A method for immunofluorescence staining of chloroplast proteins based on tissue fragmentation

Abstract

The present invention belongs to the field of biotechnology, and discloses a method for immunofluorescence staining of chloroplast proteins based on tissue fragmentation, the method comprising the following steps: 1) fragmenting plant leaf tissue; 2) performing immunofluorescence staining on the fragmented tissue; 3) treating the plant tissue that has been immunofluorescence stained with EDTA·Na2 to lyse the plant tissue; 4) observing the localization signal of the target protein under a fluorescence microscope. The present invention treats plant leaf tissue with a serrated blade, which greatly increases the number of cells stained. At the same time, the leaf tissue is treated with EDTA·Na2 to disperse the cells in the tissue block, thereby facilitating the observation of the signal. The method is of great significance for studying the localization of chloroplast proteins.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for immunofluorescence staining of chloroplast proteins based on tissue fragmentation. Background Art

[0002] Chloroplasts are the main site for photosynthesis in plants and are plant-specific organelles. In addition, chloroplasts have many other important physiological functions in plants, such as the synthesis of fatty acids, amino acids and a variety of hormones. During chloroplast division, chloroplast division proteins form a ring-shaped supercomplex located in the middle of the chloroplast. The ring-shaped complex then begins to constrict, dividing a chloroplast into two. Plants maintain the number of chloroplasts in cells through chloroplast division. The study of chloroplast proteins has always been an important aspect of biology.

[0003] Common methods for studying the localization of chloroplast proteins include fluorescent signal labeling and immunofluorescence staining. The fluorescent signal labeling method must be accompanied by a fluorescent tag, which may change the structure and function of the target protein and thus fail to reflect the true state of the protein in the plant. Fluorescent labels usually also require transgenic plants, which is difficult to achieve for many types of plants. Previous immunofluorescence staining methods required wax embedding or enzymatic hydrolysis of protoplasts and the preparation of polylysine slides in advance, which made the experiment time-consuming and inconvenient to operate. Affected by the structural characteristics of the cell walls of different plants, it is difficult to enzymatically hydrolyze protoplasts in some plants, so it is impossible to locate the target protein by enzymatic hydrolysis. Summary of the invention

[0004] In order to solve the above-mentioned technical problems and address the deficiencies of the prior art, the present invention develops a method for immunofluorescence staining of chloroplast proteins based on tissue fragmentation, so that people can better study the functions of chloroplasts. The present invention uses a serrated blade to fragment and fix leaf tissue, so that the fragmented tissue is quickly fixed with paraformaldehyde and is more easily recognized by antibodies. The fragmented tissue that has undergone immunofluorescence staining is then treated with EDTA·Na2 to lyse it, and observed under a fluorescence microscope, which can quickly, clearly and intuitively display the location of chloroplast proteins. The present invention uses a serrated blade to fragment tissue, and there is no need for embedding materials, enzymatic hydrolysis of protoplasts, and preparation of polylysine slides, etc., which greatly saves experimental time and reduces the difficulty of experimental operation. At the same time, this method has very low requirements for the amount of experimental materials, only about 1-2cm 2 The leaves are fine.

[0005] Specifically, one aspect of the present invention provides a method for immunofluorescence staining of chloroplast proteins or marking the location of chloroplast proteins in chloroplasts based on tissue fragmentation, the method comprising the following steps:

[0006] 1) Fragmentation and fixation of plant tissue: the plant tissue is immersed in a fixative solution, and the plant tissue is cut into irregular pieces with a serrated blade; the plant tissue is fixed with the fixative solution at room temperature and in the dark;

[0007] 2) Immunofluorescence staining: The fixed broken plant tissues were blocked with blocking solution; the primary antibody against chloroplast protein and the secondary antibody with fluorescent labeling were incubated in sequence;

[0008] 3) Tissue lysis: Broken plant tissues were treated with EDTA·Na2 and heated at 55°C for 0.5-1.5h;

[0009] 4) Sample storage: Add sealing medium to the sample and store it below 8°C;

[0010] 5) Sample observation: Observe using a fluorescence microscope.

[0011] In the technical solution of the present invention, the fixing solution is a fixing solution containing 4% paraformaldehyde. Preferably, the fixing solution contains 0.4M mannitol, 20mM KCl, 20mM MES at pH 5.7, and 4% paraformaldehyde.

[0012] In the technical solution of the present invention, the blocking solution is a buffer solution containing 5% BSA (w / v).

[0013] In the technical solution of the present invention, the mounting medium contains 5 mM sodium ascorbate, 15 mM Na2HPO4 pH 9.0, and 50% glycerol.

[0014] In the technical scheme of the present invention, the plant tissue is a plant material containing chloroplasts. Preferably, the plant material is selected from leaves, flowers, stems or roots having chloroplasts; more preferably, the plant material is one of Arabidopsis leaves, elm leaves, poplar leaves, paper mulberry leaves, chrysanthemum leaves, Dendrobium sphaerocephalum leaves, Epipremnum aureum leaves, Ginkgo leaves, and fern leaves.

[0015] In the technical solution of the present invention, the serrated blade used for cutting plants has a thickness of about 0.2-0.25 mm, a serration size and interval of about 0.7-1 mm, and a width range of 0.3-0.6 cm, preferably 0.3 cm.

[0016] In the technical solution of the present invention, in step 1), the broken plant tissue is fixed in the dark at room temperature for 0.5-1.5 hours; after fixation, the broken plant tissue is washed 2-4 times with PBS, each time for at least 5 minutes.

[0017] In the technical solution of the present invention, in step 2), the fixed plant tissue is blocked with 5% BSA for 20-40 minutes.

[0018] In the technical solution of the present invention, in step 2), the primary antibody of chloroplast protein is diluted with 5% BSA containing 0.5% Triton X-100, and the antibody dilution ratio is 1:100-1:400, preferably 1:100; the secondary antibody with fluorescent label is diluted with 5% BSA, and the antibody dilution ratio is 1:100-1:1000, preferably 1:100.

[0019] In the technical solution of the present invention, the incubation time of the primary antibody of chloroplast protein is 1.5-2.5h, and the incubation time of the secondary antibody with fluorescent labeling is 0.5-1.5h.

[0020] In the technical solution of the present invention, after incubation with the primary antibody and the secondary antibody, the broken plant tissue needs to be washed three times with PBS or PBS containing 5% BSA, each time for at least 5 minutes.

[0021] In the technical solution of the present invention, in step 3), the plant tissue that has been subjected to immunofluorescence staining is treated with EDTA·Na2.

[0022] In the technical solution of the present invention, in step 3), heating is performed using a water bath, a metal heating block or an oven, etc. The temperature of the heating device is adjusted to 55° C. before use.

[0023] In the technical solution of the present invention, the concentration of EDTA·Na2 is 0.1M and the pH is 9.0.

[0024] In the technical solution of the present invention, in step 4), the localization signal of the target protein is observed using a fluorescence microscope.

[0025] In the technical solution of the present invention, the plant tissue is treated with EDTA·Na2, preserved in a sealing medium and stored at a temperature below 8°C.

[0026] In the technical solution of the present invention, the chloroplast protein is selected from chloroplast division proteins, such as FtsZ1 protein, and also includes various other chloroplast proteins.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention provides a new immunofluorescence staining method for plant chloroplast proteins, especially chloroplast fission proteins. It does not require embedding materials, enzymatic hydrolysis of protoplasts, and preparation of poly-lysine slides, which saves experimental time and reduces the difficulty of experimental operation.

[0029] 2. The method of the present invention is not affected by the structural characteristics of plant cell walls and can be used to locate and stain any plant with chloroplasts. In species such as Arabidopsis, Ulmus, Poplar, Broussonetia papyrifera, Chrysanthemum, Dendrobium sphericalensis, Scindapsus scindapsus, Ginkgo biloba, and Ferns, the FtsZ1 protein is located in the middle of the chloroplast in a ring shape, with clear signals and accurate information.

[0030] 3. The present invention uses a serrated blade to break up plant tissues. After the plant tissues are fixed and dissociated, the stained cells are widely distributed in the broken plant tissues through microscopic observation. The equipment, tools and reagents are simple and easy to obtain.

[0031] 4. The method of the present invention requires very little experimental material, only about 1-2cm 2 This method is of great significance for studying the localization of chloroplast proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a technical flow chart of the chloroplast protein immunofluorescence staining method based on tissue fragmentation of the present invention.

[0033] Figure 2 This is a comparison chart of the effects of different plant tissue crushing methods. A is the effect of cutting the leaves into shreds with a blade; B is the effect of cutting the leaves into slices with a blade; C is the effect of cutting the leaves into irregular pieces with a serrated blade.

[0034] Figure 3 This is a comparison of the effects of plant tissues before and after treatment with EDTA·Na2.

[0035] Figure 4 The signal localization map of FtsZ1 protein shown by this method in wild-type Arabidopsis and arc3 and pdv2 mutants.

[0036] Figure 5 This is the signal localization map of FtsZ1 protein in other species shown by this method. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0038] Hereinafter, specific embodiments of the present invention will be described in detail with reference to the accompanying drawings. According to these detailed descriptions, those skilled in the art can clearly understand the present invention and can implement the present invention. Without violating the principles of the present invention, the features in the various embodiments can be combined to obtain new implementations, or certain features in certain embodiments can be replaced to obtain other preferred implementations.

[0039] See also Figure 1, the technical flow chart of the chloroplast fission protein immunofluorescence staining method based on tissue fragmentation of the present invention, specifically, a chloroplast protein immunofluorescence staining method based on tissue fragmentation, comprising the following steps:

[0040] 1) Leaf tissue fragmentation and fixation: The leaf tissue was immersed in the fixative and cut into irregular pieces with a serrated blade. The leaf tissue was fixed with 4% paraformaldehyde fixative for 1 hour at room temperature in the dark.

[0041] 2) Immunofluorescence staining: The fixed plant tissue blocks were blocked with 5% BSA solution for 30 minutes, and then incubated with primary antibody and secondary antibody in sequence.

[0042] 3) Tissue block lysis: The broken plant tissue blocks were treated with EDTA·Na2 and heated at 55°C for 1 h.

[0043] 4) Sample storage: Add sealing medium to the sample and store it below 8°C.

[0044] 5) Sample observation: Observe using a fluorescence microscope.

[0045] Example 1

[0046] 1) Experimental preparation: prepare leaf crushing fixative [0.4 M mannitol, 20 mM KCl, 20 mM MES (pH 5.7), 4% paraformaldehyde], 1×PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O, 2 mM KH2PO4, HCl adjusted to pH 7.4), blocking solution (5% BSA (w / v) dissolved in 1×PBS), and mounting medium (5 mM sodium ascorbate, 15 mM Na2HPO4 pH 9.0, 50% glycerin); turn on the heating equipment and adjust the temperature to 55°C.

[0047] 2) Leaf tissue fragmentation and fixation: Immerse the Arabidopsis leaves in the fixative and scrape them with a 0.3 cm long serrated blade to form irregular fragments. The leaves are fully immersed in the fixative and fixed at room temperature in the dark for 1 hour.

[0048] 3) Immunofluorescence staining: The fixed and broken tissues were washed 3 times with PBS. Each time, the solution was kept for at least 5 minutes. The fixed and broken plant tissues were blocked with 5% BSA solution (containing 0.15% Triton X-100) for 30 minutes. The primary antibody was diluted with 5% BSA (containing 0.15% Triton X-100) at a ratio of 1:100. Incubate at room temperature in the dark for 2 hours. Wash 3 times with PBS or PBS containing 5% BSA, each time for at least 5 minutes. Dilute the secondary antibody. Treat at room temperature in the dark for 1-1.5 hours. Wash 3 times with PBS or PBS containing 5% BSA, each time for at least 5 minutes. The primary antibody is an antibody that specifically binds to chloroplast proteins, and the secondary antibody is an antibody that specifically binds to the primary antibody and has a fluorescent label.

[0049] 4) Tissue lysis: Add 1 mL of 0.1 M EDTA·Na2 (pH 9.0) and heat at 55°C for 1 h.

[0050] 5) Sample preparation: After discarding EDTA·Na2, add sealing medium to completely immerse the plant material.

[0051] 6) Sample observation: Observe using a fluorescence microscope.

[0052] Example 2

[0053] The method of immunofluorescence staining of chloroplast proteins in this example is the same as that in Example 1, except that in step 1), the Arabidopsis leaves are cut into strips, slices and irregular shapes, respectively.

[0054] Example 3

[0055] The method of immunofluorescence staining of chloroplast proteins in this example is the same as that in Example 1, except that in step 3), before and after the treatment with EDTA·Na2, part of the plant tissue samples are respectively taken for observation under a microscope.

[0056] Example 4

[0057] The method of immunofluorescence staining of chloroplast proteins in this example is the same as the steps in Example 1, except that in step 2), the leaves are arc3 mutant leaves.

[0058] Example 5

[0059] The method of immunofluorescence staining of chloroplast proteins in this example is the same as the steps in Example 1, except that in step 2), the leaves are pdv2 mutant leaves.

[0060] Example 6

[0061] The method of immunofluorescence staining of chloroplast proteins in this example is the same as that in Example 1, except that in step 2), the leaves are elm leaves.

[0062] Example 7

[0063] The method of immunofluorescence staining of chloroplast proteins in this example is the same as that in Example 1, except that in step 2), the leaves are poplar leaves.

[0064] Example 8

[0065] The method of immunofluorescence staining of chloroplast proteins in this example is the same as the steps in Example 1, except that in step 2), the leaves are paper mulberry leaves.

[0066] Example 9

[0067] The method of immunofluorescence staining of chloroplast proteins in this example is the same as that in Example 1, except that in step 2), the leaves are chrysanthemum leaves.

[0068] Example 10

[0069] The method of immunofluorescence staining of chloroplast proteins in this example is the same as that in Example 1, except that in step 2), the leaves are leaves of Dendrobium sphaerocephalum.

[0070] Embodiment 11

[0071] The method of immunofluorescence staining of chloroplast proteins in this example is the same as the steps in Example 1, except that in step 2), the leaves are green radish leaves.

[0072] Example 12

[0073] The method of immunofluorescence staining of chloroplast proteins in this example is the same as that in Example 1, except that in step 2), the leaves are ginkgo leaves.

[0074] Example 13

[0075] The method of immunofluorescence staining of chloroplast proteins in this example is the same as the steps in Example 1, except that in step 2), the leaves are fern leaves.

[0076] Result analysis:

[0077] After the plant tissues were broken and fixed, the stained cells were widely distributed in the broken plant tissues under microscope observation. In wild-type Arabidopsis and other species, the FtsZ1 protein was localized in the middle of the chloroplast in a ring shape, with clear signals and accurate information. This method is time-saving, easy to operate, and requires very little experimental materials, which is of great significance for studying the localization of chloroplast proteins.

[0078] like Figure 2The photo is a photo of the immunofluorescence method of chloroplast division protein FtsZ1 by tissue fragmentation in Example 2. The left side shows different cutting methods, and the right side shows the cell state diagram observed under a bright field microscope with different cutting methods. After the leaf is irregularly broken with a serrated blade, the dispersed state of the cell is significantly higher than that when it is shredded or sliced, which is more conducive to labeling and observation.

[0079] Use EDTA·Na2 to treat plant tissues after immunofluorescence staining, such as Figure 3 The figure shows the dispersion state of plant tissue before and after treatment with EDTA·Na2 in Example 3. Before treatment with EDTA·Na2, plant cells are not easy to disperse. After treatment with EDTA·Na2 and tableting, cells are easy to disperse, which is more conducive to observing the location of chloroplast proteins.

[0080] like Figure 4 The figure shows the localization of FtsZ1 in Arabidopsis thaliana by immunofluorescence staining of chloroplast proteins in tissue fragmentation. In wild-type Arabidopsis thaliana, FtsZ1 is localized in the middle of the chloroplast as a single ring. In the arc3 mutant, FtsZ1 is localized in multiple rings and dispersedly in the large chloroplasts. In the pdv2 mutant, FtsZ1 is localized in the middle of the large chloroplast as a ring.

[0081] like Figure 5 As shown, this method is applied to elm, poplar, paper mulberry, chrysanthemum, spherical dendrobium, scindapsus, ginkgo, and fern. The results of fluorescence microscopy observation show that in these species, FtsZ1 protein is located in the middle of the chloroplast in a ring shape, indicating that the method of the present invention is generally applicable to the preparation of sample materials of different species.

[0082] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

[0083] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for immunofluorescence staining of chloroplast proteins or marking the location of chloroplast proteins in chloroplasts based on tissue fragmentation, characterized in that: The method comprises the following steps: 1) Fragmentation and fixation of plant tissue: the plant tissue is immersed in a fixative solution, and the plant tissue is cut into irregular pieces with a serrated blade; the plant tissue is fixed with the fixative solution at room temperature and in the dark; 2) Immunofluorescence staining: The fixed broken plant tissues were blocked with blocking solution; the primary antibody against chloroplast protein and the secondary antibody with fluorescent labeling were incubated in sequence; 3) Tissue lysis: The broken plant tissue after immunofluorescence staining was treated with EDTA·Na2 and heated at 55℃ for 0.5-1.5h; 4) Sample storage: Add sealing medium to the sample and store it below 8°C; 5) Observation of samples: observation using a fluorescence microscope; Among them, the thickness of the serrated blade is 0.2-0.25mm, the size and spacing of the teeth are 0.7-1mm, and the width ranges from 0.3-0.6cm.

2. The method according to claim 1, characterized in that In step 1), the broken plant tissue is fixed at room temperature in the dark for 0.5-1.5 hours; after fixation, the broken plant tissue is washed 2-4 times with PBS, each time for at least 5 minutes.

3. The method according to claim 1, characterized in that The plant tissue is a plant material containing chloroplasts.

4. The method according to claim 3, characterized in that The plant material is selected from leaves, flowers, stems or roots having chloroplasts.

5. The method according to claim 4, characterized in that The plant material is selected from one of Arabidopsis leaves, elm leaves, poplar leaves, paper mulberry leaves, chrysanthemum leaves, dendrobium spherical leaves, green radish leaves, ginkgo leaves, and fern leaves.

6. The method according to claim 1, characterized in that The fixing solution is a fixing solution containing 4% paraformaldehyde; The blocking solution is a BSA buffer containing 5% w / v; The mounting medium contained 5 mM sodium ascorbate, 15 mM Na2HPO4 pH 9.0, 50% glycerol.

7. The method according to claim 6, characterized in that The fixative solution contains 0.4 M mannitol, 20 mM KCl, 20 mM MES at pH 5.7, and 4% paraformaldehyde; 8. The method according to claim 1, characterized in that The primary antibody was diluted with 5% BSA containing 0.5% Triton X-100, and the dilution ratio of the primary antibody was 1:100-1:400; the secondary antibody was diluted with 5% BSA, and the dilution ratio of the secondary antibody was 1:25-1:

100.

9. The method according to claim 8, characterized in that The primary antibody dilution ratio was 1:100; the secondary antibody dilution ratio was 1:

100.

10. The method according to claim 1, characterized in that The primary antibody incubation time is 1.5-2.5h, and the secondary antibody incubation time is 0.5-1.5h.

11. The method according to claim 10, characterized in that After incubation with primary and secondary antibodies, the broken plant tissues need to be washed three times with PBS or PBS containing 5% BSA, each time for at least 5 minutes.

12. The method according to claim 1, characterized in that In step 3), heating is performed using a water bath, a metal heating block or an oven, and the temperature is adjusted to 55° C. before use.

13. The method according to claim 1, characterized in that The concentration of EDTA·Na2 is 0.1 M and the pH is 9.

0.

14. The method according to claim 1, characterized in that The chloroplast protein is selected from the group consisting of chloroplast division protein and other various chloroplast proteins.

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