Method for extracting protoplasts of guard cells on both surfaces of plant leaves

Abstract

The application provides a method for extracting guard cell protoplasts on the upper and lower surfaces of plant leaves, comprising: taking plant leaves, separating the upper and lower surfaces of the leaves by using a paper adhesive tape and a transparent adhesive tape; intermittently homogenizing to separate the upper and lower epidermal tissues of the leaves and the adhesive tapes; removing the adhesive tapes, intermittently homogenizing to obtain leaf upper and lower epidermal fragments; first enzymolysis treatment; placing in a mannitol solution after enzymolysis and standing on ice; second enzymolysis treatment; filtering, centrifuging the filtrate, resuspending and centrifuging each precipitate repeatedly 2-4 times, and obtaining plant leaf upper and lower surface guard cell protoplasts respectively. By using the combination peeling method of the paper adhesive tape and the transparent adhesive tape, the application realizes efficient separation of the upper and lower epidermal tissues of the plant leaves and maintains the cell activity well; by two-step enzymolysis treatment, the plant leaf upper and lower surface guard cell protoplasts are obtained in high yield and low cost respectively.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for extracting guard cell protoplasts on the upper and lower surfaces of a plant leaf. BACKGROUND

[0002] Stomata, which are composed of a pair of guard cells, are the key channels for gas exchange between plants and the environment. For most herbaceous plants, stomata are distributed on both the upper and lower surfaces of leaves. The environmental responses of stomata on the upper and lower surfaces are quite different. Guard cell protoplasts are important materials for studying stomatal movement and plant cell signal transduction processes. In order to study the differences between the upper and lower stomata, a large number of guard cell protoplasts on the upper and lower surfaces of leaves need to be obtained. However, there are currently difficulties in separating and obtaining guard cell protoplasts on the upper and lower surfaces of leaves.

[0003] Traditionally, guard cell protoplasts are obtained mainly by enzymatic digestion. Mature leaves are placed in pre-cooled distilled water, and homogenized in a low-temperature environment using a high-speed homogenizer to obtain uniform leaf epidermis fragments. Then the epidermis fragments are transferred to an enzymatic solution containing cellulase and pectinase, and shaken in a dark water bath at low speed for 2-4 hours to gradually degrade the cell wall and release the guard cell protoplasts. After enzymatic digestion, the undigested tissue fragments are removed by filtering the filtrate through a cell sieve with a pore size of less than 35 μm. The collected filtrate is transferred to a centrifuge tube and centrifuged at low speed to obtain a protoplast precipitate. This method is commonly used to obtain guard cell protoplasts. However, directly enzymatically digesting the tissue fragments of the whole leaf causes the upper and lower guard cells to mix in the same enzymatic system, making it impossible to distinguish their sources, which severely limits in-depth research on the differences between the upper and lower stomata of leaves. The upper epidermis of most plants is closely adhered to the mesophyll cells, and if an attempt is made to pre-strip the upper and lower epidermis using tweezers or other methods, the process may cause mechanical damage to the guard cells.

[0004] Laser capture microdissection technology can precisely distinguish epidermis fragments based on the morphological differences between the upper and lower surfaces of leaves, and further obtain upper and lower guard cells (including cell walls). First, the whole leaf is homogenized to obtain epidermis fragments, and then fixed, embedded, and other pretreated. The prepared sample is placed on the stage and the guard cells are precisely positioned in the field of view of the microscope. The upper and lower guard cells are labeled using a laser capture microdissection system. Then, according to the labeled positions, the laser beam is used for cutting, thereby obtaining the upper and lower guard cells. Although this method can precisely separate the upper and lower guard cells, the sample needs to be fixed, embedded, and other pretreated, which may affect the activity of the guard cells; the operation efficiency is low, and only 10 2 guard cells can be obtained at a time, which is difficult to meet the needs of large-scale experiments (for example, the number of cells required for transcriptomic analysis is usually about 10 6) and high equipment cost, complicated operation. Therefore, this method has not been widely used in research. In summary, there is no effective separation technology for the upper and lower epidermis of model plants such as Arabidopsis thaliana. SUMMARY

[0005] The purpose of the present application is to provide a method for extracting guard cell protoplasts on the upper and lower surfaces of plant leaves, to realize efficient separation of upper and lower epidermis tissues of plant leaves and maintain cell activity, and to obtain a large amount of guard cell protoplasts on the upper and lower surfaces of plant leaves with high yield and low cost. The specific technical solutions are as follows:

[0006] The present application provides a method for extracting guard cell protoplasts on the upper and lower surfaces of plant leaves, comprising:

[0007] S1. Take a plant leaf, paste the upper surface of the plant leaf on a paper tape, remove the midrib, then use a transparent tape to peel off the lower surface of the plant leaf, separate the upper and lower surfaces of the plant leaf; immerse the obtained upper epidermis tissue of the leaf and the paper tape, and the lower epidermis tissue of the leaf and the transparent tape in stomatal buffer solution in ice bath, respectively;

[0008] The stomatal buffer solution comprises 45-55 mmol / L KCl, 0.08-0.12 mmol / L CaCl2, 4-6 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES)·1,3-bis[(trihydroxymethyl)methylamino]propane;

[0009] S2. Take out the upper epidermis tissue of the leaf and the paper tape, and the lower epidermis tissue of the leaf and the transparent tape, respectively, add ice bath stomatal buffer solution, and intermittent homogenate 3-5 times, each time for 4-6 s, to separate the upper and lower epidermis tissues of each leaf and the tape;

[0010] S3. Remove the separated tape, and add ice bath stomatal buffer solution to the separated upper epidermis tissue of the leaf and the lower epidermis tissue of the leaf, respectively, and intermittent homogenate 3-5 times, each time for 24-36 s; then filter the respective obtained fragments through a 160-240 mesh filter to obtain upper epidermis fragments and lower epidermis fragments of the leaf, respectively;

[0011] S4. First enzymolysis treatment, comprising:

[0012] Respectively, the upper epidermis fragments and the lower epidermis fragments are subjected to enzymolysis filtration with enzymolysis solution I to obtain the first enzymolysis upper epidermis fragments and lower epidermis fragments;

[0013] The enzymatic hydrolysate I comprises: 0.4% (w / v) - 0.6% (w / v) cellulase R10, 0.04% (w / v) - 0.06% (w / v) dissociative enzyme R10, 0.16% (w / v) - 0.24% (w / v) bovine serum albumin, 0.08% (w / v) - 0.12% (w / v) polyvinylpyrrolidone, 0.2-0.3 mol / L mannitol, 0.8-1.2 mmol / L CaCl2, and 8-12 mmol / L MES-KOH;

[0014] S5. Place the upper and lower epidermal fragments after the first enzymatic hydrolysis into a 0.24-0.36 mol / L mannitol solution and let them stand on ice for 25-35 min.

[0015] S6. Second enzymatic hydrolysis treatment, including:

[0016] Filtration: The upper and lower epidermal fragments were subjected to a second enzymatic hydrolysis using enzymatic hydrolysis solution II, and then filtered to obtain upper and lower epidermal filtrate.

[0017] The enzymatic hydrolysate II comprises: 1.2% (w / v)-1.8% (w / v) cellulase RS, 0.4% (w / v)-0.6% (w / v) dissociative enzyme R10, 0.16% (w / v)-0.24% (w / v) bovine serum albumin, 0.32-0.48 mol / L mannitol, and 0.8-1.2 mmol / L CaCl2;

[0018] S7. Centrifuge the upper epidermal filtrate and the lower epidermal filtrate, discard the supernatant, resuspend each precipitate with washing solution II, centrifuge again, repeat 2-4 times to obtain the guard cell protoplasts on the upper surface of plant leaves and the guard cell protoplasts on the lower surface of plant leaves, respectively.

[0019] The rinsing solution II comprises 0.32-0.48 mol / L mannitol and 0.8-1.2 mmol / L CaCl2.

[0020] In one embodiment, the extraction method further includes:

[0021] S8. Purification process, including:

[0022] The guard cell protoplasts on the upper surface of the plant leaf and the guard cell protoplasts on the lower surface of the plant leaf were resuspended in a pre-cooled 0.32-0.48 mol / L mannitol solution to obtain a guard cell protoplast suspension on the upper surface of the leaf and a guard cell protoplast suspension on the lower surface of the leaf.

[0023] The guard cell protoplast suspensions on the upper and lower surfaces of the leaves were transferred to human lymphocyte separation medium, respectively, so that the guard cell protoplasts were suspended on the surface of the human lymphocyte separation medium. Then, the cells were centrifuged and separated into layers. The middle layer was the purified guard cell protoplasts on the upper and lower surfaces of the plant leaves.

[0024] In one embodiment, the extraction method further includes:

[0025] S9. Saving process, including:

[0026] The purified guard cell protoplasts from the upper and lower surfaces of plant leaves were diluted with 0.32-0.48 mol / L mannitol solution, respectively. After centrifugation, the supernatant was discarded, and 0.32-0.48 mol / L mannitol solution was added to each precipitate to suspend the guard cell protoplasts. The precipitates were then stored in the dark to obtain storage solutions for guard cell protoplasts from the upper and lower surfaces of plant leaves.

[0027] In one embodiment, in step S3, the leaf upper epidermal fragments and leaf lower epidermal fragments obtained after filtration are rinsed with pre-cooled stomatal buffer solution, so that the leaf upper epidermal fragments and leaf lower epidermal fragments are respectively concentrated in the center of the filter screen.

[0028] In one embodiment, in step S4, after obtaining the upper and lower epidermal fragments after the first enzymatic hydrolysis, they are rinsed with rinsing solution I to concentrate the upper and lower epidermal fragments after the first enzymatic hydrolysis into the center of the filter screen; the rinsing solution I includes: 0.24-0.36 mol / L mannitol and 0.8-1.2 mmol / L CaCl2.

[0029] In one embodiment, in step S7, the upper epidermal filtrate and the lower epidermal filtrate are filtered through a 400-600 mesh filter before centrifugation, the filtrates are collected, and then centrifuged; wherein, after filtration, the filter screens are rinsed with pre-cooled rinsing solution II.

[0030] In one embodiment, the volume ratio of the guard cell protoplast suspension on the upper surface of the leaf to the human lymphocyte separation solution is 1:(0.8-1.2), and the volume ratio of the guard cell protoplast suspension on the lower surface of the leaf to the human lymphocyte separation solution is 1:(0.8-1.2).

[0031] In one embodiment, the plant leaf is a herbaceous plant leaf.

[0032] In one embodiment, the herbaceous plant leaf is an Arabidopsis thaliana leaf.

[0033] In one embodiment, the Arabidopsis leaf is an Arabidopsis rosette leaf that has grown for 3-5 weeks.

[0034] The beneficial effects of this application are:

[0035] This application provides a method for extracting guard cell protoplasts from the upper and lower surfaces of plant leaves, comprising: taking plant leaves, separating the upper and lower surfaces of the plant leaves using paper tape and transparent tape; intermittent homogenization to separate the epidermal tissues of the upper and lower surfaces of each leaf from the tape; removing the tape, intermittently homogenizing to obtain fragments of the upper and lower epidermal tissues of the leaves; a first enzymatic hydrolysis treatment; placing the hydrolyzed leaves in a mannitol solution and incubating on ice; a second enzymatic hydrolysis treatment; filtering, centrifuging the filtrate, resuspending each precipitate, centrifuging again, repeating this process 2-4 times to obtain guard cell protoplasts from the upper and lower surfaces of the plant leaves, respectively. This application achieves efficient separation of the upper and lower epidermal tissues of plant leaves while maintaining cell viability through a combined paper tape and transparent tape peeling method; and obtains guard cell protoplasts from the upper and lower surfaces of plant leaves in high yield and at low cost through a two-step enzymatic hydrolysis process, which can meet the requirements of subsequent large-scale experiments (such as transcriptomics analysis) for guard cell viability, quantity, and purity. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these accompanying drawings.

[0037] Figure 1 The images show the process of separating the epidermal tissue from the upper and lower surfaces of the leaves of the Col-0 ecotype Arabidopsis thaliana in step S1 of Example 1. The upper surface of the leaf is attached to paper tape as shown in Figure a; the leaf after the midrib is removed with surgical scissors is shown in Figure b; the lower surface of the leaf is peeled off using transparent tape as shown in Figure c; and the leaves are then immersed in stomatal buffer solution as shown in Figure d. In Figure d, ad represents the epidermal tissue from the upper surface of the leaf and the paper tape, and ab represents the epidermal tissue from the lower surface of the leaf and the transparent tape.

[0038] Figure 2 Bright-field and fluorescence images of guard cells on the upper and lower surfaces of leaves of the Col-0 ecotype Arabidopsis thaliana obtained in step S3 of Example 1, as measured by fluorescence microscopy.

[0039] Figure 3This is a diagram showing the cell distribution results after mixing the storage solution of guard cell protoplasts on the upper and lower surfaces of leaves of the Col-0 ecotype Arabidopsis thaliana obtained in step S9 of Example 1;

[0040] Figure 4 The image shows the total number of guard cells collected from the upper and lower surfaces in a single experiment of Example 1, Comparative Example 1, and Comparative Example 2. Detailed Implementation

[0041] The technical solutions of this application will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0042] This application provides a method for extracting guard cell protoplasts from both the upper and lower surfaces of plant leaves, including:

[0043] S1. Take a plant leaf, attach the upper surface of the plant leaf to paper tape, remove the midrib, and then use transparent tape to peel off the lower surface of the plant leaf to separate the upper and lower surfaces of the plant leaf; immerse the obtained upper surface epidermal tissue and paper tape, and the lower surface epidermal tissue and transparent tape of the leaf in an ice bath stomatal buffer solution respectively.

[0044] The stomatal buffer solution comprises 45-55 mM (mmol / L) KCl, 0.08-0.12 mmol / L CaCl2, and 4-6 mmol / L LME S·1,3-bis[(tris(hydroxymethyl)methylamino)propane;

[0045] S2. Take out the epidermal tissue and paper tape on the upper surface of the leaf, and the epidermal tissue and transparent tape on the lower surface of the leaf, and add them to the stomatal buffer solution in an ice bath. Homogenize intermittently 3-5 times, each time for 4-6 seconds, so that the epidermal tissue and tape on the upper and lower surfaces of each leaf are separated (i.e., the epidermal tissue on the upper surface of the leaf is separated from the paper tape, and the epidermal tissue on the lower surface of the leaf is separated from the transparent tape).

[0046] S3. Remove the separated tape, add the separated upper and lower surface epidermal tissues of the leaves to the stomatal buffer solution in an ice bath, and homogenize intermittently 3-5 times, each time for 24-36 seconds; then filter the obtained fragments through a 160-240 mesh filter to obtain upper and lower surface epidermal fragments of the leaves respectively.

[0047] S4. First enzymatic hydrolysis treatment, including:

[0048] The upper epidermal fragments and the lower epidermal fragments of the leaf were enzymatically hydrolyzed with enzyme hydrolysate I, and then filtered to obtain the upper epidermal fragments and lower epidermal fragments after the first enzymatic hydrolysis.

[0049] Preferably, the first enzymatic hydrolysis treatment includes: slowly rinsing the upper epidermal fragments and the lower epidermal fragments of the leaves into a reagent bottle with enzymatic hydrolysate I, and then incubating them in a water bath at 22-26°C and 55-85 rpm for 25-35 minutes; then gently blowing the enzymatic hydrolysate I in the reagent bottle every 8-12 minutes, repeating 2-4 times; after the enzymatic hydrolysis is completed, filtering them through a 160-240 mesh filter to obtain the upper epidermal fragments and lower epidermal fragments after the first enzymatic hydrolysis.

[0050] The enzymatic hydrolysate I comprises: 0.4% (w / v) - 0.6% (w / v) cellulase R10 (i.e., cellulase R10 concentration is 0.2 g / 100 mL - 0.8 g / 100 mL), 0.04% (w / v) - 0.06% (w / v) dissociative enzyme R10, 0.16% (w / v) - 0.24% (w / v) bovine serum albumin, 0.08% (w / v) - 0.12% (w / v) polyvinylpyrrolidone, 0.2-0.3 mol / L mannitol, 0.8-1.2 mmol / L CaCl2, and 8-12 mmol / L (2-(N-morpholino)ethanesulfonic acid)-potassium hydroxide (MES-KOH);

[0051] S5. Place the upper and lower epidermal fragments after the first enzymatic hydrolysis into a 0.24-0.36M (mol / L) mannitol solution and let them stand on ice for 25-35 minutes to adjust the osmotic pressure.

[0052] S6. Second enzymatic hydrolysis treatment, including:

[0053] Filtration: The upper and lower epidermal fragments were subjected to a second enzymatic hydrolysis using enzymatic hydrolysis solution II, and then filtered to obtain upper and lower epidermal filtrate.

[0054] Preferably, the second enzymatic hydrolysis treatment includes: filtering with a 160-240 mesh filter, slowly rinsing the upper and lower epidermal fragments on the filter screen into a new reagent bottle with enzymatic hydrolysis solution II, and then oscillating at a constant temperature of 25-29°C and 40-60 rpm in a water bath until more than 80% of the guard cell protoplasts detach from the cell wall, at which point the enzymatic hydrolysis is stopped; subsequently, filtering through a 160-240 mesh filter to obtain upper epidermal filtrate and lower epidermal filtrate;

[0055] The enzymatic hydrolysate II comprises: 1.2% (w / v)-1.8% (w / v) cellulase RS, 0.4% (w / v)-0.6% (w / v) dissociative enzyme R10, 0.16% (w / v)-0.24% (w / v) bovine serum albumin, 0.32-0.48 mol / L mannitol, and 0.8-1.2 mmol / L CaCl2;

[0056] S7. Centrifuge the upper epidermal filtrate and the lower epidermal filtrate, discard the supernatant, resuspend each precipitate with washing solution II, centrifuge again, repeat 2-4 times to obtain the guard cell protoplasts on the upper surface of plant leaves and the guard cell protoplasts on the lower surface of plant leaves, respectively.

[0057] The rinsing solution II comprises 0.32-0.48 mol / L mannitol and 0.8-1.2 mmol / L CaCl2.

[0058] In this application, the pH of the enzymatic hydrolysate I is 5.2-5.6; the pH of the enzymatic hydrolysate II is 5.2-5.6. This application does not specifically limit the source of the paper tape or transparent tape, as long as it achieves the purpose of this application; for example, it can be purchased commercially. In step S1, this application does not specifically limit the method of "removing the midrib," as long as it achieves the purpose of this application; for example, the midrib can be removed by surgical scissors. The "separation of the upper and lower surfaces of the plant leaf" is based on the result that the epidermal tissue on the lower surface of the plant leaf is completely detached from the leaf without residue. In step S2, the amount of "stomatal buffer" added is based on completely submerging the surface of each tape. In steps S2-S3, intermittent homogenization is used to avoid continuous operation of the homogenizer generating heat and damaging cells. In step S4, the "gentle blowing" is based on the principle that no air bubbles are generated during blowing; for example, a glass pipette can be used for gentle blowing. In step S7, the centrifugation and re-centrifugation conditions may include centrifugation at 1800-2000 rpm and 3-5℃ for 12-16 min.

[0059] The method for extracting guard cell protoplasts from the upper and lower surfaces of plant leaves provided in this application solves the technical bottleneck of existing technologies that cannot obtain large quantities of guard cell protoplasts from the upper and lower surfaces of plant leaves separately. It employs a combination of paper tape and transparent tape for peeling, achieving efficient separation of the upper and lower epidermal tissues of plant leaves while maintaining cell viability. This overcomes the problem that traditional enzymatic digestion methods cannot distinguish the source of guard cell protoplasts from the upper and lower surfaces (i.e., cannot distinguish between upper and lower epidermal tissues). Through a two-step enzymatic digestion process, large quantities of guard cell protoplasts from the upper and lower surfaces of plant leaves can be obtained separately at low cost, overcoming the bottleneck of low yield and high cost of laser capture microdissection technology. This provides important technical support for subsequent studies such as transcriptome analysis, proteome analysis, and subcellular localization, and can meet the sample quantity and purity requirements of large-scale experiments (such as transcriptomics).

[0060] In one implementation, in step S6, the water bath and constant temperature oscillation time is not less than 40 minutes.

[0061] In one embodiment, in step S6, to ensure complete enzymatic hydrolysis, 20-25 minutes after the start of enzymatic hydrolysis (starting from the time of the start of isothermal shaking), a small amount of sample can be taken every 5 minutes for observation under an optical microscope until more than 80% of the guard cell protoplasts are observed to have detached from the cell wall, at which point the enzymatic hydrolysis is stopped.

[0062] In one embodiment, the extraction method further includes:

[0063] S8. Purification process, including:

[0064] The guard cell protoplasts on the upper surface of the plant leaf and the guard cell protoplasts on the lower surface of the plant leaf were resuspended in a pre-cooled 0.32-0.48 mol / L mannitol solution to obtain a guard cell protoplast suspension on the upper surface of the leaf and a guard cell protoplast suspension on the lower surface of the leaf.

[0065] The guard cell protoplast suspensions on the upper and lower surfaces of the leaves are transferred to human lymphocyte separation medium, respectively, so that the guard cell protoplasts are suspended on the surface of the human lymphocyte separation medium; then centrifuged and separated into layers, the middle layer being the purified guard cell protoplasts on the upper and lower surfaces of the plant leaves; preferably, in this step, the centrifugation includes centrifugation at 1000-1200 rpm and 3-5℃ for 14-16 min.

[0066] In one embodiment, the extraction method further includes:

[0067] S9. Saving process, including:

[0068] The purified guard cell protoplasts from the upper and lower surfaces of plant leaves were diluted with 0.32-0.48 mol / L mannitol solution, respectively, and then centrifuged. The supernatant was discarded, and 0.32-0.48 mol / L mannitol solution was added to each precipitate to suspend the guard cell protoplasts. The precipitates were then stored in the dark to obtain storage solutions for guard cell protoplasts from the upper and lower surfaces of plant leaves. Preferably, the centrifugation in this step includes centrifugation at 350-450 g and 3-5 °C for 10-14 min.

[0069] In one embodiment, in step S3, the leaf upper epidermal fragments and leaf lower epidermal fragments obtained after filtration are rinsed with pre-cooled stomatal buffer solution, so that the leaf upper epidermal fragments and leaf lower epidermal fragments are respectively concentrated in the center of the filter screen.

[0070] In one embodiment, in step S4, after obtaining the upper and lower epidermal fragments after the first enzymatic hydrolysis, they are rinsed with rinsing solution I to concentrate the upper and lower epidermal fragments after the first enzymatic hydrolysis into the center of the filter screen; the rinsing solution I includes: 0.24-0.36 mol / L mannitol and 0.8-1.2 mmol / L CaCl2.

[0071] In one embodiment, in step S7, the upper epidermal filtrate and the lower epidermal filtrate are filtered through a 400-600 mesh filter before centrifugation, the filtrates are collected, and then centrifuged; wherein, after filtration, the filter screens are rinsed with pre-cooled rinsing solution II. Repeated washing prevents guard cell protoplasts from remaining on the filter screen and affecting yield.

[0072] In one embodiment, the volume ratio of the guard cell protoplast suspension on the upper surface of the leaf to the human lymphocyte separation solution is 1:(0.8-1.2), and the volume ratio of the guard cell protoplast suspension on the lower surface of the leaf to the human lymphocyte separation solution is 1:(0.8-1.2).

[0073] This application does not have any particular limitation on the method of removing the separated tape in step S3, as long as it can achieve the purpose of this application; in one embodiment, in step S3, the separated tape can be removed by tweezers.

[0074] In this application, "ice bath" refers to incubation in an ice-water mixture; "pre-cooling" refers to pre-controlling the temperature to 0-4℃. "% (w / v)" means "g / 100mL".

[0075] The solvents for the stomatal buffer, the enzymatic hydrolysate I, the enzymatic hydrolysate II, the rinsing solution I, the rinsing solution II, and the mannitol solution described in this application are all water, such as distilled water.

[0076] In one embodiment, the plant leaf is a herbaceous plant leaf. In one embodiment, the herbaceous plant leaf is an Arabidopsis thaliana leaf. In one embodiment, the Arabidopsis thaliana leaf is a rosette leaf of Arabidopsis thaliana that has grown for 3-5 weeks.

[0077] Example

[0078] The embodiments and comparative examples provided below illustrate the implementation of this application in more detail. Various tests and evaluations were conducted according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" are quality standards.

[0079] Prepare stomatal buffer, enzyme digestion solution I, enzyme digestion solution II, rinsing solution I, and rinsing solution II:

[0080] Stomatal buffer: An aqueous solution containing 50 mM KCl, 0.1 mM CaCl2, and 5 mM MES·1,3-bis[(trishydroxymethyl)methylamino]propane. Preparation steps: Take a 200 mL beaker and weigh out 0.37275 g potassium chloride, 0.0015 g calcium chloride dihydrate, 0.09762 g MES, and 0.141165 g 1,3-bis[(trishydroxymethyl)methylamino]propane. Add these to the beaker, and then measure 80 mL of distilled water using a graduated cylinder and pour it into the beaker. Place the beaker on a magnetic stirrer and stir until completely dissolved. Bring the volume to 100 mL, then transfer to an Erlenmeyer flask and refrigerate at 4°C for later use.

[0081] Enzymatic hydrolysate I: An aqueous solution containing 0.5% (w / v) cellulase R10 (i.e., 0.5 g / 100 mL), 0.05% (w / v) analyte R10, 0.2% (w / v) bovine serum albumin, 0.1% (w / v) polyvinylpyrrolidone K30, 0.25 M mannitol, 1 mM CaCl2, and 10 mM MES-KOH, pH=5.4. Preparation steps include:

[0082] Step 1: Preparation of calcium chloride dihydrate stock solution and MES-KOH stock solution: 1. Weigh 7.351 g of calcium chloride dihydrate into a 50 mL centrifuge tube, add 40 mL of distilled water, invert and mix well to completely dissolve the calcium chloride dihydrate, and finally add distilled water to make up to 50 mL to obtain the calcium chloride dihydrate stock solution; 2. Weigh 2.132 g of MES into a 10 mL centrifuge tube, add 8 mL of distilled water, invert and mix well to completely dissolve the MES, then adjust the pH to 5.4 with KOH solution, and finally add distilled water to make up to 10 mL to obtain the MES-KOH stock solution.

[0083] Step 2: Prepare 100 mL of enzymatic hydrolysate I: Take a 200 mL beaker and weigh out 4.5543 g mannitol, 0.2 g bovine serum albumin, 0.1 g polyvinylpyrrolidone K30, 0.5 g cellulase R10, 0.05 g analyte R10, 100 μL calcium chloride dihydrate stock solution, and 1 mL MES-KOH stock solution. Measure 80 mL of distilled water into the beaker using a graduated cylinder. Place the beaker on a magnetic stirrer and stir until completely dissolved. Bring the volume to 100 mL and immediately seal the beaker with sealing film for later use.

[0084] Enzymatic hydrolysate II: An aqueous solution containing 1.5% (w / v) cellulase RS, 0.5% (w / v) analyte R10, 0.2% (w / v) bovine serum albumin, 0.4M mannitol, and 1mM CaCl2, pH=5.4. Preparation steps include:

[0085] Step 1: Prepare calcium chloride dihydrate stock solution: Weigh 7.351 g of calcium chloride dihydrate into a 50 mL centrifuge tube, add 40 mL of distilled water, invert the tube to mix thoroughly until the calcium chloride dihydrate is completely dissolved, and finally add distilled water to make up to 50 mL to obtain calcium chloride dihydrate stock solution.

[0086] Step 2: Prepare 100 mL of enzymatic hydrolysate II: Take a 200 mL beaker and weigh out 5.8294 g mannitol, 0.16 g bovine serum albumin, 1.2 g cellulase RS, 0.4 g analyte R10, and 80 μL of calcium chloride dihydrate stock solution. Measure 60 mL of distilled water into the beaker using a graduated cylinder. Place the beaker on a magnetic stirrer and stir until completely dissolved. Adjust the volume to 80 mL. Adjust the pH to 5.4 with 0.1 M sodium hydroxide solution (NaOH) or 0.1 M hydrochloric acid solution (HCl). Immediately seal the beaker with sealing film for later use.

[0087] Rinsing Solution I: An aqueous solution containing 0.3M mannitol and 1mM CaCl2. Preparation steps: Weigh 5.4651 g of mannitol and 0.0147 g of calcium chloride dihydrate, and place them together in a 200 mL beaker. Measure 80 mL of distilled water into the beaker using a graduated cylinder. Place the beaker on a magnetic stirrer and stir until completely dissolved. Then, bring the volume to 100 mL with distilled water.

[0088] Rinsing Solution II: An aqueous solution containing 0.4M mannitol and 1mM CaCl2. Preparation steps: Weigh 7.2868g of mannitol and 0.0147g of calcium chloride dihydrate, and place them together in a 200mL beaker. Measure 80mL of distilled water into the beaker using a graduated cylinder. Place the beaker on a magnetic stirrer and stir until completely dissolved. Then, bring the volume to 100mL with distilled water.

[0089] Example 1

[0090] S1. Weigh 10g of fully expanded Col-0 ecotype Arabidopsis rosette leaves (approximately four weeks old) as experimental material. Attach the upper surface of the leaf to 3M Scotch adhesive tape and remove the midrib with surgical scissors. Then, use transparent tape to peel off the lower surface of the leaf, thus separating the upper and lower surfaces. The separation is considered complete when the lower surface epidermis detaches from the leaf intactly without residue. After separation, the upper surface epidermis and adhesive tape, and the lower surface epidermis and transparent tape, are temporarily stored in a stomatal buffer solution in an ice bath (0°C). The procedure is as follows: Figure 1 As shown, the upper surface of the blade is attached to the paper tape, as... Figure 1 As shown in Figure a; the leaf blade after the midrib has been removed using surgical scissors, as shown in Figure a. Figure 1 As shown in Figure b; use transparent tape to peel off the lower surface of the blade, as shown. Figure 1 As shown in Figure c; they were immersed in pore buffer solution, as follows. Figure 1 As shown in Figure d, ad represents the epidermal tissue and paper tape on the upper surface of the leaf, and ab represents the epidermal tissue and transparent tape on the lower surface of the leaf.

[0091] S2. Take out the separated upper surface epidermal tissue and paper tape, and the lower surface epidermal tissue and transparent tape, and pour them into a homogenizer. Add stomatal buffer solution in an ice bath to completely submerge each tape. Start the homogenizer intermittently 4 times, with each homogenization time being about 5 seconds, thereby separating the upper and lower surface epidermal tissues of the leaf from the tape.

[0092] S3. Use tweezers to remove the separated tape from the homogenizer. Pour the obtained stomatal buffer containing the epidermal tissue from the upper and lower surfaces of the leaf back into the homogenizer, starting it intermittently four times, each time for about 30 seconds, to avoid heat damage to the cells from continuous operation. Then filter the obtained epidermal fragments from the upper and lower surfaces of the leaf through a 200-mesh filter, while rinsing with pre-cooled (0-4℃) stomatal buffer, gathering the upper and lower epidermal fragments into the center of the filter.

[0093] S4. First enzymatic hydrolysis: The obtained upper and lower epidermal fragments of the leaves were slowly rinsed into an Erlenmeyer flask with enzymatic hydrolysis solution I, and then shaken at 24°C and 70 rpm for 30 min in a water bath. Subsequently, the enzymatic hydrolysis solution I in the Erlenmeyer flask was gently agitated with a 10 mL glass pipette every 10 min, repeated three times. No bubbles were generated during agitation. After enzymatic hydrolysis, the fragments were filtered through a 200-mesh filter and rinsed with rinsing solution I, collecting the upper and lower epidermal fragments from the first enzymatic hydrolysis in the center of the filter screen.

[0094] S5. Place the upper and lower epidermal fragments after the first enzymatic hydrolysis into 0.3M mannitol solution (solvent is distilled water) and let them stand on ice for 30 minutes to adjust the osmotic pressure.

[0095] S6. Second enzymatic hydrolysis: Filter through a 200-mesh filter, and slowly rinse the upper and lower epidermal fragments from the filter into new conical flasks using enzymatic hydrolysis solution II. Incubate in a water bath at 27°C and 50 rpm for at least 40 minutes. To ensure complete hydrolysis, 20-25 minutes after the start of hydrolysis, take small samples every 5 minutes and observe them under an optical microscope (Model: MoticB5; Manufacturer: Motic Industrial Group Co., Ltd.). Stop hydrolysis when more than 80% of the guard cell protoplasts have detached from the cell wall. Then filter through a 200-mesh filter to obtain upper and lower epidermal filtrates.

[0096] S7. Filter the upper and lower epidermal filtrates separately through a 500-mesh filter and collect the filtrates into 30 mL glass centrifuge tubes. After filtration, rinse the filter repeatedly with pre-cooled (0-4℃) washing buffer II to prevent guard cell protoplasts from remaining on the filter and affecting yield. Then centrifuge at 1900 rpm and 4℃ for 14 min and discard the supernatant. Resuspend each precipitate separately with washing buffer II and centrifuge under the same conditions (1900 rpm and 4℃ for 14 min), repeating three times. The resulting precipitates are the guard cell protoplasts on the upper and lower surfaces of plant leaves, respectively.

[0097] S8. Purification treatment: The density gradient centrifugation method was used. Specifically, the above-obtained guard cell protoplasts on the upper and lower surfaces of plant leaves were resuspended in 2 mL of pre-cooled (temperature 0-4℃) 0.4M mannitol solution (solvent is distilled water) to obtain the upper and lower surface guard cell protoplast suspensions.

[0098] Add 2 mL of human lymphocyte separation medium (histopaque-1077) to two clean 10 mL glass centrifuge tubes. Using a glass dropper, slowly transfer the guard cell protoplast suspensions from the upper and lower surfaces of the leaf to the centrifuge tubes containing the human lymphocyte separation medium, ensuring the guard cell protoplasts remain suspended on the surface of the separation medium. Centrifuge at 1100 rpm and 4 °C for 15 min. It can be observed that the liquid in each tube separates into three layers, with the pale green middle layer consisting of purified guard cell protoplasts from the upper and lower surfaces of the plant leaves.

[0099] S9. Preservation: Using a glass dropper, transfer the purified guard cell protoplasts from the upper and lower surfaces of plant leaves (after intermediate layer purification) to new 10mL clean glass centrifuge tubes. Dilute with 0.4M mannitol solution (distilled water), then centrifuge at 400g and 4℃ for 12min, discarding the supernatant. Add a small amount of 0.4M mannitol solution (distilled water) to each precipitate to suspend the guard cell protoplasts, obtaining storage solutions for the upper and lower surfaces of plant leaf guard cell protoplasts. Store these solutions in the dark at 4℃ for later use.

[0100] Comparative Example 1

[0101] The method described in the reference "Guard cells on the adaxial and abaxial leaf surfaces use different compositions of potassium ion channels to drive light-induced stomatal opening" (Nature Plants, Volume 11, pp. 1260-1269) was used for extraction; specifically:

[0102] Except for replacing the pore buffer solution in steps S1-S3 with ice water, the rest is the same as in Example 1.

[0103] Comparative Example 2

[0104] Plant leaf guard cells were obtained using laser capture microdissection technology, as described in the following references: Galbiati, M., Simoni, L., Pavesi, G., et al. (2008), Gene trap lines identify Arabidopsis genes expressed in stomatal guard cells. The Plant Journal, 53: 750-762. and Nancy M. Kerk, Teresa Ceserani, S. Lorraine Tausta, Ian M. Sussex, Timothy M. Nelson, Laser Capture Microdissection of Cells from Plant Tissues, PlantPhysiology, Volume 132, Issue 1, May 2003, Pages 27–35.

[0105] S1. Experimental preparation: Use RNase-free glass staining boxes, plastic biopsy boxes, and forceps, and store them at 4°C after sterilization; prepare fixative (a 3:1 volume ratio of anhydrous ethanol-glacial acetic acid mixture, freshly prepared at 4°C or sealed and stored for no more than 1 week), a series of ethanol solutions (75%, 85%, 100%, all volume fractions, sealed and stored at room temperature, to be used within 1 month), ethanol-xylene gradient solutions (75:25, 50:50, 25:75, 0:100, volume ratios, sealed and stored at room temperature, to be used within 1 week), paraffin (melting point 55-65°C), and other reagents.

[0106] S2. Tissue Fixation and Infiltration: Select rosette leaves of Arabidopsis thaliana, place the leaf tissue containing guard cells in RNase-free distilled water, cut the target tissue block (thickness ≤4mm) with a sterile blade, immediately place it in a marked plastic biopsy box, and quickly immerse it in a 4℃ pre-cooled fixative solution to ensure complete immersion of the tissue block; apply vacuum-assisted infiltration for 15 minutes to promote the fixative solution to enter the interstitial space; remove the biopsy box and incubate sequentially with 75% ethanol at room temperature for 3 hours, 85% ethanol for 3 hours, and 100% ethanol for 3 hours (repeated 3 times, with fresh ethanol each time) to complete dehydration; subsequently, incubate sequentially with an ethanol-xylene gradient solution for 3 hours (with fresh solution each time) to achieve tissue transparency; finally, add paraffin fragments to the container, and after the paraffin is completely dissolved at room temperature, transfer the container to a 58℃ oven, replacing the liquefied paraffin every 3-6 hours until there is no xylene odor around the tissue (approximately 12-18 hours), completing paraffin infiltration.

[0107] S3. Tissue embedding: Pour molten paraffin at 58°C into a sterile embedding mold. Use sterile forceps to orient the paraffin-infiltrated tissue blocks in the molten paraffin (arrange them horizontally for easy identification of guard cell morphology laterally). Allow the tissue blocks to cool and solidify naturally at room temperature. Once the paraffin has completely hardened, trim any excess paraffin from the edges of the embedding blocks with a blade. Place the embedding blocks in a sealed container containing a desiccant and store at 4°C for no more than 4 weeks to prevent the paraffin from absorbing moisture and affecting the quality of the sections.

[0108] S4. Tissue Sectioning: Fix the embedded block on the sample holder of the rotary microtome, install the disposable microtome blade, adjust the section thickness to 10μm, and start the microtome to obtain continuous tissue sections; gently float the sections in 42℃ RNase-free distilled water, and after the sections naturally flatten, use sterile forceps to transfer the sections to ordinary glass slides; place the glass slides containing the sections in a well-ventilated place at room temperature to air dry naturally, ensuring that the sections are completely attached to the glass slides; after air drying, place the sections in a sealed glass slide box containing desiccant and store at 4℃ for no more than 2 weeks.

[0109] S5. Dewaxing: Remove the slides from 4°C and allow them to equilibrate to room temperature for 10 minutes; immerse the slides in 100% xylene for 5 minutes, then replace with fresh xylene and immerse again for 5 minutes to complete dewaxing; after dewaxing, immerse the slides in 100% ethanol, 95% ethanol, and 70% ethanol for 3 minutes each to achieve tissue gradient rehydration; remove the slides and allow them to air dry naturally at room temperature in an RNase-free environment. Immediately after dewaxing and rehydration, perform laser capture microdissection (LCM), with a maximum storage time of no more than 24 hours to avoid RNA degradation.

[0110] S6. Laser Capture: Clean the LCM microscope (e.g., Leica LMD6500) with RNase AWAY surface cleaner. Mount the dried slide onto the microscope stage. Identify the target guard cells morphologically under a 20× or 40× objective lens (power 36%, aperture 19μm, speed 32μm / s, sample equilibration 16, light intensity 76%). Add 40μl of lysis buffer (manufacturer: Thermo Fisher Scientific; catalog number: AM1931) to the collection cap. Start the laser capture program and observe in real time under the microscope to precisely cut and collect the target guard cells (mixed population of upper and lower surface guard cells). After capture, connect the collection cap to a 0.5ml centrifuge tube and centrifuge at 12000×g, 4℃ for 15 minutes to allow the cells to settle at the bottom of the tube. Remove the cap, immediately label the centrifuge tube, and seal it at -80℃ for up to one month.

[0111] S7. Quantity detection: 50-150 mixed guard cells from the upper and lower surfaces can be captured by microscopic counting.

[0112] Test Example 1: Detection of guard cell activity on both upper and lower surfaces of leaves of Col-0 ecotype Arabidopsis thaliana

[0113] In Example 1, step S3, the stomatal buffer containing epidermal fragments from both the upper and lower surfaces of the leaf, obtained by homogenization, was taken in batches of 500 μL each. An equal volume of fluorescein diacetate (FDA) working solution (0.01% m / v, acetone solvent) was added to each sample, and the mixture was incubated at 25°C in the dark for 5 min. 10 μL of the stained sample was then placed in the center of a glass slide, covered with a coverslip to avoid air bubbles and cell compression damage. The sample was observed using a fluorescence microscope (Model: ECLIPSE Ci-S, Nikon Corporation, Japan). A 488 nm excitation module was selected, and the focus was adjusted until guard cells were clearly visible. Bright-field and fluorescence images of guard cells on the upper and lower surfaces of the Col-0 leaf are shown below. Figure 2 As shown, guard cells exhibit bright green fluorescence under excitation light, and their cell membrane boundaries are clear, indicating that their activity remains intact.

[0114] The above results indicate that the extraction method described in this application can successfully separate the epidermis from both the upper and lower surfaces of Arabidopsis thaliana leaves while maintaining the complete viability of guard cells. This lays the foundation for subsequent enzymatic digestion to obtain large quantities of guard cell protoplasts from both surfaces of the leaves, as well as for subsequent physiological experiments or multi-omics analyses.

[0115] Test Example 2: Detection of Guard Cell Count

[0116] Remove a clean cell counting chamber and slowly add 6-10 μL of the storage solution containing guard cell protoplasts from the upper and lower surfaces of Col-0 ecotype Arabidopsis thaliana leaves, obtained through step S9 of Example 1, via the sample dispensing ports on both sides of the counting chamber. Place the counting chamber on an optical microscope (Model: MoticB5; Manufacturer: Motic Industrial Group Co., Ltd.) and locate the counting field. Let it stand for 2-3 minutes until the cells settle onto the counting chamber and no longer drift with the liquid, then take a photograph for counting. The cell distribution of the mixture of guard cell protoplasts from the upper and lower surfaces of Col-0 ecotype Arabidopsis thaliana leaves is shown in the figure. Figure 3 As shown, the results of a single experiment collecting guard cell counts using microscopy are as follows: Figure 4 As shown. The results of the number of guard cells collected in a single experiment in Comparative Example 1, measured using the same method, are shown below. Figure 4 As shown. According to Figure 4 It is known that, using the extraction method of this application, the number of guard cells obtained in a single experiment is approximately 0.6 × 10⁻⁶. 6 -0.9×10 6 Each, averaging approximately 0.75 × 10⁻⁶. 6 The number of guard cells obtained in a single experiment in Comparative Example 1 was approximately 0.35 × 10⁶; while the number obtained in a single experiment in Comparative Example 1 was approximately 0.35 × 10⁶. 6 The number was significantly lower than in Example 1 ( <P); In Comparative Example 2, the number of guard cells obtained in a single experiment using laser capture microdissection technique was approximately 50-150, with an average of only about 100 (0.0001×10⁻⁶). 6 ).

[0117] The above results indicate that the extraction method proposed in this application can obtain a large number of guard cell protoplasts from both the upper and lower surfaces of plant leaves in a high-yield and low-cost manner, greatly improving the acquisition level of guard cell protoplasts and laying the foundation for subsequent analyses.

[0118] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for extracting protoplasts from guard cells on both the upper and lower surfaces of plant leaves, characterized in that, include: S1. Take a plant leaf, attach the upper surface of the plant leaf to paper tape, remove the midrib, and then use transparent tape to peel off the lower surface of the plant leaf to separate the upper and lower surfaces of the plant leaf; immerse the obtained upper surface epidermal tissue and paper tape, and the lower surface epidermal tissue and transparent tape of the leaf in an ice bath stomatal buffer solution respectively. The stomatal buffer solution comprises 45-55 mmol / L KCl, 0.08-0.12 mmol / L CaCl2, and 4-6 mmol / L 2-(N-morpholino)ethanesulfonic acid·1,3-bis[(tris(hydroxymethyl)methylamino)propane; S2. Take out the epidermal tissue and paper tape on the upper surface of the leaf, and the epidermal tissue and transparent tape on the lower surface of the leaf, add them to the pore buffer solution in an ice bath, and homogenize them intermittently 3-5 times, each time for 4-6 seconds, so that the epidermal tissue and tape on the upper and lower surfaces of each leaf are separated. S3. Remove the separated tape, and add the separated upper and lower surface epidermal tissues of the leaves to the stomatal buffer solution in an ice bath. Homogenize intermittently 3-5 times, with each homogenization time being 24-36 seconds. Then, the obtained fragments were filtered through a 160-240 mesh filter to obtain upper epidermal fragments and lower epidermal fragments of the leaf, respectively. S4. First enzymatic hydrolysis treatment, including: The upper epidermal fragments and the lower epidermal fragments of the leaf were enzymatically hydrolyzed and filtered with enzymatic hydrolysate I to obtain the upper epidermal fragments and lower epidermal fragments after the first enzymatic hydrolysis. The enzymatic hydrolysate I comprises: 0.4% (w / v) - 0.6% (w / v) cellulase R10, 0.04% (w / v) - 0.06% (w / v) dissociative enzyme R10, 0.16% (w / v) - 0.24% (w / v) bovine serum albumin, 0.08% (w / v) - 0.12% (w / v) polyvinylpyrrolidone, 0.2-0.3 mol / L mannitol, 0.8-1.2 mmol / L CaCl2, and 8-12 mmol / L MES-KOH; S5. Place the upper and lower epidermal fragments after the first enzymatic hydrolysis into a 0.24-0.36 mol / L mannitol solution and let them stand on ice for 25-35 min. S6. Second enzymatic hydrolysis treatment, including: Filtration: The upper and lower epidermal fragments were subjected to a second enzymatic hydrolysis using enzymatic hydrolysis solution II, and then filtered to obtain upper and lower epidermal filtrate. The enzymatic hydrolysate II comprises: 1.2% (w / v)-1.8% (w / v) cellulase RS, 0.4% (w / v)-0.6% (w / v) dissociative enzyme R10, 0.16% (w / v)-0.24% (w / v) bovine serum albumin, 0.32-0.48 mol / L mannitol, and 0.8-1.2 mmol / L CaCl2; S7. Centrifuge the upper epidermal filtrate and the lower epidermal filtrate, discard the supernatant, resuspend each precipitate with washing solution II, centrifuge again, repeat 2-4 times to obtain the guard cell protoplasts on the upper surface of plant leaves and the guard cell protoplasts on the lower surface of plant leaves, respectively. The rinsing solution II comprises 0.32-0.48 mol / L mannitol and 0.8-1.2 mmol / L CaCl2.

2. The extraction method according to claim 1, characterized in that, The extraction method further includes: S8. Purification process, including: The guard cell protoplasts on the upper surface of the plant leaf and the guard cell protoplasts on the lower surface of the plant leaf were resuspended in a pre-cooled 0.32-0.48 mol / L mannitol solution to obtain a guard cell protoplast suspension on the upper surface of the leaf and a guard cell protoplast suspension on the lower surface of the leaf. The guard cell protoplast suspensions on the upper and lower surfaces of the leaves were transferred to human lymphocyte separation medium, respectively, so that the guard cell protoplasts were suspended on the surface of the human lymphocyte separation medium. After centrifugation and separation, the middle layer was the purified guard cell protoplasts on the upper and lower surfaces of the plant leaves.

3. The extraction method according to claim 2, characterized in that, The extraction method further includes: S9. Saving process, including: The purified guard cell protoplasts from the upper and lower surfaces of plant leaves were diluted with 0.32-0.48 mol / L mannitol solution, respectively. After centrifugation, the supernatant was discarded, and 0.32-0.48 mol / L mannitol solution was added to each precipitate to suspend the guard cell protoplasts. The precipitates were then stored in the dark to obtain storage solutions for guard cell protoplasts from the upper and lower surfaces of plant leaves.

4. The extraction method according to claim 1, characterized in that, In step S3, the leaf upper epidermal fragments and leaf lower epidermal fragments obtained after filtration are rinsed with the pre-cooled stomatal buffer solution, so that the leaf upper epidermal fragments and leaf lower epidermal fragments are concentrated in the center of the filter screen.

5. The extraction method according to claim 1, characterized in that, In step S4, after obtaining the upper and lower epidermal fragments after the first enzymatic hydrolysis, they are rinsed with rinsing solution I to concentrate the upper and lower epidermal fragments after the first enzymatic hydrolysis into the center of the filter screen; the rinsing solution I includes: 0.24-0.36 mol / L mannitol and 0.8-1.2 mmol / L CaCl2.

6. The extraction method according to claim 1, characterized in that, In step S7, the upper epidermal filtrate and the lower epidermal filtrate are filtered through a 400-600 mesh filter before centrifugation, the filtrates are collected, and then centrifuged; wherein, after filtration, the filter screens are rinsed with pre-cooled rinsing solution II.

7. The extraction method according to claim 2, characterized in that, The volume ratio of the guard cell protoplast suspension on the upper surface of the leaf to the human lymphocyte separation solution is 1:(0.8-1.2), and the volume ratio of the guard cell protoplast suspension on the lower surface of the leaf to the human lymphocyte separation solution is 1:(0.8-1.2).

8. The extraction method according to any one of claims 1-7, characterized in that, The plant leaves are those of herbaceous plants.

9. The extraction method according to claim 8, characterized in that, The herbaceous plant leaves are Arabidopsis thaliana leaves.

10. The extraction method according to claim 9, characterized in that, The Arabidopsis leaves mentioned are rosette leaves of Arabidopsis that have grown for 3-5 weeks.

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