Plant flow cytometric nuclear dissociation solution and use thereof

Abstract

The application discloses a plant flow cytometry nucleus dissociation solution and application thereof, and belongs to the technical field of biology. The nucleus dissociation solution comprises NP-40 or an analogue thereof, and further comprises sodium lauroyl glutamate, and can be used in plant DNA flow cytometry analysis such as DNA content determination, ploidy analysis and cell cycle analysis. The plant nucleus dissociation solution has high nucleus dissociation efficiency, few impurity fragments and high accuracy, and related application is based on flow cytometry, and has wide plant applicability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a plant flow cytometry nuclear dissociation solution and application thereof. BACKGROUND

[0002] Flow cytometry (FCM) is a technology for multi-parameter quantitative analysis and sorting of micro-organisms (cells, nuclei, chromosomes, etc.) in rapid flow, and plays an increasingly important role in life science research, medical research and clinical practice. FCM is mainly applied to plant genome size detection, DNA content determination, ploidy detection and cell cycle, etc., and the principle is to label nuclei with specific DNA fluorescent dye (PI, DAPI, etc.), to identify and count the stained particles (i.e. theoretically labeled nuclei) by flow cytometry, and to evaluate the DNA content of G0 / G1 phase according to the signal peak value. The experimental process includes: selecting plant tissues (generally leaves, stems, roots and seeds, etc.), preparing and optimizing the nuclear dissociation solution, cutting the tissues to prepare the nuclei suspension, staining with DNA fluorescent dye, using flow cytometry and data analysis, etc. The preparation of the nuclei suspension is a decisive step that affects the quality (accuracy and effectiveness) of FCM analysis. The key indicators of high-quality nuclei suspension include: (1) sufficient number of intact free nuclei; (2) less cell debris; (3) less interference of cell contents to the specificity of DNA fluorescent dye; (4) small coefficient of variation (CV), etc. Therefore, it is necessary to develop and optimize the formula of plant nuclear dissociation solution, so as to control these key indicators.

[0003] The roles played by different components of the plant nuclear dissociation solution should include but not limited to: (1) lysing cell wall and cell membrane; (2) maintaining osmotic pressure; (3) maintaining the integrity of the nuclei; (4) maintaining a specific range of pH; (5) dispersing cell debris and free nuclei; (6) inhibiting the interference of intracellular oxidants, pectin and polysaccharides to the dye.

[0004] The accuracy of FCM analysis is represented by the coefficient of variation (CV). CV = standard deviation / average peak value x 100%. The smaller the CV value, the more accurate the DNA content analysis. Generally, the CV should be less than 5%. Therefore, an efficient plant nuclear dissociation solution should reflect a smaller CV value of the analysis results.

[0005] At least 7 plant nuclear dissociation solutions have been screened and published since FCM was used in plants (Tian Xinmin, Zhou Xiangyan, Gong Na. Application of flow cytometry in plant research: detection of plant nuclear DNA content and ploidy level [J]. China Plant Protection, 2011, 27(09): 21-27.), and multiple authorized patents.

[0006] Due to the objective reasons such as the variety of plant species, the existence of cell wall, the large difference in plant cell tissue structure from different plants or different tissues, and the richness of plant cell contents (such as starch, polysaccharide, pectin, polyphenol, tannin and other complex secondary metabolites), the existing public formula does not have universal applicability, that is, it cannot be applied to multiple plant materials at the same time. Therefore, at present, before FCM analysis of new plants, the operator needs to test different dissociation solutions to screen the most suitable formula. DNA flow cytometry is increasingly used in plant genetics and breeding at present and in the future. Therefore, the development of plant cell nuclear dissociation solution with wide applicability or universality still has urgent application value.

[0007] The patents related to the plant nuclear dissociation solution have been granted: (1) The nuclear extraction buffer and the method for determining the genome size of the Piper plant (CN110514495A). This patent is only applicable to the Piper plant. Although the application of this nuclear extraction buffer has obtained a smaller CV value; however, it does not provide the staining observation of the nucleus and the negative control without DNA fluorescent dye, i.e., there is no evidence to show that the selection of components improves the quality of the nucleus and reduces the influence of the fragment peak. Tween-20 and Triton X-100 are commonly used detergents for nuclear dissociation solution. Moreover, only Triton X-100 is used in this formula. (2) Liquid for extracting nuclei and its application (CN111139214A). This patent first tests the improvement effect of different concentrations of PVP k12 and the detergent Tween-20 on the nuclear extraction solution. PVP (polyvinyl alcohol pyrrolidone) is used to reduce the influence of oxidative substances in plant cells, and has different molecular weights (PVP k12, PVP 40, PVP k15, PVP 30, etc.). This patent only replaces the commonly used reagents PVP 40 or PVP k15 in the existing formula with PVP k12. Tween-20 and Triton X-100 are commonly used detergents for nuclear dissociation solution. Among the 9 published formulas (Jin L, Xu W, Li X B, Liu J X, Tian D Q, Ge Y Y, Pan X Y, Wang W Y. Application of DNA flow cytometry in plant genetics and breeding [J]. Chinese Journal of Cell Biology, 2016, 38(02): 225-234.), 7 use Triton X-100 and 2 use Tween-20. Therefore, this formula does not carry out a deep comparative test of detergents. In fact, the current relatively mature plant FCM operation steps can be completely applicable to dry samples (Suda J, P. Reliable DNA ploidy determination in dehydrated tissues of vascular plants by DAPI flow cytometry—new prospects for plant research[J]. Cytometry Part A, 2006, 69(4): 273-280.) In addition, although the invention patent tested figs and blueberries, it did not include more complex plants (such as medicinal plants containing complex cellular secondary metabolic inclusions), so it cannot well prove the broad applicability of the formula.

[0008] In summary, in order to obtain high-quality cell nucleus suspension to ensure the accuracy of FCM, it is necessary to improve the plant cell nucleus dissociation solution formula, which requires: (1) more gentle and complete lysis of cells, reduction of inclusion (fragment and secondary metabolite) inhibition effect and maintenance of cell nucleus integrity, (2) more extensive applicability. Under the condition of maintaining appropriate ionic strength, it is realized by further optimizing the components and formula of the detergent.

[0009] Giant knotweed is a traditional Chinese medicinal plant. The leaves of giant knotweed are difficult to cut due to their leathery texture, and are rich in polyphenols, polysaccharides and sticky substances. These characteristics will result in the prepared cell nucleus suspension showing many impurities and fragments, low cell nucleus extraction efficiency, and low accuracy of FCM results. Therefore, on the one hand, the FCM analysis of giant knotweed needs to solve these problems, and on the other hand, the complexity of giant knotweed provides a good test material for the research and development and improvement of plant cell dissociation solution.

[0010] Among the currently disclosed cell nucleus dissociation solution formulas, the commonly used detergents are only Tween-20 and Triton X-100. NP-40 (Nonidet P-40) is a non-ionic surfactant, although it is commonly used in protein extraction, but it has not been used for the preparation of plant flow cytometry samples. SUMMARY

[0011] The purpose of the present application is to overcome the problems of low cell nucleus extraction efficiency, poor accuracy and poor applicability of the existing plant cell nucleus dissociation solution, and to provide a plant cell dissociation solution with high cell nucleus extraction efficiency, good accuracy and extensive applicability, and the application thereof in plant flow cytometry detection.

[0012] The purpose of the present application is achieved by the following technical solutions:

[0013] The present application discloses a plant cell nuclear dissociation solution prepared by using NP-40 (Nonidet P-40) as a detergent, which is used for lysing plant cells for flow cytometry analysis. The effect is significantly better than that of known detergents Tween-20 and Triton X-100. The addition of sodium lauroyl glutamate in the plant cell nuclear dissociation solution can further improve the flow cytometry analysis results. The plant cell nuclear dissociation solution containing NP-40 and sodium lauroyl glutamate is suitable for multiple plant species, including but not limited to Polygonum cuspidatum, Rehmannia glutinosa, Antirrhinum majus, Sorghum bicolor, Scrophularia ningpoensis, Ocimum basilicum, Zea mays, Nicotiana tabacum, Panacis majoris Rhizoma, Polygonatum sibiricum, Allium sativum and Lilium brownii, and plants with different characteristics.

[0014] Based on the findings of the present application, the present application provides the following uses, products or methods.

[0015] Use of NP-40 or its analogues in the preparation of a plant cell nuclear dissociation solution for flow cytometry analysis. The NP-40 analogues include CA-630 (Sigma-Aldrich), CA-630 is a substitute for NP-40, which has the same chemical properties as NP-40.

[0016] Use of NP-40 or its analogues and sodium lauroyl glutamate in the preparation of a plant cell nuclear dissociation solution for flow cytometry analysis.

[0017] A plant cell nuclear dissociation solution comprising NP-40 or its analogues and sodium lauroyl glutamate. Preferably, the concentration of NP-40 or its analogues is 0.5-2% (w / v), and the concentration of sodium lauroyl glutamate is 0.2-5% (w / v).

[0018] In some embodiments, the plant cell nuclear dissociation solution comprises the following components: 20-200 mM MgCl2, 20-50 mM sodium citrate, 5-50 mM MOPS, 5-50 mM Na2EDTA, 0.2-2% (w / v) PVP-40, 0.5-2% (w / v) NP-40, 0.2-5% (w / v) sodium lauroyl glutamate, 10-100 μL / mL β-mercaptoethanol and H2O, pH 7.0. Further, the plant cell nuclear dissociation solution comprises the following components: 45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 10 mM Na2EDTA, 1% PVP-40, 1% NP-40, 0.5% sodium lauroyl glutamate, 20 μL / mL β-mercaptoethanol and H2O, pH 7.0.

[0019] The plant cell nucleus dissociation solution is applied in plant DNA flow analysis and cell cycle analysis. The plant includes but is not limited to Polygonum cuspidatum, Rehmannia glutinosa, Antirrhinum majus, Sorghum bicolor, Scrophularia ningpoensis, Lamiophlomis rotata, Zea mays, Nicotiana tabacum, Panax japonicus, Polygonatum sibiricum, Allium sativum and Lilium brownii and plants with different characteristics.

[0020] The method for using the plant cell nucleus dissociation solution includes the following steps: placing a plant sample in a pre-cooled plant cell nucleus dissociation solution, chopping and placing on ice for 10-30 minutes.

[0021] The present application has the following advantages and effects relative to the prior art:

[0022] (1) NP-40 is used to replace Tween-20 or Triton X-100 in the existing formula, and better nucleus extraction effect and flow out peak effect are achieved.

[0023] (2) The combination of 0.5% sodium lauroyl glutamate and 1% NP-40 improves the plant nucleus extraction efficiency and FCM accuracy.

[0024] (3) The plant cell nucleus dissociation solution formula disclosed in the present application is superior to the existing formula and the commercialized formula on the market, and has more extensive applicability. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is to test and compare the effects of using different formulas to carry out flow cytometry cell nucleus suspension preparation and FCM analysis of Polygonum cuspidatum leaves. (A) The appearance of the cell nucleus suspension prepared by different formulas in a plastic test tube. (B) The fluorescence microscope photos of the cell nucleus suspension prepared by different formulas after being labeled by DNA fluorescent dye PI, showing specifically dyed cell nuclei and non-specific dyed cell (impurity) fragments. (C) The peak chart of the flow cytometer after loading the cell nucleus suspension without dye, showing the count value of the fragment particles. (D) The peak chart of the flow cytometer after loading the cell nucleus suspension after PI staining, showing the cell nucleus peak type and count value circled in the fragment particles.

[0026] Figure 2 is to test the influence of replacing the detergent with 0.5% sodium lauroyl glutamate on cell nucleus extraction and flow analysis results.

[0027] Figure 3This study investigated the effects of adding different detergents and 0.5% sodium lauroyl glutamate on cell nucleus extraction and flow cytometry analysis results. (A) Fluorescence micrographs of cell nucleus suspensions prepared with different formulations after labeling with the DNA fluorescent dye PI, showing specifically stained cell nuclei and non-specifically stained cell (impurity) debris. (B) Peak patterns of the PI-stained cell nucleus suspension loaded onto a flow cytometer, showing the peak shape and count value of cell nuclei circled in the debris particles. (C) Mean fluorescence and CV values ​​under different detergents and combinations with 0.5% sodium lauroyl glutamate.

[0028] Figure 4 The effects of the plant cell nucleus dissociation solution formulation of this invention (based on a combination of 0.5% sodium lauroyl glutamate and 1% NP-40) and a commercially available formulation on flow cytometry were tested and compared in two medicinal plants, Polygonum cuspidatum and Rehmannia glutinosa. (A) Polygonum cuspidatum. Histograms, scatter plots, and density plots are shown. (B) Rehmannia glutinosa. Histograms, scatter plots, and density plots are shown. (C) Summary table of mean fluorescence and CV values.

[0029] Figure 5 The effects of using the plant cell nucleus dissociation solution formulation of the present invention (based on a combination of 0.5% sodium lauroyl glutamate and 1% NP-40) in flow cytometry analysis on different plants.

[0030] Figure 6 Cell cycle analysis was performed on different tea varieties using the plant cell nucleus dissociation formulation of the present invention (based on a combination of 0.5% sodium lauroyl glutamate and 1% NP-40). Detailed implementation method:

[0031] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0032] The following examples illustrate the sample collection, preparation, and handling methods: Take fresh plant leaves (approximately 2×2cm in size) 2) wipe clean and place in a cell culture dish. Place the dish on ice and add 750 μL of pre-chilled nuclear dissociation solution to the dish. Quickly mince the leaves with a double-edged razor blade and place on ice for 10-30 minutes to release the nuclei and prepare a crude extract. Filter using a flow cytometry specific filter screen (100 mesh) and collect the nuclear suspension. Take a portion of the nuclear suspension and stain with 1 mg / mL PI (with RNase) and place under a fluorescence microscope to observe the staining of the nuclei. Take the remaining nuclear suspension and either do not stain or stain with 1 mg / mL PI (with RNase) and place on ice in the dark for 10 minutes before loading into a flow cytometer for analysis. Follow the flow cytometer's instructions for operation. Finally, analyze the data according to the specifics of the data recorded and perform post-data statistical analysis and interpretation.

[0033] Example 1. Test and compare the effects of using different formulations to prepare and analyze Polygonum cuspidatum leaf flow cytometry nuclear suspensions.

[0034] Seven plant nuclear dissociation solutions were prepared according to published formulations. The formulations are detailed in Table 1. Using equal amounts of P. cuspidatum leaves, seven nuclear suspensions were prepared according to the procedure. It was found that the nuclear suspensions prepared in plastic test tubes using different formulations showed different colors. In three formulations, LB01, Tris-MgCl2, and WPB, the nuclear suspensions showed a darkened brown color, indicating that the intracellular contents tended to oxidize in these formulations Figure 1 A). Under a fluorescence microscope, the PI-labeled nuclear suspensions prepared using different formulations showed that in addition to the specific staining of the nuclei (spherical), there were also non-specifically stained cell (impurity) fragments (present as irregular, poorly dispersed particles) Figure 1 B). The nuclear suspensions were then divided into a control group without dye and an observation group stained with 1 mg / mL PI (with RNase). The flow cytometer was operated according to the manufacturer's instructions. The data were recorded, and post-data statistical analysis and interpretation were performed. It was found that the control group's peak graph showed a large number of non-specific fragment particles, which would interfere with the recognition of the nuclear particles Figure 1 C). Compared with the control, the nuclear peak pattern and count value after PI staining could be circled and recorded from the fragment particles Figure 1 D). The results showed that the effects of P. cuspidatum samples prepared using different dissociation solutions for flow cytometry detection were very different. The GPB and classic Galbraith formulations could only obtain cell fragments and could not peak at all, while the mG formulation could see a larger number of free nuclei and peak significantly, showing the best performance. Of these formulations, six used Triton X-100 as a detergent, and only one used Tween-20. In the nuclear staining observation, a large number of fragments were still seen.

[0035] Table 1

[0036]

[0037]

[0038] Example 2 To test the effect of replacing the detergent Triton X-100 in the mG formulation with 0.5% sodium lauroyl glutamate in preparing a nuclei suspension.

[0039] A nuclei dissociation solution containing 0.5% sodium lauroyl glutamate was prepared. The formulation was: 45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 10 mM Na2EDTA, 1% (w / v) PVP-40, 0.5% sodium lauroyl glutamate, 20 μL / mL β-mercaptoethanol, and H2O, pH 7.0.

[0040] A flow cytometry nuclei suspension of Polygonum cuspidatum leaf pieces was prepared.

[0041] The procedure was the same as in Example 1. The results showed that 0.5% sodium lauroyl glutamate alone did not have the effect of dissociating nuclei after replacing the detergent, and could not produce a peak in FCM Figure 2 ), so it could not replace the detergent.

[0042] Example 3 To test the effect of adding different detergents and 0.5% sodium lauroyl glutamate on nuclei extraction and flow cytometry analysis results.

[0043] Different nuclei dissociation solution formulations were prepared. The common components of all formulations were: 45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 10 mM Na2EDTA, 1% (w / v) PVP-40, 20 μL / mL β-mercaptoethanol, and H2O, pH 7.0. On this basis, different detergents were added to prepare 8 comparison formulations: (1) 0.2% Triton X-100; (2) 1% Triton X-100; (3) 1% Tween-20; (4) 1% NP-40; (5) 0.2% Triton X-100 + 0.5% sodium lauroyl glutamate; (6) 1% Triton X-100 + 0.5% sodium lauroyl glutamate; (7) 1% Tween-20 + 0.5% sodium lauroyl glutamate; (8) 1% NP-40 + 0.5% sodium lauroyl glutamate.

[0044] A flow cytometry nuclei suspension of Polygonum cuspidatum leaf pieces was prepared.

[0045] The experimental operation steps are the same as those in Example 1. On the basis of the mG formula, the effects of Tween-20, Triton X-100 and NP-40 (Nonidet P-40) on the extraction of nuclei and the FCM peak results were tested. The results show that the cell nucleus suspension prepared by 1% NP-40 has the least fragments and the smallest CV value Figure 3 ). The addition of 0.5% sodium lauroyl glutamate in the plant cell nucleus dissociation solution formula does not affect the original effect of the detergent when combined with any one of Tween-20 and Triton X-100. However, when combined with NP-40, the effect of the plant cell nucleus dissociation solution formula based on 0.5% sodium lauroyl glutamate and 1% NP-40 can be significantly improved, the cell nucleus suspension has fewer fragments, and the CV value is further reduced Figure 3 ).

[0046] Example 4: Test and compare the effects of the plant cell nucleus dissociation solution formula of the present application (based on 0.5% sodium lauroyl glutamate and 1% NP-40) and commercially available dissociation solutions in flow cytometry analysis.

[0047] The commercially available dissociation solution is CyStain UV Precise P (Sysmex Partec).

[0048] The usage, dosage, and operation steps are consistent with those in Example 1.

[0049] In two medicinal plants, Polygonum cuspidatum and Rehmannia glutinosa, the effects of the two in flow cytometry analysis were compared.

[0050] The results show that compared with the commercially available flow reagent CyStain UV Precise P (Sysmex Partec), the effect of the plant cell nucleus dissociation solution formula based on 0.5% sodium lauroyl glutamate and 1% NP-40 is better than that of the commercially available flow reagent, regardless of whether the test sample is Polygonum cuspidatum or Rehmannia glutinosa Figure 4 ).

[0051] Example 5: Effects of the plant cell nucleus dissociation solution formula of the present application (based on 0.5% sodium lauroyl glutamate and 1% NP-40) in flow cytometry analysis in different plants.

[0052] In order to demonstrate the wide applicability of the plant cell nucleus dissociation solution formula of the present application in different plants, in addition to the above-mentioned Polygonum cuspidatum and Rehmannia glutinosa, plants with different characteristics such as Antirrhinum majus, Sorghum bicolor, Scrophularia ningpoensis, Ocimum basilicum, Zea mays, Nicotiana tabacum, Panax japonicus, Polygonatum sibiricum, Allium sativum, and Lilium brownii were selected. Most of them are medicinal plants, and the demand for FCM analysis will increase in the future. They have complex and diverse secondary metabolites in cells.

[0053] The results show that the plant cell nuclei dissociation solution formulation based on 0.5% sodium lauroyl glutamate combined with 1% NP-40 is suitable for multiple plant species, including but not limited to Polygonum cuspidatum, Rehmannia glutinosa, Antirrhinum majus, Sorghum bicolor, Scrophularia ningpoensis, Ocimum basilicum, Zea mays, Nicotiana tabacum, Panax japonicus, Polygonatum sibiricum, Allium sativum, and Lilium brownii, etc. plants with different characteristics Figure 5

[0054] Example 6 Cell cycle analysis of tea varieties using the plant cell nuclei dissociation solution formulation of the present application.

[0055] The plant cell nuclei dissociation solution formulation of the present application (based on 0.5% sodium lauroyl glutamate combined with 1% NP-40) was selected to test its feasibility in cell cycle analysis.

[0056] Leaf flow cytometry nuclei suspensions of two different varieties of tea were prepared.

[0057] The experimental procedure was the same as in Example 1. The results show that in addition to being suitable for plant DNA flow cytometry analysis (including DNA content determination and ploidy analysis), the plant cell nuclei dissociation solution formulation based on 0.5% sodium lauroyl glutamate combined with 1% NP-40 is suitable for plant cell cycle analysis Figure 6

[0058] In summary, the present application provides a plant cell nuclei dissociation solution based on 0.5% sodium lauroyl glutamate combined with 1% NP-40 and its application in DNA flow cytometry analysis (including DNA content determination and ploidy analysis) and cell cycle analysis. The related application is based on flow cytometry. The plant cell nuclei dissociation solution and application have wide plant applicability.

[0059] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.​​

Claims

1. A plant cell nucleus dissociation solution, characterized in that: It contains the following components: 20-200 mM MgCl2, 20-50 mM sodium citrate, 5-50 mM MOPS, 5-50 mM Na2EDTA, 0.2-2% PVP-40, 0.5-2% NP-40, 0.2-5% sodium lauroyl glutamate, 10-100 μL / mL β-mercaptoethanol, and H2O; The method of using the plant cell nucleus dissociation solution includes the following steps: place the plant sample in the pre-cooled plant cell nucleus dissociation solution, chop it up, and place it on ice for 10-30 minutes.

2. The plant cell nucleus dissociation solution according to claim 1, characterized in that: It contains the following components: 45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 10 mM Na2EDTA, 1% PVP-40, 1% NP-40, 0.5% sodium lauroyl glutamate, 20 μL / mL β-mercaptoethanol and H2O.

3. The application of the plant cell nucleus dissociation solution according to claim 1 or 2 in plant DNA flow cytometry analysis and cell cycle analysis.

4. The application according to claim 3, characterized in that: The plants mentioned include Japanese knotweed, rehmannia, snapdragon, sorghum, scrophularia, basil, corn, tobacco, ginseng, polygonatum, garlic, and lily.

5. The method of using the plant cell nucleus dissociation solution according to claim 1 or 2, characterized in that: The steps include: placing the plant sample in pre-cooled plant cell nucleus dissociation solution, chopping it, and placing it on ice for 10-30 minutes.

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