Plant flow cytometric nuclear dissociation solution based on sodium cocoyl glycinate and np-40 and application thereof

Abstract

The application discloses a plant flow cytometry nucleus dissociation solution based on sodium cocoyl glycinate and NP-40 and application thereof, and belongs to the technical field of biology. The nucleus dissociation solution comprises NP-40 or an analogue thereof and sodium cocoyl glycinate, 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] This invention belongs to the field of biotechnology, specifically relating to a plant flow cytometry cell nucleus dissociation solution based on sodium cocoyl glycinate and NP-40 and its application. Background Technology

[0002] Flow cytometry (FCM) is a technique for multi-parameter quantitative analysis and sorting of rapidly flowing microbial particles (cells, nuclei, chromosomes, etc.), and its role in life science research, medical research, and clinical practice is becoming increasingly important. FCM is mainly used for determining plant genome size, DNA content, ploidy, and cell cycle. The principle involves labeling cell nuclei with specific DNA fluorescent dyes (PI, DAPI, etc.), identifying and counting stained particles (theoretically labeled nuclei) using a flow cytometer, and assessing DNA content in the G0 / G1 phase based on signal peaks. The experimental procedure includes: selecting plant tissues (generally leaves, stems, roots, and seeds), preparing and optimizing the nuclear dissociation solution, cutting the tissue to prepare a nuclear suspension, staining with DNA fluorescent dyes, using a flow cytometer, and data analysis. The preparation of the nuclear suspension is a crucial step affecting the quality (accuracy and effectiveness) of FCM analysis. Key indicators for high-quality cell nucleus suspensions include: (1) a sufficient number of intact free cell nuclei; (2) minimal cell debris; (3) minimal interference from intracellular contents with the specificity of DNA fluorescent dyes; and (4) a low coefficient of variation (CV). Therefore, it is necessary to develop and optimize plant cell nucleus dissociation solution formulations to control these key indicators.

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

[0004] The accuracy of FCM analysis is expressed by the coefficient of variation (CV). CV = standard deviation / mean peak value × 100%. The smaller the CV value, the more accurate the DNA content analysis. Typically, the CV should be less than 5%. Therefore, a highly efficient plant cell nucleus dissociation solution should be reflected in a low CV value in the analytical results.

[0005] Since FCM was used in plants, at least seven plant cell nucleus dissociation solutions have been screened and published (Tian Xinmin, Zhou Xiangyan, Gong Na. Application of flow cytometry in plant research - detection of plant nuclear DNA content and ploidy level [J]. Chinese Agricultural Science Bulletin, 2011, 27(09):21-27.), as well as several authorized patents.

[0006] Due to the diverse plant species, the presence of cell walls, significant differences in the structural composition of plant cells derived from different plants or tissues, and the abundance of internal contents (such as complex secondary metabolites like starch, polysaccharides, pectin, polyphenols, and tannins), existing publicly available formulations lack universal applicability; that is, they cannot be simultaneously applied to multiple plant materials. Therefore, before performing FCM analysis on new plants, operators must test different dissociation solutions to select the most suitable formulation. DNA flow cytometry is increasingly used in plant genetics and breeding, both now and in the future. Therefore, the development of widely applicable or universal plant cell nuclear dissociation solutions remains of urgent practical value.

[0007] The following are the authorized invention patents related to plant cell nucleus dissociation solutions: (1) Cell nucleus extraction buffer and method for determining the genome size of Piper plants (CN110514495A). This invention patent is only applicable to Piper plants. Although the application of this cell nucleus extraction buffer resulted in a small CV value, no staining observation of the cell nucleus or a negative control without DNA fluorescent dye was provided, i.e., there is no evidence that the selection of components improved the quality of the cell nucleus and reduced the influence of fragmentation peaks. Tween-20 and Triton X-100 are detergents commonly used in cell nucleus dissociation solutions. Moreover, only Triton X-100 was used in this formulation. (2) Liquid for extracting cell nuclei and its application (CN111139214A). This invention patent is the first to test the improvement effect of different concentrations of PVP k12 and detergent Tween-20 on cell nucleus extraction solutions. PVP (polyvinylpyrrolidone) is used to reduce the effects of oxidative substances in plant cells and has different molecular weights (PVP k12, PVP 40, PVP k15, PVP 30, etc.). This invention patent simply replaces the commonly used reagents PVP 40 or PVP k15 in the existing formulation with PVP k12. Tween-20 and Triton X-100 are detergents commonly used in cell nucleus dissociation solutions. In 9 published formulations (Jin Liang, Xu Weiwei, Li Xiaobai, Liu Jianxin, Tian Danqing, Ge Yaying, Pan Xiaoyun, Wang Weiyong. Application of DNA flow cytometry in plant genetics and breeding [J]. Chinese Journal of Cell Biology, 2016, 38(02):225-234.), 7 used Triton X-100 and 2 used Tween-20. Therefore, this formulation has not conducted in-depth comparative experiments on detergents. In fact, the currently well-established plant FCM procedures are entirely applicable to dried samples (Suda J, P. Reliable DNAploidy 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 metabolites), so it cannot well prove the wide applicability of the formula. (3) Chinese patent CN104075983A discloses a method for determining the genome size of Gesneriaceae plants, and Chinese patent CN107449717A discloses a method for determining the genome size of Nymphaeaceae plants, but the applicability problem is not solved.

[0008] In summary, to obtain high-quality nuclear suspensions and ensure FCM accuracy, it is necessary to improve the formulation of plant nuclear dissociation solutions, requiring: (1) gentler and more thorough cell lysis, reducing the inhibitory effect of contents (fragments and secondary metabolites) while maintaining nuclear integrity; and (2) wider applicability. This can be achieved by further optimizing the detergent components and formulation while maintaining appropriate ionic strength.

[0009] Polygonum cuspidatum is a traditional Chinese medicinal plant. Its leaves are leathery and difficult to cut, and rich in polyphenols, polysaccharides, and mucilage. These characteristics lead to numerous impurities and fragments in the prepared nuclear suspension, resulting in low nuclear extraction efficiency and inaccurate FCM results. Therefore, while addressing these challenges is crucial for conducting FCM analysis on Polygonum cuspidatum, its complexity also provides an excellent test material for the development and improvement of plant cell dissociation solutions.

[0010] Among the currently disclosed cell nucleus dissociation solution formulations, the commonly used detergents are only Tween-20 and Triton X-100. NP-40 (Nonidet P-40) is a nonionic surfactant that, although commonly used in protein extraction, has not been used in the preparation of plant flow cytometry samples. Summary of the Invention

[0011] The purpose of this invention is to overcome the problems of low efficiency, poor accuracy, and poor applicability of existing plant cell nucleus dissociation solutions, and to provide a plant cell dissociation solution based on sodium cocoyl glycinate and NP-40 with high efficiency, good accuracy, and wide applicability, as well as its application in plant flow cytometry detection.

[0012] The objective of this invention is achieved through the following technical solution.

[0013] This invention discovers that a plant cell nucleus dissociation solution formulated with NP-40 (Nonidet P-40) as a detergent, when used for flow cytometry analysis of lysed plant cells, exhibits significantly better performance than known detergents such as Tween-20 and Triton X-100. Furthermore, the addition of sodium cocoyl glycinate to the plant cell nucleus dissociation solution further enhances the flow cytometry results. The plant cell nucleus dissociation solution containing NP-40 and sodium cocoyl glycinate is suitable for multiple plant species, including but not limited to plants with diverse characteristics such as Polygonum cuspidatum, Rehmannia glutinosa, sorghum, Chinese cabbage, Prunus armeniaca, basil, corn, tobacco, Nelumbo nucifera, Polygonatum sibiricum, and garlic.

[0014] Based on the findings of this invention, the present invention provides the following applications, products or methods.

[0015] The use of NP-40 or its analogues with sodium cocoyl glycinate in the preparation of plant cell nucleus dissociation solutions for flow cytometry analysis. The NP-40 analogues include... CA-630 (Sigma-Aldrich), CA-630 is a substitute for NP-40, and its chemical properties are the same as those of NP-40.

[0016] A plant cell nucleus dissociation solution comprising NP-40 or an analogue thereof and sodium cocoyl glycinate. The concentration of NP-40 or an analogue thereof is preferably 0.5-2% (w / v), and the concentration of sodium cocoyl glycinate is preferably 0.2-5% (w / v).

[0017] In some embodiments, the plant cell nucleus 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 cocoyl glycinate, 10-100 μL / mL β-mercaptoethanol, and H2O, with a pH of 7.0. Further, the plant cell nucleus 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 cocoyl glycinate, 20 μL / mL β-mercaptoethanol, and H2O, with a pH of 7.0.

[0018] The application of the plant cell nucleus dissociation solution in plant DNA flow cytometry and cell cycle analysis. The plants mentioned include, but are not limited to, plants with different characteristics such as Polygonum cuspidatum, Rehmannia glutinosa, sorghum, Chinese cabbage, Prunus armeniaca, basil, corn, tobacco, Nelumbo nucifera, wild Polygonatum sibiricum, and garlic.

[0019] The method of using the plant cell nucleus dissociation solution includes the following steps: placing the plant sample in the pre-cooled plant cell nucleus dissociation solution, chopping it, and placing it on ice for 10-30 minutes.

[0020] The present invention has the following advantages and effects compared with the prior art:

[0021] (1) NP-40 was used instead of Tween-20 or Triton X-100 in the existing formula, resulting in better cell nucleus extraction and flow cytometry peak elution.

[0022] (2) The combined use of 0.5% sodium cocoyl glycinate and 1% NP-40 improved the efficiency of plant kernel extraction and the accuracy of FCM.

[0023] (3) The plant cell nucleus dissociation solution formulation disclosed in this invention outperforms existing formulations and commercially available formulations, and has a wider range of applicability. Attached Figure Description

[0024] Figure 1 This study tests and compares the effectiveness of different formulations in preparing nuclear suspensions for flow cytometry analysis of Polygonum cuspidatum leaves and performing FCM analysis. (A) Appearance of nuclear suspensions prepared with different formulations in plastic test tubes. (B) Fluorescence micrographs of nuclear suspensions prepared with different formulations labeled with the DNA fluorescent dye PI, showing specifically stained nuclei and non-specifically stained cell (impurity) debris. (C) Peak diagram of the unstained nuclear suspension loaded onto the flow cytometer, showing the count values ​​of debris particles. (D) Peak diagram of the PI-stained nuclear suspension loaded onto the flow cytometer, showing the peak shape and count values ​​of nuclei circled in the debris particles.

[0025] Figure 2 This study tested the effect of replacing the detergent with 0.5% sodium cocoyl glycinate on the results of cell nucleus extraction and flow cytometry analysis.

[0026] Figure 3 This study investigated the effects of adding different detergents and 0.5% sodium cocoyl glycinate 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 cocoyl glycinate.

[0027] Figure 4 The effects of the plant cell nucleus dissociation solution formulation of this invention (based on a combination of 0.5% sodium cocoyl glycinate 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.

[0028] 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 cocoyl glycinate and 1% NP-40) in flow cytometry analysis on different plants.

[0029] Figure 6 Cell cycle analysis was performed on different tea varieties using the plant cell nucleus dissociation solution formulation of the present invention (based on a combination of 0.5% sodium cocoyl glycinate and 1% NP-40). Detailed Implementation

[0030] 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.

[0031] The following examples illustrate the sample collection, preparation, and handling methods: Take fresh plant leaves (approximately 2×2cm in size) 2 Wipe the leaves clean and place them in a cell culture dish. Place the dish on ice and add 750 μL of pre-chilled nuclear dissociation solution. Quickly chop the leaves with a double-edged blade and place on ice for 10-30 minutes to release the nuclei and prepare a crude extract. Filter the extract using a flow cytometry filter (100 mesh) to collect the nuclear suspension. Stain a portion of the nuclear suspension with 1 mg / mL PI (containing RNase) and observe the nuclear staining under a fluorescence microscope. Take the remaining nuclear suspension, either unstained or stained with 1 mg / mL PI (containing RNase), place on ice, and incubate in the dark for 10 minutes before loading into the flow cytometer for analysis. Operate the flow cytometer according to the operating instructions. Finally, perform subsequent data statistics and analysis based on the recorded data.

[0032] Example 1 tests and compares the effects of using different formulations for the preparation of nuclear suspensions for flow cytometry analysis of Polygonum cuspidatum leaf cells and for FCM analysis.

[0033] Seven plant cell nucleus dissociation solutions were prepared based on publicly available formulas. The formulas are detailed in Table 1. Following the procedure, seven nucleus suspensions were prepared using equal amounts of Polygonum cuspidatum leaves. It was found that the nucleus suspensions prepared with different formulas exhibited different colors in plastic test tubes. In the LB01, Tris-MgCl2, and WPB formulas, a deeper brown color was observed, indicating that the intracellular contents tended to oxidize in these formulas. Figure 1 A). Observation under a fluorescence microscope of cell nucleus suspensions prepared with different formulations after labeling with the DNA fluorescent dye PI showed that in addition to the cell nuclei being specifically stained (spherical), there were still non-specifically stained cell (impurity) fragments (appearing as irregular, poorly dispersed particles). Figure 1 B). Subsequently, the nuclear suspensions were evenly divided into a dye-free control group and an observation group stained with 1 mg / mL PI (containing RNase). Flow cytometry was performed according to the operating instructions. Data were recorded and subsequently statistically analyzed. The elution chromatogram of the control group showed a large number of non-specific fragment particles, which interfered with the recognition of nuclear particles. Figure 1 C). Compared to the control, the peak shape and count of PI-stained cell nuclei can be circled and recorded from the debris particles. Figure 1(D) The results showed that the flow cytometry results of Polygonum cuspidatum samples prepared with different dissociation solutions varied greatly. GPB and the classic Galbraith solution only yielded cell debris with no peaks, while the mG solution showed a large number of free nuclei and clear peaks, exhibiting the best performance. Of these solutions, six used Triton X-100 as the detergent, and only one used Tween-20. Numerous fragments were still observed in nuclear staining.

[0034] Table 1

[0035]

[0036]

[0037] Example 2 tested the effect of replacing the detergent Triton X-100 in the mG formulation with 0.5% sodium cocoyl glycinate in the preparation of cell nuclear suspension.

[0038] Prepare a cell nucleus dissociation solution containing 0.5% sodium cocoyl glycinate. The formula is: 45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 10 mM Na2EDTA, 1% (w / v) PVP-40, 0.5% sodium cocoyl glycinate, 20 μL / mL β-mercaptoethanol, and H2O, with a pH of 7.0.

[0039] Preparation of flow cytometry cell nucleus suspensions from Polygonum cuspidatum leaves.

[0040] The experimental procedure was the same as in Example 1. The results showed that using 0.5% sodium cocoyl glycinate alone to replace the detergent did not achieve the effect of dissociating the cell nucleus and did not result in FCM peak elution. Figure 2 Therefore, it cannot replace the aforementioned detergents.

[0041] Example 3 tested the effects of adding different detergents and 0.5% sodium cocoyl glycinate on cell nucleus extraction and flow cytometry results.

[0042] Different cell nucleus dissociation solutions were prepared. The common components of all solutions 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, with a pH of 7.0. Based on this, different detergents were added to prepare 8 comparative 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 cocoyl glycinate; (6) 1% Triton X-100 + 0.5% sodium cocoyl glycinate; (7) 1% Tween-20 + 0.5% sodium cocoyl glycinate; (8) 1% NP-40 + 0.5% sodium cocoyl glycinate.

[0043] Preparation of flow cytometry cell nucleus suspensions from Polygonum cuspidatum leaves.

[0044] The experimental procedures were the same as in Example 1. Based on the mG formulation, the nuclear extraction effects and FCM elution results of Tween-20, Triton X-100, and NP-40 (Nonidet P-40) were tested. The results showed that the nuclear suspension prepared with 1% NP-40 had the fewest fragments and the lowest CV value. Figure 3 Adding 0.5% sodium cocoyl glycinate to the plant cell nucleus dissociation solution formulation, the combination of 0.5% sodium cocoyl glycinate with either Tween-20 or Triton X-100 does not affect the effectiveness of the original detergent. However, the combination of 0.5% sodium cocoyl glycinate with NP-40 significantly improves the nucleus extraction effect and FCM peak results, reduces fragmentation in the cell nucleus suspension, and further decreases the CV value. Figure 3 ).

[0045] Example 4 tested and compared the effects of the plant cell nucleus dissociation solution formulation of the present invention (based on a combination of 0.5% sodium cocoyl glycinate and 1% NP-40) and a commercially available dissociation solution in flow cytometry analysis.

[0046] The commercially available dissociation solution is CyStain UV Precise P (Sysmex Partec).

[0047] The usage, dosage, and operating steps remain the same as in Example 1.

[0048] The effects of two medicinal plants, Polygonum cuspidatum and Rehmannia glutinosa, on flow cytometry analysis were compared.

[0049] The results showed that, compared with the commercially available flow cytometry reagent CyStain UV Precise P (Sysmex Partec), the plant cell nucleus dissociation solution formulation based on the combination of 0.5% sodium cocoyl glycinate and 1% NP-40 was superior to the commercially available flow cytometry reagent, regardless of whether the tested sample was Polygonum cuspidatum or Rehmannia glutinosa. Figure 4 ).

[0050] Example 5: Effects of using the plant cell nucleus dissociation solution formulation of the present invention (based on a combination of 0.5% sodium cocoyl glycinate and 1% NP-40) in flow cytometry analysis on different plants.

[0051] To highlight the broad applicability of the plant cell nucleus dissociation solution formulation of this invention to various plants, in addition to Polygonum cuspidatum and Rehmannia glutinosa mentioned above, plants with different characteristics were selected, including sorghum, Chinese cabbage, Panax notoginseng, basil, corn, tobacco, Nelumbo nucifera, wild Polygonatum sibiricum, and garlic. Some of these are medicinal plants, and the demand for FCM analysis will increase in the future, as their intracellular secondary metabolites are complex and diverse.

[0052] The results showed that the plant cell nucleus dissociation solution formulation based on the combination of 0.5% sodium cocoyl glycinate and 1% NP-40 was suitable for multiple plant species, including but not limited to plants with different characteristics such as Polygonum cuspidatum, Rehmannia glutinosa, sorghum, Chinese cabbage, Prunus armeniaca, basil, maize, tobacco, Zingiber mioga, Polygonatum sibiricum, and garlic. Figure 5 ).

[0053] Example 6 uses the plant cell nucleus dissociation solution formulation of the present invention to perform cell cycle analysis on tea varieties.

[0054] The plant cell nucleus dissociation solution formulation of this invention (based on a combination of 0.5% sodium cocoyl glycinate and 1% NP-40) was used to test its feasibility in cell cycle analysis.

[0055] Flow cytometry cell nucleus suspensions of leaves from two different tea varieties were prepared.

[0056] The experimental procedures were the same as in Example 1. The results showed that, in addition to being suitable for plant DNA flow cytometry analysis (including DNA content determination and ploidy analysis), the plant cell nucleus dissociation solution formulation based on the combination of 0.5% sodium cocoyl glycinate and 1% NP-40 was suitable for plant cell cycle analysis. Figure 6 ).

[0057] In summary, this invention provides a plant cell nucleus dissociation solution based on a combination of 0.5% sodium cocoyl glycinate and 1% NP-40, and its application in DNA flow cytometry analysis (including DNA content determination and ploidy analysis) and cell cycle analysis. The related applications are based on flow cytometry. This plant cell nucleus dissociation solution and its applications have broad applicability to various plants.

[0058] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

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% (w / v) PVP-40, 0.5-2% (w / v) NP-40, 0.2-5% (w / v) sodium cocoyl glycinate, 10-100 μL / mL β-mercaptoethanol and H2O.

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% (w / v) PVP-40, 1% (w / v) NP-40, 0.5% (w / v) sodium cocoyl glycinate, 20 μL / mL β-mercaptoethanol and H2O, with a pH of 7.

0.

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

4. The application according to claim 3, characterized in that: The plants mentioned include Japanese knotweed, rehmannia, sorghum, Chinese cabbage, purple-backed golden disc, basil, corn, tobacco, ginger lily, wild polygonatum, and garlic.

5. A method of using the plant cell nucleus dissociation solution as described in 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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