CN111139214A - Liquid for extracting cell nucleus and application thereof - Google Patents

Liquid for extracting cell nucleus and application thereof Download PDF

Info

Publication number
CN111139214A
CN111139214A CN202010009185.8A CN202010009185A CN111139214A CN 111139214 A CN111139214 A CN 111139214A CN 202010009185 A CN202010009185 A CN 202010009185A CN 111139214 A CN111139214 A CN 111139214A
Authority
CN
China
Prior art keywords
liquid
cell nucleus
content
extracting
nuclear
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202010009185.8A
Other languages
Chinese (zh)
Other versions
CN111139214B (en
Inventor
张晋丹
靳婉青
冯旻
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Botany of CAS
Original Assignee
Institute of Botany of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Botany of CAS filed Critical Institute of Botany of CAS
Priority to CN202010009185.8A priority Critical patent/CN111139214B/en
Publication of CN111139214A publication Critical patent/CN111139214A/en
Application granted granted Critical
Publication of CN111139214B publication Critical patent/CN111139214B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Botany (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a liquid for extracting cell nucleuses and application thereof. The invention firstly discloses a liquid for extracting cell nucleus, which is prepared from Na3C6H5O7·2H2O、MgCl2、MOPS、NaCl、EDTA‑Na2The liquid for extracting the cell nucleus is prepared by using PVP k12 and Tween-20 for the first time, so that the liquid can extract the cell nucleus with sufficient quantity and quality for flow cytometry detection and analysis in a very small using amount and a very short time, is suitable for extracting the cell nucleus of various plant materials including plants rich in polysaccharide and polyphenol secondary metabolites, is particularly suitable for extracting the cell nucleus of a dry sample, and has wide applicability.

Description

Liquid for extracting cell nucleus and application thereof
Technical Field
The invention relates to the field of cell nucleus extraction. In particular to a liquid for extracting cell nucleus and application thereof.
Background
The flow cytometer is mainly used for measuring the DNA content of plant cells, analyzing the ploidy of chromosomes, analyzing cell cycles and the like in the field of plant science. Obtaining high quality nuclear extract of sample is key to the determination. Since the application of flow cytometry to the field of botany, a variety of formulations have been screened for the extraction of cell nuclei from plant material of different origin. Due to the wide variety of plants and the great difference of different plant tissue structures and chemical components, the existing published formula can not be simultaneously suitable for various plant materials. Particularly for plant materials with complex secondary metabolites, the existing liquid for extracting cell nuclei can not meet the test requirements, and the components of the extracting solution are often required to be optimized according to the characteristics of a sample so as to obtain the optimal extracting effect.
Disclosure of Invention
The invention aims to solve the technical problem of how to obtain the plant cell nucleus extract with high cell nucleus content and quality, in particular to how to obtain the plant cell nucleus extract with high cell nucleus content and high quality from plant materials rich in polysaccharide and polyphenol for the detection of a flow cytometer.
In order to solve the above technical problems, the present invention first provides a liquid for extracting cell nuclei.
The liquid for extracting cell nucleus of the invention is composed of Na3C6H5O7·2H2O、MgCl2、MOPS、NaCl、EDTA-Na2Triton X-100, Tween-20, β -mercaptoethanol, PVP k12 and water, wherein the Na is sodium hydroxide3C6H5O7·2H2The content of O in the liquid is 30mmol/L, and the MgCl is adopted245mmol/L of the liquid, 20mmol/L of the MOPS, 20mmol/L of the NaCl, and EDTA-Na2The content in the liquid is 20mmol/L, the content in the liquid of the Triton X-100 is 0.1 percent by volume, the content in the liquid of the Tween-20 is 0.5 percent by volume, the content in the liquid of the β -mercaptoethanol is 2uL/mL, and the content in the liquid of the PVP k12 is 1 percent by volume.
The above liquid for extracting cell nuclei had a pH of 7.0.
The invention further provides the application of the liquid for extracting the cell nucleus in any one of the following steps:
1) the application in preparing plant cell nucleus extract;
2) the application of the plant cell nucleus extract in improving the cell nucleus content and/or quality;
3) the application of the extract in preparing products for improving the content and/or quality of cell nucleuses in the plant cell nucleus extract;
4) the application in preparing a flow cytometry detection sample.
The invention further provides a method for preparing the plant cell nucleus extract.
The method for preparing the plant cell nucleus extract comprises the following steps:
adding plant material into liquid for extracting cell nucleus, and crushing plant material in the liquid to obtain plant cell nucleus extract.
In the above method, the mass-to-volume ratio of the plant material to the liquid for extracting cell nuclei is 10-20 mg: 250 uL.
In the above method, the plant material is rich in polysaccharide and/or polyphenol, such as fructus Schisandrae chinensis.
In the above method, the plant material is dried material or fresh material; specifically, the drying material is a silica gel drying material.
The plant cell nucleus extract prepared by the method is also within the protection scope of the invention.
The invention further provides application of the plant cell nucleus extract in flow cytometry detection.
The application of PVP k12 or Tween-20 in improving the content and/or the quality of the cell nucleus in the plant cell nucleus extract or the application in preparing products for improving the content and/or the quality of the cell nucleus in the plant cell nucleus extract is also within the protection scope of the invention.
The application of PVP k12 and Tween-20 in increasing the content and/or quality of cell nucleus in plant cell nuclear extract or the application of products for increasing the content and/or quality of cell nucleus in plant cell nuclear extract is also within the protection scope of the invention.
The high quality of the cell nucleus is mainly reflected in that the peak shape is symmetrical, narrow and high when the flow cytometry is used for detecting the nuclear DNA, the fragment peak is low, and the coefficient of variation CV is less than 5 percent.
The invention firstly replaces the common reagent PVP40 or PVP k15 in the liquid for extracting the cell nucleus with PVPk12, and adds Tween-20 which is beneficial to separating the cell nucleus from viscous secondary metabolites, and the obtained liquid for extracting the cell nucleus can extract the cell nucleus with enough quantity and quality for DNA content determination and chromosome ploidy analysis in flow cytometry detection with extremely small using amount and extremely short time. The liquid for extracting the cell nucleuses is suitable for extracting the cell nucleuses of various plant materials including plants rich in polysaccharide and polyphenol secondary metabolites, is particularly suitable for extracting the cell nucleuses of dry samples, and is consistent with the detection result of fresh materials when flow cytometry is used for detection, so that the problems of destructive sampling and long-distance transportation of the fresh materials are effectively solved, and the working difficulty of DNA content determination and chromosome ploidy analysis of field plant cells is greatly reduced. Therefore, the liquid for extracting cell nuclei of the present invention has wide applicability.
Drawings
FIG. 1 is a flow chart of the effect of extracting nuclear DNA from fresh blueberry leaves with different liquids used to extract cell nuclei.
FIG. 2 shows PVPk12-mGB2 and
Figure BDA0002356477540000031
PI Absolute P extracts a flow chart of the nuclear DNA effect of fresh leaves of herbaceous plants.
FIG. 3 shows PVPk12-mGB2 and
Figure BDA0002356477540000032
PI Absolute P extracts the flow chart of the nuclear DNA effect of the fresh leaves of woody and vine plants.
FIG. 4 is a flow chart of fresh ficus carica leaf nuclear DNA content determination using fresh tomatoes as internal standards; wherein, A:
Figure BDA0002356477540000033
PI Absolute P; b: PVPk12-mGB2 is used for extracting liquid of cell nucleus; p1: fig leaves; p2: tomato leaves.
FIG. 5 is a flow chart of DNA content of silica gel dried leaves of Ficus carica using fresh tomatoes as internal standard; wherein, A:
Figure BDA0002356477540000034
PI Absolute P forExtracting DNA of the fig silica gel dried leaf nucleus extracted from the liquid of the cell nucleus; b: PVPk12-mGB2 is used for extracting liquid extracted fig silica gel dried leaf nucleus DNA of cell nucleus; p1: tomato leaves; p2: ficus carica silica gel dry blade
FIG. 6 is a flow chart of blueberry chromosome ploidy analysis; wherein, A: wild type diploid blueberry fresh leaves; b: fresh leaves of wild hexaploid blueberries; c: drying leaves of the wild hexaploid blueberry silica gel; p1: diploid; p2: hexaploid; p3: and (4) hexaploid.
FIG. 7 is a flow chart of the effect of Tween on nuclear extraction; wherein, A: a control group without Tween-20; b: 0.1% Tween-20 group; c: 0.5% Tween-20 group.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The following examples are the sources of purchase of each raw material:
PVP k15 was purchased from TCI under the cat # TCI-P0471-25G;
PVP k12 and PVP70 were purchased from ACROs, cat nos. 276142500-250 GR and 222271000-100 GR;
PVP40 was purchased from Sigma under the accession number P5288-100G.
Example 1 preparation of cell Nuclear extracts and detection of Nuclear DNA content and chromosome ploidy analysis
Liquid for extracting cell nucleus
This example provides the following liquids for extracting nuclei: PVP-mGB2, LB01, WPS, Tris-MgCl2And commercialized kit
Figure BDA0002356477540000035
PI Absolute P, specifically as follows:
1、PVP-mGB2：
this step provides the following 4 liquids for extracting cell nuclei: PVP k12-mGB2, PVP k15-mGB2, PVP40-mGB2 and PVP70-mGB 2. The method comprises the following specific steps:
1) the PVP k12-mGB2 consists of solute and water, wherein the solute comprises the following components in percentage by weight: na (Na)3C6H5O7·2H2O30mmol/L，MgCl245mmol/L，MOPS 20mmol/L，NaCl 20mmol/L，EDTA-Na220mmol/L, TritonX-1000.1% (v/v), Tween-200.5% (v/v), β -mercaptoethanol 2uL/mL, PVP k 121% (v/v), pH 7.0, 4 ℃ storage, v/v represents volume percentage content.
2) PVP k15-mGB 2: PVP k12 in 1) PVP k12-mGB2 was replaced with PVP k15, and the other components and their concentrations were the same.
3) PVP40-mGB 2: PVP k12 in 1) PVP k12-mGB2 was replaced with PVP40, and the other components and their concentrations were the same.
4) PVP70-mGB 2: PVP k12 in 1) PVP k12-mGB2 was replaced with PVP70, and the other components and their concentrations were the same.
2、LB01
LB01 is composed of solute and water, the solute comprises the following components: tris 15mmol/L, Na2EDTA·2H2O2mmol/L, spermine tetrahydrate 0.5mmol/L, KCl 80mmol/L, NaCl 20mmol/L, Triton X-1000.1% (v/v), β -mercaptoethanol 15mmol/L, pH 7.0-pH 8.0, preserving at-20 deg.C, thawing, and preserving at 4 deg.C.
3、WPS：
The WPS consists of solute and water, wherein the solute comprises the following components in percentage by weight: tris 0.2mol/L, Na2EDTA·2H2O2mmol/L，MgCl2·6H2O4 mmol/L, NaCl 86mmol/L, sodium metabisulfite 10mmol/L, PVP-101% (v/v), TritonX-1001% (v/v), pH 7.5, preservation at-20 deg.C; thawing the mixture when in use, and storing the mixture at 4 ℃.
4、Tris-MgCl2：
Tris-MgCl2The solute comprises the following components in percentage by weight: tris 0.2mol/L, MgCl24mmol/L，Trixon X-1000.5％(v/v)，pH 7.5。
5. Commercialized kit
Figure BDA0002356477540000041
PI Absolute P, brand: sysmex-partec, germany.
Secondly, preparation of cell nucleus extract, nuclear DNA content detection and chromosome ploidy analysis
1. Preparation method of cell nucleus extract
Taking 20mg of plant sample, adding 250uL of the liquid for extracting cell nucleus in the step one, and cutting the leaf blade with a sharp blade at one time (the material is immersed in the liquid for extracting cell nucleus in the whole process) to prevent cell nucleus from breaking, thereby obtaining cell nucleus extract.
2. Nuclear DNA content determination
The cell nuclear extract was filtered through a 30 μm sieve into a flow tube, and a PI stain (propidium iodide) and an RNase solution were added to each achieve a final mass concentration of 50 μ g/mL, and the mixture was left to stand in a dark environment at room temperature for 15min, and then tested on a flow cytometer of the LSRFortessa type (BD Co., U.S.A.) in three replicates per plant material. 10000 nuclei were tested 5000-.
3. Chromosome ploidy analysis
The cell nuclear extract was filtered through a 30 μm sieve into a flow tube, and DAPI stain was added to a final mass concentration of 4 μ g/mL, followed by testing on a flow cytometer of the LSRFortessa type (BD corporation, usa). Three replicates per plant material were performed. 10000 nuclei were tested 5000-.
Thirdly, screening of liquid for extracting cell nucleus
Taking fresh blueberry leaves, and respectively adding different PVP-mGB2 (namely PVP k12-mGB2, PVP k15-mGB2, PVP40-mGB2 and PVP70-mGB2), WPS and Tris-MgCl in the step one2LB01 and commercial kit
Figure BDA0002356477540000051
PIAbsolute P, preparing the nuclear extract of the fresh blueberry leaves according to the preparation method of the nuclear extract in the step two, and then preparing the nuclear extract of the fresh blueberry leaves according to the step twoAnd step two, determining the nuclear DNA content by the method for determining the nuclear DNA content, wherein the result of analysis and detection of the nuclear DNA content by a flow cytometer is shown in figure 1, and the result shows that no obvious DNA peak can be detected by the nuclear extracts extracted by PVP70-mGB2, a slope peak appears on the left side of the diploid peak of the nucleus extracted by LB01, PVP k15-mGB2, Tris-MgCl2 and PVP40-mGB2, and the conjecture is that the nuclear extracting solution has a destructive effect on the nucleus to generate a large amount of cell fragments, the number of the nuclei extracted by WPS is small, and the number of the nuclei collected at the same time is far lower than that of other nuclear extracting. Commercialized kit
Figure BDA0002356477540000052
The test result of the nuclear extract extracted by the PIAbsolute P is better, the test effect of the nuclear extract extracted by the PVPk12-mGB2 is best, the peak shape presents symmetrical distribution, the peak shape is narrow and high, the left cell fragment peak is lower, and the quality and the content of the cell nucleus are best.
Fourth, comparing the effect of the fresh herbaceous plant leaves on extracting the liquid of cell nucleus
PVPk12-mGB2 and commercial kits using step one, respectively
Figure BDA0002356477540000053
PI Absolute P is used for preparing the cell nucleus extract of the fresh leaves of the rice, the tomato and the soybean according to the preparation method of the cell nucleus extract in the second step, then the content of the nuclear DNA is determined according to the method for determining the content of the nuclear DNA in the second step, and the analysis and detection result of the flow cytometry is shown in figure 2. The CV values of the nuclear variation coefficients extracted from PVPk12-mGB2 are less than or equal to 5 percent and are all superior to commercial kits
Figure BDA0002356477540000054
The nuclear CV value extracted by PI Absolute P shows that the quality of the nucleus extracted by PVPk12-mGB2 is obviously higher than that of the commercial kit
Figure BDA0002356477540000055
PI Absolute P。
Fifthly, comparing the effect of the fresh woody and liana leaves on extracting the liquid of cell nucleus
To test the suitability of PVPk12-mGB2 for extracting fluids from cell nuclei, step one PVPk12-mGB2 and a commercial kit were used, respectively
Figure BDA0002356477540000061
PI Absolute P is used for preparing the cell nucleus extracts of the fresh leaves of the honeysuckle, the viburnum sargenti and the fructus schisandrae according to the preparation method of the cell nucleus extracts in the step two, and then the content of the nuclear DNA is determined according to the method for determining the content of the nuclear DNA in the step two. The three plant leaves differ in structure and chemical composition: the honeysuckle leaves contain a large amount of organic acids and flavonoid compounds; the fructus Schisandrae Sphenantherae is rich in lignin, polysaccharide, etc.; the viburnum sargentii has thick leathery texture, thick epidermal cell wall and developed cuticle.
The detection result analyzed by the flow cytometer is shown in figure 3, and the result shows that the kit is commercialized
Figure BDA0002356477540000062
The nuclear extract prepared from PIAbsolute P can not detect the DNA peak of the leaves of Kadsura longepedunculata. The nuclear extract prepared from PVPk12-mGB2 was able to detect DNA peaks. In addition, PVPk12-mGB2 and commercial kits
Figure BDA0002356477540000063
DNA peaks can be detected in the cell nucleus extracts of honeysuckle and viburnum sargentii prepared from PI Absolute P, the peak shapes are symmetrically distributed, the peak shapes are narrow and high, and the cell fragment peak on the left side is lower. The coefficient of variation CV values of DNA of honeysuckle and viburnum sargentii koehne in the cell nucleus extracts of honeysuckle and viburnum sargentii koehne prepared from PVPk12-mGB2 are all less than 5%, and are all superior to commercial kits
Figure BDA0002356477540000064
Extraction efficiency of PIAbsolute P. Illustrating PVPk12-mGB2 for nucleiThe extraction has wide applicability, and can be used for extracting cell nucleuses of different plant species.
Sixthly, comparing the nuclear DNA content determination accuracy of the fresh materials
Separately using PVPk12-mGB2 and a commercial kit
Figure BDA0002356477540000065
PI Absolute P the cell nucleus extract of the fresh fig leaves is prepared according to the preparation method of the cell nucleus extract in the step two, and then the nuclear DNA content is determined according to the method for determining the nuclear DNA content in the step two. Tomato genomic values of 739Mb are known using fresh tomato leaves as an internal control.
The results of the flow cytometry analysis are shown in fig. 4, and the results show that: the two liquids used to extract the nuclei extracted nuclear DNA were similar in content. However, the measured CV values of the coefficients of variation of the nuclear DNA obtained from PVPk12-mGB2 were smaller than those of the commercial kit
Figure BDA0002356477540000066
PI Absolute P shows that the accuracy of DNA content measurement of the cell nucleus obtained by PVPk12-mGB2 is higher, namely the content and the quality of the cell nucleus are higher.
Seventhly, comparison of nuclear DNA content determination accuracy of silica gel drying material
Taking fresh leaves of fig, putting into a self-sealing bag filled with silica gel, standing at room temperature, and completely drying for 12-24 hours to obtain dried leaves of fig for later use.
PVPk12-mGB2 and commercial kits using step one, respectively
Figure BDA0002356477540000067
PI Absolute P the cell nucleus extracts of the dried fig leaves and the fresh fig leaves are prepared according to the preparation method of the cell nucleus extract in the step two, and then the nuclear DNA content is determined according to the method for determining the nuclear DNA content in the step two. Tomato genomic values of 739Mb are known using fresh tomato leaves as an internal control.
The flow cytometry analysis and detection results are shown in fig. 5, and the results show that the nuclear DNA of the dried leaves is extracted from the two kinds of cell nucleus extracting solutions, the ratio of the fluorescence mean value of the G1 peak of the sample to the fluorescence mean value of the G1 peak of the internal control sample is close, the DNA content determination is basically consistent through conversion, and the DNA content determination is not obviously different from the measurement results of fresh leaves of figs and is about 280Mb (table 1).
TABLE 1 results of determination of nuclear DNA content of different materials of Ficus carica by using different plant cell extracts
Figure BDA0002356477540000071
Eighthly, chromosome ploidy analysis of silica gel drying material
Taking fresh leaves of the wild type 6-ploid blueberry, putting the fresh leaves into a self-sealing bag filled with silica gel, standing at room temperature, and completely drying the materials for 12-24 hours to obtain dried leaves of the wild type 6-ploid blueberry for later use.
Preparing the cell nucleus extracts of the wild blueberry 2-fold fresh leaves, the wild blueberry 6-fold fresh leaves and the wild blueberry 6-fold dry leaves by using the PVPk12-mGB2 in the first step according to the preparation method of the cell nucleus extracts in the second step, and then determining the chromosome ploidy by using the chromosome ploidy analysis in the second step.
The flow cytometry analysis and detection results are shown in fig. 6, and the results show that the fluorescence mean values of the silica gel dry leaf and fresh leaf cell nucleus DNA G1 peaks are all at the same position, and the chromosome ploidy analysis is 6-fold. It was demonstrated that the results of the nuclear ploidy measurements of silica gel-dried leaves obtained with PVPk12-mGB2 for the liquid used to extract the nuclei were consistent with those measured with fresh leaf nuclei. Therefore, silica gel dried samples can be used for chromosome ploidy analysis when the PVPk12-mGB2 of the patent is used for extracting liquid of cell nuclei.
Effect of Tween on extraction of cell nuclei
Taking fresh leaves of the tomatoes, putting the fresh leaves into a self-sealing bag filled with silica gel, standing at room temperature, and completely drying the materials for 12-24 hours to obtain dry leaves of the tomatoes for later use.
To optimize the liquid used for extracting the nuclei, three experiments were set, namely a control group without Tween-20 added, a 0.1% Tween-20 group and a 0.5% Tween-20 group.
Preparing a cell nucleus extract of dried leaves of the tomato in the control group by using a liquid for extracting cell nuclei without adding Tween-20 (namely, a liquid obtained by removing solutes from Tween-20 of PVPk12-mGB2 in the step one in an unchanged proportion) according to the preparation method of the cell nucleus extract in the step two; preparing a nuclear extract of dried leaves of tomatoes according to the preparation method of the nuclear extract in the second step by using a liquid for extracting nuclei, which is added in a Tween-20 adding ratio of 0.1% (v/v), in the 0.1% Tween-20 group (namely, a liquid which is obtained by setting the Tween-20 adding ratio of the PVPk12-mGB2 in the first step to 0.1% (v/v) and does not change the ratio of other components in solutes); 0.5% Tween-20 group, using a liquid for extracting cell nucleus (i.e. PVPk12-mGB2 in the first step) added with Tween-20 at a ratio of 0.5% (v/v) to prepare a cell nucleus extract of dried leaf of tomato according to the preparation method of the cell nucleus extract in the second step; then, the nuclear DNA content is determined according to the method for determining the nuclear DNA content in the step two.
The results of the flow cytometry analysis and detection are shown in fig. 7, which shows: the peak shape of the control group is wider, the coefficient of variation CV is larger, while the fluorescent peak shape obtained by the 0.5% Tween-20 group is symmetrical, narrow and high, and the coefficient of variation CV is smaller than that of the control group without Tween-20 and the 0.1% Tween-20 group.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims (10)

1. Liquid for extracting cell nuclei, characterized in that: the liquid consists of Na3C6H5O7·2H2O、MgCl2、MOPS、NaCl、EDTA-Na2Triton X-100, Tween-20, β -mercaptoethanol, PVP k12 and water, wherein the Na is sodium hydroxide3C6H5O7·2H2The content of O in the liquid is 30mmol/L, and the MgCl is adopted245mmol/L of the liquid, 20mmol/L of the MOPS, 20mmol/L of the NaCl, and EDTA-Na2The content in the liquid is 20mmol/L, the content in the liquid of the Triton X-100 is 0.1 percent by volume, the content in the liquid of the Tween-20 is 0.5 percent by volume, the content in the liquid of the β -mercaptoethanol is 2uL/mL, and the content in the liquid of the PVP k12 is 1 percent by volume.
2. Use of the liquid of claim 1 for the preparation of a plant cell nuclear extract.
3. Use of the liquid of claim 1 for increasing the nuclear content and/or quality of a plant cell nuclear extract.
4. Use of the liquid of claim 1 for the preparation of a product for increasing the nuclear content and/or quality of a plant cell nuclear extract.
5. Use of the liquid of claim 1 for preparing a flow cytometer detection sample.
6. A method of preparing a nuclear extract, comprising: the method comprises the following steps:
adding plant material to the liquid of claim 1, and disrupting the plant material in the liquid to obtain the nuclear extract.
7. The method of claim 6, wherein: the plant material is rich in polysaccharide and/or polyphenol;
or the like, or, alternatively,
the plant material is dry material or fresh material.
8. A plant cell nuclear extract prepared by the method of claim 6 or 7.
9. Use of a plant cell nuclear extract according to claim 8 in flow cytometry detection.
Use of PVP k12 and/or Tween-20 for increasing the nuclear content and/or quality in a plant nuclear extract or for the preparation of a product for increasing the nuclear content and/or quality in a plant nuclear extract.
CN202010009185.8A 2020-01-06 2020-01-06 Liquid for extracting cell nucleus and application thereof Active CN111139214B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010009185.8A CN111139214B (en) 2020-01-06 2020-01-06 Liquid for extracting cell nucleus and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010009185.8A CN111139214B (en) 2020-01-06 2020-01-06 Liquid for extracting cell nucleus and application thereof

Publications (2)

Publication Number Publication Date
CN111139214A true CN111139214A (en) 2020-05-12
CN111139214B CN111139214B (en) 2022-03-15

Family

ID=70523773

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010009185.8A Active CN111139214B (en) 2020-01-06 2020-01-06 Liquid for extracting cell nucleus and application thereof

Country Status (1)

Country Link
CN (1) CN111139214B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114277093A (en) * 2021-12-24 2022-04-05 中国农业科学院生物技术研究所 Method for extracting plant cell nucleus
CN114279786A (en) * 2021-12-24 2022-04-05 河南省农业科学院经济作物研究所 Preparation method of sample suitable for cyperus esculentus flow cytometry and cell lysate
CN114277094A (en) * 2021-12-24 2022-04-05 中国农业科学院生物技术研究所 Lysate for extracting plant cell nucleus

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104075983A (en) * 2014-06-06 2014-10-01 中国科学院华南植物园 Method for measuring size of genome of gesneriaceae plant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104075983A (en) * 2014-06-06 2014-10-01 中国科学院华南植物园 Method for measuring size of genome of gesneriaceae plant

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
赖彪 等: "流式细胞仪测定荔枝倍性和基因组大小的细胞核提取液筛选与应用", 《果树学报》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114277093A (en) * 2021-12-24 2022-04-05 中国农业科学院生物技术研究所 Method for extracting plant cell nucleus
CN114279786A (en) * 2021-12-24 2022-04-05 河南省农业科学院经济作物研究所 Preparation method of sample suitable for cyperus esculentus flow cytometry and cell lysate
CN114277094A (en) * 2021-12-24 2022-04-05 中国农业科学院生物技术研究所 Lysate for extracting plant cell nucleus
CN114277093B (en) * 2021-12-24 2024-02-27 中国农业科学院生物技术研究所 Method for extracting plant cell nucleus
CN114277094B (en) * 2021-12-24 2024-02-27 中国农业科学院生物技术研究所 Lysate for extracting plant cell nucleus

Also Published As

Publication number Publication date
CN111139214B (en) 2022-03-15

Similar Documents

Publication Publication Date Title
CN111139214B (en) Liquid for extracting cell nucleus and application thereof
van Bergen et al. Stable carbon isotopes of wood: a clue to palaeoclimate?
Minocha et al. A rapid and reliable procedure for extraction of cellular polyamines and inorganic ions from plant tissues
Ardenghi et al. Leaf wax n-alkane extraction: An optimised procedure
Polovka et al. Offline combination of pressurized fluid extraction and electron paramagnetic resonance spectroscopy for antioxidant activity of grape skin extracts assessment
Duarte et al. Influence of spatial, edaphic and genetic factors on phenols and essential oils of Myrciaria cauliflora fruits
CN107449836A (en) The quick discriminating detection method of Manuka honey
CN107941866B (en) Green vegetable freshness judgment method based on ammonia gas sensitive electrode detection of total ammonia nitrogen dissolution
Nečas et al. Evaluation of non-traditional plum cultivars for growing in the Czech conditions
CN103969323A (en) Method for determining As content in tobacco and tobacco product by utilizing <75>AS<16>O<+>
Rahman Tannins Extraction of Tea Leaves by Ultrasonic Method: Comparison with TheConventional Method
CN102621027A (en) Quantitative detection method for saponin in tea saponin
CN112683832A (en) Method for detecting numb-taste substances in fresh pepper
Andini et al. Analysis and identification of flavonoid compounds in kepok banana corm extract (musa paradisiaca L)
CN106404990A (en) A method of measuring a peroxide value of a tobacco product
CN102180987B (en) Method for extracting corncob polysaccharide
Usta et al. Effects of different site conditions on some of the wood properties of Black alder (Alnus glutinosa (L.) Gaertner subsp. barbata (CA Meyer) Yalt)
CN108645927A (en) A method of it measuring indigo plant using HPLC and spends plumbagin content in pellet
CN105601698B (en) A method of extracting saponin from pecan tree grouts and its husk
CN110954615A (en) Plant sex identification method by using characteristic metabolites
CN104820059A (en) Method for rapidly screening tea tree resource containing EGCG3''Me (Epigallocatechin Gallate)
KR20140041648A (en) Standard marker for determining place of origin or age of processed ginseng, establishing method thereof, or method for determining place of origin or age using the same
CN109293733A (en) A kind of extracting method suitable for fresh tobacco leaves holoprotein
Molnár et al. Anatomical and chemical characteristics of Miocene Taxodiaceae species from Bükkábrány (Hungary)
Vieira Propriedades físicas e mecânicas, características anatômicas e constituintes químicos da madeira de Corymbia citriodora (HooK.) KD Hill & LAS Johnson em três tipos de solo

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant