Metal ion-driven melittin nanoassemblies, methods of making and use thereof

Abstract

This invention discloses a metal ion-driven meliosteum peptide nanoassembly, its preparation method, and its applications, relating to the field of agricultural biotechnology. The meliosteum peptide nanoassembly is formed by the self-assembly of a hexahistidine-tagged meliosteum peptide with divalent metal ions through coordination. The average particle size of the meliosteum peptide nanoassembly is 20 nm–30 nm. The meliosteum peptide nanoassembly obtained by this invention through the self-assembly of a hexahistidine-tagged meliosteum peptide with zinc ions exhibits excellent antibacterial, anti-biofilm activity, stability, and biocompatibility, and can be used to control plant bacterial diseases caused by Xanthomonas and other fungi.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a metal ion-driven bee venom peptide nanoassembly, its preparation method, and its application. Background Technology

[0002] Plant pathogenic bacteria-induced diseases pose a serious threat to global food security, causing 20%-40% of yield losses in major crops annually. Among them, Xanthomonas oryzae, specifically the rice pathogenic strain Xanthomonas oryzae (… Xanthomonas oryzae pv. oryzae, Xoo Xanthomonas oryzae, a pathogen causing rice leaf streak ( Xanthomonas oryzae pv. oryzicola, Xoc These pathogens can cause bacterial leaf blight and bacterial leaf streak in rice, respectively, resulting in a 70%-80% incidence rate and causing significant economic losses to agricultural production. The bacterial biofilms formed by these pathogens can significantly enhance their resistance to conventional chemical fungicides, making the diseases extremely difficult to control.

[0003] Antimicrobial peptides (AMPs), as potential alternatives to traditional pesticides, possess broad-spectrum antimicrobial activity and a unique membrane-targeting mechanism, effectively inhibiting biofilm formation and disrupting mature biofilms without easily inducing resistance. Bee venom peptides, a 26-amino acid antimicrobial peptide derived from bee venom, are effective against various microorganisms including... Xoo and Xoc It possesses potent bactericidal effects against various plant pathogens, including those that can dissolve biofilms and embed themselves within cells. However, melitoxin faces numerous bottlenecks in agricultural applications: high toxicity to non-target species, poor environmental stability, susceptibility to protease degradation, and poor leaf adhesion. These issues severely limit its practical application value.

[0004] Nanobiotechnology offers a new approach to addressing the limitations of antimicrobial peptides in application. Integrating antimicrobial peptides into nanocarriers can improve their stability, bioavailability, and reduce off-target toxicity. While traditional nanocarriers (such as liposomes and polymer nanoparticles) can improve peptide stability, their preparation is complex, costly, and difficult to achieve controlled assembly and high loading. Summary of the Invention

[0005] The purpose of this invention is to provide a metal ion-driven bee venom peptide nanoassembly, its preparation method, and its application. The nanoassembly obtained by the self-assembly of bee venom peptide with hexahistine tag and zinc ions exhibits excellent antibacterial, anti-biofilm activity, stability, and biosafety, and can be used to prevent and control plant bacterial diseases caused by Xanthomonas and other fungi.

[0006] To achieve the above objectives, the present invention provides a metal ion-driven melittin nanoassembly, which is formed by the self-assembly of melittin with a hexahistine tag and divalent metal ions through coordination; the average particle size of the metal ion-driven melittin nanoassembly is 20 nm-30 nm.

[0007] Preferably, the divalent metal ion is a zinc ion.

[0008] Preferably, the hexahistine tag is located at the N-terminus of the bee venom peptide.

[0009] This invention provides a method for preparing a metal ion-driven bee venom peptide nanoassembly, comprising the following steps: S1. Dissolve the bee venom peptide tagged with hexahistine in double-distilled water to obtain a peptide solution; S2. Mix the peptide solution and zinc salt solution at a molar ratio of 2:1 and stir at 4°C for 8-12 hours to obtain a mixed solution; S3. After ultrafiltration and centrifugation of the mixed solution, the precipitate was collected and dried to obtain metal ion-driven bee venom peptide nanoassemblies.

[0010] Preferably, the concentration of the hexahistine-tagged bee venom peptide in the peptide solution is 1 mg / mL.

[0011] Preferably, the zinc salt solution is a zinc chloride solution; the concentration of the zinc chloride solution is 130 µM.

[0012] Preferably, the ultrafiltration centrifugation is performed at a speed of 5000 rpm / min for 1 h.

[0013] The present invention also provides an application of a metal ion-driven bee venom peptide nanoassembly, which is used to prepare a fungicide for preventing and controlling bacterial diseases of plants.

[0014] Preferably, the bacterial plant disease is a disease caused by pathogens of the genus Xanthomonas; the disease includes: rice bacterial blight and rice bacterial leaf streak.

[0015] Preferably, the effective concentration of the metal ion-driven bee venom peptide nanoassemblies in the bactericide is 100 µg / mL to 200 µg / mL.

[0016] In summary, the metal ion-driven bee venom peptide nanoassembly, its preparation method, and its application provided by this invention offer the following advantages compared to traditional technologies: This invention relates to a nanoassembly assembled via coordination between a bee venom peptide tagged with an N-terminal hexahistine residue and zinc ions. This assembly exhibits excellent antibacterial and anti-biofilm activities. Xoo , Xo c and the half-maximal effective concentration (EC50) of Escherichia coli 50 The concentration was significantly lower than that of free melitin at an effective concentration of 24 µg / mL (4 EC). 50 Under certain conditions, it achieves a clearance rate of 87.05% for mature biofilms; it exhibits strong resistance to protease degradation and is more stable in the environment; it significantly reduces the inherent toxicity of melittin and significantly reduces the toxicity to non-target organisms such as rice and zebrafish, thus improving biosafety; the one-step coordination assembly process is simple, low-cost, requires no complex equipment, and is suitable for large-scale production; it can be reused for a long time, providing a new solution for the green control of bacterial diseases in plants.

[0017] The technical method of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 This is a structural characterization diagram of the nano-assembly in an embodiment of the present invention; Figure 1 (a) Images from scanning electron microscopy (SEM) and transmission electron microscopy (TEM); Figure 1 (b) is a diagram of dynamic light scattering (DLS) analysis; Figure 2 Potential analysis, Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS) analysis, and circular dichroism (CD) spectral analysis of the nano-assemblies and meliofiber peptides. Figure 2 (a) is a potential analysis diagram; Figure 2 (b) is the FTIR spectrum analysis diagram; Figure 2 (c) is the XPS analysis chart; Figure 2 (d) is the CD spectrum analysis diagram; Figure 3 Analysis diagram of the near-edge structure of zinc K-edge X-ray absorption for atomically coordinated nanoassemblies; Figure 3(a) is the K-edge X-ray absorption near-edge structure (XANES) spectrum of zinc; Figure 3 (b) is the K-edge extended X-ray absorption fine structure (EXAFS) spectrum of zinc; Figure 3 (c) is the K-edge EXAFS Fourier transform (FT-EXAFS) spectrum of zinc; Figure 4 The spectrum of zinc is the K-side EXAFS wavelet transform (WT-EXAFS). Figure 4 (a) is the WT-EXAFS spectrum of metallic Zn; Figure 4 (b) is the WT-EXAFS spectrum of ZnO; Figure 4 (c) is the WT-EXAFS spectrum of ZnCl; Figure 4 (d) is the WT-EXAFS plot of Zn-NanoMel; Figure 5 For nano-assemblies and bee venom peptides in Xoo, Xoc and Escherichia coli ( E. coli The half-maximal effective concentration (EC) above ) 50 )picture; Figure 5 (a) For nanoassemblies and bee venom peptides in Xoo EC on 50 picture; Figure 5 (b) For nanoassemblies and bee venom peptides in Xoc EC on 50 picture; Figure 5 (c) EC on nanoassemblies and bee venom peptides in E. coli 50 picture; Figure 6 After treatment with different concentrations of trypsin Xoo Inhibition rate analysis graph; Figure 7 The pH-dependent particle size distribution and Zeta potential analysis of the nanoassemblies are shown in the figure. Figure 8 Treatment of nano-assemblies and bee venom peptides at different concentrations Xoo Electron conductivity analysis graph; Figure 9 Diagram showing the anti-biofilm and bactericidal properties of the nano-assembly and bee venom peptide; Figure 9 (a) is a graph showing the corresponding optical density values ​​of the nano-assembly and melitoxin at 595 nm for bacterial growth; Figure 9 (b) Biofilm on agar plates treated with nanoassemblies and bee venom peptides for 48 hours. Xoo A graph showing the number of bacterial colonies; Figure 9 (c) After treatment with different concentrations of bee venom peptides and nanoassemblies Xoo The average fluorescence intensity of live bacteria; Figure 10For the pre-establishment of nano-assemblies and bee venom peptide pair Xoo Comparison of biofilm eradication effects; Figure 10 (a) Images showing pre-built models of nanoassemblies with different concentrations of crystal violet staining and after treatment with bee venom peptide. Xoo Diagram showing the damage to biofilms; Figure 10 (b) Pre-built images showing different concentrations of nanoassemblies and melitoxin-treated crystal violet-stained images. Xoo Quantitative analysis diagram of biofilm; Figure 10 (c) shows the biofilm formed on agar plates after treatment with different concentrations of meliofemort peptides and nanoassemblies. Xoo Diagram showing the growth of bacterial colonies; Figure 11 After treatment with different concentrations of bee venom peptides and nanoassemblies Xoo Scenario diagram; Figure 11 (a) After treatment with different concentrations of bee venom peptides and nanoassemblies Xoo Quantity chart; Figure 11 (b) is a graph showing the average green fluorescence intensity; Figure 11 (c) is a graph showing the average intensity of red fluorescence; Figure 12 Functional diagram of the anti-biofilm mechanism of meliofacting peptides and nanoassemblies; Figure 12 (a) Different concentrations (1×EC) 50 2×EC 50 and 4×EC 50 A statistical chart of the yield of extracellular polysaccharides (EPS) treated with [treatment name missing]; Figure 12 (b) The absorbance (Optical Density, OD) of extracellular protein (PN) at 595 nm at different concentrations; Figure 12 (c) represents different concentrations (1×EC) 50 2×EC 50 and 4×EC 50 A statistical chart of extracellular protein (PN) yields after treatment; Figure 12 (d) is 2×EC 50 bee venom peptides and nanoassemblies under certain conditions Xoo Mobility plot and circular region statistical chart; Figure 12 (e) is 2×EC 50 Extracellular cellulase diagram and hydrolysis region diameter statistics of melitoxin peptide and six-nanometer assembly under certain conditions; Figure 12 (f) is 2×EC 50 Diagram of extracellular amylase induced by melitoxin and nanoassemblies under certain conditions, and statistical graph of the diameter of the hydrolysis region; Figure 12 (g) is a statistical analysis chart of leaf lesion length; Figure 13Figure showing the therapeutic effect of melitoxin peptides, ZnCl2, and nanoassemblies on bacterial wilt in rice; Figure 13 (a) A graph showing the quantified average in vivo therapeutic effect on bacterial leaf blight of rice; Figure 13 (b) A graph showing the in vivo average value of the therapeutic effect against bacterial leaf stripe disease in rice; Figure 14 Figure 1 shows the acute toxicity test results of bee venom peptides and nanoassemblies. Figure 14 (a) is a graph showing the average root length and shoot length of rice seeds at a concentration of 100 μg / mL; Figure 14 (b) is a graph showing the survival rate of zebrafish embryos at concentrations of 0 μg / mL to 60 μg / mL. Detailed Implementation

[0019] The technical method of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the scope of this application and its application or use.

[0021] In all the examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0022] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0023] The specific implementation examples are as follows: Example 1 A method for preparing a metal ion-driven bee venom peptide nanoassembly includes the following steps: (1) A bee venom peptide tagged with hexahistine, namely bee venom peptide tagged with N-terminal hexahistine (HM), was synthesized by solid-phase synthesis to obtain HM powder. In addition to HM, a bee venom peptide tagged with C-terminal hexahistine (MH) was also synthesized by solid-phase synthesis. According to activity tests, since HM maintains antibacterial activity comparable to wild-type bee venom peptide, HM was used for subsequent assembly.

[0024] (2) Dissolve HM powder in double-distilled water to obtain a peptide solution with a concentration of 1 mg / mL (calculated based on its molecular weight, its molar concentration is approximately 260 µM); add zinc chloride powder to double-distilled water to prepare a zinc chloride solution with a concentration of 130 µM.

[0025] (3) Mix the peptide solution and zinc chloride solution at a molar ratio of 2:1 and stir at 4 °C overnight (8 h-12 h) to obtain a mixed solution.

[0026] (4) Pour the mixed solution into an ultrafiltration centrifuge tube and centrifuge at 5000 rpm / min for 1 h. Collect the precipitate and freeze dry to obtain the nano-assembly, namely the bee venom peptide nano-assembly (NanoMel) with N-terminal hexahistine tag.

[0027] The metal ion-driven hexahistidine fusion melitoxin nanoassemblies consist of uniform nanoparticles with a size of approximately 20 nm-30 nm, and their structural characterization is as follows: Figure 1 As shown, the scanning electron microscope (SEM) and transmission electron microscope (TEM) images are as follows: Figure 1 As shown in (a), Dynamic Light Scattering (DLS) analysis is as follows: Figure 1 As shown in (b).

[0028] Potential analysis, Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS) analysis of the metal ion-driven hexahistine fused meliostide nanoassemblies and meliostide were performed. Figure 2 As shown.

[0029] according to Figure 2 (a) It can be seen that, compared with melitoxin, the surface charge of the metal ion-driven hexahistine fusion melitoxin nanoassembly is significantly increased.

[0030] according to Figure 2 (b) It can be seen that in the FTIR spectrum, at 3200 cm⁻¹ -1 -3600 cm -1 Within the wavenumber range, the NH stretching vibration peak associated with the amide bond / amino group in melitin exhibits a significant wavenumber shift and peak broadening in NanoMel; for the amide I band (1600 cm⁻¹)... -1 -1700 cm -1 The C=O stretching vibration peak of amide III was observed to exhibit a blue shift in wavenumber and a decrease in intensity in NanoMel; simultaneously, the amide III band (1200 cm⁻¹) showed a decrease in intensity. -1 -1400 cm -1The CN stretching vibration peak of the zinc ion also exhibits a wavenumber shift. These bond shifts originate from the coordination interactions between the N / O atoms of the amide bond in the zinc ion and the melittin molecule. These interactions modulate the electron cloud density and the bond force constants of the NH, C=O, and CN bonds, resulting in characteristic changes in its infrared vibrational modes. This confirms the successful coordination assembly of the zinc ion and the melittin.

[0031] according to Figure 2 (c) It is evident that the XPS spectrum of melittin only shows the intrinsic characteristic peaks (C 1s, N 1s, O 1s) corresponding to the peptide chain. However, the NanoMel XPS spectrum shows other characteristic peaks of zinc, including a Zn 2p spin-splitting doublet and a Zn LMM Auger electron peak, directly demonstrating that zinc ions were successfully bound to the assembly. Simultaneously, the binding energies of C 1s, N 1s, and O 1s in NanoMel are shifted relative to those in melittin. This phenomenon occurs because the coordination bonds formed between zinc ions and the N atoms (amide bonds) and O atoms (carboxyl / hydroxyl groups) in melittin alter the electron cloud density of C, N, and O atoms, thus changing the photoelectron binding energy. This result corroborates the changes in functional group vibrational modes observed in FTIR spectroscopy.

[0032] according to Figure 2 (d) It can be seen that, through circular dichroism (CD) spectroscopy analysis, the peptide secondary structure of NanoMel has undergone a significant shift: in the far ultraviolet region (180 nm-260 nm), the negative peak is closer to 208 nm and is deeper and sharper, indicating a higher α-helix content or a more ordered structure; using the CONTINLL algorithm in CD Pro software to analyze the detailed structural contents of meliostein and NanoMel, it can be seen that α-helices are dominant in both meliostein and NanoMel. Among them, NanoMel has a higher total α-helix content (84.3%), and regular α-helices (50.2%) are more than twisted α-helices (34.1%), while the total α-helix content of meliostein (82.7%) is characterized by twisted α-helices (42.3%) being more than regular α-helices (40.4%). The disordered structure of NanoMel was reduced by nearly half (from 8.2% to 4.2%), indicating that NanoMel has lower structural order and more regular and stable peptide secondary structures, as shown in Table 1.

[0033] Table 1 shows the secondary structure analysis of melitrix venom peptide and NanoMel.

[0034] To further understand the composition and structure of NanoMel, the atomic-level coordination structure was analyzed using Zn K-edge X-ray absorption near-edge structures (XANES), such as... Figure 3 and Figure 4 As shown. According to Figure 3 (a) It can be seen that the normalized XANES confirms that Zn 2+ Oxidation state (absorption edge 9659.7 eV), the white line asymmetry indicates its coordination heterogeneity relative to crystalline ZnCl2 and ZnO. According to Figure 3 (b) It can be seen that the k of NanoMel 3 The weighted extended X-ray absorption fine structure (EXAFS) oscillations do not conform to the strong crystallinity characteristics of ZnO (amplitude ±12) nor the simple salt pattern of ZnCl2, clearly ruling out the possibility of oxide formation, residual ZnCl2, or metal aggregation. According to Figure 3 (c) It can be seen that the R-space Fourier transform reveals that the combined parameter R+α≈2.355 Å (1.25 Å) for characterizing the radial distance R of the first coordination layer of metal atoms and the structural disorder α -3 ·k 3 A significant primary first shell peak exists at (R+α), corresponding to an atomic spacing of 2.0 Å–2.4 Å, consistent with a mixed Zn-N (histidine) / Zn-Cl coordination mode; while the broadened second shell feature (R+α > 3 Å) reflects the conformational flexibility of the peptide backbone. According to Figure 4 (a) Figure 4 (b) Figure 4 (c) and Figure 4 (d) shows that wavelet transform (WT) analysis provides key coordination fingerprint features. NanoMel exhibits broad WT features at R+α≈1.5 Å, k=5 Å. -1 -10 Å -1 Where k represents the K-edge (a XANES analysis term) clearly indicates the simultaneous presence of Zn-Cl (k=5 Å) in the mixed first coordination shell. -1 -7 Å -1 ) and Zn-N / O (k=8 Å) -1 -10 Å -1The contribution of this extended k-range coverage confirms that the His-tag (a protein purification tag consisting of 6-10 consecutive histidine residues) assembles partially but not completely substitutes for chloride, forming a heterogeneous coordination sphere estimated to contain 2-3 imidazole nitrogens and 1-2 chloride / water ligands, exhibiting a distorted octahedral geometry. Weak secondary signatures (R+α≈3.5 Å, k≈5 Å) are also observed. -1 This originates from the scattering of the disordered second shell within the flexible peptide chain framework.

[0035] NanoMel activity, stability, and driving force detection: By measuring NanoMel and melitin in Xoo, Xoc and Escherichia coli ( E. coli The half-maximal effective concentration (EC) above ) 50 To characterize its activity, such as Figure 5 As shown. According to Figure 5 (a) Figure 5 (b) and Figure 5 (c) It can be seen that NanoMel in Xoo, Xoc and EC on E. coli 50 The concentrations were 3.795 µg / mL, 3.202 µg / mL, and 2.722 µg / mL, respectively, which were 37.2%, 27.6%, and 31.6% higher than those of melittin.

[0036] In practical applications, exposure to proteases leads to peptide digestion. This study compared the effects of NanoMel and melitrix incubated with different concentrations of trypsin on... Xoo The inhibition rate was used to characterize and compare its protease resistance, such as... Figure 6 As shown, with increasing protease concentration, melittin's effect on... Xoo The inhibition rate decreased significantly. In contrast, NanoMel maintained high activity even at the highest concentration of 2 mg / mL, which was significantly better than free melitin alone.

[0037] To verify that the interaction of hexahistine metal affinity mediates the NanoMel assembly process, pH-dependent assembly tests were performed. The particle size distribution and zeta potential of the nanoparticles were monitored using dynamic light scattering (DLS) and a zeta particle size analyzer. Figure 7 As shown, the diameter of the nanoparticles continuously increases with increasing pH. However, no nanoparticles were detected at pH 6. This is because when pH < 7, the coordinating groups of the protein undergo protonation, and H... + Competing binding sites and the repulsion between positively charged proteins hinder Zn binding. 2+- Formation and assembly of protein nanoparticles. At pH 7, nanoparticles with a size of approximately 25 nm formed; simultaneously, their zeta potential was significantly higher than that under other pH conditions, indicating the formation of stable nanoassemblies. In contrast, larger aggregates formed at pH 8 and pH 10. This is because increasing pH leads to protein deprotonation and Zn... 2+ Enhanced cross-linking and weakened electrostatic repulsion between proteins promote the aggregation and enlargement of nanoparticles.

[0038] To further clarify the relevant mechanisms of cell membrane disruption, we tested cells treated with NanoMel and melitrix. Xoo The conductivity. For example... Figure 8 As shown, the relative conductivity of bacterial samples treated with low concentrations of NanoMel and melittin gradually increased over 8 hours of co-culture. However, when the concentration exceeded 24 μg / mL, the conductivity value increased sharply in the first hour, indicating large-scale cell leakage. Compared to the same concentration of melittin, the samples treated with NanoMel showed significantly higher conductivity values, suggesting that NanoMel is more effective at disrupting bacterial cell membranes and altering cell membrane permeability than melittin.

[0039] NanoMel exhibits enhanced inhibitory activity against bacterial biofilms: Xoo It is the most important plant pathogen in the occurrence of biofilm-associated infections. Melitoxin, by acting on the cell membrane, exhibits a significant potential to inhibit biofilm formation and clearance. Therefore, it is used... Xoo As a pathogen model, the activity of NanoMel in inhibiting biofilm formation and clearance was evaluated using crystal violet staining. Figure 9 As shown.

[0040] like Figure 9 As shown in (a), with increasing concentrations of melittin and NanoMel, the effect on... Xoo The inhibitory effect on biofilm formation gradually increased. At lower doses (3 μg / mL, 6 μg / mL, and 12 μg / mL), NanoMel achieved biofilm inhibition rates of 29.10%, 55.87%, and 79.71%, respectively, significantly higher than that of melittin (16.06%, 38.54%, and 66.28%). These results indicate that nanoassembly can enhance the anti-biofilm activity of melittin. Notably, when the NanoMel concentration increased to 24 µg / mL, the inhibition rate reached 95.61%.

[0041] To further investigate the inhibitory effect of NanoMel on bacterial growth, in culture... Xoo Following the biofilm treatment, the results showed that the samples were treated with NanoMel and melitoxin. XooInoculate the colonies. For example... Figure 9 As shown in (b), NanoMel significantly reduced Xoo The inhibitory effect on bacterial colony count within the biofilm exceeded that of melitoxin. (1.0-4.0 × EC50) 50 Within the specified concentration range, the NanoMel-treated group exhibited significantly lower colony-forming units, indicating a stronger inhibitory effect on the growth of bacteria protecting biofilms compared to melittin. This result is consistent with the findings of confocal laser scanning microscopy (CLSM) three-dimensional imaging, such as... Figure 9 As shown in (c), where Xoo Live bacteria in the biofilm were visualized using acridine orange (AO) staining.

[0042] NanoMel removes pre-existing... Xoo It has significant therapeutic effects on biomembranes.

[0043] Removing established mature biofilms not only helps to eliminate pathogens more thoroughly but also effectively prevents their recurrence. To evaluate the efficacy of NanoMel in removing mature biofilms and inhibiting bacterial growth within them, a series of experiments were designed: Xoo The strain was pre-cultured in 96-well plates for 48 hours to form a mature biofilm, followed by co-culturing with different concentrations of NanoMel (or melitrix) for another 48 hours. The eradication effect of the fungicide was analyzed by crystal violet staining.

[0044] Crystal blue staining experiment as follows Figure 10 (a) and Figure 10 As shown in (b), the eradication effects of both NanoMel and melitoxin increased with increasing concentration. The biofilm disruption rate of NanoMel was significantly higher than that of the melitoxin-treated group.

[0045] In addition to the known biofilm scavenging function, based on colony formation analysis, such as Figure 10 As shown in (c), administration of NanoMel and melivitin also triggered changes in biofilms. Xoo The concentration-dependent decrease in cell number was observed. Furthermore, the inhibitory potency of melittin was consistently lower than that of NanoMel. After treatment with NanoMel, compared to melittin, the inhibitory potency was 2.0 × EC50. 50 4.0×EC 50 and 8.0×EC 50 The colony counts at the different concentrations decreased by 23.28%, 24.29%, and 72.62%, respectively, demonstrating a significantly improved clearance rate.

[0046] To verify NanoMel's biofilm-killing ability, CLSM three-dimensional imaging technology was used, and acridine orange (AO) and propidium iodide (PI) were mixed for staining. Xoo Live and dead bacteria in the biofilm are labeled. For example... Figure 11 As shown in (a), NanoMel can effectively kill dead cells within biofilms. Xoo Cells. With increasing dosage, the intensity of red fluorescence obtained from propidium iodide (PI) staining showed a gradient increase. In contrast, the control group still had a large number of surviving bacteria, whose acridine orange (AO) staining produced strong green fluorescence accompanied by weak red fluorescence. Statistical analysis of the average green and red fluorescence intensities showed... Figure 11 (b) and Figure 11 (c) NanoMel gel exhibits a more significant killing effect than melitoxin, indicating that it has stronger biofilm scavenging activity.

[0047] NanoMel's potential anti-biomembrane mechanism: To clarify the anti-biofilm mechanism of NanoMel, the phenol-sulfuric acid method, gravimetric method, and Coomassie brilliant blue staining method were used to investigate the effects of different staining methods on the biofilm structure. Xoo The influence of key structural components of biological membranes—extracellular polysaccharides (EPS) and extracellular proteins (PN)—was investigated.

[0048] like Figure 12 As shown in (a), at 1×EC 50 and 2×EC 50 At the specified concentrations, the EPS yields in the NanoMel-treated group were only 41.8% and 15.43% of those in the control group, respectively, both significantly lower than those in the melilotinib-treated group (75.63% and 25.06%). This result was verified by gravimetric analysis. Figure 12 (b). When the concentration reaches 4×EC 50 At the same time, both NanoMel and melitoxin-treated samples showed strong and similar EPS inhibition effects.

[0049] Regarding PN generation, the values ​​in the NanoMel-treated groups were 72.46%, 34.36%, and 15.50% of the control group, respectively, all significantly lower than those in the melitoxin-treated groups (48.65%, 21.75%, and 10.68%). Figure 12 (c). These results indicate that NanoMel is more effective than melitoxin in inhibiting the formation or generation of key biofilm components (EPS and PN).

[0050] NanoMel and bee venom peptides Xoo The effects of movement, such as Figure 12As shown in (d), compared with the melitoxin-treated group, the NanoMel-treated group... Xoo The diameter of the cell was significantly reduced, measuring only 20.50 mm.

[0051] During plant infection by pathogens, the secretion levels of extracellular enzymes (such as cellulase and amylase) directly determine their pathogenicity, migration ability, and colonization efficiency. Further utilization of 2×EC 50 The effective concentration of NanoMel (12 μg / mL) characterized the effect of NanoMel on... Xoo The effects of these key virulence factors. For example... Figure 12 (e) and Figure 12 As shown in (f), the control group Xoo The average colony diameter reached 25.5 mm, while the NanoMel treatment group limited it to 6.83 mm, demonstrating superior inhibitory effect compared to the melitoxin treatment group (12.7 mm). Regarding extracellular enzyme activity, at 2×EC... 50 At a concentration of 12 μg / mL, the diameter of the cellulase hydrolysis zone in the NanoMel-treated group (12.1 mm) was smaller than that in the melittin-treated group (15.0 mm), while the control group had a diameter of 17.8 mm, which was smaller than that of the treated group. The diameter of the amylase hydrolysis zone in the NanoMel-treated group (11.13 mm) was also smaller than that in the melittin-treated group (13.1 mm). Both were larger than that in the control group (17.4 mm).

[0052] NanoMel's potent anti-biofilm function manifests in its inhibition of extracellular polysaccharide (EPS) and nanoparticle (NP) formation, reduction of extracellular enzyme (cellulase, amylase) activity, and inhibition of bacterial motility, potentially reducing pathogen infection and transmission to plants. This was further validated by results from rice pathogenicity experiments. In this experiment, NanoMel at a concentration of 12 μg / mL and melitrix venom were treated using a leaf-cutting inoculation method. Xoo Infecting rice leaves, such as Figure 12 As shown in (g), the average lesion lengths in the control group, zinc chloride (ZnCl2) treatment group, melitoxin treatment group, and NanoMel treatment group were 20.43 cm, 19.13 cm, 13.69 cm, and 8.73 cm, respectively. After co-culturing with NanoMel, Xoo The pathogenicity of the disease is significantly reduced, specifically manifested in a significant reduction in the length of the lesions.

[0053] NanoMel can effectively control bacteria caused by Xanthomonas spp. ( Xoo and Xoc Bacterial diseases of plants caused by: Based on NanoMel's excellent bactericidal and anti-biofilm activities, its in vivo antibacterial and anti-biofilm effects were evaluated. Xoo / Xoc The effect, such as Figure 13 As shown. Figure 13 As shown in (a), rice in the control group and the zinc chloride (ZnCl2) treatment group exhibited typical and severe symptoms of bacterial wilt in rice. The disease control effect of the NanoMel treatment group was significantly improved, with a protective activity of 69.9% and a curative activity of 68.6%, both of which were superior to the melitoxin treatment group (protective activity 56.4% and curative activity 56.3%).

[0054] Considering NanoMel's... Xoo The effects of induced bacterial leaf blight on rice and its influence on Xoc Excellent in vitro activity, such as Figure 13 As shown in (b), NanoMel's performance in responding to pressure osmosis was evaluated using the pressure osmosis method. Xoc Efficacy in inducing bacterial leaf streak. Severe disease symptoms were observed on rice leaves in both the control and zinc chloride (ZnCl2) treatment groups, with lesion lengths of 13.1 mm and 13.0 mm, respectively. NanoMel treatment reduced lesion length to 3.9 mm, achieving a control effect of 69.6%, significantly exceeding that of melitrix (48.6%). As a control, the commercial fungicide thiabendazole (20%) was used, which showed efficacy against... Xoo and Xoc The cure rates were 26.6% and 28.98%, respectively. These results indicate that NanoMel has significant potential in controlling bacterial wilt in rice.

[0055] NanoMel exhibits excellent biocompatibility: The biosafety of fungicides is crucial for sustainable plant protection. One limitation of melitoxin application is its non-specific target site and high toxicity; therefore, the effects of NanoMel on rice and a representative non-target organism (zebrafish) need to be evaluated. First, the potential toxicity of NanoMel to rice seedlings was investigated. Rice seedlings treated with 500 μg / mL NanoMel did not show obvious symptoms or abnormalities of plant poisoning, such as leaf chlorosis or wilting. Compared with the control group, the melitoxin-treated group, and the ZnCl2-treated group, the overall growth status of the plants was not significantly affected.

[0056] Then, the effects of NanoMel on rice seed germination rate and root-shoot growth were investigated. Figure 14 As shown in (a), the NanoMel and bee venom treatment groups exhibited similar growth performance to the control group. There were no significant differences in seed germination rate and root-cap growth, indicating that the toxicity of NanoMel to rice seed germination and early seedling growth was negligible.

[0057] Finally, zebrafish embryos were used as representative non-target organisms to evaluate the acute toxicity of each component at different concentrations. Figure 14 (b) Each component was co-cultured with zebrafish embryos for 48 hours. At a concentration of 45 μg / mL, the survival rates of zebrafish in the NanoMel and melittin treatment groups were 74% and 26%, respectively. No zebrafish mortality was observed in the other control groups, with a survival rate of 100%.

[0058] The NanoMel constructed in this invention exhibits excellent biosafety, environmental friendliness, and biocompatibility. It not only effectively kills planktonic bacteria but also penetrates the barrier of stubborn biofilms, inhibiting bacteria encapsulated by biofilms. At a concentration of 24 μg / mL, it shows a 95.61% inhibition rate against Xanthomonas biofilms, superior to free bee venom. Its bactericidal mechanism lies in its ability to effectively penetrate... Xoo NanoMel can disrupt cell membranes, leading to intracellular electrolyte leakage. Furthermore, it may disrupt biomembrane structure by inhibiting the expression of key biomembrane components such as extracellular polysaccharides and proteins. In addition, NanoMel can limit bacterial motility and extracellular enzyme secretion, thereby reducing bacterial virulence, migration ability, and pathogenicity. At a concentration of 200 μg / mL, NanoMel exhibited 68.6% therapeutic activity and 69.9% protective activity against bacterial leaf blight in rice, significantly superior to free melitin. Moreover, NanoMel demonstrated excellent biosafety against both the target plant (rice) and non-target organisms. This invention provides important ecological safety data support for the agricultural application of NanoMel as a green fungicide.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical methods of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical methods of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical methods to deviate from the spirit and scope of the technical methods of the present invention.

Claims

1. A metal ion-driven bee venom peptide nanoassembly, characterized in that, The metal ion-driven melitoxin nanoassemblies are self-assembled by melitoxin with a hexahistine tag and divalent metal ions through coordination; the average particle size of the metal ion-driven melitoxin nanoassemblies is 20 nm-30 nm.

2. The metal ion-driven bee venom peptide nanoassembly according to claim 1, characterized in that, The divalent metal ion is a zinc ion.

3. The metal ion-driven bee venom peptide nanoassembly according to claim 1, characterized in that, The hexahistine tag is located at the N-terminus of the bee venom peptide.

4. A method for preparing a metal ion-driven bee venom peptide nanoassembly, characterized in that, The method for preparing a metal ion-driven bee venom peptide nanoassembly as described in any one of claims 1-3 comprises the following steps: S1. Dissolve the bee venom peptide with a hexahistine tag in double-distilled water to obtain a peptide solution; S2. Mix the peptide solution and zinc salt solution at a molar ratio of 2:1 and stir at 4°C for 8-12 hours to obtain a mixed solution; S3. After ultrafiltration and centrifugation of the mixed solution, the precipitate was collected and dried to obtain metal ion-driven bee venom peptide nanoassemblies.

5. The method for preparing a metal ion-driven bee venom peptide nanoassembly according to claim 4, characterized in that, The concentration of bee venom peptide with a hexahistine tag in the peptide solution is 1 mg / mL.

6. The method for preparing a metal ion-driven bee venom peptide nanoassembly according to claim 4, characterized in that, The zinc salt solution is a zinc chloride solution; the concentration of the zinc chloride solution is 130 µM.

7. The method for preparing a metal ion-driven bee venom peptide nanoassembly according to claim 4, characterized in that, The ultrafiltration centrifugation was performed at a speed of 5000 rpm / min for 1 h.

8. An application of a metal ion-driven bee venom peptide nanoassembly, characterized in that, The metal ion-driven bee venom peptide nanoassembly according to any one of claims 1-3 is used to prepare a fungicide for preventing and controlling bacterial diseases of plants.

9. The application of the metal ion-driven bee venom peptide nanoassembly according to claim 8, characterized in that, The plant bacterial diseases mentioned are diseases caused by pathogens of the genus Xanthomonas; the diseases include: rice bacterial blight and rice bacterial leaf streak.

10. The application of the metal ion-driven bee venom peptide nanoassembly according to claim 8, characterized in that, The effective concentration of the metal ion-driven bee venom peptide nanoassemblies in the bactericide is 100 μg / mL-200 μg / mL.

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