A kit and method for extracting plant tissue nuclei

Abstract

This invention discloses a kit and method for extracting cell nuclei from plant tissues, belonging to the field of biotechnology. The kit includes a nuclear lysis buffer and a nuclear washing buffer. The nuclear lysis buffer is prepared using nuclease-free water with sucrose, Tris-HCl (pH 7-8), NaCl, MgCl2, a surfactant, DTT, an RNase inhibitor, and spermidine. The nuclear washing buffer is prepared using nuclease-free water with sucrose, Tris-HCl (pH 7-8), NaCl, MgCl2, DTT, an RNase inhibitor, and spermidine. This kit is suitable for extracting cell nuclei from fresh yew stem tissue, yielding cell nuclei with intact nuclear membranes, stable gene expression, high purity, and few impurities, suitable for single-cell sequencing.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a kit and method for extracting plant tissue cell nuclei. Background Technology

[0002] Single-cell transcriptome sequencing (scRNA-seq) is a technology that performs high-throughput sequencing analysis of the transcriptome at the single-cell level, revealing the expression of all genes within a single cell and intercellular heterogeneity. In recent years, single-cell RNA sequencing has enabled the analysis of gene expression patterns in heterogeneous tissues and organs at the single-cell level, better addressing the cellular heterogeneity problem inherent in traditional research. It provides excellent solutions for cell type identification and rare cell type discovery, Marker gene screening, exploration of cell developmental trajectories, and cell function analysis, and has been widely applied in human and animal research. Due to continuous breakthroughs in single-cell sequencing technology, it has become possible in plant research to study the function and molecular mechanisms of individual plant cells from a single-cell perspective, at the genomic, transcriptomic, and epigenomic levels.

[0003] Although significant progress has been made in recent years in using protoplast isolation (PI) technology for single-cell transcriptome sequencing of plants, single-cell sequencing of plant tissues and cells remains more challenging compared to mammalian tissues and cells.

[0004] 1. Plant cells are fixed in a rigid cell wall matrix, which needs to be removed when separating single cells;

[0005] 2. Enzymatic digestion of plant cell walls is an important stress source that easily stimulates stress responses in plant cells, induces the expression of stress-response genes, and artificially introduces transcriptional bias.

[0006] 3. The size of plant protoplasts is variable (protoplast swelling due to osmotic pressure);

[0007] 4. Secondary metabolites that can interact with RNA are present in plastid organelles and vacuoles;

[0008] 5. Different enzyme combinations are suitable for different tissue types, and it is necessary to optimize the enzymes required for digesting cell walls;

[0009] 6. The prepared protoplasts are very fragile and their activity fluctuates greatly.

[0010] In animal studies, single-cell nuclear sequencing (snRNA-seq) is often used because some sample types have large cells or cells are sensitive to enzyme dissociation. Therefore, single-cell nuclear sequencing of plants can overcome the above difficulties. However, there are currently few universal reagents and methods for extracting plant cell nuclei that can effectively extract plant cell nuclei.

[0011] The yew is a globally recognized rare and endangered species, an ancient relict tree species that survived the Quaternary glaciation. Paclitaxel and its derivatives, extracted from the bark of the yew, are among the most widely used anti-tumor drugs in the world, giving the yew significant economic value. However, due to its plant characteristics, protoplasts cannot be easily extracted like those from animal cells for in-depth single-cell research. Currently, there is no technology for the large-scale and efficient extraction of cell nuclei from yew stems. Summary of the Invention

[0012] To solve at least one of the above-mentioned technical problems, the present invention adopts the following technical solution:

[0013] A first aspect of the present invention provides a kit for extracting plant tissue cell nuclei, comprising a nucleus lysis buffer and a nucleus washing buffer, wherein the nucleus lysis buffer is prepared by using nuclease-free water with sucrose, Tris-HCl (pH 7-8), NaCl, MgCl2, a surfactant, DTT, an RNase inhibitor, and spermidine; and the nucleus washing buffer is prepared by using nuclease-free water with sucrose, Tris-HCl (pH 7-8), NaCl, MgCl2, DTT, an RNase inhibitor, and spermidine.

[0014] In some embodiments of the present invention, the concentration of sucrose in the nuclear lysis buffer is 350–450 mM, the concentration of Tris-HCl is 5–12 mM, the concentration of NaCl is 15–30 mM, the concentration of MgCl2 is 5–12 mM, the concentration of surfactant is 0.01–0.2%, the concentration of DTT is 0.2–2 mM, the concentration of RNase inhibitor is 0.1–0.6 U / μL, and the concentration of spermidine is 0.1–1 mM.

[0015] In some specific embodiments of the present invention, the concentration of sucrose in the nuclear lysis buffer is 400 mM, the concentration of Tris-HCl is 10 mM, the concentration of NaCl is 20 mM, the concentration of MgCl2 is 10 mM, the concentration of surfactant is 0.10%, the concentration of DTT is 1 mM, the concentration of RNase inhibitor is 0.4 U / μL, and the concentration of spermidine is 0.4 mM.

[0016] In some embodiments of the present invention, the concentration of sucrose in the nuclear washing solution is 350–450 mM, the concentration of Tris-HCl is 5–12 mM, the concentration of NaCl is 15–30 mM, the concentration of MgCl2 is 5–12 mM, the concentration of DTT is 0.2–2 mM, and the concentration of RNase inhibitor is 0.1–0.6 U / μL.

[0017] In some specific embodiments of the present invention, the concentration of sucrose in the nuclear washing solution is 400 mM, the concentration of Tris-HCl is 10 mM, the concentration of NaCl is 20 mM, the concentration of MgCl2 is 10 mM, the concentration of DTT is 1 mM, and the concentration of RNase inhibitor is 0.4 U / μL.

[0018] In this invention, the above components can be pre-mixed together and dissolved in the nuclease-free water, or they can be packaged separately and mixed before use. If pre-mixed, DTT and RNase inhibitor are added before use.

[0019] In some embodiments of the present invention, the surfactant is selected from at least one of the group consisting of SDS, Triton X-100, Tween 20, and Tween 80.

[0020] In some specific embodiments of the present invention, the surfactant is Triton X-100.

[0021] A second aspect of the present invention provides a method for providing plant tissue cell nuclei using the kit described in the first aspect of the present invention, comprising the following steps:

[0022] S1, obtain plant tissue and cut it into 1mm3 pieces;

[0023] S2, transfer the tissue fragments to a homogenizer, add the pre-cooled nuclear lysis buffer, and grind the tissue using the homogenizer;

[0024] S3, filter using a cell sieve, wash the sieve with the aforementioned cell nucleus washing solution, collect the filtrate, centrifuge and discard the supernatant.

[0025] S4, add the cell nuclear washing solution to the precipitate again and resuspend the precipitate;

[0026] S5. Cell nuclei were sorted using flow cytometry and the sorted nuclei were collected.

[0027] In some embodiments of the present invention, between steps S3 and S4, the process further includes adding the cell nuclear washing solution to the precipitate, resuspending the precipitate, and centrifuging to remove the supernatant.

[0028] In some embodiments of the present invention, the centrifugation conditions are 2-8°C, 300-800g, and 3-8min. In some embodiments of the present invention, the centrifugation conditions are 4°C, 500g, and 5min.

[0029] In some embodiments of the present invention, the plant tissue is characterized as stem tissue.

[0030] In some specific embodiments of the present invention, the plant is characterized in that it is a yew.

[0031] Beneficial effects of the present invention

[0032] Compared with the prior art, the present invention achieves the following beneficial effects:

[0033] The reagent kit of this invention uses readily available and inexpensive raw materials, and the reagent components have good biocompatibility, contain no harmful ingredients, are safe and environmentally friendly, and are suitable for the extraction of cell nuclei from fresh stem tissues of plants such as yew.

[0034] This invention provides for the first time a method for preparing cell nuclear suspensions from fresh stem tissues of plants such as yew. The method is simple and easy to operate, and the obtained cell nuclei have intact nuclear membranes, stable gene expression, high purity and few impurities. It can be used for single-cell sequencing and has good practical application value. Attached Figure Description

[0035] Figure 1 The microscopic results of the extraction and purification of cell nuclei from fresh yew stem tissue in Example 2 of the present invention are shown.

[0036] Figure 2 The results of fluorescence counting of purified cell nuclei extracted from fresh yew stem tissue in Example 2 of this invention are shown.

[0037] Figure 3 The results of single-cell sequencing of nuclear extraction and purification from fresh yew stem tissue in Example 3 of the present invention are shown.

[0038] Figure 4 The results of nuclear microscopy of fresh tissue cells extracted and purified from Taxus stems in Example 4 of this invention are shown.

[0039] Figure 5 The results of nuclear fluorescence counting of fresh tissue cells extracted and purified from Taxus stems in Example 4 of this invention are shown. Detailed Implementation

[0040] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, in particular the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0041] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. A numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly described in this application.

[0042] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0043] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0044] Example

[0045] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and referenced by them are incorporated herein by reference.

[0047] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0048] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed according to the specific methods listed in *Molecular Cloning: A Laboratory Manual* (4th Edition) (J. Sambrook & MR. Green, 2017), or according to the kit and product instructions. Other experimental methods, unless otherwise specified, were conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples were standard laboratory equipment; the experimental materials used in the following examples, unless otherwise specified, were purchased from a regular biochemical reagent store.

[0049] Example 1: Extraction and purification reagents for Taxus stem cell nuclei

[0050] This embodiment provides the formulation and preparation method of the reagent for extracting and purifying the nuclei of fresh yew cells.

[0051] 1. Cell nuclear lysate

[0052] The cell nuclear lysis buffer was prepared by adding the components as shown in Table 1:

[0053] Table 1 Cell nuclear lysate

[0054] Serial Number Component Name concentration 1 sucrose 400mM 2 Tris-HCl, pH 7.4 10mM 3 NaCl 20mM 4 <![CDATA[MgCl2]]> 10mM 5 Triton X-100 0.10％ 6 DTT 1mM 7 RNase inhibitor 0.4 U / μL 8 Spermidine 0.4mM 9 Nuclease-free water Make up for the volume

[0055] Mix the above components except for DTT and RNase inhibitor and store at 2-8°C. Add DTT and RNase inhibitor before use.

[0056] 2. Cell nuclear washing solution

[0057] The cell nucleus washing solution is used for cell nucleus purification and is prepared by adding components as shown in Table 2:

[0058] Table 2 Cell nucleus washing solution

[0059] Serial Number Component Name concentration 1 sucrose 400mM 2 Tris-HCl, pH 7.4 10mM 3 NaCl 20mM 4 <![CDATA[MgCl2]]> 10mM 5 RNase inhibitor 0.4 U / μL 6 DTT 1mM 7 Nuclease-free water Make up for the volume

[0060] Mix the above components except for DTT and RNase inhibitor and store at 2-8°C. Add DTT and RNase inhibitor before use.

[0061] Example 2: Extraction and purification of Taxus stem cell nuclei

[0062] The reagents prepared in Example 1 were used to extract and purify cell nuclei from fresh yew stem tissue. The specific steps are as follows:

[0063] ① Take 1g of fresh yew stem tissue, place it in a petri dish, and use a blade to cut the tissue into small pieces (1-2mm). 3 size);

[0064] ② Transfer the tissue to a homogenizer, add 5 mL of pre-cooled lysis buffer, and homogenize the tissue using the homogenizer;

[0065] ③ Filter using a 30μm cell sieve, add 5mL of washing solution to clean the sieve, and collect the filtrate;

[0066] ④ Centrifuge at 4℃ and 500g for 5 minutes, then carefully discard the supernatant;

[0067] ⑤ Add 5 mL of washing buffer, resuspend the cell nuclei, and centrifuge at 500 g for 5 min at 4 °C. Discard the supernatant.

[0068] ⑥ Add 1 mL of cell washing buffer and resuspend the precipitate;

[0069] ⑦ Sort cell nuclei using flow cytometry and collect the sorted nuclei.

[0070] The inventors used trypan blue staining to observe cell nucleus morphology under a microscope, and the results were as follows: Figure 1 As shown. The inventors further used a CountStar cell counter to perform AOPI fluorescence staining and counting on the cell nucleus suspension, and the results are as follows. Figure 2 As shown, combined with Figure 1 and Figure 2 The total number of cell nuclei obtained was 530,000, with good morphology, intact nuclear membranes, clean background of suspension, few impurities, and no cell nuclei clumping, meeting the requirements for single-cell sequencing of 10×genomics.

[0071] Example 3 Single-cell nuclear sequencing

[0072] The concentration of cell nuclei prepared in Example 2 was adjusted to 1000 nuclei / μL using the washing solution prepared in Example 1. The samples were then subjected to 10×Genomics single-cell transcriptome sequencing, aiming to capture 8000 nuclei. The results are as follows... Figure 3 As shown in Table 3, from Figure 3 As shown in Table 3, the single-cell sequencing results were of high quality, capturing 8411 cell nuclei (Estimated Number of Cells), with a median gene count of 2981 per cell and a fractionation read rate of 89.30%.

[0073] Table 3 Single-cell sequencing results

[0074]

[0075] Example 4: Extraction of cell nuclei from fresh yew stem tissue using different cell nucleus extraction reagents and methods

[0076] To verify the superiority of the cell nucleus extraction reagent and method of the present invention, the inventors used the reagent and method disclosed in Chinese Invention Patent CN109371017A to extract cell nuclei from fresh stem tissue of Taxus chinensis.

[0077] (1) Plant cell nucleus extraction reagent disclosed in CN109371017A

[0078] The Buffer A solution contained 20 mM Tris-HCl, 10 mM KCl, 250 mM sucrose, 1.5 mM MgCl2, 5 mM β-Mercaptoethanol, and 1 mM protease inhibitor.

[0079] The Buffer B solution contained 20 mM Tris-HCl, 10 mM KCl, 250 mM sucrose, 1.5 mM MgCl2, 1 mM protease inhibitor, 5 mM β-Mercaptoethanol, and 0.1% Triton X-100.

[0080] (2) CN109371017A discloses the steps for cell nucleus extraction and purification.

[0081] ①Pretreatment: Take fresh tissue from the stem of the yew tree and remove impurities;

[0082] ② First extraction: Take 1g of the pretreated material from step ① and 4-6g of zirconium beads and put them into a grinding tube. Add 10mL of Buffer A solution with pH 7.5 to the grinding tube, mix, and grind 2-4 times. Centrifuge and collect the supernatant and precipitate separately. The grinding conditions are: grinding speed 6.5m / s, grinding time 1min per grinding, and grinding interval 1min per grinding. The centrifugation conditions are: centrifugal force 125g and centrifugation time 7min.

[0083] ③ Crude extraction of cell nuclei: The precipitate from step ② was extracted 5-6 times. The extraction conditions were as follows: 8-12 mL of Buffer A solution was added to the precipitate for mixing and grinding, followed by centrifugation. The grinding conditions were: grinding speed 6.5 m / s, grinding time 1 min each time; centrifugation conditions were: centrifugation force 125 g, centrifugation time 7 min; the supernatant obtained each time was collected and mixed to obtain the crude extract of cell nuclei.

[0084] ④ Filtration of crude cell nuclear extract: The supernatant obtained in step ① and the crude cell nuclear extract obtained in step ③ are mixed and then filtered through an N-layer 200-mesh nylon mesh. The filtrate is centrifuged twice at a temperature of 4℃, a centrifugal force of 400g, and a centrifugation time of 10min each time. The precipitate is then collected.

[0085] ④ Cell nucleus purification: The precipitate from step four was resuspended and combined with 20 mL of Buffer B solution at pH 7.5, washed twice, and the cell nuclei were collected by centrifugation to obtain plant cell nuclei. Each washing step was to shake on a shaker for 30 seconds, in an ice bath for 2 minutes, and then centrifuged at 4°C with a centrifugal force of 400 g for 10 minutes.

[0086] After purification, the cell nuclei were examined under a microscope and counted using an AOPI staining and fluorescence counting instrument. The results are as follows: Figure 4 and Figure 5 As shown.

[0087] The results showed that the extraction of cell nuclei from yew stem cells using the reagents and methods provided in CN109371017A was not very effective. The cell nuclei were mostly broken and fragmented, and the number of nuclei was low, only about 150,000. The integrity and number of nuclei were not sufficient for effective sorting by flow cytometry.

[0088] Example 5: Validation of the reagent kit performance (Example 1)

[0089] To further verify the performance of the kit in Example 1, the inventors adjusted other components or ratios and extracted cell nuclei from fresh yew stem tissue according to the method in Example 2. The scheme and results are shown in Table 5.

[0090] Table 5 Results after different combinations of reagent kit substitutions

[0091]

[0092]

[0093] As shown in Table 5, any substitution or adjustment of the proportion of any component in the kit of Example 1 of the present invention will result in a significant decrease in the number of extracted cell nuclei and a serious impact on the integrity of the cell nuclei.

[0094] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for extracting plant tissue cell nuclei, characterized in that, Includes the following steps: S1, Obtain plant tissue and cut to 1mm. 3 Small fragments, the plant tissue being stem tissue, the plant being yew; S2, the tissue fragments are transferred to a homogenizer, pre-cooled nuclear lysis buffer is added, and the tissue is homogenized using the homogenizer. The nuclear lysis buffer is prepared with nuclease-free water containing sucrose, Tris-HCl (pH 7.4), NaCl, MgCl2, Triton X-100, DTT, RNase inhibitor, and spermidine. The concentrations of sucrose, Tris-HCl, NaCl, MgCl2, Triton X-100, DTT, RNase inhibitor, and spermidine in the nuclear lysis buffer are 400 mM, 10 mM, 20 mM, 10 mM, 0.1%, 1 mM, 0.4 U / μL, and 1 mM. S3, filter using a cell sieve, wash the sieve with nuclear washing solution, collect the filtrate, centrifuge and discard the supernatant. The nuclear washing solution is prepared using nuclease-free water with sucrose, Tris-HCl (pH=7.4), NaCl, MgCl2, DTT, and RNase inhibitor. The concentration of sucrose in the nuclear washing solution is 400 mM, the concentration of Tris-HCl is 10 mM, the concentration of NaCl is 20 mM, the concentration of MgCl2 is 10 mM, the concentration of DTT is 1 mM, and the concentration of RNase inhibitor is 0.4 U / μL. S4, add the cell nuclear washing solution to the precipitate again and resuspend the precipitate; S5. Cell nuclei were sorted using flow cytometry and the sorted nuclei were collected.

2. The method according to claim 1, characterized in that, Between steps S3 and S4, the process further includes adding the cell nucleus washing solution to the precipitate, resuspending the precipitate, and centrifuging to remove the supernatant.

3. The method according to claim 1, characterized in that, The centrifugation conditions are 2-8℃, 300-800g, and 3-8min.

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