CN114236136A - Plant plasma membrane fluorescence labeling method and plasma membrane protein positioning and dynamic monitoring method - Google Patents
Plant plasma membrane fluorescence labeling method and plasma membrane protein positioning and dynamic monitoring method Download PDFInfo
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Abstract
The invention provides a plant plasma membrane fluorescence labeling method and a plasma membrane protein positioning and dynamic monitoring method, belonging to the technical field of plasma membrane dyeing. The FM4-64 dye is adopted to dye the plasma membrane structure, and the FM4-64 dye can be embedded in a plasma membrane bilayer and follows the plasma membrane to carry out dynamic transport, so that the dye can be used in subsequent related researches such as plasma membrane endocytosis and transport. In addition, the fluorescence labeling method of the invention can perform the fluorescence labeling of the plasma membrane in genetic transformation or transient transformation, thereby saving the experimental time and avoiding the interference problem of fluorescence signals.
Description
Technical Field
The invention belongs to the technical field of plasma membrane dyeing, and particularly relates to a fluorescent labeling method for a plant plasma membrane and a positioning and dynamic monitoring method for a plasma membrane protein.
Background
The cell membrane is a layer of a limiting membrane that the cell surrounds the contents. Can protect the relative stability of the microenvironment in the cells, prevent the random entering of foreign matters and sense the change of the external environment. The cell membrane is firstly a membrane structure with active transport function and high selectivity, maintains the concentration difference of substances inside and outside the cell, and controls the transport of nutrient components into the cell and the discharge of metabolic wastes and secretions in the cell. Secondly, it has the function of sensing external signals of cells and transmitting signals, and mediates various physiological and biochemical reactions of extracellular factors to the cells. It is also an intermediary in the connection of cells to adjacent cells and the extracellular matrix. Therefore, the study of plasma membranes is an important aspect in today's biological and biotechnological fields, and has important significance in both basic and application research.
The fluorescence imaging technology has the advantages of high sensitivity, good biocompatibility and the like, so the fluorescence imaging technology is widely applied to the field of imaging of the biological plasma membrane. The traditional plant mass membrane fluorescence labeling method is characterized in that reported plasma membrane localized protein and fluorescent protein are fused and transformed into plants for fluorescence observation, and the traditional plant mass membrane fluorescence labeling method has the defects of complex operation, high difficulty, long period and the like, so that the application of the traditional plant mass membrane fluorescence labeling method in a plurality of experiments is limited. What is needed and needed in the art is a method for rapidly and easily labeling plasma membrane fluorescence.
Disclosure of Invention
In view of the above, the present invention provides a plasma membrane fluorescence labeling method for a plant and a method for locating and dynamically monitoring plasma membrane proteins, which can effectively, accurately and rapidly research the location and dynamics of the plasma membrane proteins of the plant.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a plasma membrane fluorescence labeling method of plants, which comprises the following steps: and (3) dyeing the separated plasma membrane structure by using FM4-64 dye, and washing the dyed plasma membrane structure by using buffer solution to obtain the plasma membrane structure of the plasma membrane fluorescence label.
Preferably, the working solution concentration of the FM4-64 dye is 4 mu M.
Preferably, the plasma membrane structure of said plant comprises root hair cells and/or mesophyll protoplasts.
Preferably, the plant comprises arabidopsis thaliana.
Preferably, the method for isolating root hair cells of arabidopsis thaliana comprises: the method comprises the steps of (1) disinfecting arabidopsis thaliana seeds, sowing the seeds on a solid 1/2MS culture medium, standing at 4 ℃ for 2 days, then carrying out 7-day light-dark alternate culture, and cutting roots of arabidopsis thaliana seedlings as a dyeing material;
the separation method of the mesophyll protoplast of the arabidopsis thaliana comprises the following steps: cutting leaves of 3 rd to 4 th rounds of arabidopsis thaliana, removing the lower skins of the leaves, performing enzymolysis, placing the leaves subjected to enzymolysis in a W5 solution, and collecting precipitates to obtain separated mesophyll protoplasts.
Preferably, the culturing temperature of the light-dark alternate culture is 22 ℃, the photoperiod is 12h of light/12 h of dark, and the light intensity is 150 mu E m-2s-1；
The enzymolysis liquid for enzymolysis comprises the following raw materials in percentage by mass and volume: 1.5% cellulase R10, 0.25% pectinase R10, 0.05% beta-mercaptoethanol and 1% BSA, and further comprising the following raw materials in quantitative concentrations: 0.4M mannitol, 20mM KCl, 20mM MES and 10mM CaCl2，pH＝5.7。
Preferably, the dyeing temperature is 20-25 ℃, and the dyeing time is 1 min.
Preferably, the method further comprises the step of utilizing ddH after the plasma membrane structure of the plant is stained2And O, washing the stained root hair cells, and washing the stained mesophyll protoplasts by using a W5 solution.
The invention also provides a method for monitoring the localization and dynamics of plasma membrane proteins in plants, comprising the steps of: and observing and photographing the plasma membrane structure of the plasma membrane fluorescence mark obtained by the plasma membrane fluorescence marking method by using a laser confocal microscope.
Preferably, the fluorescence excitation wavelength is set to 515nm and the emission wavelength is set to 640 nm.
Has the advantages that: the invention provides a plant plasma membrane fluorescence labeling method, which adopts FM4-64 dye to dye a plasma membrane structure, wherein the FM4-64 dye can be embedded in a plasma membrane bilayer and can carry out dynamic transport along with a plasma membrane, so that the plant plasma membrane fluorescence labeling method can also be used in subsequent related researches such as plasma membrane endocytosis and transport. In addition, the fluorescence labeling method of the invention can perform fluorescence labeling of the plasma membrane in genetic transformation or transient transformation, thereby saving experimental time. In addition, since the FM4-64 dye is red fluorescent, the dye is suitable for the co-localization of the plasma membrane with double fluorescent markers, such as the co-localization experiment with the plasma membrane protein marked by green fluorescent protein.
In the embodiment of the invention, the root hair cells and the mesophyll protoplasts of arabidopsis are subjected to plasma membrane fluorescence labeling, as the arabidopsis mesophyll protoplasts have more chloroplasts and the chloroplasts have red autofluorescence, fluorescence signals can be distinguished according to the fluorescence style and shape, the chloroplast autofluorescence is red spherical or ellipsoidal and is distributed in the mesophyll protoplasts, and the red plasma membrane fluorescence labeling is linear and is only distributed at the periphery of the mesophyll protoplasts; the root hair cells of the plant have no chloroplast, so the interference problem of a fluorescent signal does not exist.
Drawings
FIG. 1 is a graph showing the FM4-64 staining of the plasma membrane of Arabidopsis root hair cell (DsSWEET12-GFP), in which red is the fluorescent signal of FM4-64 stained plasma membrane and green is the fluorescent signal of the plasma membrane protein DsSWEET 12-GFP;
FIG. 2 is a photograph of the coloration of Arabidopsis mesophyll protoplasts FM4-64 (DsSWEET12-GFP) wherein green is the fluorescence signal of the plasma membrane protein DsSWEET12-GFP, red as indicated by yellow arrows is the fluorescence signal of FM4-64 stained plasma membrane, and red as indicated by white arrows is the chlorophyll (Chl) autofluorescence signal.
Detailed Description
The invention provides a plasma membrane fluorescence labeling method of plants, which comprises the following steps: and (3) dyeing the separated plasma membrane structure by using FM4-64 dye, and washing the dyed plasma membrane structure by using buffer solution to obtain the plasma membrane structure of the plasma membrane fluorescence label.
The present invention is not limited to the kind of the plant, and in the examples, the model plant Arabidopsis thaliana is used for illustration, but it is not to be construed as the full scope of the present invention.
The working solution concentration of the FM4-64 dye is preferably 4 mu M, and the preparation method of the working solution of the FM4-64 dye preferably comprises the steps of dissolving 1mg of FM4-64 in 0.5mL of DMSO (dimethyl sulfoxide) and utilizing ddH (ddH)2Diluting O until the concentration of FM4-64 is 4 μ M. The content of FM4-64 in the working solution is preferably 4 mu M.
The FM4-64 dye is used for dyeing a plasma membrane structure, the plasma membrane structure preferably comprises root hair cells and/or mesophyll protoplasts, the dyeing temperature is preferably 20-25 ℃, and the dyeing time is preferably 1 min. The FM4-64 dye is a red dye, can be embedded in a plasma membrane bilayer and can follow the plasma membrane for dynamic transport, so that the dye can be used in subsequent related researches such as plasma membrane endocytosis and transport.
The method for separating and extracting the plasma membrane structure is not particularly limited, and the conventional root hair cell separation method and the mesophyll protoplast separation method in the field can be used. In the embodiment of the present invention, the method for isolating root hair cells of arabidopsis preferably includes: the arabidopsis thaliana seeds are sterilized and then sowed on a solid 1/2MS culture medium, placed for 2 days at 4 ℃, then subjected to 7-day light-dark alternate culture, and roots of arabidopsis thaliana seedlings are cut to be used as dyeing materials. The disinfection of the invention preferably comprises the steps of disinfecting arabidopsis seeds for 30s by 70% (v/v) alcohol, disinfecting for 3min by sodium hypochlorite, and then cleaning for 5 times by using sterile water. According to the invention, after the disinfection, the arabidopsis seeds are sown, and the solid 1/2MS culture medium used for sowing preferably comprises the following raw materials in concentration: 10.3mM NH4NO3，9.4mM KNO3，0.6mM KH2PO4，0.8mM MgSO4，1.5mM CaCl2，2.5μM KI，50μM MnSO4，50μM H3BO3，15μM ZnSO4，0.5μM Na2MoO4，0.05μM CuSO4，0.06μM CoCl2，0.1mM FeSO4，0.1mM Na2-EDTA, 1% agar powder and 3% sucrose, pH 5.8. The invention stands for 2 days at 4 ℃ after sowing, then is placed in a light incubator for vertical culture for 7 days, the culture temperature is set to be 22 ℃, the photoperiod is 12h of light/12 h of darkness, and the light intensity is 150 mu E m-2s-1。
In an embodiment of the present invention, the method for separating mesophyll protoplasts of arabidopsis preferably comprises: cutting leaves of 3 rd to 4 th rounds of arabidopsis thaliana, removing the lower skins of the leaves, performing enzymolysis, placing the leaves subjected to enzymolysis in a W5 solution, and collecting precipitates to obtain separated mesophyll protoplasts. According to the invention, an arabidopsis thaliana plant in an 8-leaf stage with a good growth state is selected preferentially, leaves of 3 rd to 4 th rounds are cut, the lower epidermis of the leaves is removed by using a double-sided adhesive, and the back of the whole leaf is placed downwards into an enzymolysis solution to be dissociated for 1 hour at room temperature. After the dissociation is finished, taking out the leaves, sucking off excessive enzymolysis liquid, adding a W5 solution, naturally precipitating to obtain separated mesophyll protoplasts, and then cleaning the dyed root hair cells and mesophyll protoplasts by adopting a buffer solution.
The enzymolysis liquid for enzymolysis preferably comprises the following raw materials in percentage by mass and volume: 1.5% cellulase R10, 0.25% pectinase R10, 0.05% beta-mercaptoethanol and 1% BSA, and further comprising the following raw materials in quantitative concentrations: 0.4M mannitol, 20mM KCl, 20mM MES and 10mM CaCl2，pH＝5.7。
The present invention preferably further comprises, after said dyeing, the use of ddH2And O, washing the stained root hair cells, and washing the stained mesophyll protoplasts by using a W5 solution. The formula of the W5 solution is preferably as follows: 0.4M mannitol, 20mM KCl and 20mM MES, pH 5.7.
The invention also provides a method for monitoring the localization and dynamics of plasma membrane proteins in plants, comprising the steps of: and observing and photographing the plasma membrane structure obtained by the plasma membrane fluorescence labeling method by using a laser confocal microscope.
The invention uses a laser confocal microscope to observe and photograph the cleaned root hair cells and mesophyll protoplasts, wherein the excitation wavelength is 515nm, and the emission wavelength is 640 nm. At this wavelength, the red autofluorescence of chloroplast can be detected as well, and the fluorescence signals are distinguished and photographed according to the fluorescence pattern and shape, the chloroplast autofluorescence is red spherical or ellipsoidal and is distributed in the mesophyll protoplast, while the red plasma membrane fluorescence label is linear and is distributed only at the periphery of the mesophyll protoplast, which explains the accuracy of the FM4-64 dye labeling the plasma membrane of arabidopsis mesophyll protoplast.
The following examples are provided to describe the method for fluorescent labeling of plasma membrane and the method for locating and dynamically monitoring plasma membrane protein of plant in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Materials: transgenic Columbia Arabidopsis thaliana (Zhou et al, A Novel Sugar Transporter from Dianthus spicifolius, DsSWEET12, Affercts Sugar Metabolism and transfer organization and Oxidative Stress Tolerance in Arabidopsis thaliana, molecular sciences.2018) with plasma membrane localized protein DsSWEET12(GenBank accession number: MG737823)
1. Staining the arabidopsis root hair cytoplasmic membrane;
step one, sterilizing transgenic arabidopsis seeds for 30s by 70% alcohol, sterilizing for 3min by sodium hypochlorite, cleaning for 5 times by sterile water, then paving the seeds on a solid 1/2MS culture medium, and standing for 2 days at 4 ℃;
step two, taking out the culture dish with the seeds, placing the culture dish in an illumination incubator for vertical culture for 7 days, wherein the culture temperature is 22 ℃, the photoperiod is 12h of illumination/12 h of darkness, and the light intensity is 150 mu E m-2s-1；
Step three, cutting the whole root of the arabidopsis thaliana seedling vertically growing for 7 days in a culture dish by using scissors, then putting the arabidopsis thaliana seedling into a 1.5ml centrifuge tube, adding 1ml FM4-64 working solution (the final concentration is 4 mu M) for dyeing for 1min, discarding the dyeing solution, and immediately using ddH2Cleaning twice;
and step four, tabletting, observing by using a laser confocal microscope, finding the root hair cells in the root mature region and taking a picture. As a result, as shown in FIG. 1, the red fluorescent FM4-64 signal can be coincided with the plasma membrane localized green fluorescent DsSWEET-GFP signal, indicating the accuracy of the FM4-64 dye in labeling the plasma membrane of Arabidopsis root hair cells.
2. Staining the protoplast plasma membrane of arabidopsis mesophyll;
step one, separating arabidopsis mesophyll protoplasts: (1) selecting Arabidopsis thaliana plant with good growth state at 8-leaf stage, cutting leaf blade of 3-4 th round, sticking the lower epidermis of leaf blade with double-sided adhesive tape, placing the whole leaf blade into 6-well plate with its back facing downwards, adding 5ml of enzymolysis solution (1.5% (M/v) cellulase R10, 0.25% (M/v) pectinase R10, 0.4M mannitol, 20mM KCl, 20mM MES, 10mM CaCl)2Carrying out enzymolysis on 0.05% (m/v) of beta-mercaptoethanol and 1% (m/v) of BSA at the pH value of 5.7 for 1h at room temperature, and slightly shaking once every 10 min; (2) after enzymolysis, carefully sucking off the enzyme digestion solution by using a liquid shifter, wherein the dissociated mesophyll protoplast is green and is precipitated at the bottom of a pore plate, and the protoplast is not sucked during sucking;
step two, add 200 μ l W5 solution (0.4M mannitol, 20mM KCl, 20mM MES, pH 5.7) to the well plate followed by FM4-64 dye (final concentration 4 μ M) and stain for 1min at room temperature;
step three, carefully sucking off the W5 solution, adding the 200 mu l W5 solution again, washing once, and then resuspending the protoplast by using the 200 mu l W5 solution;
and step four, cutting off the front end of a 1ml blue gun head, carefully sucking 10 mul of the dyed mesophyll protoplast, performing light volume operation to avoid the protoplast from being broken, adding the protoplast onto a glass slide, and preparing the protoplast into a slice.
And fifthly, observing and photographing by using a laser confocal microscope, wherein the excitation wavelength is 515nm, and the emission wavelength is 640 nm. Since the red autofluorescence of chloroplast can be detected at this wavelength as well, the fluorescence signal is distinguished and photographed according to the fluorescence pattern and morphology, the chloroplast autofluorescence is red spherical or ellipsoidal and is distributed in the inner part of mesophyll protoplast, and the red plasma membrane fluorescence label is linear and is distributed only in the periphery of mesophyll protoplast.
As shown in FIG. 2, the FM4-64 signal of red fluorescence can coincide with the DsSWEET-GFP signal of plasma membrane localization, and is located at the periphery of the cell and is linear, while the red spontaneous signal of chlorophyll is spherical or ellipsoidal and is distributed in the protoplast, which illustrates the accuracy of the FM4-64 dye labeling the plasma membrane of the Arabidopsis mesophyll protoplast.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A plant plasma membrane fluorescence labeling method is characterized by comprising the following steps: and (3) dyeing the separated plasma membrane structure by using FM4-64 dye, and washing the dyed plasma membrane structure by using buffer solution to obtain the plasma membrane structure of the plasma membrane fluorescence label.
2. The method for fluorescence labeling of a plasma membrane according to claim 1, wherein the working solution concentration of the FM4-64 dye is 4 μ M.
3. The method for fluorescent labeling of a plasma membrane according to claim 1, wherein the plasma membrane structure of the plant comprises root hair cells and/or mesophyll protoplasts.
4. The plasma membrane fluorescent labeling method of claim 3, wherein the plant comprises Arabidopsis thaliana.
5. The plasma membrane fluorescent labeling method according to claim 4, wherein the method for isolating root hair cells of Arabidopsis thaliana comprises: the method comprises the steps of (1) disinfecting arabidopsis thaliana seeds, sowing the seeds on a solid 1/2MS culture medium, standing at 4 ℃ for 2 days, then carrying out 7-day light-dark alternate culture, and cutting roots of arabidopsis thaliana seedlings as a dyeing material;
the separation method of the mesophyll protoplast of the arabidopsis thaliana comprises the following steps: cutting leaves of 3 rd to 4 th rounds of arabidopsis thaliana, removing the lower skins of the leaves, performing enzymolysis, placing the leaves subjected to enzymolysis in a W5 solution, and collecting precipitates to obtain separated mesophyll protoplasts.
6. The plasma membrane fluorescent labeling method according to claim 5, wherein the culturing temperature of the light-dark alternate culture is 22 ℃, the photoperiod is 12h of light/12 h of dark, and the light intensity is 150 μ E m-2s-1；
The enzymolysis liquid for enzymolysis comprises the following raw materials in percentage by mass and volume: 1.5% cellulase R10, 0.25% pectinase R10, 0.05% beta-mercaptoethanol and 1% BSA, and further comprising the following raw materials in quantitative concentrations: 0.4M mannitol, 20mM KCl, 20mM MES and 10mM CaCl2，pH＝5.7。
7. The plasma membrane fluorescent labeling method according to claim 1, wherein the dyeing temperature is 20 to 25 ℃ and the dyeing time is 1 min.
8. The method for fluorescent labeling of plasma membrane according to claim 3, further comprising the step of staining the plasma membrane structure of the plant with ddH2And O, washing the stained root hair cells, and washing the stained mesophyll protoplasts by using a W5 solution.
9. A method of monitoring the localization and dynamics of plasma membrane proteins in a plant comprising the steps of: observing and photographing a plasma membrane structure of a plasma membrane fluorescently labeled by the plasma membrane fluorescently labeling method according to any one of claims 1 to 8 by using a confocal laser microscope.
10. The method of claim 9, wherein the fluorescence excitation wavelength is set to 515nm and the emission wavelength is set to 640 nm.
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