CN111139214B - Liquid for extracting cell nucleus and application thereof - Google Patents
Liquid for extracting cell nucleus and application thereof Download PDFInfo
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- 239000007788 liquid Substances 0.000 title abstract description 48
- 239000000284 extract Substances 0.000 claims abstract description 68
- 239000000463 material Substances 0.000 claims abstract description 32
- 229920001213 Polysorbate 20 Polymers 0.000 claims abstract description 25
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims abstract description 25
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims abstract description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 238000002360 preparation method Methods 0.000 claims abstract description 15
- 229910001629 magnesium chloride Inorganic materials 0.000 claims abstract description 10
- 239000011780 sodium chloride Substances 0.000 claims abstract description 8
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007993 MOPS buffer Substances 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000013504 Triton X-100 Substances 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims abstract description 5
- 229920004890 Triton X-100 Polymers 0.000 claims abstract description 3
- 239000001509 sodium citrate Substances 0.000 claims abstract description 3
- 241000196324 Embryophyta Species 0.000 claims description 49
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- 240000000851 Vaccinium corymbosum Species 0.000 claims description 16
- 235000003095 Vaccinium corymbosum Nutrition 0.000 claims description 16
- 235000017537 Vaccinium myrtillus Nutrition 0.000 claims description 16
- 235000021014 blueberries Nutrition 0.000 claims description 16
- 210000004027 cell Anatomy 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 10
- 241000205585 Aquilegia canadensis Species 0.000 claims description 9
- 235000013250 Viburnum opulus var sargentii Nutrition 0.000 claims description 8
- 240000004861 Viburnum opulus var. sargentii Species 0.000 claims description 8
- 244000068988 Glycine max Species 0.000 claims description 5
- 235000010469 Glycine max Nutrition 0.000 claims description 5
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- 238000004458 analytical method Methods 0.000 abstract description 12
- 150000004676 glycans Chemical class 0.000 abstract description 5
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- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 abstract description 3
- 108091093105 Nuclear DNA Proteins 0.000 description 26
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 14
- 240000003768 Solanum lycopersicum Species 0.000 description 14
- 239000000741 silica gel Substances 0.000 description 13
- 229910002027 silica gel Inorganic materials 0.000 description 13
- 210000000349 chromosome Anatomy 0.000 description 12
- 238000000684 flow cytometry Methods 0.000 description 12
- 210000004940 nucleus Anatomy 0.000 description 11
- 230000000694 effects Effects 0.000 description 9
- 238000001035 drying Methods 0.000 description 8
- 238000000605 extraction Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 244000025361 Ficus carica Species 0.000 description 4
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- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000561709 Kadsura longipedunculata Species 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- PFNFFQXMRSDOHW-UHFFFAOYSA-N Spermine Natural products NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 1
- 244000071378 Viburnum opulus Species 0.000 description 1
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- 210000001339 epidermal cell Anatomy 0.000 description 1
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- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 229940001584 sodium metabisulfite Drugs 0.000 description 1
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- 229940063675 spermine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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- 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
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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‑Na2、Triton X-100, Tween-20, beta-mercaptoethanol, PVP k12 and water. The invention further discloses application of the plant cell nucleus extract in preparation of plant cell nucleus extracts. The invention firstly uses PVP k12 and Tween-20 to prepare the liquid for extracting the cell nucleus, so that the liquid can extract the cell nucleus with enough quantity and quality for the detection and analysis of the flow cytometer 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
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, beta-mercaptoethanol, PVP k12 and water; the Na is3C6H5O7·2H2The content of O in the liquid is 30mmol/L, and the MgCl is adopted2The content of the MOPS in the liquid is 45mmol/LThe content of (A) is 20mmol/L, the content of the NaCl in the liquid is 20mmol/L, and the EDTA-Na2The content of the beta-mercaptoethanol in the liquid is 20mmol/L, the content of the Triton X-100 in the liquid is 0.1 percent by volume, the content of the Tween-20 in the liquid is 0.5 percent by volume, the content of the beta-mercaptoethanol in the liquid is 2uL/mL, and the content of the PVP k12 in the liquid 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 PVP k12, 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
extraction of grass from PI Absolute PFlow chart of nuclear DNA effect of fresh leaves of the plant.
FIG. 3 shows PVPk12-mGB2 and
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:
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:
PI Absolute P is used for extracting liquid extracted fig silica gel dry leaf nucleus DNA of 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
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)3C6H5O7·2H2O 30mmol/L，MgCl2 45mmol/L，MOPS 20mmol/L，NaCl 20mmol/L，EDTA-Na220mmol/L, Triton X-1000.1% (v/v), Tween-200.5% (v/v), beta-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·2H2O2 mmol/L, spermine tetrahydrate 0.5mmol/L, KCl 80mmol/L, NaCl 20mmol/L, Triton X-1000.1% (v/v), beta-mercaptoethanol 15mmol/L, pH 7.0-pH 8.0, preservation at-20 deg.C; thawing the mixture when in use, and storing the mixture at 4 ℃.
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·2H2O 2mmol/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
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
PI Absolute P, preparing the cell nucleus extract of the blueberry fresh leaves according to the preparation method of the cell nucleus extract in the step two, then determining the content of the nuclear DNA according to the method for determining the content of the nuclear DNA in the step two, and analyzing and detecting the result by a flow cytometer as shown in figure 1, wherein the result shows that the cell nucleus extract extracted by PVP70-mGB2 cannot detect an obvious DNA peak, and a slope peak appears on the left side of a cell nucleus diploid peak extracted by LB01, PVP k15-mGB2, Tris-MgCl2 and PVP40-mGB2, and the cell nucleus extract is supposed to have a destructive effect on the cell nucleus and generate a large amount of cell fragments, the number of the cell nucleus extracted by WPS is small, and the number of the cell nuclei collected at the same time is far lower than that of other cell nucleus extracts. Commercialized kit
The test result of the nuclear extract extracted by PI Absolute P is better, the test effect of the nuclear extract extracted by PVPk12-mGB2 is best, the peak shape is symmetrically distributed, 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-m using step one, respectivelyGB2 and commercial kit
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
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
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
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 results of the flow cytometry analysis and detection are shown in FIG. 3, and the results are displayed and commercializedReagent kit
The DNA peak of the leaves of Kadsura longipedunculata can not be detected by the nuclear extract prepared by PI Absolute P. The nuclear extract prepared from PVPk12-mGB2 was able to detect DNA peaks. In addition, PVPk12-mGB2 and commercial kits
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
Effect of extraction of PI Absolute P. The PVPk12-mGB2 is proved to have wide applicability to the extraction of cell nuclei and can be used for the extraction of the cell nuclei of different plant species.
Sixthly, comparing the nuclear DNA content determination accuracy of the fresh materials
Separately using PVPk12-mGB2 and a commercial kit
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
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
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
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 (7)
1. An extract of cell nuclei, comprising: the extract is composed of Na3C6H5O7·2H2O、MgCl2、MOPS、NaCl、EDTA-Na2Triton X-100, Tween-20, beta-mercaptoethanol, PVP k12 and water; the Na is3C6H5O7·2H2The content of O in the extracting solution is 30mmol/L, and the MgCl is adopted245mmol/L of the extractive solution, 20mmol/L of the MOPS, 20mmol/L of the NaCl, and EDTA-Na2The content of the extracting solution is 20mmol/L, the volume percentage content of the Triton X-100 in the extracting solution is 0.1 percent, the volume percentage content of the Tween-20 in the extracting solution is 0.5 percent, the content of the beta-mercaptoethanol in the extracting solution is 2uL/mL, and the volume percentage content of the PVP k12 in the extracting solution is 1 percent.
2. Use of the extract of claim 1 for the preparation of a plant cell nuclear extract; the plant is blueberry, rice, tomato, soybean, honeysuckle, viburnum sargentii koehne, fructus Schisandrae Sphenantherae or fig.
3. Use of the extract of claim 1 for increasing the nuclear content and/or quality of a plant cell nuclear extract; the plant is blueberry, rice, tomato, soybean, honeysuckle, viburnum sargentii koehne, fructus Schisandrae Sphenantherae or fig.
4. Use of the extract of claim 1 for the preparation of a product for increasing the nuclear content and/or quality of a plant cell nuclear extract; the plant is blueberry, rice, tomato, soybean, honeysuckle, viburnum sargentii koehne, fructus Schisandrae Sphenantherae or fig.
5. Use of the extract 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 extract of claim 1, and disrupting the plant material in the extract to obtain a nuclear extract;
the plant is blueberry, rice, tomato, soybean, honeysuckle, viburnum sargentii koehne, fructus Schisandrae Sphenantherae or fig.
7. The method of claim 6, wherein: the plant material is dry material or fresh material.
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