Extraction method and application of extracellular vesicles in camellia oleifera leaf protoplast lotion

Abstract

The invention discloses an extraction method and application of extracellular vesicles in camellia oleifera leaf protoplast lotion. The method comprises the following steps: (1) taking complete disease-free camellia oleifera leaves, cutting off petioles, cleaning with ultrapure water, and absorbing water with filter paper; (2) putting the cleaned leaves into a needleless injector cylinder added with an infiltrating liquid, infiltrating in a vacuumizing manner, and centrifuging the infiltrated leaves to obtain a camellia oleifera leaf protoplast washing liquid; and (3) carrying out graded ultracentrifugation on the protoplast washing liquid, and collecting supernate. (4) filtering out cell debris from the supernate through a needle filter to obtain filtrate; (5) centrifuging the filtrate again, removing large vesicles and / or microparticle precipitates, and collecting supernate; (6) adding the supernatant into an ultracentrifuge tube for centrifugation, and removing the supernatant to obtain extracellular vesicle precipitate; and (7) gently resuspending the extracellular vesicle precipitate by using the precooled PBS, centrifuging the resuspended solution, and removing the supernatant to obtain the washed extracellular vesicle precipitate.

Description

Technical Field

[0001] This invention belongs to the field of extracellular vesicle biotechnology, and particularly relates to a method for extracting extracellular vesicles from the protoplast washing solution of tea leaves and its application. Background Technology

[0002] Extracellular vesicles mainly include exosomes and microvesicles. They are nanoscale membrane-bound vesicles secreted by cells into the extracellular space, containing substances such as proteins, lipids, and nucleic acids, and play an important role in the physiological and pathological processes of animals and plants.

[0003] Camellia oleifera is a unique woody oil-bearing plant in my country with significant economic value. Anthracnose and other diseases are major threats in Camellia oleifera producing areas, leading to substantial yield reductions. Extracellular vesicles in plant protoplast washes have been proven to be an important component of plant resistance mechanisms against pathogens, and their contents show promise for development as screening markers for high-yield, disease-resistant germplasm.

[0004] Existing techniques for the so-called destructive (crushing and juicing) extraction of extracellular vesicles from plant tissues mainly include: a purification method for plant exosomes disclosed in patent CN114540271 A; a plant exosome, its preparation method, and its application in anti-skin aging products disclosed in patent CN 115584337 A; a method for tangential flow extraction and preparation of camellia exosomes disclosed in patent CN 118185853 A; a method for preparing and applying berry tea exosomes disclosed in patent CN 117821363 A; the application of tea leaf exosomes disclosed in patent CN 111297790 A; the therapeutic use of tea leaf extracellular vesicles for atopic dermatitis disclosed in patent CN 118021883 A; and the application of perilla leaf-derived nanovesicles and their preparation in anti-inflammatory products disclosed in patent CN 118593561 A. All of the above-disclosed methods involve collecting the filtrate from the enzymatic hydrolysis or mechanical crushing of various plant tissues, and the extracted extracellular vesicles are mainly used in cosmetics or medicine.

[0005] In other words, current methods for extracting plant extracellular vesicles or exosomes involve destructively pulverizing or homogenizing plant tissues, and then extracting the extracellular vesicles or exosomes from the filtrate for use in cosmetics or medicine. There is currently no method for extracting extracellular vesicles from the protoplast wash of intact tea leaf petals that reflects the real-time physiological and pathological state of the plant. Summary of the Invention

[0006] The present invention aims to overcome the technical problem in the prior art that the extraction of extracellular vesicles from tea leaves is not pure due to the crushing and juicing of plant tissues, and thus cannot reflect the real-time physiological and pathological conditions of the plant. The present invention provides a method for extracting extracellular vesicles from protoplast washings of tea leaves.

[0007] To achieve the above objectives, the technical solution provided by this invention is as follows: The method for extracting extracellular vesicles from the protoplast washing solution of tea leaves includes the following steps: (1) Take whole, disease-free tea leaves, cut off the petioles, wash with ultrapure water, and then dry with filter paper. (2) Place the cleaned leaves into a needleless syringe barrel containing the wetting solution, and use vacuum wetting. Then centrifuge the wetting leaves to obtain the protoplast washing solution of tea leaves. (3) The protoplast washing solution collected in step (2) is fractionated by ultracentrifugation, and the supernatant is collected; (4) The supernatant collected in step (3) is filtered through a needle filter to remove cell debris, and the filtrate is obtained; (5) Centrifuge the filtrate obtained in step (4) again to remove large vesicles and / or microparticle precipitates, and collect the supernatant; (6) Add the supernatant collected in step (5) into an ultracentrifuge tube and centrifuge to remove the supernatant and obtain extracellular vesicle precipitate; (7) Gently resuspend the extracellular vesicle precipitate in pre-cooled PBS, centrifuge the resuspended solution to remove the supernatant, and obtain the washed extracellular vesicle precipitate.

[0008] Preferably, the wetting solution in step (2) contains 2-morpholine ethanesulfonic acid, calcium chloride, and sodium chloride, wherein the concentrations of 2-morpholine ethanesulfonic acid, calcium chloride, and sodium chloride in the wetting solution are 20-30 mM, 1.5-2.5 mM, and 0.05-0.15 M, respectively, and the pH value of the wetting solution is 5.5-6.5.

[0009] More preferably, the concentrations of 2-morpholine ethanesulfonic acid, calcium chloride, and sodium chloride in the impregnation solution are 25 mM, 2 mM, and 0.1 M, respectively, and the pH value of the impregnation solution is 6.

[0010] Preferably, the centrifugation conditions in step (2) are a centrifugal force of 500-1500 × 10⁻⁶. g Centrifuge for 15 minutes. The step (3) step of the fractional ultracentrifugation is performed at 4°C with a centrifugal force of 1000-3000 × 1000. g Centrifuge for 20 minutes. The centrifugation conditions described in step (5) are 4℃ and a centrifugal force of 5000-15000 × 10⁻⁶. g Centrifuge for 30 minutes. The ultracentrifugation conditions described in step (6) are 4℃ and a centrifugal force of 50,000-150,000 × 10⁻⁶. g Centrifuge for 60 minutes. The centrifugation conditions described in step (7) are 4℃ and a centrifugal force of 50,000-150,000 × 10⁻⁶. g Centrifuge for 60 minutes.

[0011] More preferably, the centrifugation conditions in step (2) are a centrifugal force of 1000 × ...g Centrifuge for 15 minutes. The step (3) step of the fractional ultracentrifugation is performed at 4°C and a centrifugal force of 2000 × 10⁻⁶. g Centrifuge for 20 minutes. The centrifugation conditions described in step (5) are 4℃ and a centrifugal force of 10000 × 10000. g Centrifuge for 30 minutes. The ultracentrifugation conditions described in step (6) are 4℃ and a centrifugal force of 100,000 × 10000 × 100000. g Centrifuge for 60 minutes. The centrifugation conditions described in step (7) are 4℃ and a centrifugal force of 100,000 × 10⁻⁶. g Centrifuge for 60 minutes.

[0012] Preferably, the needle filter in step (4) is a 0.22 or 0.45 μm needle filter.

[0013] More preferably, the needle filter in step (4) is a 0.45 μm needle filter.

[0014] This invention also provides extracellular vesicles in a protoplast washing solution of *Camellia oleifera* leaves, which are extracted and prepared by the aforementioned method for extracting extracellular vesicles from protoplast washing solution of *Camellia oleifera* leaves. The obtained extracellular vesicles in the protoplast washing solution of *Camellia oleifera* leaves can be used to prepare an inhibitor of *Camellia oleifera* anthrax infection.

[0015] The present invention will be further described below: The method for extracting extracellular vesicles from the protoplast washing solution of tea leaves according to the present invention includes the following: ① Extraction of protoplasts from tea leaves (1) Collect fresh tea leaves with intact petioles and free from disease (collected from March to May, preferably in April). Immediately after collection, place them in a pre-cooled ultrapure water insulated container until the required sample volume is collected. Usually, more than 200g is collected each time.

[0016] (2) Cut off the petiole of the leaf with clean scissors, wash the leaf three times with ultrapure water, and place it on filter paper to remove excess water.

[0017] (3) Take a needle-free syringe, pull out the plunger, plug the injection port with waterproof tape, add the wetting buffer (25mM 2-morpholine ethanesulfonic acid, 2mM calcium chloride, 0.1M sodium chloride, and adjust the pH to 6 with sodium hydroxide solution), and then carefully add the treated tea leaves, allowing the leaves to slide naturally into the solution to avoid bending and damaging the leaves. After the bottom is filled with leaves, continue to add wetting buffer to submerge all the leaves, put the plunger back on, tear off the tape, gently push the plunger to expel the air from the top of the syringe, and reseal the injection port with waterproof tape to form an airtight seal between the leaves and the wetting buffer in the syringe.

[0018] (4) Without damaging the syringe airtight seal, pull the syringe piston outward as far as possible and hold for 5 seconds. Then let the syringe piston slowly and naturally pull back. Repeat this step 2-4 times until all the tea leaves in the syringe are wetted by the buffer solution and become translucent.

[0019] (5) Repeat steps (3)-(4) until all leaves are saturated. Remove the leaves and gently wipe them dry with a paper towel to remove any remaining saturation buffer from the leaf surface.

[0020] (6) Take a piece of transparent tape about 1 cm wide and lay it flat on the table. Place about 30 soaked leaves in layers on the adhesive surface of the tape. Make sure the bottom of the leaves are aligned with the bottom of the 1ml syringe sleeve and wrap them around the tube so that the petiole ends of all the leaves face the bottom of the 50ml centrifuge tube.

[0021] (7) Place the soaked leaves into a 50ml centrifuge tube and centrifuge at a force of 500-1500× g 1000× preferred g Centrifuge for 15 minutes and collect the liquid at the bottom of the centrifuge tube. This liquid is the protoplast washing solution of the tea leaves. The collected liquid should not be green or light green.

[0022] (8) Repeat steps (6)-(7) until all protoplast washes from the soaked tea leaves have been collected.

[0023] ② Extraction of extracellular vesicles from protoplast washings of tea leaves (1) The collected protoplast washing solution was heated at 4℃ and incubated at 1000-3000× g (Preferred 2000×) g Centrifuge for 20 min to remove detached cell pellet, collect the supernatant, and filter through a (0.22 or 0.45 μm, preferably 0.45 μm) needle filter to remove cell debris. Incubate the filtrate at 4°C, 5000-15000 × 10⁻⁶ rpm. g (Preferred 10000×) g Centrifuge for 30 minutes to remove large vesicle precipitates and collect the supernatant.

[0024] (2) Add the supernatant obtained in step (1) into an ultracentrifuge tube and incubate at 4°C for 50,000-150,000 × 10⁻⁶ rpm. g (Preferred 100000×) g Centrifuge for 60 min, remove supernatant, gently resuspend the extracellular vesicle pellet in pre-cooled PBS, and wash. Incubate the resuspended solution at 4°C, 50,000-150,000 × 10⁻⁶ ppm. g (Preferred 100000×) gCentrifuge for 60 minutes, remove the supernatant, and the precipitate is the extracellular vesicle. The extracellular vesicles in the protoplast washing solution of Camellia oleifera leaves can be used to prepare anthrax-inhibiting agents from Camellia oleifera. They can also serve as a characteristic indicator for screening Camellia oleifera germplasm resistant to anthrax.

[0025] This invention discloses for the first time an extraction method for protoplasts from *Camellia oleifera* leaf washings, including the formulation of the infiltration buffer and centrifugation conditions, resulting in high-purity extracellular vesicles from *Camellia oleifera* leaves. Simultaneously, this invention is the first to evaluate the anti-anthrax infection ability of extracellular vesicles in the protoplast washings of *Camellia oleifera* leaves. This invention, by collecting protoplast washings from intact leaves and extracting the extracellular vesicles, can reflect the physiological and pathological state of living tissue, and its main application is in anti-anthrax infection of *Camellia oleifera*, which can be correlated with the physiological state of diseased *Camellia oleifera*.

[0026] In summary, this invention addresses the current lack of methods for extracting extracellular vesicles from protoplast washings of Camellia oleifera leaves. It develops a suitable method for extracting extracellular vesicles from protoplast washings of Camellia oleifera leaves and applies it to the resistance of Camellia oleifera to anthracnose infection. This helps to elucidate the reasons for differences in disease resistance among Camellia oleifera varieties and provides reference characteristic markers for cultivating high-yielding, disease-resistant Camellia oleifera germplasm. Attached Figure Description

[0027] Figure 1 Flowchart for extraction of protoplasts from tea leaves; Figure 2 Flowchart for the extraction of extracellular vesicles from protoplast washings of tea leaves; Figure 3 Electron micrographs of Example 1 and Comparative Example 1; Figure 4 Nanoparticle tracking analysis graphs for Example 1 and Comparative Example 1; Figure 5 Example 1: Effect of extracellular vesicles extracted in Example 1 on anti-anthrax infection; Figure 6 Transmission electron micrographs of leaf lesions after simulated inoculation and inoculation with anthracnose; Figure 7 Example 1: Extraction and analysis of the number of extracellular vesicles in simulated inoculation and inoculated anthrax leaves. Detailed Implementation

[0028] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0029] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0030] Example 1: A method for extracting extracellular vesicles from a protoplast washing solution of tea leaves. The specific steps of this method are as follows: (1) Take 50g of whole, disease-free tea leaves, cut off the petioles, wash them 3 times with ultrapure water, and then dry them with filter paper.

[0031] (2) Place the cleaned leaflets into a needleless syringe barrel and add the wetting solution (25 mM 2-morpholine ethanesulfonic acid, 2 mM calcium chloride, 0.1 M sodium chloride, pH=6). Figure 1 As shown, the protoplast washing solution of tea leaves was obtained by vacuum impregnation-centrifugation method.

[0032] (3) The collected protoplast washings were subjected to fractional ultracentrifugation to extract and purify the extracellular vesicles, such as... Figure 2 As shown, at 4℃, 2000× g Centrifuge for 20 minutes to remove detached cell pellet and collect the supernatant.

[0033] (4) The collected supernatant was filtered with a 0.45 μm needle filter to remove cell debris.

[0034] (5) The filtrate was kept at 4℃ and 10000× g Centrifuge for 30 minutes to remove large vesicles or microparticle precipitates and collect the supernatant.

[0035] (6) Add the collected supernatant to an ultracentrifuge tube, and centrifuge at 4°C and 100,000 × 10⁻⁶ rpm using a horizontal basket rotor SW55Ti. g Centrifuge for 60 minutes and remove the supernatant.

[0036] (7) Gently resuspend the extracellular vesicle pellet in pre-cooled PBS. The resuspension should be incubated at 4°C and 100,000 × 10⁻⁶ ppm. g Centrifuge for 60 min, wash the precipitate, remove the supernatant, and collect the extracellular vesicle precipitate.

[0037] Example 2: A method for extracting extracellular vesicles from a protoplast washing solution of tea leaves. The specific steps of step (2) of the comparative example 1 using the infiltration solution formulation (1×PBS buffer, pH=7.4) are as follows: (1) Take 50g of whole, disease-free tea leaves, cut off the petioles, wash them 3 times with ultrapure water, and then dry them with filter paper.

[0038] (2) Place the cleaned leaves into a needle-free syringe and add the wetting solution (1×PBS buffer, pH=7.4), as follows: Figure 1 As shown, the protoplast washing solution of tea leaves was obtained by vacuum impregnation-centrifugation method.

[0039] (3) The collected protoplast washings were subjected to fractional ultracentrifugation to extract and purify the extracellular vesicles, such as... Figure 2 As shown, at 4℃, 2000× g Centrifuge for 20 minutes to remove detached cell pellet and collect the supernatant.

[0040] (4) The collected supernatant was filtered with a 0.45 μm needle filter to remove cell debris.

[0041] (5) The filtrate was kept at 4℃ and 10000× g Centrifuge for 30 minutes to remove large vesicles or microparticle precipitates and collect the supernatant.

[0042] (6) Add the collected supernatant to an ultracentrifuge tube, and centrifuge at 4°C and 100,000 × 10⁻⁶ rpm using a horizontal basket rotor SW55Ti. g Centrifuge for 60 minutes and remove the supernatant.

[0043] (7) Gently resuspend the extracellular vesicle pellet in pre-cooled PBS. The resuspension should be incubated at 4°C and 100,000 × 10⁻⁶ ppm. g Centrifuge for 60 min, wash the precipitate, remove the supernatant, and collect the extracellular vesicle precipitate.

[0044] Example 3: A method for extracting extracellular vesicles from a protoplast washing solution of tea leaves. In comparison with Example 1, step (4) of this method uses a needle filter with a pore size of 0.22 μm. The specific steps are as follows: (1) Take 50g of whole, disease-free tea leaves, cut off the petioles, wash them 3 times with ultrapure water, and then dry them with filter paper.

[0045] (2) Place the cleaned leaflets into a needleless syringe barrel and add the wetting solution (25 mM 2-morpholine ethanesulfonic acid, 2 mM calcium chloride, 0.1 M sodium chloride, pH=6). Figure 1 As shown, the protoplast washing solution of tea leaves was obtained by vacuum impregnation-centrifugation method.

[0046] (3) The collected protoplast washings were subjected to fractional ultracentrifugation to extract and purify the extracellular vesicles, such as... Figure 2 As shown, at 4℃, 2000× g Centrifuge for 20 minutes to remove detached cell pellet and collect the supernatant.

[0047] (4) The collected supernatant was filtered with a 0.22 μm needle filter to remove cell debris.

[0048] (5) The filtrate was kept at 4℃ and 10000× g Centrifuge for 30 minutes to remove large vesicles or microparticle precipitates and collect the supernatant.

[0049] (6) Add the collected supernatant to an ultracentrifuge tube, and centrifuge at 4°C and 100,000 × 10⁻⁶ rpm using a horizontal basket rotor SW55Ti. g Centrifuge for 60 minutes and remove the supernatant.

[0050] (7) Gently resuspend the extracellular vesicle pellet in pre-cooled PBS. The resuspension should be incubated at 4°C and 100,000 × 10⁻⁶ ppm. g Centrifuge for 60 min, wash the precipitate, remove the supernatant, and collect the extracellular vesicle precipitate.

[0051] Example 4: A method for extracting extracellular vesicles from a protoplast washing solution of tea leaves. The centrifugal force parameter in steps (6) and (7) of the comparative embodiment 1 is 50000 × g， The specific steps are as follows: (1) Take 50g of whole, disease-free tea leaves, cut off the petioles, wash them 3 times with ultrapure water, and then dry them with filter paper.

[0052] (2) Place the cleaned leaflets into a needleless syringe barrel and add the wetting solution (25 mM 2-morpholine ethanesulfonic acid, 2 mM calcium chloride, 0.1 M sodium chloride, pH=6). Figure 1 As shown, the protoplast washing solution of tea leaves was obtained by vacuum impregnation-centrifugation method.

[0053] (3) The collected protoplast washings were subjected to fractional ultracentrifugation to extract and purify the extracellular vesicles, such as... Figure 2 As shown, at 4℃, 2000× g Centrifuge for 20 minutes to remove detached cell pellet and collect the supernatant.

[0054] (4) The collected supernatant was filtered with a 0.22 μm needle filter to remove cell debris.

[0055] (5) The filtrate was kept at 4℃ and 10000× g Centrifuge for 30 minutes to remove large vesicles or microparticle precipitates and collect the supernatant.

[0056] (6) Add the collected supernatant to an ultracentrifuge tube, and centrifuge at 4°C and 50,000 × 10⁻⁶ rpm using a horizontal basket rotor SW55Ti. g Centrifuge for 60 minutes and remove the supernatant.

[0057] (7) Gently resuspend the extracellular vesicle pellet in pre-cooled PBS. The resuspension should be incubated at 4°C and 50,000 × 10⁻⁶ ppm. g Centrifuge for 60 min, wash the precipitate, remove the supernatant, and collect the extracellular vesicle precipitate.

[0058] Comparative Example 1: Method for extracting extracellular vesicles from the filtrate of tea leaf juice This method is a method for extracting extracellular vesicles from plant tissue juice filtrate. Take 50g of whole, disease-free tea leaves and add them to 800ml of soaking solution (25mM 2-morpholinoethanesulfonic acid, 2mM calcium chloride, 0.1M sodium chloride, pH=6).

[0059] After the juicer crushes the food, it is filtered through sterile gauze. The filtrate is then heated at 4°C and 2000× g Centrifuge for 20 minutes and collect the supernatant.

[0060] The collected supernatant was filtered through a 0.45 μm needle filter to remove cell debris.

[0061] The filtrate was heated at 4℃ and 10000× g Centrifuge for 30 min to remove large vesicles or microparticle precipitates, collect the supernatant, and concentrate it using an ultrafiltration tube with a 100 kDa pore size.

[0062] The collected supernatant concentrate was added to an ultracentrifuge tube and centrifuged at 100,000 × 10⁻⁶ rpm using a horizontal basket rotor SW41Ti at 4°C. g Centrifuge for 60 minutes and remove the supernatant.

[0063] Gently resuspend the extracellular vesicle pellet in pre-chilled PBS. Incubate the resuspended solution at 4°C and 100,000 × 10⁻⁶ ppm. g Centrifuge for 60 min, wash the precipitate, remove the supernatant, and collect the extracellular vesicle precipitate.

[0064] Example 5: Characterization of extracellular vesicles Total protein concentration determination Take 10 μL of extracellular vesicles isolated from Examples 1, 2, 3, 4, and Comparative Example 1, respectively, and add an equal volume of RIPA lysis buffer containing 1% protease inhibitor. Mix thoroughly and sonicate on ice for 1 min. Prepare standard and working solutions according to the BCA total protein assay kit instructions. Take 2 μL of each sample and mix with the working solution. After incubation at 37°C for 30 min, measure the OD value of each well at OD630 nm using a microplate reader. Six sample wells were set up. Calculate the total protein concentration of each sample group according to the standard curve equation. The results are shown in Table 1.

[0065] Compared with Examples 2, 3 and 4, the total protein content of extracellular vesicles in Example 1 was higher than that in Examples 2, 3 and 4 (Table 1). Therefore, the wetting solution formulation, needle filter pore size and centrifugation conditions of the method in Example 1 were preferred for subsequent comparative experiments.

[0066] Table 1 Total protein content of extracellular vesicles Transmission electron microscopy characterization 10 μL of extracellular vesicles isolated in Example 1 and Comparative Example 1 were respectively added to a copper grid for transmission electron microscopy. After drying, they were stained with uranium acetate, dried, and washed three times with ultrapure water to remove excess staining. They were then dried on a paraffin tray for later use. Transmission electron microscopy was performed at 100 kV to obtain information on the size and morphology of the extracellular vesicles. The analysis results are as follows: Figure 3 As shown, the obtained extracellular vesicles are membrane-bound vesicles of about 100 nm, all conforming to the general morphology of extracellular vesicles.

[0067] Nanoparticle tracking analysis 20 μL of extracellular vesicles isolated from Example 1 and Comparative Example 1 were taken respectively, diluted with PBS to a measurable concentration, and after instrument calibration, the particle size distribution of each group of samples was measured using a nanoparticle tracking analyzer. The analysis results are as follows. Figure 4 As shown.

[0068] The extracellular vesicles in the Camellia oleifera protoplasm washing solution obtained by the method in Example 1 of this invention exhibit a typical membrane-bound vesicle morphology under transmission electron microscopy, and nanoparticle tracking analysis shows a single peak, mainly distributed in the 100-200 nm range. This indicates that the method of this invention can obtain extracellular vesicles of Camellia oleifera leaves with high purity. Compared with Comparative Example 1, although Comparative Example 1 has a higher total protein content (Table 1), the nanoparticle tracking analysis of Comparative Example 1 showed two peaks (…). Figure 4 This indicates the presence of two types of substances with low purity, mainly distributed in the 100-500 nm range, resulting in larger extracellular vesicles. Therefore, the method of this invention is more suitable for extracting extracellular vesicles from the protoplast washings of tea leaves.

[0069] Example 6: Analysis of the resistance of extracellular vesicles of tea leaves to anthrax infection Extracellular vesicles extracted in Example 1 of this invention were sprayed onto one side of the leaves of Camellia oleifera seedlings infected with *Anthracnose glomeratus*. A control group (CK) was sprayed onto the other side of the same leaf, using the centrifuged supernatant. After 6 days of incubation in an artificial climate chamber, the size of the lesions was measured, and samples were collected for qPCR detection. The analysis results are as follows: Figure 5 As shown, the extracellular vesicles (EVs) extracted in this invention can significantly inhibit the size of anthrax plaques and the biomass of anthrax bacteria compared to the control group.

[0070] Example 7: Extracellular vesicle secretion from tea leaves can be associated with the disease status of tea oil plants. Leaf tissues from the fungal patches on *Camellia oleifera* leaves were collected after simulated inoculation and 48 hours after inoculation with *Bacillus anthracis*. The tissues were fixed with 2.5% glutaraldehyde, dehydrated, embedded, sectioned, stained, and the amount of extracellular vesicles secreted by *Bacillus anthracis* inoculated leaves was observed using transmission electron microscopy. Results are as follows: Figure 6 As shown, anthracnose inoculation significantly promotes the secretion of extracellular vesicles in leaves.

[0071] Simultaneously, using the method of Example 1 of this invention, extracellular vesicles of tea leaves were collected 48 hours after simulated inoculation and after anthrax inoculation, respectively. Figure 7 As shown, the morphology of extracellular vesicles did not change significantly under electron microscopy. However, nanoparticle tracking analysis showed that the number of extracellular vesicles in the protoplast washings of Camellia oleifera leaves increased significantly after anthrax infection. This indicates that the secretion of extracellular vesicles can be associated with the disease status of Camellia oleifera and is an important component of the disease resistance mechanism of Camellia oleifera. It can be used as a potential disease resistance characteristic indicator for the screening of Camellia oleifera germplasm.

Claims

1. A method for extracting extracellular vesicles from a protoplast washing solution of tea leaves, characterized in that, The method includes the following steps: (1) Take whole, disease-free tea leaves, cut off the petioles, wash with ultrapure water, and then dry with filter paper. (2) Place the cleaned leaves into a needleless syringe barrel containing the wetting solution, and use vacuum wetting. Then centrifuge the wetting leaves to obtain the protoplast washing solution of tea leaves. (3) The protoplast washing solution collected in step (2) is fractionated by ultracentrifugation, and the supernatant is collected; (4) The supernatant collected in step (3) is filtered through a needle filter to remove cell debris, and the filtrate is obtained; (5) Centrifuge the filtrate obtained in step (4) again to remove large vesicles and / or microparticle precipitates, and collect the supernatant; (6) Add the supernatant collected in step (5) into an ultracentrifuge tube and centrifuge to remove the supernatant and obtain extracellular vesicle precipitate; (7) Gently resuspend the extracellular vesicle precipitate in pre-cooled PBS, centrifuge the resuspended solution to remove the supernatant, and obtain the washed extracellular vesicle precipitate.

2. The method for extracting extracellular vesicles from the protoplast washing solution of tea leaves as described in claim 1, characterized in that, The immersion solution in step (2) contains 2-morpholine ethanesulfonic acid, calcium chloride, and sodium chloride. The concentrations of 2-morpholine ethanesulfonic acid, calcium chloride, and sodium chloride in the immersion solution are 20-30 mM, 1.5-2.5 mM, and 0.05-0.15 mM, respectively. The pH value of the immersion solution is 5.5-6.

5.

3. The method for extracting extracellular vesicles from the protoplast washing solution of tea leaves as described in claim 2, characterized in that, The concentrations of 2-morpholine ethanesulfonic acid, calcium chloride, and sodium chloride in the impregnation solution are 25 mM, 2 mM, and 0.1 M, respectively, and the pH value of the impregnation solution is 6.

4. The method for extracting extracellular vesicles from the protoplast washing solution of tea leaves as described in claim 1, characterized in that, The centrifugation conditions described in step (2) are a centrifugal force of 500-1500 × 10⁻⁶. g Centrifuge for 15 minutes. The step (3) step of the fractional ultracentrifugation is performed at 4°C with a centrifugal force of 1000-3000 × 1000. g Centrifuge for 20 minutes. The centrifugation conditions described in step (5) are 4℃ and a centrifugal force of 5000-15000 × 10⁻⁶. g Centrifuge for 30 minutes. The ultracentrifugation conditions described in step (6) are 4℃ and a centrifugal force of 50,000-150,000 × 10⁻⁶. g Centrifuge for 60 minutes. The centrifugation conditions described in step (7) are 4℃ and a centrifugal force of 50,000-150,000 × 10⁻⁶. g Centrifuge for 60 minutes.

5. The method for extracting extracellular vesicles from the protoplast washing solution of tea leaves as described in claim 4, characterized in that, The centrifugation conditions described in step (2) are a centrifugal force of 1000 × ... g Centrifuge for 15 minutes. The step (3) step of the fractional ultracentrifugation is performed at 4°C and a centrifugal force of 2000 × 10⁻⁶. g Centrifuge for 20 minutes. The centrifugation conditions described in step (5) are 4℃ and a centrifugal force of 10000 × 10000. g Centrifuge for 30 minutes. The ultracentrifugation conditions described in step (6) are 4℃ and a centrifugal force of 100,000 × 10000 × 100000. g Centrifuge for 60 minutes. The centrifugation conditions described in step (7) are 4℃ and a centrifugal force of 100,000 × 10⁻⁶. g Centrifuge for 60 minutes.

6. The method for extracting extracellular vesicles from the protoplast washing solution of tea leaves as described in claim 1, characterized in that, The needle filter in step (4) is a 0.22 or 0.45 μm needle filter.

7. The method for extracting extracellular vesicles from the protoplast washing solution of tea leaves as described in claim 6, characterized in that, The needle filter mentioned in step (4) is a 0.45μm needle filter.

8. Extracellular vesicles in a protoplast washing solution of tea leaf oil, characterized in that, The extracellular vesicles in the tea leaf protoplast washing solution are obtained by the extraction method for extracellular vesicles in the tea leaf protoplast washing solution according to any one of claims 1 to 7.

9. The application of extracellular vesicles in the protoplast washing solution of tea leaves as described in claim 8 in the preparation of anthrax-inhibiting agents from tea leaves.

10. An infiltration solution for extracting extracellular vesicles from the washing solution of protoplasts in tea leaf extract, characterized in that, The impregnation solution contains 2-morpholine ethanesulfonic acid, calcium chloride, and sodium chloride, with concentrations of 20-30 mM, 1.5-2.5 mM, and 0.05-0.15 mM, respectively. The pH value of the impregnation solution is 5.5-6.5.

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