FSC) signal, which is related to the size and morphological complexity of the nuclei.

[0094] 2.1.4, Data analysis: Use FlowJo and other software to compare the distribution of FSC signals of the two groups of samples.

[0095] Results as shown in Figure 2: The median value and range of FSC signal of the cucumber-pumpkin grafted callus sample were slightly larger than that of the cucumber stem tissue sample, indicating that the average volume of the grafted callus nuclei might be larger, which is consistent with the expectation of its highly active cell division and differentiation state. Figure 3

[0096] 2.2, Optimization of density gradient (concentration of lower iodixanol layer) for grafted callus nuclei isolation:

[0097] Given that the grafted callus nuclei might be larger in volume and different in density, to ensure their effective enrichment and separation from impurities, this part focuses on optimizing the concentration of lower iodixanol layer in the density gradient.

[0098] 2.2.1, Take cucumber-pumpkin grafted callus (about 1 g / sample) and process it according to the steps in Example 1.1.2-1.1.5 to obtain the filtrate (use 1.0% Triton X-100 concentration).

[0099] 2.2.2, Mix the filtrate with 50% (v / v) iodixanol solution at a volume ratio of 1:1 to obtain the sample layer (final iodixanol concentration 25%).

[0100] 2.2.3, Construct a three-layer iodixanol density gradient: the upper layer is 600 μL of the sample layer, the middle layer is fixed at 1000 μL of 30% (v / v) iodixanol solution, and the lower layer (200 μL) is set at 30%, 35%, 40%, 45%, 50%, and 55% (v / v) iodixanol concentrations.

[0101] 2.2.4, Centrifuge at 4°C and 10000 x g for 30 minutes.

[0102] 2.2.5, Carefully pipette the nuclei enrichment band at the interface between the 30% iodixanol layer and the lower iodixanol layer, process it according to the steps in 1.2.1.6-1.2.1.7, and count it.

[0103] Table 4 Comparison of density gradient optimization for grafted callus nuclei isolation

[0104]

[0105] From Table 4, for cucumber-pumpkin grafted callus, to minimize the retention effect of the lower gradient on tissue debris, when the concentration of the lower iodixanol layer is increased to 45% (v / v), the recovery rate and purity of the nuclei reach a good balance. Therefore, the recommended iodixanol gradient is: sample layer 25% / middle layer 30% / lower layer 45%, as shown in Figure 2. Figure 4 ​​

[0106] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0107] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0108] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0109] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0110] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0111] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0112] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0113] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0114] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0115] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0116] 2.3.2, Place the tissue in a pre-chilled glass petri dish and add 3 mL of pre-chilled nuclei extraction solution (containing 1.0% Triton X-100).

[0117] Example 3, Extraction of cucumber stem tissue nuclei using Percoll density gradient centrifugation:

[0118] 3.1, The treatment of cucumber stem tissue (pre-treatment, mechanical disruption, filtration) is the same as the steps of Example 1.1.2-1.1.5, and the concentration of Triton X-100 in the nuclei extraction buffer is 1.0%.

[0119] 3.2, Prepare 10%, 20%, 30% (v / v) Percoll dilutions using the basic nuclear extraction buffer, and place them in three 2 mL centrifuge tubes, respectively. Add 200 μL of 2.3 M sucrose solution to the bottom of each centrifuge tube to complete the construction of the three gradient solutions.

[0120] 3.3, Place the filtrate directly on the pre-formed Percoll gradient to form three layers.

[0121] 3.4, Centrifuge at 1500 x g, 4°C for 15 minutes.

[0122] 3.5, Collect 100 μL of the cell nucleus layer between the sucrose and Percoll solutions.

[0123] 3.6, Add 400 μL of the basic nuclear extraction buffer, mix gently, and centrifuge at 800 x g, 4°C for 5 minutes. Discard 450 μL of the supernatant.

[0124] 3.7, Count the obtained cell nuclei, examine the purity and integrity by DAPI staining under a microscope, and compare the results with those of Example 1. The results are shown in Table 3 and Table 5. Figure 5

[0125] Example 4, Extraction of cell nuclei from cucumber stem tissue using sucrose density gradient centrifugation:

[0126] 4.1, The treatment of cucumber stem tissue is the same as the procedures of Example 1.1.2-1.1.5, and the concentration of Triton X-100 in the nuclear extraction buffer is 1.0%.

[0127] 4.2, Construct the sucrose density gradient: sequentially place 200 μL of high-concentration sucrose layer (2.0 M) and 1 mL of low-concentration sucrose layer (1.3 M) in the centrifuge tube, and carefully place the filtrate on the gradient.

[0128] 4.3, Centrifuge at 10000 x g, 4°C for 30 minutes.

[0129] 4.4, Collect the cell nucleus layer at the interface between the 2.0 M and 1.3 M sucrose concentrations.

[0130] 4.5, Add 400 μL of the basic nuclear extraction buffer, mix gently, and centrifuge at 800 x g, 4°C for 5 minutes. Discard 450 μL of the supernatant.

[0131] 4.6, Count the obtained cell nuclei, examine the purity and integrity by DAPI staining under a microscope, and compare the results with those of Example 1. The results are shown in Table 3 and Table 5. Figure 6

[0132] ​​The purity, integrity, and operation complexity of the nuclei extracted in Example 3 and Example 4 were compared with those of Example 1, and the results are shown in Table 5. Figure 6 and Table 5:

[0133] Purity and integrity evaluation: under a microscope, as shown in D of Figure 6 , under bright field and fluorescence conditions, the sucrose gradient separation effect was limited, and high-concentration sucrose caused the nuclei to shrink; as shown in A / B / C of Figure 6 , under the same imaging parameters of exposure and gain, the Percoll group had a more obvious blue fluorescence background in the DAPI fluorescence field, and under bright field and fluorescence conditions, it could be seen that the Percoll density gradient centrifugation method for extracting nuclei had more tissue fragments or other organelle contamination, and the nuclei aggregation phenomenon, indicating that the Percoll density gradient centrifugation method for extracting nuclei had a higher proportion of damaged nuclear membranes; compared with the iodixanol density gradient method shown in E and F of Figure 6 Figure 6 , the yield of nuclei by the Percoll method was improved, but the effect was poor in removing impurities unique to Cucurbitaceae stem tissues (such as sticky substances and calcium oxalate crystals), and it was difficult to separate the precipitate formed during the operation.

[0134] In terms of operation complexity, the viscosity of the sucrose solution in Example 4 was high, and the gradient preparation operation was relatively complicated and time-consuming.

[0135] Table 5 Comparison of the number and quality of nuclei extracted in Example 3, Example 4, and Example 1

[0136]

[0137] As can be seen from Table 5, the effect of the Cucurbitaceae plant nucleus extraction method described in the present application is relatively stable, and compared with the traditional Percoll method, although the yield is reduced, the purity is greatly guaranteed, which is beneficial to various downstream experimental techniques based on high-purity nuclei; at the same time, compared with the high-osmotic sucrose method, the isotonicity of the iodixanol solution can also ensure the integrity of the nuclei on the premise of maintaining a high yield.

[0138] Through the above examples and the preset comparative example framework, the present application describes in detail a non-flow cytometry-dependent Cucurbitaceae plant tissue nucleus separation and purification method and its optimization process. This method uses iodixanol as a density gradient medium and optimizes the key parameters for different tissue types (stem, grafted callus) in detail, which can stably and efficiently obtain high-purity and high-integrity nuclei, providing a reliable sample preparation scheme for subsequent molecular biology research, especially high-throughput single-cell omics analysis. The setting of the comparative example will further highlight the superiority of the method of the present application over the traditional Percoll method or sucrose method, especially in the treatment of Cucurbitaceae plant materials rich in impurities and secondary metabolites.

[0139] It will be obvious to a person skilled in the art that the application is not limited to the details of the foregoing exemplary embodiments and can be implemented in other concrete forms without departing from the spirit or essential characteristics of the application. The embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

[0140] Furthermore, it should be understood that although the description is made on the basis of the embodiments, not every embodiment contains only one independent technical solution, and the description is made in this way only for the sake of clarity, and a person skilled in the art should consider the description as a whole, and the technical solutions in each embodiment can also be properly combined to form other embodiments that can be understood by a person skilled in the art.

Claims

1. A method for separating and purifying cell nuclei from Cucurbitaceae plant tissues, characterized in that: The following steps are involved: S1. Take Cucurbitaceae plant tissue and wash it thoroughly with pre-cooled nuclear extraction buffer; S2. Place the thoroughly cleaned plant tissue in a pre-cooled grinding container and cut the tissue until a fine and uniform tissue homogenate is formed; S3, filtering the tissue homogenate obtained in S2 through two cell sieves with decreasing pore sizes, and collecting all the filtrates that pass through the cell sieves; S4. Density gradient centrifugation: S4-1. Sample preparation: The filtrate collected in S3 was mixed evenly with a 50% iodixanol solution at a volume ratio of 1:1 to form a sample; S4-2. Gradient construction: Construct a three-layer discontinuous iodixanol density gradient in a pre-cooled 2 mL centrifuge tube, where: For Cucurbitaceae stem tissue: first, transfer 600 μL of sample into a centrifuge tube as the sample layer. Then, add 1000 μL of 30% iodixanol solution as the middle layer below the sample layer, and then add 600 μL of 40% iodixanol solution as the bottom layer. For callus tissue of the grafted part of Cucurbitaceae plants: first take 600 μL of sample and transfer it into a centrifuge tube as the sample layer. Then, add 1000 μL of 30% iodixanol solution as the middle layer under the sample layer, and then add 600 μL of 45% iodixanol solution as the bottom layer. S4-3, centrifugation: centrifuge the centrifuge tube with the constructed density gradient at 4°C, 10,000 × g for 30 min; S4-5, targeted collection: After centrifugation, aspirate the milky white, cloudy band located at or adjacent to the interface between the middle and lower iodixanol solutions. This is the cell nucleus enrichment solution, and the aspirated volume is 100-150 μL. S4-6, washing and resuspending: The cell nucleus enrichment solution collected in S4-5 is washed, centrifuged, and then resuspended to obtain a preliminarily purified cell nucleus suspension.

2. The method for separating and purifying cell nuclei from Cucurbitaceae plant tissues according to claim 1, wherein: The base solution of the cell nuclear extraction buffer was from the CelLytic PN isolation / extraction kit, and Triton X-100, a reducing agent, a protease inhibitor, and an RNase inhibitor were additionally added to a final concentration of 1.0%.

3. The method for separating and purifying cell nuclei from Cucurbitaceae plant tissues according to claim 1, wherein: In S2, the thoroughly cleaned plant tissue is placed in a pre-cooled grinding container, 1-3 mL of pre-cooled nuclear extraction buffer is added per gram of tissue, and the tissue is quickly and thoroughly cut using a sharp cutting tool in an ice bath or at 0-4°C for 8-12 minutes until a fine and uniform tissue homogenate is formed.

4. The method for separating and purifying cell nuclei from Cucurbitaceae plant tissues according to claim 1, wherein: In S3, the tissue homogenate obtained in S2 is filtered sequentially through two cell sieves with decreasing pore sizes, first through a first cell sieve with a pore size of 30-50 μm to remove large cell clumps and unbroken tissue fragments, and then through a second cell sieve with a pore size of 10-30 μm to further remove small cell fragments and some organelles, and all filtrates passing through the cell sieves are collected.

5. The method for separating and purifying cell nuclei from Cucurbitaceae plant tissues according to claim 1, wherein: The S4-5 targeted collection step is as follows: after centrifugation, aspirate the milky white cloudy strip located at or adjacent to the interface between the middle and lower iodixanol solutions using a wide-mouth pipette tip. This is the cell nucleus enrichment solution, and the aspirated volume is 100-150 μL.

6. The method for separating and purifying cell nuclei from Cucurbitaceae plant tissues according to claim 1, wherein: The S4-6 washing and resuspension step is as follows: the cell nucleus enrichment solution collected in S4-5 is transferred to a new pre-cooled centrifuge tube, 3-5 times the volume of pre-cooled cell nucleus extraction buffer is added, mixed and centrifuged at low speed at 4°C for 5 minutes, the supernatant is discarded, and the cell nucleus pellet is resuspended with cell nucleus extraction buffer to obtain a preliminarily purified cell nucleus suspension.

7. The method for separating and purifying cell nuclei from Cucurbitaceae plant tissues according to claim 1, wherein: Before filtering the S3, the mesh can be pre-wetted with a small amount of cell nucleus extraction buffer.

8. The method for separating and purifying cell nuclei from Cucurbitaceae plant tissues according to claim 1, wherein: The composition of the cell nuclear extraction buffer is: 1× basic solution, 0.1 mM dithiothreitol, 0.4 U / μL RNase inhibitor, 1× Cocktail protease inhibitor mixture, and Triton X-100 with a final concentration of 1.0%.

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