CN110595988A - Preparation method and application of cell nucleus suitable for detecting plant C value by flow cytometry - Google Patents

Preparation method and application of cell nucleus suitable for detecting plant C value by flow cytometry Download PDF

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CN110595988A
CN110595988A CN201910973618.9A CN201910973618A CN110595988A CN 110595988 A CN110595988 A CN 110595988A CN 201910973618 A CN201910973618 A CN 201910973618A CN 110595988 A CN110595988 A CN 110595988A
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plant
value
flow cytometer
steel balls
standing
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杨俊波
贾艳霞
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Kunming Institute of Botany of CAS
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
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    • G01—MEASURING; TESTING
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    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

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Abstract

The invention provides a preparation method of cell nucleuses suitable for detecting a plant C value by a flow cytometer, belonging to the technical field of genetics and genomics, and comprising the following steps: mixing fresh plant leaves, dissociation liquid and steel balls, grinding for 30-45 seconds under the condition of 25-40 HZ, standing, filtering with a filter membrane, and collecting filtrate to obtain cell nucleuses; the mass of the fresh plant leaves, the volume of the dissociation liquid and the quantity ratio of the steel balls are 0.015-0.025 g, 0.6-1.0 mL and 10-15. The method can quickly and efficiently obtain the complete cell nucleus required by the plant C value detection of the flow cytometer, thereby greatly improving the efficiency of the flow cytometer for detecting the plant C value and ploidy; the method is simple, convenient, feasible, efficient and reliable, is suitable for various plant samples, and greatly reduces the consumption of plant materials compared with materials required by hand cutting.

Description

Preparation method and application of cell nucleus suitable for detecting plant C value by flow cytometry
Technical Field
The invention relates to the technical field of genetics and genomics, in particular to a preparation method and application of a cell nucleus suitable for detecting a plant C value by a flow cytometer.
Background
Since the 50s of the 20 th century, it became increasingly clear that there was a large difference in the amount of nuclear DNA between organisms and attempts to explain this phenomenon, the most well-known of which was the "C-value paradox" theory, i.e., there was no correlation between genome size and the complexity of the organisms. The C-value is the basic data for determining the size of a species genome, and knowledge of these data will facilitate the formulation of genetic and genomic research protocols and the analysis of the data.
The haploid genome of an organism contains the total amount of DNA, called the C-value, which represents the size of the organism's genome and is an important feature of the organism. Flow Cytometry (FCM) is a technique that uses a flow cytometer to analyze and sort fluorescently labeled microparticles in a liquid flow state and perform rapid, accurate, qualitative, quantitative determination and analysis, and in research in botany, FCM is mainly used to detect the DNA content of plant cell nuclei and its ploidy analysis. Detecting the DNA content of the cells by using a flow cytometer, generally dyeing the cells by using a specific fluorescent dye, wherein the quantity of the DNA combined by fluorescent molecules is in direct proportion to the DNA content in the cells, obtaining the DNA content of the detected cells by detecting the fluorescence intensity emitted by the excited dyed cells, and obtaining the genome size of the detected plants by comparing the DNA content with an internal standard with known genome size.
The cell wall of the plant cell not only causes the cell character to be out of specification, but also has fluorescence, thereby disturbing the sample injection liquid flow and being incapable of well detecting and separating the plant cell. Direct staining of plant protoplasts results in fluorescence peak patterns that do not accurately reflect nuclear DNA content because of the fluorescence of plant cytoplasm while the low permeability of plasma membranes. Thus, it is necessary to free intact nuclei. At present, the general method for dissociating cell nuclei is to cut tissues into pieces by using a blade and release the cell nuclei by utilizing the osmosis of a dissociation solution, and the method has the defects of low efficiency, time and labor waste and the like and is a limiting factor in the process of realizing the batch detection of the plant C value by a flow cytometer. With the development of plant genomics, the requirement of batch detection of plant C values by using a flow cytometer is more and more common, so that the efficiency of detecting the plant C values by using the flow cytometer can be greatly improved by establishing a preparation method for quickly and efficiently obtaining cell nucleuses required by detecting the plant C values by using the flow cytometer.
Disclosure of Invention
The invention aims to provide a preparation method and application of a cell nucleus suitable for detecting a plant C value by a flow cytometer.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of cell nucleuses suitable for detecting a plant C value by a flow cytometer, which comprises the following steps: mixing fresh plant leaves, dissociation liquid and steel balls, grinding for 30-45 seconds under the condition of 25-40 HZ, standing, filtering with a filter membrane, and collecting filtrate to obtain cell nucleuses;
the mass of the fresh plant leaves, the volume of the dissociation liquid and the quantity ratio of the steel balls are 0.015-0.025 g, 0.6-1.0 mL and 10-15;
the diameter of the steel ball is 0.15-0.25 cm;
the dissociation liquid comprises the following components in concentration: 40 to 50mM MgCl2·6H2O, 15-25 mM MOPS, 25-35 mM sodium citrate, 0.5-1.5% of PVP-40 with the mass volume concentration of 0.5-1.5%, 0.1-0.3% of Tritonx-100 with the volume concentration of 8-12 mM Na2EDTA。
Preferably, the ratio of the mass of the fresh plant leaves, the volume of the dissociation liquid and the number of steel balls is 0.02g:0.8mL: 12.
Preferably, the frequency of the grinding is 30 HZ; the grinding time is 40S; the grinding temperature is 0-4 ℃.
Preferably, the standing time is 10-15 min; the standing temperature is 0-4 ℃.
Preferably, the diameter of the filter membrane used for filtering by the filter membrane is 30-50 μm.
The invention also provides a cell nucleus obtained by the preparation method in the scheme.
The invention also provides application of the cell nucleus in the scheme in detecting the C value of the plant, which comprises the following steps:
and mixing the cell nucleus with propidium iodide and RNAase to obtain a mixed solution, standing the mixed solution, and detecting on a flow cytometer to obtain a plant C value.
Preferably, the concentration of the propidium iodide in the mixed solution is 45-55 mu g/mL; the concentration of the RNAase in the mixed solution is 45-55 mu g/mL.
Preferably, the standing time is 0.5-2 h; the standing temperature is 0-4 ℃.
The invention has the beneficial effects that: the invention provides a preparation method of cell nucleuses suitable for detecting a plant C value by a flow cytometer, which comprises the following steps: mixing fresh plant leaves, dissociation liquid and steel balls, grinding for 30-45 seconds under the condition of 25-40 HZ, standing, filtering with a filter membrane, and collecting filtrate to obtain cell nucleuses; the mass of the fresh plant leaves, the volume of the dissociation liquid and the quantity ratio of the steel balls are 0.015-0.025 g, 0.6-1.0 mL and 10-15. The method can quickly and efficiently obtain the complete cell nucleus required by the plant C value detection of the flow cytometer, thereby greatly improving the efficiency of the flow cytometer for detecting the plant C value and ploidy; the method is simple, convenient, feasible, efficient and reliable, is suitable for various plant samples, and greatly reduces the consumption of plant materials compared with materials required by hand cutting.
Drawings
FIG. 1 shows the flow cytometry results of the samples after grinding in example 1;
FIG. 2 is a graph showing the flow cytometry results of the samples after grinding in example 2;
FIG. 3 shows the flow cytometry results of the milled samples of comparative example 1;
FIG. 4 shows the flow cytometry results of the milled samples of comparative example 2;
FIG. 5 shows the flow cytometry results of the samples after grinding in example 3;
FIG. 6 shows the flow cytometry results of the samples after grinding in example 4;
FIG. 7 shows the flow cytometry results of the milled samples of comparative example 3;
FIG. 8 is a flow cytometer measurement of the milled sample of comparative example 4;
FIG. 9 is a Miscanthus flow histogram of example 5;
FIG. 10 is a flow chart of Miscanthus strawberries in example 5;
FIG. 11 is a flow histogram of China rose in example 5;
FIG. 12 is a flow chart of China rose in example 5;
FIG. 13 is a water lily flow histogram of example 5;
FIG. 14 is a water lily flow chart of example 5;
FIG. 15 is a mulberry flow histogram in example 5;
FIG. 16 is a flow chart of mulberry in example 5;
FIG. 17 is an Arabidopsis thaliana flow histogram of example 5;
FIG. 18 is an Arabidopsis thaliana flow scattergram in example 5;
FIG. 19 is a flow histogram of Physcomitrella patens in example 5;
FIG. 20 is a flow chart of Physcomitrella patens in example 5.
Detailed Description
The invention provides a preparation method of cell nucleuses suitable for detecting a plant C value by a flow cytometer, which comprises the following steps: mixing fresh plant leaves, dissociation liquid and steel balls, grinding for 30-45 seconds under the condition of 25-40 HZ, standing, filtering with a filter membrane, and collecting filtrate to obtain cell nucleuses;
the ratio of the mass of the fresh plant leaves, the volume of the dissociation liquid and the quantity of the steel balls is 0.015-0.025 g: 0.6-1.0 mL: 10-15, preferably 0.02g:0.8mL: 12;
the diameter of the steel ball is 0.15-0.25 cm, and preferably 0.2 cm;
the dissociation liquid comprises the following components in concentration: 40 to 50mM MgCl2·6H2O, 15-25 mM MOPS, 25-35 mM sodium citrate, 0.5-1.5% of PVP-40 with the mass volume concentration of 0.5-1.5%, 0.1-0.3% of Tritonx-100 with the volume concentration of 8-12 mM Na2EDTA; preferably, the dissociation liquid preferably comprises the following components: 45mM MgCl2·6H2O, 20mM MOPS, 30mM sodium citrate, 1% PVP-40 by mass volume concentration, 0.2% Tritonx-100 by volume concentration and 10mM Na2EDTA。
In the invention, the mass of the fresh plant leaves, the volume of the dissociation liquid and the quantity of the steel balls have a specific ratio, the fresh plant leaves are too few to dissociate enough cell nucleuses, and the fresh plant leaves are too many to generate too many fragments, thus being not beneficial to dissociation.
The types of plants to which the methods of the invention are applicable include: nymphaeaceae, Moraceae, Rosaceae, Mangiferaceae and Cruciferae.
In the invention, the fresh plant leaves comprise young leaves and mature leaves, and preferably the young leaves.
In the invention, the size of the fresh plant leaves is preferably 0.2cm by 0.2-0.3 cm, and more preferably 0.2cm by 0.2 cm.
In the invention, the mixing temperature is preferably 0-4 ℃, and the temperature and the mode of mixing are not particularly limited, and the uniform mixing is taken as the standard; the frequency of the grinding is preferably 30 Hz; the grinding time is preferably 40S; the grinding temperature is preferably 0-4 ℃; the equipment of grinding is preferably the machine of grinding, more preferably the quick grinding appearance of the full-automatic sample of shanghai net letter science and technology, model: tissue lyser-48.
In the invention, the standing time is preferably 10-15 min; the preferred temperature of the standing is 0-4 ℃, and the low temperature is favorable for ensuring the stability of a cell nuclear membrane; the diameter of the filter membrane adopted by the filter membrane filtration is preferably 30-50 mu m, more preferably 40-42 mu m, so that fragments generated by dissociation can be effectively filtered, and complete cell nuclei can be obtained at maximum efficiency.
The invention also provides a cell nucleus obtained by the preparation method in the scheme.
The invention also provides application of the cell nucleus in the scheme in detecting the C value of the plant, which comprises the following steps:
and mixing the cell nucleus with propidium iodide and RNAase to obtain a mixed solution, standing the mixed solution, and detecting on a flow cytometer to obtain a plant C value.
In the invention, the mixing temperature is preferably 0-4 ℃, and the temperature and the mode of mixing are not particularly limited, and the uniform mixing is taken as the standard; the concentration of the propidium iodide in the mixed solution is preferably 45-55 mu g/mL, and more preferably 50 mu g/mL; the concentration of the RNAase in the mixed solution is 45-55 mug/mL, and more preferably 50 mug/mL; the standing time is preferably 0.5-2 h, and more preferably 1-1.5 h; the temperature of standing is preferably 0-4 ℃; the flow cytometer is preferably a flow cytometer BD FACSCalibur.
In the invention, Propidium Iodide (PI) can be combined with intracellular nucleic acid, and emits fluorescence of about 600nm under the excitation of 488nm exciting light, and the strength of a fluorescence signal is in direct proportion to the DNA content of cells; RNAase can remove RNA interference in a sample and reduce detection errors.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Selecting materials: cutting about 0.02g of fresh young Gramineae plant Amberryptisia angustifolia leaf into 0.2cm by 0.2cm square with scissors, placing into 2mL centrifuge tube precooled on ice, adding 0.8mL dissociation liquid (45mM MgCl) precooled on ice2·6H2O, 20mM MOPS, 30mM sodium citrate, 1% (W/V) PVP-40, 0.2% (V/V) Tritonx-100, 10mM Na2EDTA), 12 steel balls with a diameter of 0.2cm were added.
(2) Material grinding conditions: the centrifuge tube with the materials and steel balls placed therein is placed on a pre-cooled adapter to keep the plant materials in a low temperature state, and then the centrifuge tube is placed on a grinder (Shanghai Jingxin science and technology full-automatic sample rapid grinder, model: tissue laser-48), the program is set to 30HZ and 40S, and the centrifuge tube is started.
(3) Filtering and dyeing a sample: the samples were placed on ice in that order and allowed to stand for 12min, filtered through a 42 μm diameter filter into a fresh 1.5mL centrifuge tube, and stained on ice for 1h with Propidium Iodide (PI) (50 μ g/mL) and RNAase (50 μ g/mL).
(4) And (3) detection: the sample was transferred to the sample tube, tested on a flow cytometer (BD FACSCalibur), and 5000 particles were collected, and the test result showed a sample fluorescence value of 51.56 and a CV value of 2.49. The obtained cell nuclei were collected for detection on the machine, and the results are shown in FIG. 1.
Example 2
(1) Selecting materials: cutting about 0.02g of fresh young Gramineae plant Amberryptisia angustifolia leaf into 0.2cm by 0.2cm square with scissors, placing into 2mL centrifuge tube precooled on ice, adding 0.8mL dissociation liquid precooled on ice (45mM MgCl)2·6H2O, 20mM MOPS, 30mM sodium citrate, 1% (W/V) PVP-40, 0.2% (V/V) Tritonx-100, 10mM Na2EDTA), 12 steel balls with a diameter of 0.2cm were added.
(2) Material grinding conditions: the centrifuge tube with the materials and steel balls placed therein is placed on a pre-cooled adapter to keep the plant materials in a low temperature state, and then is placed on a grinder (Shanghai Jingxin science and technology full-automatic sample rapid grinder, model: tissue laser-48), the program is set to 40HZ and 40S, and the centrifuge tube is started.
(3) Filtering and dyeing a sample: the samples were placed on ice in that order and allowed to stand for 12min, filtered through a 42 μm diameter filter into a fresh 1.5mL centrifuge tube, and stained on ice for 1h with Propidium Iodide (PI) (50 μ g/mL) and RNAase (50 μ g/mL).
(4) And (3) detection: the sample was transferred to the sample tube and tested on a flow cytometer (BD FACSCalibur) to collect 5000 particles, which showed 51.77 sample fluorescence and 5.28 CV value. The obtained cell nuclei were collected for detection on the machine, and the results are shown in FIG. 2.
Comparative example 1
(1) Selecting materials: cutting about 0.02g of fresh young Gramineae plant Amberryptisia angustifolia leaf into 0.2cm by 0.2cm square with scissors, placing into 2mL centrifuge tube precooled on ice, adding 0.8mL dissociation liquid precooled on ice (45mM MgCl)2·6H2O, 20mM MOPS, 30mM sodium citrate, 1% (W/V) PVP-40, 0.2% (V/V) Tritonx-100, 10mM Na2EDTA), 12 steel balls with a diameter of 0.2cm were added.
(2) Material grinding conditions: the centrifuge tube with the materials and steel balls placed therein is placed on a pre-cooled adapter to keep the plant materials in a low temperature state, and then the centrifuge tube is placed on a grinder (Shanghai Jingxin science and technology full-automatic sample rapid grinder, model: tissue laser-48), the program is set to 20HZ and 40S, and the centrifuge tube is started.
(3) Filtering and dyeing a sample: the samples were placed on ice in that order and allowed to stand for 12min, filtered through a 42 μm diameter filter into a fresh 1.5mL centrifuge tube, and stained on ice for 1h with Propidium Iodide (PI) (50 μ g/mL) and RNAase (50 μ g/mL).
(4) And (3) detection: the sample was transferred to the sample tube and tested on a flow cytometer (BD FACSCalibur) to collect 5000 particles, which showed 52.47 sample fluorescence and 5.78 CV. The obtained cell nuclei were collected for detection on the machine, and the results are shown in FIG. 3.
Comparative example 2
(1) Selecting materials: cutting about 0.02g of fresh young Gramineae plant Amberryegrass leaf into 0.2cm by 0.2cm square with scissors, placing into ice pre-cooled 2mL centrifuge tube, adding 0.8mL ice pre-cooled dissociation liquid (45mM MgCl. sub.L. sub.MgCl.)2·6H2O, 20mM MOPS, 30mM sodium citrate, 1% (W/V) PVP-40, 0.2% (V/V) Tritonx-100, 10mM Na2EDTA), 12 steel balls with a diameter of 0.2cm were added.
(2) Material grinding conditions: the centrifuge tube with the materials and steel balls placed therein is placed on a pre-cooled adapter to keep the plant materials in a low temperature state, and then the centrifuge tube is placed on a grinder (Shanghai Jingxin science and technology full-automatic sample rapid grinder, model: tissue laser-48), the program is set to be 50HZ and 40S, and the centrifuge tube is started.
(3) Filtering and dyeing a sample: the samples were placed on ice in that order and allowed to stand for 12min, filtered through a 42 μm diameter filter into a fresh 1.5mL centrifuge tube, and stained on ice for 1h with Propidium Iodide (PI) (50 μ g/mL) and RNAase (50 μ g/mL).
(4) And (3) detection: the sample was transferred to the sample tube, tested on a flow cytometer (BD FACSCalibur), and 5000 particles were collected, and the test results showed a sample fluorescence value of 46.08 and a CV value of 6.56. The obtained cell nuclei were collected for detection on the machine, and the results are shown in FIG. 4.
As can be seen from the above examples 1 and 2 and comparative examples 1 and 2, the grinding effect of example 1 is the best, specifically, CV is the smallest, and under the condition of collecting the same data particles, the peak value is the highest, which indicates that the most effective and complete cell nuclei are obtained; the grinding effect of example 2 is the second, which is expressed as CV 5.26, but the CV of the effective result is less than 5%, and the result error of the CV measurement is larger; most of the samples obtained in comparative example 1 were not ground efficiently, so the results showed more debris and a larger CV; the CV value of the results obtained in comparative example 2 was the highest because most of the nuclei were fragmented due to too high a frequency of milling, so that the peak was broadened and less intact nuclei were obtained.
Example 3
(1) Selecting materials: cutting about 0.02g of fresh young Gramineae plant Amberryegrass leaf into 0.2cm by 0.2cm square with scissors, placing into ice pre-cooled 2mL centrifuge tube, adding 0.8mL ice pre-cooled dissociation liquid (45mM MgCl. sub.L. sub.MgCl.)2·6H2O, 20mM MOPS, 30mM sodium citrate, 1% (W/V) PVP-40, 0.2% (V/V) Tritonx-100, 10mM Na2EDTA), 12 steel balls with a diameter of 0.2cm were added.
(2) Material grinding conditions: the centrifuge tube with the materials and steel balls placed therein is placed on a pre-cooled adapter to keep the plant materials in a low temperature state, and then the centrifuge tube is placed on a grinder (Shanghai Jingxin science and technology full-automatic sample rapid grinder, model: tissue laser-48), the program is set to 30HZ and 30S, and the centrifuge tube is started.
(3) Filtering and dyeing a sample: the samples were placed on ice in that order and allowed to stand for 12min, filtered through a 42 μm diameter filter into a fresh 1.5mL centrifuge tube, and stained on ice for 1h with Propidium Iodide (PI) (50 μ g/mL) and RNAase (50 μ g/mL).
(4) And (3) detection: the sample was transferred to the sample tube and tested on a flow cytometer (BD FACSCalibur) to collect 5000 particles, which showed 56.28 sample fluorescence and 3.54 CV. The obtained cell nuclei were collected for detection on the machine, and the results are shown in FIG. 5.
Example 4
(1) Selecting materials: cutting about 0.02g of fresh young Gramineae plant Amberryptisia angustifolia leaf into 0.2cm by 0.2cm square with scissors, placing into 2mL centrifuge tube precooled on ice, adding 0.8mL dissociation liquid precooled on ice (45mM MgCl)2·6H2O, 20mM MOPS, 30mM sodium citrate, 1% (W/V) PVP-40, 0.2% (V/V) Tritonx-100, 10mM Na2EDTA), adding steel ball with diameter of 0.2cm12 particles.
(2) Material grinding conditions: the centrifuge tube with the materials and steel balls placed therein is placed on a pre-cooled adapter to keep the plant materials in a low temperature state, and then the centrifuge tube is placed on a grinder (Shanghai Jingxin science and technology full-automatic sample rapid grinder, model: tissue laser-48), the program is set to 30HZ and 40S, and the centrifuge tube is started.
(3) Filtering and dyeing a sample: the samples were placed on ice in that order and allowed to stand for 12min, filtered through a 42 μm diameter filter into a fresh 1.5mL centrifuge tube, and stained on ice for 1h with Propidium Iodide (PI) (50 μ g/mL) and RNAase (50 μ g/mL).
(4) And (3) detection: the sample was transferred to the sample tube, tested on a flow cytometer (BD FACSCalibur), and 5000 particles were collected, and the test results showed a sample fluorescence value of 55.89 and a CV value of 2.83. The obtained cell nuclei were collected for the detection effect on the machine, and the results are shown in FIG. 6.
Comparative example 3
(1) Selecting materials: cutting about 0.02g fresh young Gramineae plant caulis et folium Tritici Aestivi into 0.2cm by 0.2cm square with scissors, placing into 2mL centrifuge tube precooled on ice, adding 0.8mL dissociation liquid (45mM MgCl) precooled on ice2·6H2O, 20mM MOPS, 30mM sodium citrate, 1% (W/V) PVP-40, 0.2% (V/V) Tritonx-100, 10mM Na2EDTA), 12 steel balls with a diameter of 0.2cm were added.
(2) Material grinding conditions: the centrifuge tube with the materials and steel balls placed therein is placed on a pre-cooled adapter to keep the plant materials in a low temperature state, and then the centrifuge tube is placed on a grinder (Shanghai Jingxin science and technology full-automatic sample rapid grinder, model: tissue laser-48), the program is set to 30HZ and 20S, and the centrifuge tube is started.
(3) Filtering and dyeing a sample: the samples were placed on ice in that order and allowed to stand for 12min, filtered through a 42 μm diameter filter into a fresh 1.5mL centrifuge tube, and stained on ice for 1h with Propidium Iodide (PI) (50 μ g/mL) and RNAase (50 μ g/mL).
(4) And (3) detection: the sample was transferred to the sample tube and tested on a flow cytometer (BD FACSCalibur) to collect 5000 particles, which showed 53.21 sample fluorescence and 4.87 CV. The resulting nuclei were collected for detection on the machine, and the results are shown in FIG. 7.
Comparative example 4
(1) Selecting materials: cutting about 0.02g of fresh young Gramineae plant Amberryptisia angustifolia leaf into 0.2cm by 0.2cm square with scissors, placing into 2mL centrifuge tube precooled on ice, adding 0.8mL dissociation liquid precooled on ice (45mM MgCl)2·6H2O, 20mM MOPS, 30mM sodium citrate, 1% (W/V) PVP-40, 0.2% (V/V) Tritonx-100, 10mM Na2EDTA), 12 steel balls with a diameter of 0.2cm were added.
(2) Material grinding conditions: the centrifuge tube with the materials and steel balls placed therein is placed on a pre-cooled adapter to keep the plant materials in a low temperature state, and then is placed on a grinder (Shanghai Jingxin science and technology full-automatic sample rapid grinder, model: tissue laser-48), the program is set to 30HZ and 50S, and the centrifuge tube is started.
(3) Filtering and dyeing a sample: the samples were placed on ice in that order and allowed to stand for 12min, filtered through a 42 μm diameter filter into a fresh 1.5mL centrifuge tube, and stained on ice for 1h with Propidium Iodide (PI) (50 μ g/mL) and RNAase (50 μ g/mL).
(4) And (3) detection: the sample was transferred to the sample tube and tested on a flow cytometer (BD FACSCalibur) to collect 5000 particles, which showed 54.27 sample fluorescence and 4.65 CV value. The obtained cell nuclei were collected for detection on the computer, and the results are shown in FIG. 8.
As can be seen from the above examples 3 and 4 and comparative examples 3 and 4, the grinding effects were better for the grinding times of 30s and 40s and the grinding effect was best for 40s at the same grinding frequency (30 Hz); the lower main peak for two grind 20s indicates less effective cells were obtained, probably due to insufficient grinding, and the increased debris in front of the main peak was found for two grind 50s, probably due to more cell disruption over the course of the grinding time.
Example 5
General verification of the grinding method of the invention
Nymphaeaceae plant; moraceae family; rosaceae; gramineae; cruciferae family; cucurbitaceae plant.
(1) Selecting materials: respectively cutting about 0.02g of fresh tender plant (Gramineae old mango wheat; Rosaceae plant rose; Nymphaeaceae plant water lily) with scissors(ii) a Moraceae plant Morus alba; arabidopsis thaliana of the family Brassicaceae; physcomitrella patens (Thunb.) Moencnakai of Dictamaceae) leaves were cut into 0.2cm by 0.2cm cubes, placed in 2mL ice-precooled centrifuge tubes, and 0.8mL ice-precooled dissociation fluid (45mM MgCl. RTM. MgCl.) was added2·6H2O, 20mM MOPS, 30mM sodium citrate, 1% (W/V) PVP-40, 0.2% (V/V) Tritonx-100, 10mM Na2EDTA), 12 steel balls with a diameter of 0.2cm were added.
(2) Material grinding conditions: the centrifuge tube with the materials and steel balls placed therein is placed on a pre-cooled adapter to keep the plant materials in a low temperature state, and then the centrifuge tube is placed on a grinder (Shanghai Jingxin science and technology full-automatic sample rapid grinder, model: tissue laser-48), the program is set to 30HZ and 40S, and the centrifuge tube is started.
(3) Filtering and dyeing a sample: the samples were placed on ice in that order and allowed to stand for 12min, filtered through a 42 μm diameter filter into a fresh 1.5mL centrifuge tube, and stained on ice for 1h with Propidium Iodide (PI) (50 μ g/mL) and RNAase (50 μ g/mL).
(4) And (3) detection: moving to an upper sample tube, detecting on a flow cytometer (BD FACSCalibur), collecting 5000-10000 particles, wherein the detection result shows that CV values in histograms of the five selected plants are less than 5%, and an obvious main cell mass can be seen in a scatter diagram, which shows that the cell nucleus obtained by the method has good completeness and the measured plant C value or ploidy is reliable. Collecting the obtained on-machine detection effect of the cell nucleus, and referring to the results of fig. 9-20, wherein fig. 9 is a ramrod flow type histogram; FIG. 10 is a Miscanthus strawberries flow chart; FIG. 11 is a rose flow histogram; FIG. 12 is a flow chart of Chinese rose; FIG. 13 is a water lily flow histogram; FIG. 14 is a water lily flow scattergram; FIG. 15 is a mulberry flow histogram; FIG. 16 is a mulberry flow chart; FIG. 17 is an Arabidopsis flow histogram; FIG. 18 is an Arabidopsis thaliana flow scatterplot; FIG. 19 is a physcomitrella patens flow histogram; FIG. 20 is a flow chart of Physcomitrella patens.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (9)

1. A preparation method of cell nucleuses suitable for detecting C values of plants by a flow cytometer comprises the following steps: mixing fresh plant leaves, dissociation liquid and steel balls, grinding for 30-45 seconds under the condition of 25-40 HZ, standing, filtering with a filter membrane, and collecting filtrate to obtain cell nucleuses;
the mass of the fresh plant leaves, the volume of the dissociation liquid and the quantity ratio of the steel balls are 0.015-0.025 g, 0.6-1.0 mL and 10-15;
the diameter of the steel ball is 0.15-0.25 cm;
the dissociation liquid comprises the following components in concentration: 40 to 50mM MgCl2·6H2O, 15-25 mM MOPS, 25-35 mM sodium citrate, 0.5-1.5% of PVP-40 with the mass volume concentration of 0.5-1.5%, 0.1-0.3% of Tritonx-100 with the volume concentration of 8-12 mM Na2EDTA。
2. The preparation method according to claim 1, wherein the ratio of the mass of the fresh plant leaves, the volume of the dissociation liquid and the number of steel balls is 0.02g:0.8mL: 12.
3. The method according to claim 1 or 2, wherein the frequency of the milling is 30 HZ; the grinding time is 40S; the grinding temperature is 0-4 ℃.
4. The preparation method according to claim 1 or 2, wherein the standing time is 10 to 15 min; the standing temperature is 0-4 ℃.
5. The method according to claim 1 or 2, wherein the diameter of the filter membrane used for the membrane filtration is 30 to 50 μm.
6. A cell nucleus obtained by the production method according to any one of claims 1 to 5.
7. The use of the cell nucleus of claim 6 for detecting the C value of a plant, comprising the steps of:
and mixing the cell nucleus with propidium iodide and RNAase to obtain a mixed solution, standing the mixed solution, and detecting on a flow cytometer to obtain a plant C value.
8. The use according to claim 6, wherein the concentration of propidium iodide in the mixed solution is 45-55 μ g/mL; the concentration of the RNAase in the mixed solution is 45-55 mu g/mL.
9. The use according to claim 6, wherein the standing time is 0.5-2 h; the standing temperature is 0-4 ℃.
CN201910973618.9A 2019-10-14 2019-10-14 Preparation method and application of cell nucleus suitable for detecting plant C value by flow cytometry Pending CN110595988A (en)

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