A method for preparing a plant cell suspension for reducing secondary metabolites

Abstract

The present invention provides a method for preparing a plant cell suspension with reduced secondary metabolites, belonging to the technical field of nucleus extraction. The method for preparing the plant cell suspension of the present invention includes: soaking plant leaves in a treatment solution for 5 - 15 h, and then dissociating in an mG<supgt;b< / supgt; dissociation solution to obtain a nucleus suspension. The treatment solution of the present invention can fully dissociate the nuclei of plant tissues with high contents of polyphenols, polysaccharides or other secondary metabolites in plant leaves, and reduce the influence of secondary metabolites on the detection of nuclear DNA content. The nucleus suspension obtained by the method of the present invention has the characteristics of a large number of injected particles, a large number of collected particles, a small coefficient of variation (CV), and high accuracy in DNA content detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleus extraction, and particularly relates to a method for preparing a plant cell suspension for reducing secondary metabolites. Background Art

[0002] In the process of studying plants, it is often necessary to detect and study factors such as their growth and development and genes. Among them, the study of the genome size and DNA ploidy of plants is extremely closely related to the strain, traits, nutritional and medicinal values of the plant. A flow cytometer uses laser technology and photoelectric measurement technology, combines computer technology and hydrodynamics, and is based on cell immunofluorescence chemistry technology to detect the size, internal structure, DNA content, specific proteins, etc. of cells. This method includes the extraction and fluorescence staining of intact nuclear DNA, and analyzes the DNA content according to the relative fluorescence intensity.

[0003] During the detection process, certain substances in plant tissues have a certain impact on the determination of DNA content. Such substances are small molecule organic compounds that are non-essential for the normal operation of cell life activities or plant growth and development, which are metabolized by plants in the long-term evolution process through interaction with biological or abiotic factors to adapt to the environment, that is, secondary metabolites. They are mainly divided into three categories: nitrogen-containing organic compounds, terpenoids and phenolic compounds, such as pigments, alkaloids, terpenoids, antibiotics, etc. These secondary metabolites are not only difficult to remove, but also mediate the degradation of genomic DNA and wrap and adsorb on the surface of genomic DNA, making the detection of DNA content inaccurate.

[0004] At present, the conventional method for preparing a nucleus suspension has certain limitations for plant tissues with high secondary metabolite content. For example: 1. The cell wall permeability of some reagents is low; 2. Many cell structures may non-specifically bind to the fluorescent label, so that the fluorescence emitted by the fluorescent pigment is difficult to distinguish from the measurement signal. And the secondary metabolites often present in plant cells may interfere with the staining of fluorescent dyes and / or the fluorescence signal; 3. The autofluorescence, viscous substances, etc. of secondary metabolites interfere with the detection of nuclei (driving the irregular movement of particles), thus affecting the determination of the DNA content of plant nuclei; 4. The nylon membrane can only filter some large molecular cell debris and impurities, and it is difficult to filter and remove more small molecular secondary metabolites; 5. Commonly used dissociating solutions such as mG b, WPB, etc., and adding a certain concentration of DTT (a reducing agent and deprotecting agent for thiolated DNA, which reduces the efficiency of some coupling reaction experiments; adding DTT to the DNA solution and reacting for a period of time can reduce the dimerization of DNA), reducing agents such as β-mercaptoethanol, etc., cannot well dissociate the cell nuclei of plant tissues with high polyphenol, polysaccharide or other secondary metabolite contents such as bayberry and rhododendron, resulting in the detection of DNA content being affected. Therefore, there is an urgent need to invent a treatment method to reduce the influence of secondary metabolites in plant tissues on the detection of DNA content. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing a plant cell suspension for reducing secondary metabolites, which can significantly reduce the influence of secondary metabolites in plant tissues on the detection of nuclear DNA content.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] A method for preparing a plant cell suspension for reducing secondary metabolites, the method includes: soaking plant leaves in a treatment solution for 5 - 15 h, and then dissociating in an mG b dissociation solution to obtain a cell nucleus suspension; the treatment solution includes an ethanol solution and / or a PVP10 solution.

[0008] Preferably, the secondary metabolites include polyphenols and / or polysaccharides.

[0009] Preferably, when the secondary metabolite is polyphenol, the treatment solution is an ethanol solution, and the volume percentage content of the ethanol solution is 70% - 80%.

[0010] Preferably, the plants include bayberry and rhododendron.

[0011] Preferably, when the secondary metabolite is polysaccharide, the treatment solution includes an ethanol solution and a PVP10 solution; the preparation method of the treatment solution includes: adding PVP10 to an ethanol solution with a volume percentage content of 70% - 80%; the mass-volume ratio of PVP10 in the treatment solution is 0.5% - 2%.

[0012] Preferably, the plants include kelp.

[0013] Preferably, the mG b dissociation solution includes components with the following concentrations: 40 - 50 mM MgCl2·6H2O, 15 - 25 mM MOPS, 25 - 35 mM sodium citrate, PVP40 with a mass-volume ratio of 0.5% - 2%, Tritonx-100 with a volume ratio of 0.1% - 0.2%, 8 - 12 mM Na2EDTA.

[0014] Preferably, the plant leaves and mG b The mass-volume ratio of the dissociation solution is (30 - 40):(0.5 - 1) mg / mL; the dissociation time is 8 - 12 min.

[0015] Preferably, the dissociation process further includes chopping the plant leaves.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The method for preparing a plant cell suspension of the present invention includes: soaking plant leaves in a treatment solution for 5 - 15 h, and then dissociating in an mG b dissociation solution to obtain a nucleus suspension. The treatment solution of the present invention can fully dissociate the nuclei of plant tissues with high polyphenol, polysaccharide or other secondary metabolite contents in plant leaves, reducing the influence of secondary metabolites on the detection of nuclear DNA content. The nucleus suspension obtained by the method of the present invention has the characteristics of a large number of sample injection particles, a large number of collected particles, a good peak shape, beautiful mapping, and accurate DNA content detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Results of DNA content detection by flow cytometry for Example 1;

[0019] Figure 2 Results of DNA content detection by flow cytometry for Example 3;

[0020] Figure 3 Results of DNA content detection by flow cytometry for Example 4;

[0021] Figure 4 Results of DNA content detection by flow cytometry for Comparative Example 1;

[0022] Figure 5 Results of DNA content detection by flow cytometry for Comparative Example 2;

[0023] Figure 6 Results of DNA content detection by flow cytometry for Comparative Example 3;

[0024] Figure 7 Results of DNA content detection by flow cytometry for Comparative Example 4;

[0025] Figure 8 Results of DNA content detection by flow cytometry for Comparative Example 5;

[0026] Figure 9 Results of DNA content detection by flow cytometry for Comparative Example 6;

[0027] Figure 10 Result of DNA content detection by flow cytometry for Comparative Example 7;

[0028] Figure 11 Result of DNA content detection by flow cytometry for Comparative Example 8;

[0029] Figure 12 Result of DNA content detection by flow cytometry for Comparative Example 9;

[0030] Figure 13 Result of DNA content detection by flow cytometry for Comparative Example 10. Detailed implementation mode

[0031] The present invention provides a method for preparing a plant cell suspension with reduced secondary metabolites, comprising: soaking plant leaves in a treatment solution for 5 - 15 h, and then dissociating in a dissociation solution to obtain a nucleus suspension; the treatment solution comprises an ethanol solution and / or a PVP10 solution. b The dissociation solution is used to dissociate to obtain a nucleus suspension; the treatment solution comprises an ethanol solution and / or a PVP10 solution.

[0032] The method of the present invention can reduce secondary metabolites in the plant cell suspension; the secondary metabolites preferably include polyphenols and / or polysaccharides.

[0033] When the secondary metabolite is polyphenol, the treatment solution is preferably an ethanol solution; the volume percentage content of the ethanol solution is preferably 70% - 80%, more preferably 75%; the soaking time is preferably 5.5 - 7 h, more preferably 6 h; as an implementation mode, the plants that can be selected include Myrica rubra and Rhododendron simsii.

[0034] When the secondary metabolite is polysaccharide, the treatment solution comprises an ethanol solution and a PVP10 solution; the volume percentage content of the ethanol solution is preferably 70% - 80%, more preferably 75%; the mass - volume ratio of the PVP10 solution is preferably 0.5% - 2%, more preferably 1%; as an implementation mode, the preparation method of the treatment solution includes adding 0.02 g of PVP10 powder to 2 mL of an ethanol solution with a volume percentage content of 75%. The soaking time is preferably 10 - 14 h, more preferably 12 h.

[0035] In the present invention, the ethanol solution can effectively fix DNA to prevent cell aggregation and reduce adhesion. The secondary metabolites in the leaves can dissolve in the ethanol solution, which can reduce the influence of secondary metabolites on the detection of nuclear DNA content; PVP10 can strip phenolic substances from proteins and DNA before oxidation and inhibit oxidation. In the present invention, different treatment solutions and different treatment times are used for different plant tissues with different sizes and thicknesses and different secondary metabolites. After treating the leaves, the secondary metabolites in the leaves can be dissolved, the oxidation of DNA can be inhibited, and the influence of secondary metabolites on the nuclear DNA content can be reduced.

[0036] In the present invention, the mG b The dissociation solution preferably comprises components with the following concentrations: 40 - 50 mM MgCl2·6H2O, 15 - 25 mM MOPS, 25 - 35 mM sodium citrate, PVP40 with a mass - volume ratio of 0.5% - 2%, Tritonx - 100 with a volume ratio of 0.1% - 0.2%, 8 - 12 mM Na2EDTA,; more preferably 45 mM MgCl2·6H2O, 20 mM MOPS, 30 mM sodium citrate, PVP40 with a mass - volume ratio of 1%, Tritonx - 100 with a volume ratio of 0.2%, 10 mM Na2EDTA, pH 7.5; The mass - volume ratio of the plant leaves to the mG b The mass - volume ratio of the dissociation solution is preferably (30 - 40):(0.5 - 1) mg / mL, more preferably 35:0.8 mg / mL; The dissociation time is preferably 8 - 12 min, more preferably 10 min; The dissociation process preferably further includes chopping the plant leaves. The dissociation step of the present invention can effectively dissociate cell tissues and release the cell nuclei.

[0037] After the dissociation step, it is preferably further included to filter with a nylon membrane filter with a pore size of 35 - 45 μm, more preferably 40 μm.

[0038] The present invention has no special limitation on the source of the raw materials, and conventional commercially available products in the art can be used.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] The mG used in the examples and comparative examples b The dissociation solution is composed of components with the following concentrations: 45 mM MgCl2·6H2O, 20 mM MOPS, 30 mM sodium citrate, 1% (W / V) PVP40, 0.2% (v / v) Tritonx - 100, 10 mM Na2EDTA, pH 7.5.

[0041] Example 1

[0042] (1) Selection of materials: Select mature bayberry leaves with a length of 8 - 10 cm, gently wipe the surface clean, cut 35 mg of plant tissue, and place it in a clean EP tube for standby;

[0043] (2) Preparation of treatment solution and pretreatment of materials: Add the prepared ethanol solution with a volume percentage of 75% into the tube; place the weighed plant tissue in the ethanol solution and soak for 6 h;

[0044] (3) Take out the treated bayberry leaves, wipe the surface clean and rinse with UP water, place them in 0.8 mL of precooled mG b dissociation solution, quickly and vertically chop the tissue with a sharp blade, place the culture dish on ice for dissociation for 10 min, and gently pipette twice during this period;

[0045] (4) Aspirate the liquid in step (3) and filter it through a nylon membrane filter with a pore size of 40 μm to obtain a nuclear suspension.

[0046] Example 2

[0047] (1) Selection of materials: Select mature rhododendron leaves with a length of 9 - 11 cm, gently wipe the surface clean, cut 35 mg of plant tissue, and place it in a clean EP tube for standby;

[0048] (2) Preparation of treatment solution and pretreatment of materials: Add the prepared ethanol solution with a volume percentage of 75% into the tube; place the weighed plant tissue in the ethanol solution and soak for 6 h;

[0049] (3) Take out the treated rhododendron leaves, wipe the surface clean and rinse with UP water, place them in 0.8 mL of precooled mG b dissociation solution, quickly and vertically chop the tissue with a sharp blade, place the culture dish on ice for dissociation for 10 min, and gently pipette twice during this period;

[0050] (4) Aspirate the liquid in step (3) and filter it through a nylon membrane filter with a pore size of 40 μm to obtain a nuclear suspension.

[0051] Example 3

[0052] (1) Selection of materials: Select the root of mature kelp leaves with a thickness of about 0.3 mm, gently wipe the surface clean, cut 35 mg of plant tissue, and place it in a clean EP tube for standby;

[0053] (2) Preparation of treatment solution and pretreatment of materials: Add 0.02 g of PVP10 powder into 2 mL of ethanol solution with a volume percentage of 75% to obtain a treatment solution; place the weighed plant tissue in the treatment solution and soak for 12 h;

[0054] (3) Take out the processed kelp leaves, wipe their surfaces clean and rinse with UP water, and place them in 0.8 mL of pre-cooled mG b dissociation solution. Use a sharp blade to quickly and vertically chop the tissue, place the petri dish on ice for dissociation for 10 min, and gently pipette twice during this period;

[0055] (4) Aspirate the liquid from step (3) and filter it through a nylon membrane filter with a pore size of 40 μm to obtain a nuclear suspension.

[0056] Example 4

[0057] The specific implementation method is the same as that of Example 1, except that the bayberry leaves are changed to rhododendron leaves.

[0058] Comparative Example 1

[0059] The specific implementation method is the same as that of Example 1, except that the treatment solution is absolute ethanol.

[0060] Comparative Example 2

[0061] The specific implementation method is the same as that of Example 1, except that the treatment solution is Carnoy's fixative.

[0062] Comparative Example 3

[0063] The specific implementation method is the same as that of Example 1, except that step (2) is not carried out.

[0064] Comparative Example 4

[0065] The specific implementation method is the same as that of Example 3, except that the treatment solution is 75% ethanol solution.

[0066] Comparative Example 5

[0067] The specific implementation method is the same as that of Example 3, except that the treatment solution is 75% ethanol solution and 1.5% PVP10.

[0068] Comparative Example 6

[0069] The specific implementation method is the same as that of Example 3, except that the treatment solution is 75% ethanol solution and 2% PVP10.

[0070] Comparative Example 7

[0071] The specific implementation method is the same as that of Example 3, except that step (2) is not carried out.

[0072] Comparative Example 8

[0073] The specific implementation method is the same as that of Example 4, except that the treatment solution is absolute ethanol.

[0074] Comparative Example 9

[0075] The specific implementation manner is the same as that of Example 4, except that the treatment solution is Carnoy's fixative.

[0076] Comparative Example 10

[0077] The specific implementation manner is the same as that of Example 4, except that step (2) is not carried out.

[0078] Experimental Example 1

[0079] Determining the genome size by flow cytometry (FCM) has been developed in recent years and has gradually become a commonly used method. The intact cell nuclei of the sample pass through the laser of the flow cytometer in a fast-flowing state, so as to obtain data parameters such as the size, shape, internal complexity and fluorescence signal of the sample. By comparing the fluorescence intensities of the reference plant and the sample, the DNA content of the sample to be measured is calculated. The specific calculation formula is as follows: Genome size of the sample to be measured = Fluorescence intensity of the sample to be measured / Fluorescence intensity of the reference plant × Genome size of the reference plant. This method is simple and easy to operate, and the detection results are relatively stable and reliable.

[0080] (1) Add 25 μL of pre-cooled propidium iodide PI (working concentration 50 μg / mL) and 25 μL of RNase solution (working concentration 50 μg / mL) to 0.5 mL of the cell nucleus suspension of Example 1, Example 3, and Comparative Examples 1-7 respectively, and place it on ice for dark staining for 0.5-1 h;

[0081] (2) Use a BD FACScalibur flow cytometer to detect the stained cell nucleus suspension sample on the machine, excite it with 488 nm blue light, and detect the fluorescence intensity of the emitted light of propidium iodide. Each detection is carried out at the same speed and the fluorescence intensity of the emitted light of propidium iodide is collected within the same time. Use Cell Quest analysis software to plot and analyze;

[0082] (3) Obtain a scatter plot, a flow cytometer plot of plant cells (R1 cell population), and a single-parameter histogram (with a high and sharp peak at the position) through plotting. The specific results are shown in Tables 1-2 and Figures 1-13 .

[0083] Table 1 Collection of waxberry cell nuclei in each treatment

[0084]

[0085] Table 2 Collection of kelp cell nuclei in each treatment

[0086]

[0087] From Table 1 and Figure 1 、 Figures 4-6It can be seen that the material is waxberry. Compared with Comparative Examples 1-3, Example 1 has a faster sample loading speed, more target cell nuclei collected, a smaller CV (coefficient of variation, the smaller the CV, the more complete and specific the cell nuclei), the best effect of effectively separating cells, and more accurate DNA content detection; from Table 2 and Figures 7-10 It can be seen that the material is kelp. Compared with Comparative Examples 4-7, Example 3 has a faster sample loading speed, more target cell nuclei collected, a smaller CV (coefficient of variation, the smaller the CV, the more complete and specific the cell nuclei), the best effect of effectively separating cells, and more accurate DNA content detection.

[0088] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a plant cell suspension for reducing secondary metabolites, characterized in that, The method includes: soaking plant leaves in a treatment solution for 5 - 15 h, and then dissociating in a dissociation solution b to obtain a nuclear suspension; b ​ The treatment solution includes an ethanol solution and / or a PVP10 solution; The secondary metabolites include polyphenols and / or polysaccharides; When the secondary metabolite is polyphenol, the treatment solution is an ethanol solution, and the volume percentage content of the ethanol solution is 70%-80%; When the secondary metabolite is polysaccharide, the treatment solution includes an ethanol solution and a PVP10 solution; the preparation method of the treatment solution includes: adding PVP10 to an ethanol solution with a volume percentage content of 70%-80%; the mass-volume ratio of PVP10 in the treatment solution is 0.5%-2%.

2. The method for preparing a plant cell suspension according to claim 1, wherein When the secondary metabolite is polyphenol, the plants include Chinese bayberry and rhododendron.

3. The method for preparing a plant cell suspension according to claim 1, wherein When the secondary metabolite is polysaccharide, the plant includes kelp.

4. The method for preparing a plant cell suspension according to claim 1, wherein The mG b The dissociation solution comprises components with the following concentrations: 40 - 50 mM MgCl2·6H2O, 15 - 25 mM MOPS, 25 - 35 mM sodium citrate, PVP40 with a mass - volume ratio of 0.5% - 2%, Tritonx - 100 with a volume ratio of 0.1% - 0.2%, and 8 - 12 mM Na2EDTA.

5. The method for preparing a plant cell suspension according to claim 4, wherein, The plant leaf and mG b The mass-volume ratio of the dissociation solution is (30-40):(0.5-1) mg / mL; the dissociation time is 8-12 min.

6. The method for preparing a plant cell suspension according to claim 1, characterized in that, The dissociation process also includes chopping the plant leaves.

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