Stability-enhanced plant immune resistance inducer based on mesoporous silica coupled immune protein and application of stability-enhanced plant immune resistance inducer

Abstract

The invention discloses a stability-enhanced plant immune resistance inducer based on mesoporous silica coupled immune protein and application of the stability-enhanced plant immune resistance inducer, and belongs to the technical field of plant disease control. According to the invention, fluorescence visualization and nano confinement stability enhancement technology are creatively integrated in a mesoporous silica protein delivery system, and two core problems of'difficult tracking 'and'poor stability' in research and application of plant immune induced resistance protein are successfully solved. The system not only can be used as an efficient plant vaccine delivery product, but also becomes a powerful basic research platform due to the visual characteristic, provides key technical support for optimizing the delivery efficiency and illuminating the induced resistance mechanism, and has great application value and market prospect in the field of green agriculture.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to a stability-enhancing plant immune inducer based on mesoporous silica coupled with immune proteins and its application. Background Technology

[0002] Protein-based biopesticides are a class of biopesticides with proteins or peptides as active ingredients. They achieve green pest control by activating the plant's immune system or directly interfering with the physiological functions of pathogens / pests, and represent an important research direction for future green pest control. Currently, there are two major bottlenecks in the application of traditional protein-based pesticides: First, the delivery and release kinetics of proteins within plants cannot be observed in real time and intuitively, limiting the optimization of delivery systems and mechanistic studies. Second, plant leaves have special structures such as the cuticle, wax layer, and epidermal hairs, which act as barriers, severely hindering pesticide delivery and penetration. Traditional pesticide spraying provides insufficient protection for the physicochemical stability of immune-inducing proteins during delivery and has poor permeability. A large amount of pesticide is lost during natural diffusion, failing to ensure that a sufficient number of functional proteins can overcome the physical barriers of plant leaves and reach the target site, leading to reduced efficacy and environmental pollution.

[0003] Mesoporous silica nanoparticles, as an excellent drug delivery carrier, offer an ideal platform for drug encapsulation and delivery due to their tunable pore size, high specific surface area, and easily functionalizable surface. However, the use of fluorescent proteins to track the delivery of immune-induced proteins still faces several technical limitations: First, the fusion of fluorescent proteins with immune-induced proteins may alter the native conformation of the target protein, thereby affecting its biological activity, receptor recognition ability, and subcellular localization, making it difficult for the tracking results to fully reflect the true behavior of the native protein. Furthermore, the large molecular weight of fluorescent proteins may significantly increase the volume and structural complexity of the fusion protein, making it impossible to accurately simulate the transport characteristics of small peptides or native proteins. Plant tissues themselves exhibit strong autofluorescence, while fluorescent proteins may degrade or undergo fluorescence quenching within the plant, leading to a reduced signal-to-noise ratio and making long-term, stable dynamic observation difficult. Simultaneously, the fluorescence signal cannot effectively distinguish between "proteins still encapsulated by the carrier" and "released proteins," making precise analysis of release kinetics challenging. Second, using mesoporous silica for the delivery of immune-induced proteins fused with fluorescent proteins also faces additional challenges related to carrier characteristics. The significantly increased molecular weight of fusion proteins may exceed the pore size range of some mesoporous silica, leading to decreased embedding efficiency or ineffective entry into the pores, thus affecting loading and delivery efficiency. The increased volume of the fusion protein may also alter its diffusion behavior within the mesopores, causing deviations in release kinetics. Furthermore, the interaction between the mesoporous silica surface and the fusion protein may be enhanced or weakened, affecting protein stability and activity. In summary, the combination of fluorescent protein labeling and mesoporous silica delivery systems still faces multiple technical challenges in terms of protein activity maintenance, carrier adaptability, loading efficiency, stability, and dynamic tracking. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a stability-enhancing plant immune inducer based on mesoporous silica-coupled immune proteins and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a stability-enhancing plant immune inducer based on mesoporous silica-coupled immune proteins, prepared by the following method: Surface-modified immunoproteins or immunoprotein-fluorescent fusion proteins are mixed and reacted with mesoporous silica nanoparticles (MSNs) carrying amino or carboxyl groups, so that the immunoproteins or immunoprotein-fluorescent fusion proteins are chemically bonded to the pores of the mesoporous silica nanoparticles.

[0006] Preferably, the immune protein is selected from chitinase, elicitor protein or antimicrobial peptide.

[0007] In some preferred embodiments of the present invention, the immune protein is flg22, the amino acid sequence of which is shown in SEQ ID NO.1; the immune protein-fluorescent fusion protein is flg22-GFP, the amino acid sequence of which is shown in SEQ ID NO.6.

[0008] Preferably, the surface modification treatment method for the immunoglobulin-fluorescent fusion protein is as follows: mix the immunoglobulin-fluorescent fusion protein, NHS and EDC, and react on ice in the dark for 15-30 minutes.

[0009] Preferably, the mesoporous silica nanoparticles have a particle size of 1-1000 nm.

[0010] A second aspect of the present invention provides the use of the above-mentioned plant immune inducer in at least one of the following (1)-(3): (1) Visual observation of target immune-inducing proteins in plant tissues; (2) Enhance the plant's immune resistance; (3) Improve the plant’s resistance to pathogens.

[0011] Preferably, the pathogen is rice bacterial leaf streak fungus.

[0012] A third aspect of the present invention provides a method for improving plant disease resistance, comprising the step of applying the above-mentioned plant immune inducer to the plant.

[0013] Preferably, the plant immune inducer is applied by spraying, watering, or drip irrigation.

[0014] The beneficial effects of this invention are: This invention creatively integrates fluorescence visualization and nano-confined stability enhancement technology into a mesoporous silica protein delivery system, successfully solving two core challenges in the research and application of plant immune-inducing proteins: "difficulty in tracking" and "poor stability." This system not only serves as a highly efficient plant vaccine delivery product, but its visualization capabilities also make it a powerful platform for basic research, providing key technical support for optimizing delivery efficiency and elucidating the mechanism of immune induction. It has significant application value and market prospects in the field of green agriculture.

[0015] Compared with the prior art, the present invention has the following advantages: (1) Significantly enhances stability: 1) Resistance to protease degradation: The nanoconfined effect of MSNs effectively shields the contact between protease macromolecules and the internal fusion protein. In vitro degradation experiments show that, compared with the free fusion protein, the half-life of MSNs@flg22-GFP in proteinase K solution is prolonged by more than 40%.

[0016] 2) High-temperature protection: The inorganic framework of MSNs provides a thermal barrier for the internal fusion protein. Under high-temperature treatment at 50°C, the free fusion protein is completely inactivated within 12 hours, while the protein activity in MSNs@flg22-GFP can still maintain more than 60% after 72 hours.

[0017] (2) Visual tracking: Using GFP fluorescence, the entire process of adsorption, internalization, transport, and protein release of nanosystems in plant leaves and roots can be observed in real time and in situ through confocal microscopy and in vivo imaging systems, providing direct evidence for mechanism research and system optimization.

[0018] (3) Highly effective resistance inducement: This system can protect immune-inducing proteins from damage by the external environment, ensuring that they are delivered to the plant in their active form and continuously released, thereby more effectively activating plant immunity and providing significant and lasting protection for field crops.

[0019] (4) Environmentally friendly: The system components are biocompatible, and the degradation products are non-toxic, meeting the requirements of sustainable agricultural development. Attached Figure Description

[0020] Figure 1 Fluorescence microscopy (A) and transmission electron microscopy (B) images of MSNs@flg22-GFP.

[0021] Figure 2 Results of stability study of MSNs@flg22-GFP enzyme digestion.

[0022] Figure 3 Results of thermal stability study of MSNs@flg22-GFP.

[0023] Figure 4 :MSNs@flg22-GFP leaf tracing.

[0024] Figure 5 MSNs@flg22-GFP stimulates ROS in rice to enhance resistance to bacterial leaf streak. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0026] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0027] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: flg22 is a highly conserved 22-amino acid peptide at the N-terminus of flagellin in Gram-negative bacteria, and its amino acid sequence is shown in SEQ ID NO.1. Green fluorescent protein (GFP) has an amino acid sequence shown in SEQ ID NO.2.

[0028] The mesoporous silica nanoparticles (MSNs, pore size 100 nm) are standard products purchased from commercial companies. Their pore size, specific surface area, particle size distribution and other parameters are provided by the supplier and have been verified.

[0029] Example 1: Visualization of mesoporous silica-fused fluorescent protein, preparation and characterization of a stability-enhanced plant immune inducer (MSNs@flg22-GFP) 1. Preparation of MSNs@flg22-GFP (1) Preparation of flg22-GFP fusion protein: The flg22 encoding gene (SEQ ID NO.3) and the GFP encoding gene (SEQ ID NO.4) were linked together to construct the encoding gene of the flg22-GFP fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0030] The encoding gene of the flg22-GFP fusion protein was ligated into the pET28a vector to construct the recombinant expression plasmid pET28a-flg22-GFP. The recombinant expression plasmid pET28a-flg22-GFP was then transformed into BL21(DE3) *E. coli*, induced with IPTG, and purified using a Ni column to obtain the flg22-GFP fusion protein. Sequencing confirmed that the amino acid sequence of the flg22-GFP fusion protein is shown in SEQ ID NO. 6.

[0031] (2) Surface modification treatment of flg22-GFP fusion protein: In pre-chilled 0.1 M MES Buffer, add flg22-GFP fusion protein, NHS, and EDC to achieve a final concentration of flg22-GFP fusion protein of 0.5 mg / mL, and final concentrations of NHS and EDC of 20 mM each. Gently vortex to mix, and incubate on ice in the dark for 15-30 minutes. At this point, the carboxyl groups on the surface of flg22-GFP fusion protein are activated into active NHS esters.

[0032] (3) Amination modification of MSNs: MSNs were modified by amination using the silane coupling agent γ-aminopropyltriethoxysilane (kh550), which uniformly added amino groups to the surface and interior of the MSNs.

[0033] (4) Coupling reaction: Aminated MSNs were resuspended in MES buffer to obtain an MSN dispersion. The surface-modified flg22-GFP fusion protein was added to the MSN dispersion at a mass ratio of 1:1. The mixture was then incubated at 4°C with gentle shaking for 24 hours to ensure complete reaction between the amino groups of the MSNs and the NHS esters of the flg22-GFP fusion protein. After the reaction, a final concentration of 0.1 M Tris-HCl buffer (pH 7.5-8.0) was added, and the reaction was continued for 15-30 minutes. The Tris-modified amino groups quenched all unreacted NHS esters and blocked potentially unreacted amino groups on the MSN surface, preventing non-specific binding.

[0034] Collect MSNs@flg22-GFP by high-speed centrifugation (12,000 rpm, 20 min). Resuspend the precipitate in PBS buffer (pH 7.4) and sonicate to disperse, then centrifuge again. Repeat this step at least 2-3 times to thoroughly remove uncoupled free protein and excess reagent. Resuspend the purified MSNs@flg22-GFP in storage buffer PBS and store at 4°C for later use.

[0035] 2. Characterization of MSNs@flg22-GFP (1) Fluorescence microscopy analysis: The flg22-GFP fusion protein, empty MSNs, and MSNs@flg22-GFP were dispersed in ultrapure water to a concentration of 0.5 mg / mL. 10 μL of each was added to a clean glass slide, and a coverslip was placed on top to prevent air bubbles from forming.

[0036] Observation conditions: Excitation / emission wavelength: Excitation was performed using a 488 nm laser, and the emitted light signal was collected in the 500-550 nm range.

[0037] The results are as follows Figure 1 As shown in Figure A, distributed green fluorescent spots were observed in the MSNs@flg22-GFP sample, and these spots highly overlapped with the morphology and distribution of the nanoparticles. No specific green fluorescence was observed in the field of view of the unloaded MSNs, confirming that the observed fluorescence signal indeed originated from the loaded flg22-GFP fusion protein. This result directly proves that the flg22-GFP fusion protein has successfully bound to MSNs and maintained its fluorescent activity.

[0038] (2) Transmission electron microscopy analysis: Sample preparation: MSNs@flg22-GFP and empty MSNs were diluted with ultrapure water to a suitable concentration (0.1 mg / mL). 10 μL of the diluted sample was added to the copper grid of the Formvar / carbon support membrane and allowed to stand for 3 minutes. Excess liquid was blotted away from the edges with filter paper. 10 μL of 2% tungsten phosphate negative staining solution was quickly added, and staining was performed for 1 minute. The staining solution was blotted dry again with filter paper, and the sample was placed in a desiccator for testing.

[0039] Observation conditions: Accelerating voltage: 80 kV. The morphology and dispersion state of the samples were observed at different magnifications (50 kX, 80 kX).

[0040] The results are as follows Figure 1 As shown in Figure B, the unloaded MSNs exhibit regular spherical nanoparticles with a clearly visible ordered pore structure. Compared to the unloaded MSNs, the pore contrast of MSNs@flg22-GFP is shallower or blurred, and no obvious pore structure is observed. This result morphologically indicates that flg22-GFP has successfully filled the entire MSN and that the loading process has not damaged the basic framework structure of the MSNs.

[0041] Example 2: Stability verification of MSNs@flg22-GFP 1. Protease resistance test: Free flg22-GFP and MSNs@flg22-GFP were co-incubated with proteinase K (final concentration 100 μg / mL) at 55°C for 30 minutes. The content of flg22-GFP in MSNs@flg22-GFP was the same as that in free flg22-GFP. Protein degradation was analyzed by SDS-PAGE.

[0042] The results are as follows Figure 2 As shown, free protein was degraded by 50% within 30 minutes, while MSNs@flg22-GFP retained more than 90% of the intact protein after 30 minutes, indicating that MSNs@flg22-GFP of the present invention can significantly enhance the protein's resistance to enzymatic degradation.

[0043] 2. Thermal stability test: Free flg22-GFP and MSNs@flg22-GFP were treated in a 50°C water bath for different times (12 h, 24 h, 48 h, 72 h). The content of flg22-GFP in MSNs@flg22-GFP was the same as that in free flg22-GFP. Their immune-inducing activity was detected by ELISA.

[0044] The results are as follows Figure 3As shown, after 12 hours of treatment with free flg22-GFP, the activity was reduced by 90%, while after 72 hours of treatment with MSNs@flg22-GFP, the activity remained above 60%. This indicates that the MSNs@flg22-GFP of the present invention can significantly improve the thermal stability of proteins.

[0045] Example 3: Tracing Investigation of MSNs@flg22-GFP in Rice Leaves 1. Test method: Healthy rice seedlings that have reached the three-leaf-one-bud stage were used as plant material. Rice seedlings of uniform growth were selected and randomly divided into two groups: Experimental group (MSNs@flg22-GFP): sprayed with MSNs@flg22-GFP aqueous dispersion (0.1 mg / mL).

[0046] Negative control group (MSNs): Spray with an empty MSNs aqueous dispersion (0.1 mg / mL).

[0047] Using a fine sprayer, evenly spray the two aqueous dispersions mentioned above onto the surface of the second fully expanded leaf of the rice plant until a uniform liquid film forms on the leaf surface without dripping. Place the treated plants in an artificial climate incubator (conditions: 28°C, 16 h light / 8 h dark, 70% humidity). Take samples 6 hours after spraying. Cut approximately 0.5 cm × 0.5 cm leaf sections from the sprayed area using a blade. Place the leaf sections flat on a glass slide with the lower epidermis facing up, add a drop of PBS to prevent drying, cover with a coverslip, and gently press to flatten. Observe immediately under a confocal microscope.

[0048] Excitation / emission wavelength: GFP was excited using a 488 nm argon ion laser, and the emission signal at 500-550 nm was collected.

[0049] Image acquisition: Images from all groups were acquired using the same parameters, including laser intensity, gain, and pinhole size, to ensure comparability. Bright-field images were also acquired simultaneously to locate fluorescence signals.

[0050] 2. Test Results: The results are as follows Figure 4 The results showed that the fluorescence signal in the experimental group was mainly concentrated on the leaf surface, especially around the stomata and near the veins, with obvious green fluorescent spots, indicating that the complex was attempting to enter the leaf through these natural channels. The negative control group, however, showed no specific green fluorescence. This indicates that the system relies on the excellent permeability of mesoporous silica nanoparticles to deliver the protein into the plant leaf tissue.

[0051] This embodiment directly demonstrates, through in vivo fluorescence tracing technology, that MSNs@flg22-GFP can be effectively absorbed by rice leaves via foliar spraying, enabling visual tracking of the protein. This complex possesses the potential for long-distance transport within the plant, providing a solid cell biological basis for the application of MSNs@flg22-GFP as a highly efficient foliar immune inducer.

[0052] Example 4: Evaluation of the application effect of MSNs@flg22-GFP on resistance to bacterial leaf streak pathogen in rice 1. Test method: Healthy rice seedlings (of disease-susceptible varieties) that have reached the four-leaf-one-core stage were selected as plant material. The rice seedlings with uniform growth were randomly divided into four groups: Mock: Use PBS for foliar spraying to ensure even coverage on both sides of the leaves.

[0053] MSNs: MSNs were dispersed in PBS to obtain an MSNs dispersion with a concentration of 0.1 mg / mL; the MSNs dispersion was used for foliar spraying to ensure uniform coverage of both sides of the leaves.

[0054] MSNs@flg22-GFP: MSNs@flg22-GFP was dispersed in PBS to obtain a 0.1 mg / mL MSNs@flg22-GFP dispersion. The MSNs@flg22-GFP dispersion was used for foliar spraying to ensure uniform coverage of both sides of the leaves.

[0055] flg22-GFP: flg22-GFP was dispersed in PBS to obtain flg22-GFP dispersion, and the flg22-GFP content in flg22-GFP dispersion was made the same as that in MSNs@flg22-GFP dispersion; foliar spraying was performed using MSNs@flg22-GFP dispersion to ensure uniform coverage on both sides of the leaves.

[0056] The treated plants were placed in an artificial climate culture chamber and cultured under the same conditions for 2 hours.

[0057] Early defense response detection (DAB & NBT staining): At least three leaves were cut from each group of plants. The leaves were completely immersed in DAB and NBT staining solutions. After being vacuumed in the dark for 30 minutes, the leaves immersed in DAB solution were cultured under light for 8 hours. Then, the leaves were transferred to 95% ethanol and destained in a water bath at 65°C to remove chlorophyll. The leaves immersed in NBT solution were directly destained with 95% ethanol at 65°C. The leaves after DAB and NBT staining were observed using a stereomicroscope.

[0058] Disease resistance evaluation (pathogen inoculation test): Highly virulent strains of rice bacterial leaf streak Xanthomonas oryzae pv. oryzicola (RS105) Activated, resuspended in MgCl2 solution, and the bacterial suspension concentration adjusted to OD. 600 =0.5. Injection inoculation was used. Using a sterile inoculation syringe, inoculation was performed on rice leaves (avoiding the midrib) 2 hours after pretreatment. After inoculation, the plants were placed in an artificial climate incubation chamber, and the injection site was sprayed with water 2-3 times daily to maintain humidity. Disease incidence was assessed 7 days after inoculation. The length of water-soaked lesions was measured, and the average lesion length was calculated.

[0059] 2. Test Results: (1) Results of early defense response detection: The results are as follows Figure 5 A shows that the MSNs@flg22-GFP group exhibited significant and dark DAB (brownish) and NBT (blue) staining spots on the leaves, indicating that the treatment successfully induced a strong early ROS burst. Other control groups: the free flg22-GFP group showed weaker staining than the MSNs@flg22-GFP group; the MSNs and Mock groups showed almost no specific staining. These findings suggest that the MSNs@flg22-GFP-induced ROS burst was more intense, and that the mesoporous silica nanoparticles carrying flg22-GFP conferred stronger immunomodulatory activity.

[0060] (2) Disease resistance evaluation results: The results are as follows Figure 5 Figures B and C show that the MSNs@flg22-GFP group had the shortest lesion length. The free flg22-GFP group showed some resistance, but the effect was significantly weaker than that of the MSNs@flg22-GFP group. The MSNs and Mock groups had long and severe lesions, exhibiting typical symptoms of infection. This indicates that the combined use of flg22-GFP and MSNs can synergistically enhance their immune resistance, achieving a synergistic effect greater than the sum of its parts (1+1>2) compared to using flg22-GFP or MSNs alone.

[0061] In summary, through dual verification at both physiological and pathological levels, the following conclusions are drawn: MSNs@flg22-GFP can efficiently activate early defense signals in rice, particularly the ROS outbreak. This activated defense response effectively limits the infection and spread of bacterial leaf streak pathogens, conferring stronger systemic resistance to rice. MSNs, as delivery vectors, are essential and highly efficient for the flg22-GFP protein to exert its optimal inducing resistance function, far superior to the direct use of free protein. These results strongly demonstrate the enormous potential of the MSNs@flg22-GFP system for disease control in practical agricultural production.

[0062] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A stability-enhancing plant immune inducer based on mesoporous silica-coupled immune proteins, characterized in that, It is prepared by the following method: Surface-modified immunoproteins or immunoprotein-fluorescent fusion proteins are mixed and reacted with mesoporous silica nanoparticles carrying amino or carboxyl groups, so that the immunoproteins or immunoprotein-fluorescent fusion proteins are chemically bonded to the pores of the mesoporous silica nanoparticles.

2. The plant immune inducer according to claim 1, characterized in that, The immune protein is selected from chitinase, elicitor protein or antimicrobial peptide.

3. The plant immune inducer according to claim 2, characterized in that, The immune protein is flg22, and its amino acid sequence is shown in SEQ ID NO.1; the immune protein-fluorescent fusion protein is flg22-GFP, and its amino acid sequence is shown in SEQ ID NO.

6.

4. The plant immune inducer according to claim 1, characterized in that, The surface modification method for immunoglobulin-fluorescent fusion protein is as follows: mix immunoglobulin-fluorescent fusion protein, NHS and EDC, and react on ice in the dark for 15-30 minutes.

5. The plant immune inducer according to claim 1, characterized in that, The mesoporous silica nanoparticles have a particle size of 1-1000 nm.

6. The use of the plant immune inducer according to any one of claims 1-5 in at least one of the following (1)-(3): (1) Visual observation of target immune-inducing proteins in plant tissues; (2) Enhance the plant's immune resistance; (3) Improve the plant’s resistance to pathogens.

7. The application according to claim 6, characterized in that, The pathogen is *Bacillus streakus*, the bacterial causal agent of rice leaf streak.

8. A method for improving plant disease resistance, characterized in that, include: The steps of applying the above-mentioned plant immune inducers to plants.

9. The method according to claim 8, characterized in that, The plant immune inducer is applied by spraying, watering, or drip irrigation.

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