CN115678965A - Kit and method for extracting plant tissue cell nucleus - Google Patents
Kit and method for extracting plant tissue cell nucleus Download PDFInfo
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- CN115678965A CN115678965A CN202211396825.0A CN202211396825A CN115678965A CN 115678965 A CN115678965 A CN 115678965A CN 202211396825 A CN202211396825 A CN 202211396825A CN 115678965 A CN115678965 A CN 115678965A
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Abstract
The invention discloses a kit and a method for extracting plant tissue cell nucleuses, belonging to the technical field of biology. The kit comprises a cell nucleus lysate and a cell nucleus cleaning solution, wherein the cell nucleus lysate is prepared from sucrose, tris-HCl with pH = 7-8, naCl and MgCl 2 The surfactant, the DTT, the RNase inhibitor and the spermidine are prepared by nuclease-free water; the cell nucleus cleaning solution is prepared from sucrose, tris-HCl with the pH = 7-8, naCl, mgCl2, DTT, RNase inhibitor and spermidine by using nuclease-free water. The kit is suitable for extracting the cell nucleus of the fresh tissue of the taxus chinensis stems, the obtained cell nucleus nuclear membrane is complete, the gene expression is stable, the purity is high, the impurities are few, and the kit can be used for single cell sequencing.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit and a method for extracting plant tissue cell nucleuses.
Background
Single cell transcriptome sequencing technology (scRNA-seq) is a technology that performs high throughput sequencing analysis of transcriptomes at the single cell level, revealing all gene expression and intercellular heterogeneity within a single cell. In recent years, the single cell RNA sequencing technology realizes the analysis of gene expression patterns of heterogeneous tissues and organs on the single cell level, better solves the problem of cell heterogeneity in the traditional research, provides a good solution for cell type identification, discovery of rare cell types, marker gene screening, cell development track discussion, cell function analysis and the like, and is widely applied to human and animal research. Just because of the continuous breakthrough of single cell sequencing technology, in plant research, from the single cell point of view, the research on the functions and molecular mechanisms of individual cells of plants at the genome, transcriptome and epigenome levels is possible.
Although recent advances have been made in sequencing plant single cell transcriptomes using Protoplast Isolation (PI), single cell sequencing in plant tissues and plant cells has been more challenging than in mammalian tissues and cells:
1. plant cells are fixed in a rigid cell wall matrix and need to be removed when isolating single cells;
2. the enzymatic digestion of plant cell walls is an important stress source, which is easy to stimulate plant cells to generate stress response, induce the expression of stress response genes and artificially introduce transcription deviation;
3. variability in plant protoplast size (protoplast swelling due to osmotic pressure);
4. the presence of secondary metabolites in plastid organelles, and vacuoles, that can interact with RNA;
5. the different tissue types differ in the combination of enzymes that are suitable, requiring optimization of the enzymes needed to digest the cell wall;
6. the prepared protoplast is fragile and has large activity fluctuation.
In animal research, single cell nucleus sequencing (snRNA-seq) is often adopted due to the fact that cells in certain sample types are large or the cells are sensitive to enzyme dissociation, so that the single cell nucleus sequencing carried out by plants can better overcome the difficulties.
Taxus chinensis is a rare endangered species recognized all over the world and is an ancient mosquito larvae tree species left to date by the fourth era glacier. The taxol and the derivatives thereof extracted from the stem bark of the taxus chinensis are one of the most widely applied anti-tumor medicaments in the world today, so that the taxus chinensis has great economic value. However, due to its plant characteristics, protoplasts cannot be extracted as conveniently as animal cells, and intensive single cell studies have been carried out. At present, no related technology for extracting a large amount of cell nucleuses of the stems of the taxus chinensis efficiently exists.
Disclosure of Invention
In order to solve at least one of the above technical problems, the technical solution adopted by the present invention is as follows:
the invention provides a kit for extracting plant tissue cell nuclei, which comprises cell nucleus lysate and cell nucleus cleaning solution, wherein the cell nucleus lysate is prepared from sucrose, tris-HCl with pH = 7-8, naCl and MgCl 2 The surfactant, the DTT, the RNase inhibitor and the spermidine are prepared by nuclease-free water; the cell nucleus cleaning solution is prepared from sucrose, tris-HCl with the pH = 7-8, naCl, mgCl2, DTT, RNase inhibitor and spermidine by using nuclease-free water.
In some embodiments of the invention, the cell nuclear lysate has a sucrose concentration of 350 to 450mM, tris-HCl concentration of 5 to 12mM, naCl concentration of 15 to 30mM, mgCl 2 The concentration of (A) is 5-12 mM, the concentration of surfactant is 0.01-0.2%, the concentration of DTT is 0.2-2mM, the concentration of RNase inhibitor is 0.1-0.6U/uL, and the concentration of spermidine is 0.1-1 mM.
In some embodiments of the invention, the cell nucleus lysate has a sucrose concentration of 400mM, a Tris-HCl concentration of 10mM, a NaCl concentration of 20mM, and MgCl 2 The concentration of (2) was 10mM, the concentration of the surfactant was 0.10%, the concentration of DTT was 1mM, the concentration of RNaseinhibitor was 0.4U/. Mu.L, and the concentration of spermidine was 0.4mM.
In some embodiments of the invention, the nuclear wash has a sucrose concentration of 350 to 450mM, tris-HCl concentration of 5 to 12mM, naCl concentration of 15 to 30mM, mgCl 2 The concentration of (A) is 5 to 12mM, the concentration of DTT is 0.2 to 2mM, and the concentration of RNase inhibitor is 0.1 to 0.6U/. Mu.L.
In some embodiments of the inventionIn the cell nucleus washing solution, the concentration of sucrose is 400mM, the concentration of Tris-HCl is 10mM, the concentration of NaCl is 20mM, and MgCl is adopted 2 The concentration of (3) was 10mM, the concentration of DTT was 1mM, and the concentration of RNase inhibitor was 0.4U/. Mu.L.
In the present invention, the above components may be mixed together in advance and dissolved in the nuclease-free water, or may be packaged separately and mixed at the time of use. If pre-mixed together, DTT and RNase inhibitor are added prior to use.
In some embodiments of the invention, the surfactant is selected from at least one of the group consisting of SDS, triton X-100, tween20, tween 80.
In some embodiments of the invention, the surfactant is Triton X-100.
In a second aspect, the present invention provides a method for providing plant tissue cell nuclei using the kit of the first aspect of the present invention, comprising the steps of:
s1, obtaining plant tissues and cutting the plant tissues into 1mm & lt 3 & gt pieces;
s2, transferring the tissue crumbs into a homogenizer, adding the precooled cell nucleus lysate, and grinding the tissue by using the homogenizer;
s3, filtering by using a cell sieve, adding the cell nucleus cleaning solution to clean the screen, collecting filtrate, centrifuging and discarding supernatant,
s4, adding the cell nucleus cleaning solution into the sediment again, and resuspending the sediment;
and S5, sorting the cell nucleuses by using a flow cytometer, and collecting the sorted cell nucleuses.
In some embodiments of the present invention, between step S3 and step S4, adding the nuclear wash solution to the pellet, resuspending the pellet, and centrifuging the supernatant.
In some embodiments of the invention, the centrifugation conditions are 2-8 ℃, 300-800 g, 3-8 min. In some embodiments of the invention, the centrifugation conditions are 4 ℃, 500g, 5min.
In some embodiments of the invention, the plant tissue is stem tissue.
In some embodiments of the invention, characterized in that said plant is Taxus chinensis.
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
the kit has the advantages of easily available raw materials, low price, good biocompatibility of reagent components, no harmful components, safety and environmental protection, and is suitable for extracting cell nucleuses of fresh stem tissues of plants such as taxus chinensis and the like.
The invention provides the preparation method of the cell nucleus suspension of the fresh plant stem tissues such as the taxus chinensis for the first time, the preparation method is simple and easy to operate, the obtained cell nucleus has complete nuclear membrane, stable gene expression and high purity and few impurities, can be used for single cell sequencing, and has good practical application value.
Drawings
FIG. 1 shows the microscope results of the extraction and purification of nuclei from fresh tissue of Taxus chinensis in example 2 of the present invention.
FIG. 2 shows the fluorescence counting results of the extraction and purification of nucleus from fresh tissue of Taxus chinensis in example 2 of the present invention.
FIG. 3 shows the sequencing results of single cell extraction and purification from fresh tissue nuclei of Taxus chinensis in example 3 of the present invention.
FIG. 4 shows the result of nuclear microscopy on fresh tissue extracted from Taxus chinensis as an example 4 of the present invention.
FIG. 5 shows the fluorescence count of cell nuclei from fresh tissue extracted from purified Taxus chinensis in example 4 of the present invention.
Detailed Description
Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. Where applicable, the contents of any patent, patent application, or publication referred to in this application are incorporated herein by reference in their entirety, and the equivalent family of patents is also incorporated by reference, especially where relevant in the art to which these documents disclose definitions of relevant terms. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definition provided herein, the definition of the term provided herein controls.
The numerical ranges in this application are approximations that can include values outside of the ranges unless otherwise specified. A numerical range includes all numbers from a lower value to an upper value, in increments of 1 unit, provided that there is a separation of at least 2 units between any lower value and any higher value. For ranges containing a numerical value less than 1 or containing a fraction greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is considered to be 0.0001,0.001,0.01, or 0.1, as appropriate. For ranges containing single digit numbers less than 10 (e.g., 1 to 5), 1 unit is typically considered 0.1. These are merely specific examples of what is intended to be presented, and all possible combinations of numerical values between the lowest value and the highest value enumerated, are to be considered to be expressly stated in this application.
The terms "comprising," "including," "having," and derivatives thereof do not exclude the presence of any other component, step or procedure, and are not intended to exclude the presence of other elements, steps or procedures not expressly disclosed herein. To the extent that any doubt is eliminated, all compositions herein containing, including, or having the term "comprise" may contain any additional additive, adjuvant, or compound, unless expressly stated otherwise. Conversely, the term "consisting essentially of 8230%, \8230composition" excludes any other components, steps or processes from the scope of any of the terms hereinafter recited, insofar as those terms are necessary for operational performance. The term "consisting of 8230%" \8230comprises "does not include any components, steps or processes not specifically described or listed. Unless explicitly stated otherwise, the term "or" refers to the listed individual members or any combination thereof.
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, 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 belongs and the disclosures and materials cited therein are hereby incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The molecular biological experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in the manual of molecular cloning, laboratory manual (fourth edition) (j. Sambrook, m.r. green, 2017), or according to the kit and product instructions. Other experimental methods, unless otherwise specified, are conventional. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1 extraction and purification of Taxus chinensis Stem cell nucleus
The embodiment provides a formula and a preparation method of a new sequoia neobrevifolia nucleus extraction reagent and a new sequoia neobrevifolia nucleus purification reagent.
1. Cell nucleus lysate
The cell nucleus lysate is prepared by adding the following components according to the formula shown in table 1:
TABLE 1 cell nucleus lysates
| Serial number | Component name | Concentration of |
| 1 | Sucrose | 400mM |
| 2 | Tris-HCl，pH 7.4 | 10mM |
| 3 | NaCl | 20mM |
| 4 | MgCl 2 | 10mM |
| 5 | Triton X-100 | 0.10％ |
| 6 | DTT | 1mM |
| 7 | RNase inhibitor | 0.4U/μL |
| 8 | Spermidine (Spermidine) | 0.4mM |
| 9 | Nuclease-free water | Make up volume |
Mixing the above components except DTT and RNase inhibitor, storing at 2-8 deg.C, and adding DTT and RNase inhibitor before use.
2. Cell nucleus cleaning solution
The cell nucleus cleaning solution is used for purifying cell nuclei, and the components are added and prepared according to the following table 2:
TABLE 2 cell nucleus washing solution
| Serial number | Component name | Concentration of |
| 1 | Sucrose | 400mM |
| 2 | Tris-HCl，pH 7.4 | 10mM |
| 3 | NaCl | 20mM |
| 4 | MgCl 2 | 10mM |
| 5 | RNase inhibitor | 0.4U/μL |
| 6 | DTT | 1mM |
| 7 | Nuclease-free water | Make up volume |
Mixing the above components except DTT and RNase inhibitor, storing at 2-8 deg.C, and adding DTT and RNase inhibitor before use.
Example 2 Taxus chinensis Stem cell nucleus extraction and purification
The reagent prepared in the embodiment 1 is used for extracting and purifying cell nucleuses of fresh taxus chinensis stem tissues, and the specific steps are as follows:
(1) taking 1g of fresh taxus chinensis stem tissue, placing the fresh taxus chinensis stem tissue in a culture dish, and cutting the tissue into pieces (1-2 mm) by a blade 3 Size);
(2) transferring the tissue to a homogenizer, adding 5mL of precooled lysate, and grinding the tissue by using the homogenizer;
(3) filtering with a 30-micron cell sieve, adding 5mL of cleaning solution to clean the sieve, and collecting the filtrate;
(4) centrifuging at 4 deg.C for 5min at 500g, and carefully discarding the supernatant;
(5) adding 5mL of cleaning solution, resuspending cell nucleus, centrifuging at 4 deg.C and 500g for 5min, and discarding supernatant;
(6) adding 1mL of cell washing liquid, and resuspending the precipitate;
(7) and (4) sorting cell nucleuses by a flow cytometer, and collecting the sorted cell nucleuses.
The inventors observed the morphology of the cell nuclei under a microscope with trypan blue staining, and the results are shown in FIG. 1. The inventor further uses a countstar cell counter to perform AOPI fluorescence staining counting on the cell nucleus suspension, and the result is shown in figure 2, and the result is combined with figures 1 and 2, so that the total amount of the obtained cell nucleus is 53 thousands, the form is good, the nuclear membrane state is complete, the suspension background is clean, the impurities are few, the cell nucleus does not agglomerate, and the requirement of 10 Xgenomics on single-cell computer is met.
Example 3 Single cell Nuclear sequencing
The cell nucleus concentration prepared in example 2 was adjusted to 1000 cells/. Mu.L using the wash solution prepared in example 1. Samples were subjected to 10 × genomics single cell transcriptome sequencing with 8000 capture targets. As shown in FIG. 3 and Table 3, it can be seen from FIG. 3 and Table 3 that the sequencing result of single Cells is high in quality, 8411 Cell nuclei (optimized Number of Cells) are captured, the Median gene (media Genes per Cell) is 2981, and the proportion of high-quality Cell Reads (Fraction Reads in Cells) is 89.30%.
TABLE 3 Single cell sequencing results
Example 4 extraction of cell nuclei from fresh stem tissue of Taxus chinensis by different cell nuclei extraction reagents and methods
In order to verify the superiority of the reagent and the method for extracting the cell nucleus, the inventor utilizes the reagent and the method disclosed in the Chinese patent invention CN109371017A to extract the cell nucleus of the fresh stem tissue of the taxus chinensis.
(1) Plant cell nucleus extraction reagent disclosed in CN109371017A
The Tris-HCl concentration in the Buffer A solution is 20mM, the KCl concentration is 10mM, the sucrose concentration is 250mM, and MgCl is adopted 2 The concentration is 1.5mM, the concentration of beta-Mercaptoethanol is 5mM, and the concentration of protease inhibitor is 1mM.
The Tris-HCl concentration in the Buffer B solution is 20mM, the KCl concentration is 10mM, the sucrose concentration is 250mM, and MgCl is adopted 2 Concentration of 1.5mM, protease inhibitor concentration of 1mM, beta-Mercaptoethanol concentration of 5mM, triton X-100 concentrationThe degree was 0.1%.
(2) CN109371017A discloses a cell nucleus extraction and purification step
(1) Pretreatment: taking fresh tissue of taxus chinensis stems, and removing impurities;
(2) extracting for the first time: putting 1g of the material pretreated in the step (1) and 4-6 g of zirconium beads into a grinding tube, adding 10mL of Buffer A solution with the pH value of 7.5 into the grinding tube, mixing, grinding for 2-4 times, centrifuging, and collecting a supernatant and a precipitate respectively; the grinding conditions are that the grinding speed is 6.5m/s, the grinding time is 1min each time, and the grinding interval time is 1min each time; the centrifugation condition is that the centrifugal force is 125g, and the centrifugation time is 7min;
(3) crude extraction of cell nucleus: extracting the precipitate in the step (2) for 5-6 times, wherein the extraction condition is to add 8-12 mL of Buffer A solution into the precipitate, mix, grind and centrifuge, the grinding speed is 6.5m/s, and the grinding time is 1min each time; the centrifugation condition is that the centrifugal force is 125g, and the centrifugation time is 7min; collecting the supernatant obtained each time, and mixing to obtain a crude cell nucleus extract;
(4) filtering a crude extract of cell nuclei: mixing the supernatant obtained in the step (1) with the crude cell nucleus extract obtained in the step (3), filtering with N layers of 200-mesh nylon net, centrifuging the filtrate for 2 times at 4 ℃, 400g and 10min each time, and collecting the precipitate;
(4) and (3) purifying cell nuclei: the precipitate in the fourth step is resuspended and merged by 20mL of Buffer B solution with the pH value of 7.5, washed for 2 times and centrifuged to collect cell nucleus, thus obtaining the plant cell nucleus; each washing step is shaking with oscillator for 30s, ice-cooling for 2min, and centrifuging at 4 deg.C and centrifugal force of 400g for 10min.
The purified cell nuclei were examined under microscope and counted by a countstar counter using AOPI staining fluorescence, and the results are shown in FIGS. 4 and 5, respectively.
The result shows that the reagent and the method provided by CN109371017A have very undesirable effect on the extraction of the stem cell nucleus of the taxus chinensis, a large amount of damaged fragments of the cell nucleus and a small amount of nucleus, only about 15 ten thousand, and the nuclear integrity and the nuclear amount cannot be effectively sorted in an up-flow manner.
Example 5 verification of the Performance of the kit of example 1
To further verify the performance of the kit of example 1, the inventors adjusted other components or ratios, and performed cell nucleus extraction on fresh tissue of taxus chinensis stems according to the method of example 2, wherein the scheme and the results are shown in table 5:
TABLE 5 results of different combinations of the kit
As can be seen from Table 5, the substitution or ratio adjustment of any component in the kit of example 1 of the present invention resulted in a significant decrease in the number of extracted nuclei and a severe influence on the nuclear integrity.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. The kit for extracting plant tissue cell nuclei is characterized by comprising cell nucleus lysate and cell nucleus cleaning solution, wherein the cell nucleus lysate is prepared from sucrose, tris-HCl with pH = 7-8, naCl and MgCl 2 The surfactant, the DTT, the RNase inhibitor and the spermidine are prepared by nuclease-free water; the cell nucleus cleaning solution is prepared from sucrose, tris-HCl with pH = 7-8, naCl and MgCl 2 DTT, RNase inhibitor and spermidine are prepared by using nuclease-free water.
2. The kit of claim 1, wherein the kit is characterized byIn the cell nucleus lysate, the concentration of sucrose is 350 to 450mM, the concentration of Tris-HCl is 5 to 12mM, the concentration of NaCl is 15 to 30mM, and MgCl is 2 The concentration of (A) is 5-12 mM, the concentration of surfactant is 0.01-0.2%, the concentration of DTT is 0.2-2mM, the concentration of RNase inhibitor is 0.1-0.6U/uL, and the concentration of spermidine is 0.1-1 mM.
3. The kit according to claim 1, wherein the washing solution for cell nuclei contains sucrose at a concentration of 350 to 450mM, tris-HCl at a concentration of 5 to 12mM, naCl at a concentration of 15 to 30mM, mgCl at a concentration of 15 to 30mM, and 2 the concentration of (2) is 5 to 12mM, the concentration of DTT is 0.2 to 2mM, and the concentration of RNase inhibitor is 0.1 to 0.6U/. Mu.L.
4. The kit according to any one of claims 1 to 3, wherein the surfactant is at least one selected from the group consisting of SDS, triton X-100, tween20, tween 80.
5. The kit of claim 4, wherein the surfactant is Triton X-100.
6. A method for providing plant tissue cell nuclei using the kit of claim 1, comprising the steps of:
s1, obtaining plant tissues and shearing the plant tissues to 1mm 3 Crushing into powder with different sizes;
s2, transferring the tissue crumbs into a homogenizer, adding the precooled cell nucleus lysate, and grinding the tissue by using the homogenizer;
s3, filtering by using a cell sieve, adding the cell nucleus cleaning solution to clean the screen, collecting filtrate, centrifuging and discarding supernatant,
s4, adding the cell nucleus cleaning solution into the sediment again, and resuspending the sediment;
and S5, sorting the cell nucleuses by using a flow cytometer, and collecting the sorted cell nucleuses.
7. The method of claim 6, further comprising, intermediate step S3 and step S4, adding the nuclear wash to the pellet, resuspending the pellet and centrifuging the supernatant.
8. The method of claim 6, wherein the centrifugation conditions are 2-8 ℃, 300-800 g, 3-8 min.
9. The method of any one of claims 6-8, wherein the plant tissue is stem tissue.
10. The method of any one of claims 6 to 8, wherein the plant is Taxus chinensis.
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