CN118703596A

CN118703596A - Application of a cell nucleus extraction kit in the extraction and purification of plant tissue cell nuclei

Info

Publication number: CN118703596A
Application number: CN202411160256.9A
Authority: CN
Inventors: 杨欢欢; 张汉虞; 潘煜; 陈天翔; 林建雄
Original assignee: Hangzhou Lianchuan Biotechnology Co ltd
Current assignee: Hangzhou Lianchuan Biotechnology Co ltd
Priority date: 2022-08-19
Filing date: 2022-08-19
Publication date: 2024-09-27
Also published as: CN115433764B; CN115433764A

Abstract

The present invention discloses a kit for extracting plant cell nuclei, which is used to extract cell nuclei of frozen or fresh plant tissues, including extraction reagents and purification reagents; the extraction reagents include ion buffer, metal cations, acid anions, EDTA, spermidine, spermine and DTT; the purification reagents include Tris-HCl, sucrose, potassium chloride, magnesium chloride, sodium chloride and BSA. The kit provided by the present invention is used for the extraction and purification of plant cell nuclei, and the obtained cell nuclei have intact nuclear membranes and stable gene expression, which can be used for gene detection and research. The method for using the kit provided by the present invention is simple and easy to operate, the raw materials are easily available, the price is low, the reagent components have good biocompatibility, no harmful components, and are safe and environmentally friendly.

Description

Application of cell nucleus extraction kit in extraction and purification of plant tissue cell nuclei

Cross Reference to Related Applications

The application is based on the application number 2022110034174, the application date 2022, the month 08 and the 19 date, and the application is named as follows: a kit for extracting plant cell nucleus and its application are provided.

Technical Field

The invention belongs to the technical field of cell nucleus extraction, and particularly relates to application of a cell nucleus extraction kit in extraction and purification of plant tissue cell nuclei.

Background

In recent years, single cell transcriptome sequencing technology has been vigorously developed and has been widely used in different types of tissues and cell lines of various species (particularly human, mouse, etc.), including single cell transcriptome sequencing of normal and diseased cells. However, single cell transcriptome sequencing for plant tissues and plant cells also presents a significant challenge: plant cells are immobilized in a rigid cell wall matrix, which needs to be removed when isolating single cells; enzymatic digestion of plant cell walls is an important stress source, which is easy to stimulate plant cells to generate stress reaction, induce the expression of stress response genes and artificially introduce transcription bias; there is variability in plant protoplast size (osmotic pressure induced protoplast swelling); the presence of secondary metabolites in plastid organelles, and vacuoles that can interact with RNA; the enzyme combinations applicable between different tissue types are different, and the enzymes required for digesting the cell wall need to be optimized; the protoplast prepared is fragile and has large activity fluctuation. The single cell nuclear transcriptome sequencing of the plant can better overcome the difficulty. However, there is currently no general plant cell nucleus extraction reagent and method, especially cryopreserved plant cell nucleus extraction reagent and method. In view of the above, the present invention provides a method for extracting and purifying single cell nuclei suitable for frozen plants or fresh plants, which aims at the defects existing in the prior art.

Disclosure of Invention

In order to solve the problems that the extraction of cell nuclei cannot be immediately carried out after the collection of fresh plant samples, the resource utilization after the extraction of frozen retrospective sample cell nuclei and the re-detection after the freezing of residual samples are carried out, the invention aims to provide a plant cell nucleus extraction kit and application thereof.

In order to achieve the above purpose, the invention adopts the following technical means:

The first aspect of the invention is to provide a kit for extracting plant cell nuclei, for extracting cell nuclei of frozen or fresh plant tissues, comprising an extraction reagent and a purification reagent;

the extraction reagent comprises an ion buffer, a metal cation, an acid radical anion, EDTA, spermidine (spermidine), spermine (spermine) and DTT;

The purification reagent comprises Tris-HCl, sucrose, chloride ion, potassium ion, magnesium ion, sodium ion and BSA;

Preferably, the ion buffer in the extraction reagent is MES with the concentration of 20-30mM; MES is a zwitterionic buffer solution, the pKa value of which is close to the physiological pH value, the water solubility is good, and the MES is not easy to dissolve in other solvents and is not easy to chelate salt ions;

Preferably, the metal cations in the extraction reagent include sodium, magnesium and potassium ions; the acid radical anion is selected from at least one of chloride ion, nitrate ion and sulfate ion; the concentration of sodium ions is 45-55mM; the acid radical anion is chloride ion, and the concentration of the chloride ion is 150-190mM; the concentration of potassium ions is 95-105mM, and the concentration of magnesium ions is 5-15mM;

Preferably, the concentration of EDTA in the extraction reagent is 2-6mM; EDTA can prevent metal ions from activating protease, so that the influence of metal ions on the quality of nucleic acid is reduced;

Preferably, the concentration of spermidine in the extraction reagent is 0.2-0.6mM; the concentration of spermine in the extraction reagent is 0.20-0.30mM; the concentration of DTT in the extraction reagent is 0.3-0.7mM;

preferably, the concentration of sucrose in the purification reagent is 0.2-0.6mM; the pH of Tris-HCl in the purifying reagent is 7.4, and the concentration of the Tris-HCl is 5-15mM; the concentration of potassium ions in the purification reagent is 15-25mM; the concentration of magnesium ions in the purification reagent is 5-15mM; the concentration of sodium ions in the purification reagent is 5-15mM; the chloride ions in the purifying reagent are from sodium chloride, magnesium chloride and potassium chloride;

preferably, the concentration of BSA in the purification reagent is 0.1wt%;

The second aspect of the present invention is to provide a preparation method of an extraction reagent and a purification reagent for extracting plant cell nuclei, respectively:

the extraction reagent is prepared by adding the components according to the following table:

preparing solution according to serial numbers of 1-8 shown in the table, uniformly mixing, storing at 2-8deg.C, and adding DTT before use;

the purification reagent was formulated by component addition as shown in the following table:

the solutions were mixed according to the above table and stored at 2-8deg.C.

A third aspect of the present invention provides a method for using the kit according to the first aspect of the present invention, comprising the steps of:

S1, taking plant tissues in a mortar precooled by dry ice, and putting the plant tissues on the dry ice for grinding to be in a powder shape;

S2, transferring the ground plant tissues into a pre-cooled extraction reagent at the temperature of 2-8 ℃, filtering by a 70 mu m cell filter screen, and collecting filtrate;

S3, centrifuging the filtrate obtained in the step S2 at the temperature of 4 ℃, and discarding the supernatant after the centrifugation is finished to obtain a first precipitate;

S4, re-suspending the first precipitate obtained in the step S3 by using a purifying reagent, filtering by using a 20 mu m cell filter screen, and collecting filtrate;

S5, centrifuging the filtrate obtained in the step S4 at the temperature of 4 ℃, and discarding the supernatant after the centrifugation is finished to obtain a second precipitate;

S6, further re-suspending the second precipitate obtained in the step S5 by using a purifying reagent to obtain a mixture;

s7, separating the cell nucleus from the mixture obtained in the S6 by using a flow cytometer, collecting the separated cell nucleus, observing the morphology of the cell nucleus by using trypan blue staining under a microscope, and performing fluorescent staining counting.

Preferably, in step S1, the plant tissue may be freshly harvested plant tissue or frozen plant tissue;

Preferably, in step S2, the mass-to-volume ratio of plant tissue to extraction reagent is 1:20 (g/mL);

preferably, in step S4, the mass to volume ratio of the first precipitation to the purification reagent is 1:10 (g/mL)

Preferably, in step S6, the mass to volume ratio of the second precipitation to the purification reagent is 1:10 (g/mL);

In a fourth aspect, the invention provides the use of the kit according to the first aspect of the invention for the extraction and purification of nuclei in frozen or fresh plant tissue.

The beneficial effects of the invention are that

Compared with the prior art, the invention has the following beneficial effects:

(1) The kit for extracting the plant cell nucleuses provided by the invention is used for extracting and purifying the plant cell nucleuses, and the obtained cell nucleuses have complete membranes and stable gene expression and can be used for gene detection and research.

(2) The application method of the kit provided by the invention is simple and easy to operate, raw materials are easy to obtain, the price is low, the reagent components have good biocompatibility, no harmful components are generated, and the kit is safe and environment-friendly.

(3) The application method of the kit provided by the invention is different from the conventional fresh plant cell nucleus extraction and purification method, and the method not only can extract fresh plant cell nuclei for extraction and purification, but also can effectively extract plant cell nuclei frozen by liquid nitrogen, so that a plurality of samples which are not easy to obtain and can not be immediately subjected to experimental treatment, retrospective, dynamic monitoring, traceability detection and the like are effectively utilized.

Drawings

FIG. 1 shows a graph of microscopic results of extraction and purification of nuclei of Taxus stems frozen for 7 days in example 2 of the present invention;

FIG. 2 is a graph showing the result of fluorescence count of extraction and purification of the nuclei of Taxus chinensis stem cells frozen for 7 days in example 2 of the present invention;

FIG. 3 is a graph showing the results of extraction and purification microscopy of nuclei of Taxus stems frozen for 30 days in example 2 of the present invention;

FIG. 4 is a graph showing the result of fluorescence count of extraction and purification of stem cell nuclei of Taxus chinensis frozen for 30 days in example 2 of the present invention;

FIG. 5 is a graph showing the result of the extraction and purification microscope of the stem cell nuclei of fresh Taxus chinensis in example 2 of the present invention;

FIG. 6 is a graph showing the result of fluorescence counts of fresh Taxus chinensis stem cell nuclei extraction and purification in example 2 of the present invention;

FIG. 7 is a graph showing the results of the sequencing of nuclei obtained by cryopreservation of fresh Taxus stems for 7 days, 30 days and purification in example 2 of the present invention;

FIG. 8 is a graph showing the result of a microscope for extracting and purifying nuclei of Taxus chinensis leaf frozen for 30 days in example 3 of the present invention;

FIG. 9 is a graph showing the result of fluorescence count of the extraction and purification of the nuclei of Taxus chinensis leaf frozen for 30 days in example 3 of the present invention;

FIG. 10 is a graph showing the result of the extraction and purification microscope of the fresh yew leaf nuclei in example 3 of the present invention;

FIG. 11 is a graph showing the result of fluorescence counts of fresh yew leaf nuclei extraction and purification in example 3 of the present invention;

FIG. 12 is a graph showing the result of the cryopreservation of rice root cell nuclei extraction and purification microscope in example 4 of the present invention;

FIG. 13 is a graph showing the result of fluorescence counts extracted and purified from the root nuclei of cryopreserved rice in example 4 of the present invention;

FIG. 14 is a graph showing the result of a fresh rice root cell nucleus extraction and purification microscope in example 4 of the present invention;

FIG. 15 is a graph showing the result of fluorescence count of fresh rice root cell nuclei extraction and purification in example 4 of the present invention;

FIG. 16 is a graph showing the result of the cryopreservation of rice root nuclei extraction purification microscope in comparative example 1;

FIG. 17 is a graph showing the result of fluorescence counts of cryopreserved rice root nuclei extraction in comparative example 1;

FIG. 18 is a graph showing the result of a fresh rice root cell nucleus extraction and purification microscope in comparative example 1;

FIG. 19 is a graph showing the result of fluorescence counts of fresh rice root cell nuclei extraction and purification in comparative example 1;

FIG. 20 is a graph showing the results of the cryopreserved alfalfa root cell nuclei extraction purification microscope of example 5 of the present invention;

FIG. 21 is a graph showing the result of fluorescence counts extracted and purified from the frozen alfalfa root cell nuclei in example 5 of the present invention;

FIG. 22 is a graph showing the result of the extraction and purification microscope of the frozen wheat root cell nuclei in example 6 of the present invention;

FIG. 23 is a graph showing the result of fluorescence counts extracted and purified from the nuclei of cryopreserved wheat roots in example 6 of the present invention;

FIG. 24 is a graph showing the result of the extraction and purification microscope of the frozen wheat leaf cell nuclei in example 6 of the present invention;

FIG. 25 is a graph showing the result of fluorescence counts extracted and purified from the nuclei of cryopreserved wheat leaves in example 6 of the present invention;

Detailed Description

Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this disclosure is incorporated herein by reference in its entirety, and the equivalent patents are incorporated herein by reference, especially with respect to the definitions of synthetic techniques, product and process designs, polymers, comonomers, initiators or catalysts, etc. in the art, as disclosed in these documents. If the definition of a particular term disclosed in the prior art is inconsistent with any definition provided in the present application, the definition of the term provided in the present application controls.

The numerical ranges in the present application are approximations, so that it may include the numerical values outside the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For example, if a component, physical or other property (e.g., molecular weight, melt index, etc.) is recited as being 100 to 1000, it is intended that all individual values, e.g., 100, 101, 102, etc., and all subranges, e.g., 100 to 166, 155 to 170, 198 to 200, etc., are explicitly recited. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, 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 disclosure.

As used with respect to chemical compounds, the singular includes all isomeric forms and vice versa unless explicitly stated otherwise (e.g., "hexane" includes all isomers of hexane, either individually or collectively). In addition, unless explicitly stated otherwise, the use of the terms "a," "an," or "the" include plural referents.

The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or process, and are not related to whether or not such other component, step or process is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising", "including" or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant or compound. Rather, the term "consisting essentially of … …" excludes any other component, step, or process from the scope of any of the terms recited below, as those out of necessity for performance of the operation. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.

In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.

Examples

The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of 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, the disclosure of which is incorporated herein by reference as is commonly understood 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 claims.

Example 1

The present embodiment provides a preparation method of an extraction reagent and a purification reagent in a kit for extracting plant cell nuclei:

The extraction reagents were formulated as shown in table 1;

TABLE 1 extraction reagent formulation Table

The purification reagents were formulated as shown in table 2;

Table 2 purification reagent formulation table

The solutions were mixed according to the above table and stored at 2-8deg.C.

Example 2

The embodiment provides the application of the kit obtained in the embodiment 1 in the freezing and the fresh yew stem cell nucleus extraction;

Extraction of cryopreserved Douglas fir stem cell nuclei for 7 days at-80 ℃ in experiment I:

S1, taking 0.5g of Taxus chinensis stems stored at-80 ℃ for 7 days after quick freezing by liquid nitrogen, placing the Taxus chinensis stems in a mortar precooled by dry ice, and grinding the Taxus chinensis stems on the dry ice to be powder;

S2, transferring the ground plant tissues into the extraction reagent prepared in the example 1, filtering the plant tissues by using a 70 mu m cell filter screen, and collecting filtrate;

S3, centrifuging 300g of the filtrate obtained in the step S2 for 5min at the temperature of 4 ℃, and discarding the supernatant after the centrifugation is finished to obtain a first precipitate;

S4, re-suspending the first precipitate obtained in the step S3 by using the purification reagent prepared in the embodiment 1, filtering the solution by using a 20 mu m cell filter screen, and collecting filtrate;

s5, centrifuging 300g of the filtrate obtained in the step S4 for 5min at the temperature of 4 ℃, and discarding the supernatant after the centrifugation is finished to obtain a second precipitate;

s6, adding 1mL of the purification reagent prepared in the example 1 into the second precipitate obtained in the step S5 for resuspension to obtain a mixture;

s7, separating the cell nucleus of the mixture obtained in the S6 by using a flow cytometer, collecting the separated cell nucleus, staining the cell nucleus by trypan blue, observing the morphology of the cell nucleus under a microscope (see figure 1), and performing fluorescent staining counting (see figure 2);

extraction of frozen Abies stigmariurus stem cell nuclei stored at-80℃for 30 days in experiment II:

S1, taking 0.5g of Taxus chinensis stems stored for 30 days at the temperature of minus 80 ℃ after quick freezing by liquid nitrogen, placing the Taxus chinensis stems in a mortar precooled by dry ice, and grinding the Taxus chinensis stems on the dry ice to be powder;

s7, separating the cell nucleus of the mixture obtained in the S6 by using a flow cytometer, collecting the separated cell nucleus, staining the cell nucleus by trypan blue, observing the morphology of the cell nucleus under a microscope (see figure 3), and performing fluorescent staining counting (see figure 4).

Extraction of stem cell nuclei of fresh taxus chinensis in experiment III:

s1, taking 0.5g of fresh taxus stems in a mortar precooled by dry ice, and putting the mortar on the dry ice for grinding to be powdery;

S4, carrying out resuspension precipitation on the first precipitate obtained in the step S3 by using the purification reagent prepared in the example 1, filtering by using a 20 mu m cell filter screen, and collecting filtrate;

s7, sorting the cell nuclei of the mixture obtained in the step S6 by using a flow cytometer, collecting the sorted cell nuclei, staining the cell nuclei by trypan blue, observing the morphology of the cell nuclei under a microscope (see figure 5), and performing fluorescent staining counting (see figure 6);

10000 cell nuclei extracted and purified in the first experiment, the second experiment and the third experiment are respectively taken for single cell transcriptome sequencing, and the result is shown in figure 7;

Experimental results: the nuclei of the frozen yew stems and the fresh yew stems after extraction and purification are complete; the total quantity of the cell nucleuses is frozen for 7 days, 30 days and the fresh plants are 35 ten thousand, 30 ten thousand and 45 ten thousand respectively, and the integrity and the quantity of the cell nucleuses can meet the single-cell on-machine requirement; sequencing results showed that 8000 cells were expected to be captured and that the samples were consistent.

Example 3

The embodiment provides the application of the kit obtained in the embodiment 1 in freezing and extracting fresh yew leaf cell nuclei;

Taking 0.5g of frozen 30-day yew leaves and 0.5g of fresh yew leaves, and respectively carrying out experiments according to the experiment II and the experiment III of the example 2;

The experimental results are shown in fig. 8, 9, 10 and 11: the cell nucleus extracted from the frozen and fresh yew leaves is complete; the total quantity of the cell nucleuses is 32 ten thousand and 50 ten thousand respectively, and the nuclear integrity and the number can meet the single-cell on-machine requirement.

Example 4

The present example provides the use of the kit obtained in example 1 in the extraction of frozen rice roots and fresh rice root nuclei;

Taking 1.0g of frozen rice root tissue and 1.0g of fresh rice root tissue respectively, and performing experiments according to the experiment II and the experiment III in the example 2;

The experimental results are shown in fig. 12, 13, 14 and 15: the cell nucleus of the frozen rice root after extraction and purification is complete, the total quantity of the cell nucleus is 15 ten thousand, and the requirements of the machine are met; the cell nucleus of the fresh rice root after extraction and purification is complete, and the total amount of the cell nucleus is 36 ten thousand.

Comparative example 1

Comparative example 1a plant cell nucleus extraction reagent was formulated according to patent application CN109371017 a: tris-HCl concentration of 20mM, KCl concentration of 10mM, sucrose concentration of 250mM, mgCl ₂ concentration of 1.5mM, beta-Mercaptoethanol concentration of 5mM and protease inhibitor concentration of 1mM in Buffer A solution; the concentration of Tris-HCl in the Buffer B solution is 20mM, the concentration of KCl is 10mM, the concentration of sucrose is 250mM, the concentration of MgCl ₂ is 1.5mM, the concentration of protease inhibitor is 1mM, the concentration of beta-Mercaptoethanol is 5mM, and the mass concentration of Tritonx-100 is 0.1%;

the patent application CN109371017A cell nucleus extraction and purification steps:

1. pretreatment: impurity removal from plant material

2. Extracting for the first time: 1-2 g of the pretreated material in the step one and 4-6 g of zirconium beads are put into a grinding pipe, 10mL of Buffer A solution with pH of 7.5 is added into the grinding pipe, mixed and ground for 2-4 times, and the mixture is centrifuged, and the supernatant and the precipitate are respectively collected; 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 centrifugal condition is that the centrifugal force is 125g and the centrifugal time is 7min;

3. Crude extraction of cell nuclei: extracting the precipitate for 5-6 times under the extraction condition that 8-12 mL of Buffer A solution is added into the precipitate for mixing, grinding and centrifuging, wherein the grinding condition is that the grinding speed is 6.5m/s and the grinding time is 1min each time; the centrifugal condition is that the centrifugal force is 125g and the centrifugal time is 7min; collecting and mixing the supernatant obtained each time to obtain a crude cell nucleus extract;

4. Filtering the crude cell nucleus extract: mixing the supernatant obtained in the first step with the crude cell nucleus extract obtained in the third step, filtering with N layers of nylon mesh with 200 meshes, centrifuging the filtrate for 2 times at a centrifugal temperature of 4 ℃ and a centrifugal force of 400g for 10min each time, and collecting the precipitate;

5. And (3) cell nucleus purification: re-suspending the precipitate in the fourth step with 20mL Buffer B solution with pH of 7.5, washing for 2 times, and centrifugally collecting cell nuclei to obtain plant cell nuclei; each washing step is that an oscillator oscillates for 30s, an ice bath is carried out for 2min, and then the centrifugal force is 400g and the centrifugal force is carried out for 10min at the temperature of 4 ℃.

Taking 1.0g of frozen rice root tissue and 1.0g of fresh rice root tissue respectively, extracting and purifying according to the step of extracting and purifying cell nuclei of the patent application CN109371017A, observing the morphology of the cell nuclei under a microscope by trypan blue staining (see figure 16 and figure 18), and performing fluorescent staining counting (see figure 17 and figure 19);

Experimental results: the frozen rice roots are extracted and purified, so that the number of broken cell nucleus fragments is large, the total cell nucleus amount is only 5 ten thousand, and the cell nucleus integrity and the nuclear amount cannot be effectively separated in an up-flow mode; the total amount of the cell nucleus after the extraction and purification of the fresh rice roots is 20 ten thousand, which is far less than that of the fresh rice roots in the embodiment 4.

As is clear from the corresponding graphs of the experimental results of comparative example 4 and comparative example 1, the cell nucleus impurities extracted in example 4 were less and the cell nucleus was more intact.

Example 5

The present example provides the use of the kit obtained in example 1 in the extraction of cryopreserved alfalfa root nuclei;

0.6g of frozen alfalfa root tissue was taken and tested according to test two in example 2;

the experimental results are shown in fig. 20 and 21: the detection result of a microscope after the sorting by a flow cytometer shows that the cell nucleus is complete, the number of the fluorescence count cell nucleus reaches 24 ten thousand, and the requirement of single cell transcriptome on-line is met.

Example 6

The embodiment provides the application of the kit obtained in the embodiment 1 in the extraction of frozen wheat roots and leaf nuclei;

taking 0.5g of frozen wheat root tissue and 0.5g of frozen wheat leaf tissue respectively, and performing an experiment according to the experiment II in the example 2;

The experimental results are shown in fig. 22, 23, 24 and 25: the cell nucleus after the frozen wheat root is extracted and purified is complete, the total cell nucleus is 22 ten thousand, and the number of the cell nucleus and the nuclear integrity meet the requirement of single cell transcriptome sequencing on-machine; the cell nucleus extracted and purified from the frozen wheat leaves is complete, the total cell nucleus is 28 ten thousand, and the number of the cell nuclei and the nuclear integrity meet the requirement of single cell transcriptome sequencing on-machine.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. An application of a cell nucleus extraction kit in the extraction and purification of cell nuclei of plant tissues, characterized in that the plant tissues include fresh plant samples and frozen plant samples;

The cell nucleus extraction kit comprises an extraction reagent and a purification reagent;

The extraction reagent comprises an ion buffer, a metal cation, an acid anion, EDTA, spermidine, spermine and DTT;

The purification reagents include Tris-HCl, sucrose, chloride ions, magnesium ions, sodium ions, potassium ions and BSA;

The ion buffer in the extraction reagent is MES, with a concentration of 20-30mM;

The metal cations in the extraction reagent include sodium ions, magnesium ions and potassium ions; the acid anions in the extraction reagent are selected from at least one of chloride ions, nitrate ions, sulfate ions and carbonate ions;

The concentration of sodium ions in the extraction reagent is 45-55 mM; the concentration of potassium ions in the extraction reagent is 95-105 mM, and the concentration of magnesium ions in the extraction reagent is 5-15 mM; the acid anions in the extraction reagent are chloride ions, and the concentration of chloride ions is 150-190 mM;

The concentration of EDTA in the extraction reagent is 2-6 mM; the concentration of spermidine in the extraction reagent is 0.2-0.6 mM; the concentration of spermine in the extraction reagent is 0.20-0.30 mM; the concentration of DTT in the extraction reagent is 0.3-0.7 mM;

The concentration of sucrose in the purification reagent is 0.2-0.6 mM; the pH of Tris-HCl in the purification reagent is 7.4, and the concentration is 5-15 mM; the concentration of potassium ions in the purification reagent is 15-25 mM; the concentration of magnesium ions in the purification reagent is 5-15 mM; and the concentration of sodium ions in the purification reagent is 5-15 mM.

2. The application according to claim 1, characterized in that it comprises the following steps:

S1, take the plant tissue in a mortar pre-cooled with dry ice, place it on dry ice and grind it into powder;

S2, transfer the ground plant tissue to the extraction reagent precooled at 2-8°C, filter with a 70 μm cell filter, and collect the filtrate;

S3, centrifuging the filtrate obtained in S2 at 4°C, discarding the supernatant after the centrifugation to obtain a first precipitate;

S4, resuspending the first precipitate obtained in S3 with a purification reagent, filtering with a 20 μm cell filter, and collecting the filtrate;

S5, centrifuging the filtrate obtained in S4 at 4°C, discarding the supernatant after the centrifugation to obtain a second precipitate;

S6, further resuspending the second precipitate obtained in S5 with a purification reagent to obtain a mixture;

S7, sorting the cell nuclei of the mixture obtained in S6 by flow cytometry, and collecting the sorted cell nuclei.