Preparation method of rice vesicles wrapped with sulfokine-derived peptides and application of rice vesicles in prevention and treatment of rice blast

Abstract

The invention discloses a sulphonin-derived peptide, a method for preparing rice vesicles containing the sulphonin-derived peptide and application of the rice vesicles in rice blast prevention and control, and belongs to the field of rice blast prevention and control. After being wrapped, the sulfopeptide derivative peptide (SEQ ID NO.2) and the rice vesicles have the function of resisting rice blast bacteria, and can be used for preventing and treating the rice blast bacteria. According to the prepared rice vesicles wrapping the sulfopeptide derived peptide, the cost is greatly reduced, meanwhile, the rice blast resistance effect of the rice vesicles is improved, and therefore the rice vesicles can be used as potential biopesticide for preventing and controlling the rice blast, and a new way is provided for achieving green and efficient prevention and control of the field rice blast.

Description

Technical Field

[0001] This invention belongs to the field of rice blast control, specifically relating to the application of a sulfopeptide-derived peptide and rice vesicles encapsulating the sulfopeptide-derived peptide in rice blast control. Background Technology

[0002] Rice is one of my country's three staple grains, and its yield and quality directly affect the country's food security. However, rice is often susceptible to devastating diseases during its growth and development. Rice blast is one of the most devastating diseases in rice production, widely distributed in major rice-producing areas of Asia, Africa, and the Americas. For a long time, the control of rice blast both domestically and internationally has relied mainly on traditional methods such as chemical agents, agricultural cultivation practices, and the breeding and promotion of disease-resistant varieties. In terms of chemical control, since the 1960s, agents such as tricyclazole, isoprothiolane, and fludioxonil have been widely used to control leaf blast and neck blast, especially during the epidemic period, where they can rapidly inhibit the spread of the pathogen (Kato, 2020). Agricultural control measures include field sanitation, timely sowing, reasonable planting density, controlling nitrogen application, and improving field ventilation, creating an ecological environment unfavorable to the spread of pathogens to suppress the disease (Li Chunying et al., 2016). In addition, the breeding and application of disease-resistant varieties are also important means of controlling rice blast. For example, rice varieties carrying disease-resistant genes such as Pi-ta, Pi-b, and Pi9 have been widely planted in China, Japan, and the Philippines, effectively slowing the occurrence and spread of the disease (Liu et al., 2014; Wang et al., 2020). However, these traditional methods have gradually revealed significant limitations in long-term application. First, pathogens have a strong ability to mutate, often breaking through the barrier formed by a single resistance gene, leading to frequent "resistance collapse" phenomena, which seriously affect the sustainable utilization value of disease-resistant varieties (Liu et al., 2014). Second, long-term reliance on chemical pesticides not only induces drug resistance in pathogens but also brings environmental pollution and food safety risks (Li et al., 2019). In addition, agricultural management measures are highly dependent on human labor and have a low degree of operational standardization, making them difficult to implement under large-scale planting conditions, resulting in large fluctuations in actual control efficacy (Wang et al., 2020). In summary, although traditional control methods have alleviated the threat of rice blast to rice production to some extent, their limitations are becoming increasingly prominent, making it difficult to meet the current needs of green agriculture and sustainable development. Therefore, there is an urgent need to develop safer, more efficient, and environmentally friendly new control technologies, such as nanocarrier pesticides, biological control, RNA interference, and molecular breeding, to provide new strategies for the precise control of rice blast.

[0003] Phytosulfokine (PSK) is a small peptide hormone first isolated and identified from asparagus mesophyll cell suspension culture medium in 1996. PSK peptides play important roles in plant growth, development, stress resistance, and disease resistance. The active peptide that exerts its effects is primarily sulfated PSK-α, formed through the translation and modification of precursor genes. It is known that plant PSK-α regulates plant growth, development, and stress resistance by activating specific plant cell surface receptors (PSKRs) (He et al., 2024). Current research focuses on the mechanisms of natural PSK and its receptor recognition signal transduction in different plant species. Although research in this field has provided valuable insights into the function of PSK, there is still a lack of research on the application of PSK-based products and related derivative peptides as plant immune activators. Meanwhile, although the highly active PSK-α has shown good effects in enhancing plant disease resistance, the synthesis cost of PSK-α (200 yuan / mg) is much higher than 40 times that of unmodified PSK (4.5 yuan / mg), which seriously restricts its application in scientific research, especially in the field of disease resistance.

[0004] Extracellular vesicles (EVs), as natural nanoscale vesicular bilayer membrane structures, possess excellent biocompatibility and safety, and can cross biological barriers, making them a potentially highly efficient carrier material. Animal EVs, in particular, have been extensively studied in various scientific fields, including drug delivery, targeted therapy, and disease diagnosis and treatment. EVs can be loaded with drugs using methods such as ultrasound, chemical transfection, freeze-thaw cycles, electroporation, extrusion, co-incubation, saponin-assisted loading, low-osmotic analysis, and pH gradients. However, complex extraction processes, potential side effects, limited yields, and high costs restrict their widespread clinical application. In recent years, plant EV-like vesicles (EVLVs) have gradually become a research hotspot in nanomedicine delivery systems due to their excellent biocompatibility, low immunogenicity, wide availability, ease of acquisition, and transmembrane transport capabilities. Several research teams abroad have successfully developed drug delivery platforms based on plant EV-like vesicles. For example, Zhang et al. (2016) first reported that turmeric-derived vesicles could be efficiently taken up by mouse intestinal epithelial cells and modulate the host immune response by loading small RNA molecules. Furthermore, turmeric-derived vesicles were also used to load doxorubicin, significantly inhibiting tumor growth in mouse tumor models with low toxicity. Ju et al. (2013) successfully delivered anti-inflammatory siRNA to macrophages using grape-derived vesicles, significantly alleviating DSS-induced colitis symptoms in mice. Wang et al. (2013) developed grapefruit-derived nanovesicles that could efficiently load siRNA, DNA, and small molecule drugs, achieving targeted delivery to the liver and tumor tissues. In comparison, domestic research started slightly later but has developed rapidly. For example, Liu et al. (2020) reported that green tea-derived EVs could load epigallocatechin gallate (EGCG) for tumor treatment, and vesicle packaging significantly improved the stability and cellular uptake of EGCG. Furthermore, Cao et al. (2022) constructed PDEVs using ginseng extract to deliver miRNA and anticancer drugs, demonstrating good anti-proliferative and pro-apoptotic effects in a lung cancer model. These studies indicate that plant EV-like vesicles play a crucial role in regulating cross-biological communication between humans and plants, and that plant EV-like vesicles have shown great potential as sustainable, green, and efficient drug delivery nanocarriers. However, there are currently no reports on the application of plant EV-like vesicles as drug delivery nanocarriers in plant disease resistance. Summary of the Invention

[0005] One object of the present invention is to provide a sulfopeptide-derived peptide with the function of resisting rice blast fungus.

[0006] Another object of the present invention is to provide the application of sulfopeptide-derived peptides in resistance to rice blast fungus.

[0007] Another object of the present invention is to provide a method for preparing rice vesicles containing sulfopeptide-derived peptides.

[0008] Another object of the present invention is to provide the application of rice vesicles containing sulfopeptide-derived peptides in resistance to rice blast fungus.

[0009] This invention is based on a natural, unmodified sulfopeptide PSK obtained from rice, with the amino acid sequence Tyr-Ile-Tyr-Thr-Gln (SEQ ID NO.1). Amino acid mutations were performed at points 2, 4, and 5 of the PSK sequence. Six PSK-derived peptides with the lowest binding energies were screened using Autodock CrankPep molecular docking: Tyr-Trp-Tyr-Thr-Gln (SEQ ID NO.2), Tyr-Ile-Tyr-Arg-Gln (SEQ ID NO.3), Tyr-Ile-Tyr-Trp-Gln (SEQ ID NO.4), Tyr-Ile-Tyr-Tyr-Gln (SEQ ID NO.5), Tyr-Ile-Tyr-Phe-Gln (SEQ ID NO.6), and Tyr-Ile-Tyr-Thr-Trp (SEQ ID NO.1). NO.7), these 6 sulfopeptide small molecule peptides were chemically synthesized in vitro, and the disease resistance effect of PSK-derived peptides was determined in combination with rice blast disease resistance experiments. Finally, the disease resistance effect of the derived peptide Tyr-Trp-Tyr-Thr-Gln (SEQ ID NO.2) was significantly better than that of unmodified natural PSK.

[0010] This invention provides a sulfopeptide-derived peptide with the amino acid sequence Tyr-Trp-Tyr-Thr-Gln (SEQ ID NO. 2), which has the function of resisting rice blast fungus.

[0011] This invention provides the application of sulfopeptide-derived peptides in the prevention and control of rice blast fungus.

[0012] This invention also provides the application of sulfopeptide-derived peptides in the preparation of agents against rice blast fungus.

[0013] The present invention further provides a rice vesicle containing sulfopeptide-derived peptides for resistance to rice blast fungus, wherein the concentration of sulfopeptide-derived peptides in the rice vesicles is 5-20 μM, preferably 10 μM.

[0014] This invention provides a method for preparing rice vesicles containing sulfopeptide-derived peptides, the method comprising the following steps: (1) Select rice seedlings that have been cultured for 2-3 weeks, cut them into pieces, place them in a mortar, add extraction buffer and grind them thoroughly until homogeneous, filter, and centrifuge at low temperature of 2000-2500 rpm; (2) Aspirate the supernatant, add 40-55% sucrose buffer, and centrifuge at high speed and low temperature; (3) Take the intermediate membrane component from the centrifuge tube, add 10-20% sucrose buffer, mix well, and then centrifuge at high speed and low temperature; (4) Discard the supernatant, keep the precipitate, and suspend it in PBS buffer; (5) Add the sulfopeptide-derived peptide as described in claim 1; (6) Rice vesicles encapsulating sulfopeptide-derived peptides were obtained by repeated freeze-thaw cycles.

[0015] Furthermore, the method for preparing rice vesicles containing sulfopeptide-derived peptides includes the following steps: (1) Select rice seedlings that have been cultured for 2-3 weeks, cut them into pieces, place them in a mortar, add extraction buffer and grind them thoroughly until homogeneous, filter, and centrifuge at 2000-2500 rpm and 4 ℃ for 20-40 min; (2) Take 5 ml of supernatant, add 4 ml of 40-55% sucrose buffer, and centrifuge at 100000g and 4℃ for 2 h; (3) Take the intermediate membrane component from the centrifuge tube, add 10-20% sucrose buffer, mix well, and centrifuge at 100000g and 4℃ for 1.5 h; (4) Discard the supernatant, keep the precipitate, and suspend it in PBS buffer; (5) Add sulfopeptide-derived peptides to a concentration of 5-20 μM; (6) Frozen at -80℃ for 1 h, thawed at 37℃ for 10 min, and repeatedly freeze-thawed to obtain rice vesicles encapsulating small molecule peptides.

[0016] Furthermore, the method for preparing rice vesicles containing sulfopeptide-derived peptides includes the following steps: (1) Select rice seedlings that have been cultured for 2-3 weeks, cut them into pieces, place them in a mortar, add extraction buffer and grind them thoroughly until homogeneous, filter, and centrifuge at 2500 rpm and 4℃ for 20 min; (2) Take 5 ml of supernatant, add 4 ml of 55% sucrose buffer, and centrifuge at 100000g and 4℃ for 2 h; (3) Take the intermediate membrane component from the centrifuge tube, add 10% sucrose buffer, mix well, and centrifuge at 100000g and 4℃ for 1.5h; (4) Discard the supernatant, keep the precipitate, and suspend it in PBS buffer; (5) Add sulfopeptide-derived peptides at a concentration of 10 μM; (6) Frozen at -80℃ for 1 h, thawed at 37℃ for 10 min, and subjected to three cycles of freeze-thaw incubation to obtain rice vesicles encapsulating small molecule peptides.

[0017] The application of rice vesicles in the prevention and control of rice blast fungus.

[0018] Advantages of this invention: This invention involves point mutation of the amino acid unit of the rice small molecule peptide sulfopeptide (PSK), and screening with plant phenotypic experiments to obtain a PSK-derived peptide with better resistance to rice blast. The obtained PSK-derived peptide is encapsulated by cyclic freeze-thaw incubation with rice vesicles. The synthesis cost of the encapsulated PSK-derived peptide Y38 is reduced by more than 40 times compared with PSK-α. At the same time, the encapsulated PSK-derived peptide Y38 reduces the leaf lesion area of ​​rice by 79.1% compared with the control group Mock (PSB buffer), and the control effect is also improved by 8.5% compared with sulfate-modified PSK-α.

[0019] In this invention, PSK stands for unmodified sulfopeptide small molecule peptide synthesized in vitro; PSK-α represents a sulfated modified sulfopeptide small molecule peptide synthesized in vitro. PSK-FITC represents the N-terminal fluorescein isothiocyanate labeling of PSK synthesized in vitro; Y38 represents the substitution of the second lle amino acid in in vitro chemically synthesized PSK by Trp; Y73 represents the 4th Thr amino acid of PSK synthesized in vitro, which is replaced by Arg. Y76 represents the 4th Thr amino acid of PSK synthesized in vitro, which is replaced by Trp. Y77 represents the 4th Thr amino acid of PSK synthesized in vitro, which is replaced by Tyr. Y82 represents the 4th Thr amino acid of PSK synthesized in vitro, which is replaced by Phe. Y95 indicates that the 5th Gln amino acid in PSK synthesized in vitro has been replaced by Trp. Attached Figure Description

[0020] Figure 1 This is a schematic diagram showing the effect of exogenous spraying of PSK-α and PSK small peptides on promoting the defense of rice against rice blast fungus; in the figure, A represents rice leaves 8 days after rice blast fungus infection; B represents the statistical analysis of the lesion area of ​​rice leaves 8 days after rice blast fungus infection; Mock represents spraying with PBS buffer as a negative control; PSK-α represents the exogenously sprayed modified sulfopeptide small molecule peptide; PSK represents the exogenously sprayed unmodified sulfopeptide small molecule peptide.

[0021] Figure 2 This is a schematic diagram showing the significant improvement in rice resistance after exogenous spraying of PSK-derived peptides; in the diagram, A represents rice leaves 8 days after infection with rice blast fungus; B represents the statistical analysis of the lesion area on rice leaves 8 days after infection with rice blast fungus; Mock represents spraying with PBS buffer as a negative control; PSK represents exogenously sprayed unmodified sulfopeptide small molecule peptides; Y77, Y82, Y95, Y73, Y38, and Y76 represent exogenously sprayed modified and optimized sulfopeptide derivative peptides, respectively.

[0022] Figure 3 This is a schematic diagram of the rice vesicle extraction process and electron microscopy; A in the figure represents a simplified flowchart of rice vesicle extraction; B represents a schematic diagram of the components of the rice intermediate membrane during the extraction process and the rice vesicles obtained by ultracentrifugation, as well as a morphological diagram of the rice vesicles observed under a transmission electron microscope.

[0023] Figure 4 This is a schematic diagram of the co-localization observation of PSK-FITC small molecule peptides and rice vesicles; the two columns below are magnified views of parts of the image above.

[0024] Figure 5 This is a schematic diagram illustrating the effect of rice vesicle-encapsulated Y38-derived peptides on rice resistance to rice blast. In the diagram, A represents rice leaves 8 days after infection with *Bacillus oryzae*, and B represents the statistical analysis of lesion area on rice leaves 8 days after infection. The control group (Mock) represents sprayed with PBS buffer as a negative control; PSK represents exogenously sprayed unmodified sulfopeptide small molecule peptides; Y38 represents exogenously sprayed optimized sulfopeptide-derived peptides; and Y38+EV represents exogenously sprayed rice vesicle-encapsulated Y38-derived peptides. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0026] Example 1: Screening for derived peptides based on the amino acid sequence of natural, unmodified sulfopeptide PSK Virtual screening process for PSK-derived peptides with receptor binding potential: (1) Target receptor structure determination: Based on unpublished experimental data, the receptor OsPSKR for PSK peptides in rice was determined. Subsequently, the amino acid sequence of the target receptor OsPSKR was used to predict the protein structure in Alphafold 3, and the domains were annotated and visualized using PyMOL software.

[0027] (2) Establishment of a polypeptide ligand library: First, based on the unmodified PSK (Tyr-Ile-Tyr-Thr-Gln) pentapeptide sequence of rice, the second, fourth and fifth unit amino acid mutations were performed on the natural PSK sequence. The target of the amino acid mutations was the 20 common amino acids that constitute proteins or polypeptides. Finally, a ligand library consisting of 96 pentapeptide sequences was established.

[0028] (3) Molecular docking: The docking prediction of peptides in the peptide ligand library with the target receptor structure is performed. The docking prediction uses the software Autodock CrankPep to score and predict the binding between macromolecular receptors and small molecule ligands. Specifically, the spatial fit of different conformations of each ligand with the receptor structure is calculated and evaluated using an atomic-level scoring function. The affinity is then determined by the free energy of ligand-receptor binding. The smaller the binding free energy, the more stable the binding (as shown in Table 1).

[0029] Table 1: Energy values ​​of Autodock CrankPep molecular docking virtual screening of PSK-derived peptides

[0030] Example 2: In vitro chemical synthesis of sulfopeptide PSK sequence-related peptides The synthetic peptide sequences are shown in Table 2 and were synthesized by Wuhan Dangang Biotechnology Co., Ltd. Based on the mass and relative molecular weight of the peptide powder, the appropriate amount of PBS buffer was added, vortexed, and diluted to an appropriate concentration for storage at -20°C. The synthesis cost of the modified PSK-α (200 RMB / mg) is more than forty times that of the PSK-derived peptide (4.5 RMB / mg).

[0031] Table 2 Information on artificially synthesized peptides

[0032] Example 3: Determination of the efficacy of exogenous application of sulfopeptide PSK and PSK-α polypeptide against rice blast Select healthy rice leaves that are about 4 weeks old. Attach approximately 4 cm long (2 cm wide) of transparent tape to the underside of each leaf. Use a pressure inoculator to make a circular mark on the tape-covered leaf. Cover the circular mark with a blast fungus (5 mm in diameter) cultured on PDA medium for 7 days. Add 10 μl of sterile water, then wrap the remaining tape around the fungus block and press it tightly to seal. Dilute the synthesized PSK-α and PSK to 10 μM with PBS buffer, using PBS buffer as a blank control. Spray the inoculated rice plants evenly around the edges of the inoculated plants. Repeat the spraying treatment after 2 days. After 8 days of inoculation, retrieve the leaves, remove the tape, count the area of ​​lesions, and photograph them.

[0033] Vaccination status as follows Figure 1 The results showed that exogenous spraying of PSK-α reduced the leaf lesion area of ​​rice by 70.6% compared to Mock (PSB buffer), demonstrating the best disease resistance effect. Secondly, unmodified PSK also reduced the leaf lesion area to some extent by 42.6%, but its inhibitory effect on rice blast fungus infection was significantly weaker than that of sulfate-modified PSK-α. These results indicate that both PSK-α and PSK can inhibit rice blast fungus infection in rice leaves, but the activity of unmodified PSK against rice blast fungus is significantly lower than that of sulfate-modified PSK-α.

[0034] Example 4: Determination of the effect of exogenous spraying of PSK and its derivative peptides on rice blast resistance Based on the molecular docking results analyzed in Example 1, six PSK-derived peptides (Y38: Tyr-Trp-Tyr-Thr-Gln; Y73: Tyr-Ile-Tyr-Arg-Gln; Y76: Tyr-Ile-Tyr-Trp-Gln; Y77: Tyr-Ile-Tyr-Tyr-Gln; Y82: Tyr-Ile-Tyr-Phe-Gln; Y95: Tyr-Ile-Tyr-Thr-Trp) with the lowest binding free energy were chemically synthesized in vitro. After exogenous spraying onto rice leaves, the phenotypic infection of *Strombus haematocephala* was determined. The in vivo inoculation experiment was conducted as described in Example 2.

[0035] Vaccination status as follows ( Figure 2The results showed that all six PSK-derived peptides could inhibit the infection of rice leaves by *Strombus oryzae* to a certain extent. In addition, the four optimized PSK-derived peptides, Y38, Y73, Y77, and Y95, showed significantly better inhibitory effects on *Strombus oryzae* infection than the unmodified original PSK peptides. After exogenous spraying, the leaf lesion area was reduced by 64.7%, 62.9%, 52.8%, and 64.7%, respectively. Among them, the spraying of Y38 and Y95 derivative peptides showed the best control effect and could significantly inhibit the infection of *Strombus oryzae*. However, since the chemically synthesized derivative peptide Y95 is not easy to dissolve, its subsequent application will also be somewhat limited. Therefore, derivative peptide Y38 can be regarded as the best PSK-derived peptide for later application.

[0036] Example 5: Extraction and Encapsulation Method of Rice Vesicles Method 1: (1) Select rice seedlings that have been cultured for 2-3 weeks, cut them into pieces and place them in a mortar. Add extraction buffer (50 mM HEPES + 0.45 M Sucrose + 5 mM MgCl2 + 1 mM DTT + 0.5% PVP) and grind them thoroughly until homogeneous. Filter and centrifuge at 2000 rpm and 4 ℃ for 40 min. (2) Take 5 ml of supernatant, add 4 ml of 40% sucrose buffer (40 g Sucrose dissolved in 100 ml of 50 mM HEPES), and centrifuge at 100000 g and 4 ℃ for 2 h; (3) Take the intermediate membrane component from the centrifuge tube, add 20% sucrose buffer (20 g Sucrose dissolved in 100 ml 50 mM HEPES), mix well, and centrifuge at 100000 g and 4 ℃ for 1.5 h; (4) Discard the supernatant, keep the precipitate, and suspend it in PBS buffer; (5) Add sulfopeptide-derived peptides by volume, at a concentration of 5 μM; (6) Frozen at -80 ℃ for 1 h, thawed at 37 ℃ for 10 min, and subjected to three cycles of freeze-thaw incubation to obtain rice vesicles encapsulating sulfopeptide-derived peptides.

[0037] Method 2: (1) Select rice seedlings that have been cultured for 2-3 weeks, cut them into pieces and place them in a mortar. Add extraction buffer (50 mM HEPES + 0.45 M Sucrose + 5 mM MgCl2 + 1 mM DTT + 0.5% PVP) and grind them thoroughly until homogeneous. Filter and centrifuge at 2000 rpm and 4 ℃ for 40 min. (2) Take 5 ml of supernatant, add 4 ml of 40% sucrose buffer (40 g Sucrose dissolved in 100 ml of 50 mM HEPES), and centrifuge at 100000 g and 4 ℃ for 2 h; (3) Take the intermediate membrane component from the centrifuge tube, add 20% sucrose buffer (20 g Sucrose dissolved in 100 ml 50 mM HEPES), mix well, and centrifuge at 100000 g and 4 ℃ for 1.5 h; (4) Discard the supernatant, keep the precipitate, and suspend it in PBS buffer; (5) Add sulfopeptide-derived peptides by volume to a concentration of 20 μM; (6) Frozen at -80 ℃ for 1 h, thawed at 37 ℃ for 10 min, and subjected to three cycles of freeze-thaw incubation to obtain rice vesicles encapsulating sulfopeptide-derived peptides.

[0038] Method 3: (1) Select rice seedlings that have been cultured for 2-3 weeks, cut them into pieces and place them in a mortar. Add extraction buffer (50 mM HEPES + 0.45 M Sucrose + 5 mM MgCl2 + 1 mM DTT + 0.5% PVP) and grind them thoroughly until homogeneous. Filter and centrifuge at 2500 rpm and 4 ℃ for 20 min. (2) Take 5 ml of supernatant, add 4 ml of 55% sucrose buffer (55 g Sucrose dissolved in 100 ml of 50 mM HEPES), and centrifuge at 100000 g and 4 ℃ for 2 h; (3) Take the intermediate membrane component from the centrifuge tube, add 10% sucrose buffer (10 g Sucrose dissolved in 100 ml 50 mM HEPES), mix well, and centrifuge at 100000 g and 4 ℃ for 1.5 h; (4) Discard the supernatant, keep the precipitate, and suspend it in PBS buffer; (5) Add sulfopeptide-derived peptides by volume, at a concentration of 10 μM; (6) Frozen at -80 ℃ for 1 h, thawed at 37 ℃ for 10 min, and subjected to three cycles of freeze-thaw incubation to obtain rice vesicles encapsulating sulfopeptide-derived peptides.

[0039] Example 6: Electron Microscopy Observation Take 10 μl of the above rice vesicles and drop them onto the carbonaceous contact surface of a 300-mesh copper mesh. Incubate at room temperature for 1 min. Place strips of absorbent paper at the edge of the copper mesh to absorb the liquid until the surface of the copper mesh is dry and free of droplets. Stain with 10 µl of 1% uranium acetate in the dark for 15 s, dry overnight, and observe under a JEOL transmission electron microscope. The rice vesicles were observed to be "tea tray-shaped or hemispherical with one side concave". Figure 3 The morphology and structure of the rice vesicles match those of the cells, which also proves the reliability of the rice vesicle extraction method in this invention.

[0040] Example 7: Confocal laser scanning microscopy fluorescence observation of PSK-derived peptides encapsulated in rice vesicles The vesicles extracted according to Example 5 were mixed with the in vitro chemically synthesized PSK-FITC small peptide (Tyr (FITC)-Trp-Tyr-Thr-Gln), and then further prepared into rice vesicles encapsulated with PSK-derived peptides through multiple freeze-thaw cycles (freezing at -80℃ for 1 h, thawing at 37℃ for 10 min). Appropriate encapsulated vesicles were co-incubated with FM4-64 (a fluorescent marker that specifically binds to cell membrane and endometrial organelles) for 10 min, and the fluorescence localization was observed using a confocal laser scanning microscope. During the observation, a large number of co-localizations were found between FITC-labeled PSK (green fluorescence) and FM4-64-labeled cell vesicles (red fluorescence), such as (…). Figure 4 However, these fluorescence overlaps also demonstrate that rice vesicles can successfully load PSK peptides.

[0041] Example 8: Phenotypic determination of rice blast resistance by rice vesicle-encapsulated PSK-derived peptides Following the method in Example 5, rice vesicles containing the derived peptide Y38 were encapsulated with PSK and unencapsulated Y38. These were then used as an exogenous spray on rice leaves for blast fungus infection phenotypic determination. The in vivo inoculation experiment was conducted as described in Example 2.

[0042] The inoculation results are shown in Figure (5): after exogenous spraying of rice vesicle-encapsulated derivative peptide Y38, the leaf lesion area of ​​rice decreased by 79.1% compared with Mock (PSB buffer), showing the best disease resistance effect; secondly, the unencapsulated derivative peptide Y38 also reduced the leaf lesion area by 67.3%. These results indicate that the encapsulation of rice vesicles improves the stability and activity of small molecule peptides to a certain extent, thereby further enhancing their control effect on rice blast.

Claims

1. A sulfopeptide-derived peptide, characterized in that: The amino acid sequence is Tyr-Trp-Tyr-Thr-Gln.

2. The application of the sulfopeptide-derived peptide according to claim 1 in the prevention and control of rice blast fungus.

3. The application of the sulfopeptide-derived peptide according to claim 1 in the preparation of a biological pesticide against rice blast fungus.

4. A rice vesicle resistant to rice blast fungus, characterized in that: The rice vesicles contain the sulfopeptide-derived peptide as described in claim 1.

5. The rice vesicles resistant to rice blast fungus according to claim 4, characterized in that: The concentration of the sulfopeptide-derived peptide is 5-20 μM.

6. A method for preparing rice vesicles containing the sulfopeptide-derived peptide of claim 1, characterized in that: The method includes the following steps: (1) Select rice seedlings that have been cultured for 2-3 weeks, cut them into pieces, place them in a mortar, add extraction buffer and grind them thoroughly until homogeneous, filter, and centrifuge at low temperature of 2000-2500 rpm; (2) Aspirate the supernatant, add 40-55% sucrose buffer, and centrifuge at high speed and low temperature; (3) Take the intermediate membrane component from the centrifuge tube, add 10-20% sucrose buffer, mix well, and then centrifuge at high speed and low temperature; (4) Discard the supernatant, keep the precipitate, and suspend it in PBS buffer; (5) Add the sulfopeptide-derived peptide as described in claim 1; (6) Rice vesicles encapsulating sulfopeptide-derived peptides were obtained by repeated freeze-thaw cycles.

7. The method for preparing rice vesicles according to claim 6, characterized in that: Includes the following steps: (1) Select rice seedlings that have been cultured for 2-3 weeks, cut them into pieces, place them in a mortar, add extraction buffer and grind them thoroughly until homogeneous, filter, and centrifuge at 2000-2500 rpm and 4 ℃ for 20-40 min; (2) Take 5 ml of supernatant, add 4 ml of 40-55% sucrose buffer, and centrifuge at 100000g and 4℃ for 2 h; (3) Take the intermediate membrane component from the centrifuge tube, add 10-20% sucrose buffer, mix well, and centrifuge at 100000g and 4℃ for 1.5h; (4) Discard the supernatant, keep the precipitate, and suspend it in PBS buffer; (5) Add the sulfopeptide-derived peptide as described in claim 1; (6) Frozen at -80 ℃ for 1 h, thawed at 37 ℃ for 10 min, and repeatedly freeze-thawed to obtain rice vesicles encapsulating sulfopeptide-derived peptides.

8. The method for preparing rice vesicles according to claim 6, characterized in that: Includes the following steps: (1) Select rice seedlings that have been cultured for 2-3 weeks, cut them into pieces, place them in a mortar, add extraction buffer and grind them thoroughly until homogeneous, filter, and centrifuge at 2500 rpm and 4 ℃ for 20 min; (2) Take 5 ml of supernatant, add 4 ml of 55% sucrose buffer, and centrifuge at 100000g and 4℃ for 2 h; (3) Take the intermediate membrane component from the centrifuge tube, add 10% sucrose buffer, mix well, and centrifuge at 100000g and 4℃ for 1.5 h; (4) Discard the supernatant, keep the precipitate, and suspend it in PBS buffer; (5) Add the sulfopeptide-derived peptide as described in claim 1; (6) Frozen at -80 ℃ for 1 h, thawed at 37 ℃ for 10 min, and subjected to three cycles of freeze-thaw incubation to obtain rice vesicles encapsulating sulfopeptide-derived peptides.

9. The method for preparing rice vesicles according to claim 6, characterized in that: In step (5), the concentration of sulfopeptide-derived peptides added is 5-20 μM.

10. The application of the rice vesicles according to claim 4 or 5 or the rice vesicles prepared by the method according to any one of claims 6-8 in the prevention and control of rice blast fungus.

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