A method for improving stability of dsRNA by using nano-mesoporous carbon to load dsRNA

Abstract

This invention discloses a method for improving the stability of dsRNA by loading double-stranded RNA onto nanoporous carbon, belonging to the interdisciplinary field of agricultural biotechnology and nanomaterials. This invention employs a specific ceramic template method to prepare nanoporous carbon with a regular mesoporous structure and a high degree of graphitization. Through mechanisms such as mesoporous confinement effect, full-band UV shielding, and enhanced leaf adhesion, the stability of dsRNA under environmental stresses such as UV irradiation, nuclease degradation, and rain washout is significantly improved. This composite carrier exhibits high loading efficiency and is environmentally friendly, suitable for RNA interference control of various pests, and provides key technical support for the development of efficient and long-lasting nanoRNA pesticides.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of agricultural biotechnology and nanomaterials science, specifically involving a nanoporous carbon (NMG) prepared based on a specific ceramic template method and its application as a double-stranded RNA (dsRNA) carrier in the preparation of photostable, enzyme-stable and rain-resistant nanopesticides. Background Technology

[0002] RNA interference (RNAi) technology, which silences key genes in pests through sequence-specific silencing, is considered the third agricultural technological revolution after chemical pesticides and genetically modified crops. However, as a biological macromolecule, dsRNA faces extreme environmental challenges in its field application: Poor photolysis stability: Strong ultraviolet radiation (UV-A and UV-B) in the field can directly affect the phosphodiester bonds and bases of RNA, causing them to break rapidly or undergo dimerization and cross-linking, thus losing their silencing activity.

[0003] Enzymatic degradation is extremely rapid: Plant secretions, soil, and the digestive tracts of insects are teeming with various nucleases (RNases). Naked dsRNA is typically degraded within minutes, with a bioavailability of less than 1%.

[0004] Short shelf life: dsRNA is highly hydrophilic and has difficulty adhering to the surface of hydrophobic plant waxy layers, making it easily washed away by dew or rain.

[0005] Existing carrier technologies have significant shortcomings: cationic polymers (such as PEI) can compress dsRNA, but their high charge density often leads to plant cell toxicity and lacks UV protection; liposomes have poor storage stability, are prone to oxidation and leakage, and are expensive, making them unsuitable for the pesticide field. While mesoporous materials offer certain advantages as nucleic acid delivery carriers, existing systems still have significant limitations. Although mesoporous silica (MSN) possesses a porous structure, its framework has high transmittance in the ultraviolet region, failing to shield against light damage. Furthermore, in agricultural environments (often accompanied by weakly acidic or weakly alkaline water films), the silica matrix is ​​highly susceptible to hydrolysis (dissolution rate >25% within 30 days), leading to premature release of the carrier. In addition, the silanol groups (Si-OH) on the MSN surface are difficult to modify in a controlled manner, often causing non-specific aggregation of the carrier, clogging nozzles, or resulting in uneven distribution on the leaf surface. Its limitations are prominently manifested in the following aspects: (1) The contradiction between pore size and loading efficiency: the molecular size of dsRNA is usually >10 nm (e.g., the length of a 500 bp fragment is about 170 nm), which is much larger than the pore size of most MSN (<10 nm), resulting in an effective loading rate of less than 15%, and large fragments of dsRNA are prone to shearing during loading; (2) Uncontrollable surface chemistry: the surface groups mainly composed of silanol (Si-OH) are difficult to modify in a directional manner, and cationic modification (e.g., amination) can easily cause carrier aggregation (the degree of polymerization increases by 47% when the Zeta potential is > +30 mV), affecting the uniform spreading of the leaf surface; (3) Poor plant environmental adaptability: MSN is prone to dissolution in the pH range of leaf surface (5.0-7.0) (silicon dissolution within 30 days >25%), resulting in premature leakage of dsRNA.

[0006] This invention designs a nanoporous carbon carrier, utilizing its regular morphology and mesoporous structure to achieve efficient embedding of dsRNA. Simultaneously, the mesoporous structure increases the surface roughness of the nanoporous carbon, thereby enhancing its adhesion to plant leaves. This technology not only overcomes the limitation of traditional carrier loading rates (<30%), but its unique mesoporous confinement effect also shields against nuclease attack, extending the dsRNA retention time on the leaf surface by more than three times. This provides key technological support for the development of next-generation environmentally friendly nanopesticides. Summary of the Invention

[0007] This invention proposes a novel method to enhance the anti-degradation ability of dsRNA by using nanoporous carbon-loaded double-stranded RNA (dsRNA), aiming to improve the stability of dsRNA under harsh environmental conditions, thereby enhancing its ability to enter plant cells, and solving the problem of poor dsRNA stability in the prior art.

[0008] This invention first uses in vitro transcription or chemical synthesis to synthesize double-stranded RNA (dsRNA) of the target gene.

[0009] This invention utilizes mesoporous carbon nanomaterials as a carrier for dsRNA. The mesoporous carbon nanomaterials can be nanoparticles with a highly porous structure composed of microcrystalline carbon nanosheets. The surface of the mesoporous carbon nanomaterials can be further modified chemically or physically to improve their binding affinity to dsRNA, thereby enhancing the stability and delivery efficiency of the complex. The average diameter of the microcrystalline carbon nanomaterials is adjustable, typically ranging from 100 to 400 nm, and their morphology is near-spherical to ensure good biocompatibility and efficient intracellular uptake.

[0010] This invention involves mixing the prepared dsRNA with nanoporous carbon materials at a specific mass ratio. The specific preparation steps are as follows: Select a suitable solvent, such as sterile water or PBS buffer, to prepare a dsRNA solution of a certain concentration.

[0011] Ultrasonic treatment is used to promote the dispersion of nanoporous carbon, enabling it to bind better to dsRNA. The ultrasonic frequency is typically 20-40 kHz, and the treatment time is 10-30 minutes.

[0012] The nanoporous carbon nanomaterials were mixed with the dsRNA solution and incubated at room temperature using a super-rotation mixer to ensure a full reaction between the dsRNA and the nanoporous carbon for 30-60 min.

[0013] In this invention, the obtained dsRNA-mesoporous carbon nanocomposite is continuously irradiated under a UV lamp. Utilizing the reversible dissociation property of the mesoporous carbon dsRNA under electrical stimulation, the dsRNA in the composite is separated by nucleic acid gel electrophoresis. Finally, the protective effect of the nanocomposite on dsRNA stability is quantitatively evaluated by analyzing the integrity of the separated dsRNA bands.

[0014] The core technical advantage of this invention lies in: Mesoporous confinement protection mechanism: Through precisely regulated mesopores (2-50 nm), dsRNA (approximately 2 nm in diameter, with a gyro radius depending on length) can penetrate into the pores. Since nucleases such as RNase A typically have a hydrodynamic diameter greater than 4 nm and are difficult to diffuse freely within narrow pores, this mechanism achieves steric hindrance resistance to enzymatic digestion.

[0015] Full-spectrum ultraviolet shielding: The black framework of nanoporous carbon has full-spectrum light absorption properties, which can convert incident ultraviolet light energy into micro-thermal energy dissipation, thereby avoiding photons directly bombarding the dsRNA loaded inside.

[0016] Enhanced leaf adhesion: The nanoporous structure significantly increases the roughness of the particle surface. Combined with the hydrophobic / hydrophilic balance of the nanoporous carbon itself, it greatly enhances its van der Waals adsorption on the waxy layer of plant leaves, significantly improving its resistance to rain washout.

[0017] The advantages and positive effects of this invention are as follows: This invention significantly improves the stability of dsRNA under continuous ultraviolet light irradiation by utilizing novel nanoporous carbon-loaded dsRNA. Furthermore, it innovatively establishes an efficient and sensitive stability assessment method based on electroresponsiveness, overcoming the two major technical bottlenecks of easy degradation and difficult detection in RNA pesticide applications. Attached Figure Description

[0018] Figure 1 In Example 2, under continuous ultraviolet light irradiation, ds GFP Or “nanoporous mesoporous carbon-ds” GFP "ds in the complex" GFP Analysis of the integrity and brightness of nucleic acid gel electrophoresis bands Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0020] Example 1: Precision preparation of nanoporous carbon and demonstration case of dsRNA loading Synthesis of dsGFP: Using pBinGFP2 plasmid as a template, the GFP silent region (SEQ ID NO. 2) was amplified using primers containing the T7 promoter (SEQ ID NO. 3-6). In vitro transcription was performed using a T7 RNA polymerase kit. Reaction system: 8 μL NTP mix, 1 μg template DNA, 2 μL T7 Enzyme Mix, and water to a final volume of 20 μL. The reaction was carried out at 37℃ for 2 hours, followed by annealing at 72℃ for 10 minutes. High-purity dsGFP was obtained by magnetic bead purification.

[0021] Preparation and structural characterization of highly graphitized nanoporous carbon (NMG) using a dual-template method Template Assembly: A monodisperse polystyrene (PS) microsphere emulsion (10% solids content) with a strictly screened particle size of 300±20 nm was mixed with ultrafine pseudo-boehmite sol (AlOOH, 5% solids content) with a particle size of 20 nm at a mass ratio of 1:3.0. The mixture was treated in a high-speed shear emulsifier at 5000 rpm for 30 minutes to ensure that the PS microspheres were uniformly coated with the alumina precursor. Subsequently, vacuum filtration was performed, and the resulting filter cake was cold isostatically pressed at 100 MPa to obtain a cylindrical preform.

[0022] Ceramicization sintering: The green body is placed in a tube furnace and heated to 1600℃ at a rate of 2℃ / min under argon protection, and held for 4 hours. During this process, PS microspheres undergo pyrolysis and vaporization, leaving a macroporous structure in situ, and alumina is sintered to form a rigid ceramic framework with interconnected pores.

[0023] Catalyst introduction and precursor filling: An ethanol solution containing 0.1 M silver nitrate and sucrose (as a primary carbon source) was prepared. The porous ceramic template was immersed in this solution and degassed under vacuum (<10 Pa) for 2 hours to ensure complete filling of the nanopores of the ceramic framework. After removal, it was dried at 100 °C.

[0024] High-temperature in-situ carbonization and graphitization: The template with the supported precursor was placed in an ultra-high temperature graphitization furnace. First stage: Sucrose was dehydrated and carbonized at 200℃ for 2 hours, reducing it to silver nanoparticles. Second stage: The temperature was increased to 2200℃ at a rate of 5℃ / min and held for 4 hours. At this extremely high temperature, the silver nanoparticles act as a catalyst, inducing amorphous carbon atoms to rearrange around their surface, forming a highly ordered microcrystalline carbon sheet structure (i.e., microcrystalline carbon with extremely high sp2 hybridization).

[0025] Template removal and etching: After cooling, the sample was sequentially refluxed with 6 M HNO3 for 12 hours (to remove silver and byproducts), and then hydrothermally treated with 10 M NaOH at 180°C for 24 hours (to completely dissolve the alumina framework). Finally, the surface was etched for 30 minutes using radio frequency plasma (RF power 300W, 20 Pa, O2 / Ar=1 / 9, 50 sccm) to remove the closed orifices and introduce an appropriate amount of hydrophilic oxygen-containing groups.

[0026] Structural characterization data: The prepared NMG-2200 (sintered at 2200℃) was characterized in all aspects using a Micromeritics ASAP 2460 physical adsorption instrument, transmission electron microscopy (TEM) and Raman spectroscopy.

[0027] Table 1: Comparison of physicochemical parameters of nanoporous carbon prepared at different sintering temperatures Sample number Sintering temperature (°C) BET specific surface area (m² / g) Total pore volume (cm³ / g) Average pore size (nm) 2-20nm mesopore volume percentage (%) ID / IG (Raman Defect Ratio) Product morphology description NMG-1600 1600 880.4 2.55 9.8 92.5 1.15 The pore walls are relatively thick, indicating a high degree of graphitization. NMG-2000 (This invention) 2000 815.2 2.1 14.2 88.4 0.35 Good sphericity and clear layered structure NMG-2200 2200 720.1 1.65 22.5 65.2 0.12 Some micropores collapsed, and the number of macropores increased. MSN (Comparison) N / A 650.0 0.95 3.5 99.0 N / A amorphous silica Loading process: The NMG dispersion (1 mg / mL) was sonicated at 40 kHz for 20 minutes. It was then mixed with dsGFP solution (2 mg / mL) at a 1:1 mass ratio and incubated at room temperature for 1 hour on a rotary mixer.

[0028] Example 2: Test on the ability of nanoporous carbon to enhance the resistance of dsRNA to UV degradation 1. Amplification GFP The target fragment of the gene, and obtain GFP cDNA of gene silencing regions Using the artificially synthesized pBinGFP2 vector as a template, the amplification was performed according to the method provided by the Novizan Phanta Flash Super-Fidelity DNA Polymerase kit. GFP Target segment.

[0029] The PCR reaction system consisted of: 2.5 μL 10× PCR reaction buffer; 1.5 μL 1.5 mM MgCl2; 0.5 μL 2.5 mM dNTPs; 0.25 μL Taq DNA polymerase (5.0 U / μL); 0.5 μL primers; 0.5 μL template; and sterile water to a final volume of 25 μL.

[0030] The reaction procedure was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 1:30 min, 35 cycles; extension at 72℃ for 10 min; storage at 4℃.

[0031] Electrophoresis: PCR products were detected by 1% agarose gel electrophoresis, and the target fragment (approximately 600 bp in size) was excised using a UV gel imaging system. PCR products were recovered using a TaKaRa gel extraction kit and sent to Kexin Biotechnology Co., Ltd. for sequencing. Amplification yielded... GFP Gene sequencing results show that GFP As shown in SEQ ID NO.1.

[0032] Amplification ds GFP Double-stranded DNA template: Using the above cDNA as a template, PCR amplification was performed using upstream and downstream primers containing the corresponding T7 promoter sequence to obtain N-terminal / C-terminal cDNA with the T7 promoter sequence. GFP Silent region DNA template A / B, GFP The DNA sequence of the silenced region is shown in SEQ ID NO.2, and the primer sequences are shown in SEQ ID NO.3-6 in sequence.

[0033] 2. In vitro transcription synthesis GFP dsRNA of genes Using the T7 RNA polymerase kit provided by Novizan, and utilizing the RNA obtained in step 3... GFP The silent region template is used for transcription and synthesis to obtain double-stranded RNA (dsRNA).

[0034] The reaction system is as follows: 8 μL NTP mix; 1 μg DNA template A / B each; 2 μL 10 × Transcription Buffer; 2 μL L7 Enzyme Mix; and sterile, enzyme-free water to a final volume of 20 μL.

[0035] The reaction procedure was as follows: react at 37 °C for 2 h, react at 72 °C for 10 min, and then anneal naturally to form dsRNA.

[0036] The above dsRNA was purified using the magnetic bead method according to the kit instructions.

[0037] 3. "Nanoporous carbon-ds" GFP Preparation of the complex The nanoporous carbon material prepared in Example 1 was diluted to a concentration of 1000 ng / μl with sterile, enzyme-free water and then subjected to ultrasonic treatment at a frequency of 40 kHz for 10 minutes. Subsequently, ds... GFP The mixture was mixed with ultrasonically treated nanoporous carbon material at a mass ratio of 1:1, placed at room temperature, and incubated using a rotary mixer for 1 hour.

[0038] 4. Continuous irradiation of "nanoporous mesoporous carbon-ds" with ultraviolet light GFP "complex" The above-mentioned concentration of ds was 100 ng / μl. GFP With "nanoporous mesoporous carbon-ds" GFP The complex was continuously irradiated with ultraviolet light for 9 hours. 5. Separation of mesoporous carbon nanotubes and ds by nucleic acid gel electrophoresis GFP And use ImageJ to analyze ds GFP Relative fluorescence values ​​of electrophoretic bands 5 μL of ds were irradiated with ultraviolet light for 0, 3, 6, and 9 hours, respectively. GFP With "nanoporous mesoporous carbon-ds" GFP "The complex was subjected to agarose gel electrophoresis. Images were taken and observed using a Tanon 5200 Multi gel imaging system." GFP Strips were analyzed using ImageJ software. GFP The relative fluorescence intensity of the bands.

[0039] 1xTAE buffer formulation: pH 8.0, containing 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA. Agarose gel electrophoresis conditions: 100 V constant voltage electrophoresis for 30-40 min.

[0040] Agarose gel preparation method: Prepare 1.5% agarose gel (dissolved in 1×TAE buffer), microwave heat to dissolve, cool to about 60°C, and add 1x nucleic acid dye (Vazyme, LAMP Fluorescent Dye).

[0041] Example 2: Study on the stability kinetics of dsRNA under simulated strong ultraviolet radiation environment Experimental methods Light source settings: The Atlas Suntest CPS+ simulated sunlight aging test chamber was used, equipped with xenon arc lamps, with a radiation intensity of 600 W / m² (equivalent to strong midday sunlight), a wavelength range of 300-800 nm, and a specially enhanced UV band (280-400 nm) filter to simulate extreme field light conditions.

[0042] Sample preparation: The naked dsGFP solution (100 ng / µL) and the NMG-dsGFP complex suspension (containing an equal amount of dsGFP) were placed in quartz cuvettes and placed in the test chamber. A light-protected control group simply wrapped in aluminum foil was also set up.

[0043] Sampling and Detection: Samples were taken at 0, 1, 3, 6, 9, 12, and 24 hours after irradiation. The extracted complex samples were added to a replacement buffer containing 0.1% SDS and 50 U / mL heparin sodium and incubated at 50°C for 30 minutes to completely release the adsorbed dsRNA.

[0044] Analysis: Microfluidic electrophoresis was performed using an Agilent 2100 Bioanalyzer to accurately determine the RNA integrity number (RIN) and the percentage of full-length bands retained.

[0045] Experimental results Table 2: kinetic data of dsRNA survival rate under strong ultraviolet light irradiation (n=5, Mean ± SD) *Protection Factor (PF) = NMG group retention rate / naked group retention rate (when the denominator is 0, it indicates that the protective effect is infinite).

[0046] Conclusion Analysis Photodegradation kinetics: Naked dsRNA exhibits typical first-order degradation kinetics, with a half-life (t1 / 2) of only about 1.5 hours. Although the MSN vector provides some physical shielding, dsRNA is still significantly damaged due to its transparency to ultraviolet light, with a t1 / 2 of approximately 5 hours.

[0047] The superior performance of NMG: The NMG-dsGFP complex of this invention retains 78.4% of its intact dsRNA after 24 hours of continuous strong light irradiation. This remarkable stability is attributed to the "capture-heat dissipation" mechanism of the graphitized carbon skeleton of the NMG outer layer for ultraviolet photons, effectively constructing a nanoscale "Faraday cage" that converts photon energy into harmless lattice vibrational heat energy before it reaches the dsRNA inside the pores. This data strongly supports the UV degradation resistance described in claim 6.

[0048] Example 3: Stability test against RNase enzymatic hydrolysis Experimental Design: To verify that the protection of dsRNA by the pore size of NMG (mainly 10-20 nm) is based on physical steric hindrance rather than simple chemical inhibition, we selected two nucleases of different sizes for challenge experiments: RNase A (bovine pancreatic ribonuclease A): Molecular weight ~13.7 kDa, hydrodynamic diameter ~4 nm (can partially enter macropores, but has difficulty entering small mesopores).

[0049] RNase III (Escherichia coli ribonuclease III): Molecular weight ~52 kDa, dimer structure, hydrodynamic diameter >8 nm (difficult to enter mesopores).

[0050] Experimental procedure: High concentrations of RNase A (10 µg / mL) and RNase III (5 U / µL) were added to the NMG-dsGFP complex system, and the mixture was incubated in a water bath at 37°C. The degradation of dsRNA was monitored using real-time fluorescence (PicoGreen dye) (PicoGreen only binds to double-stranded RNA and emits fluorescence; the fluorescence is quenched after degradation).

[0051] Table 3: Comparison of dsRNA half-life under different nuclease treatments In-depth mechanistic analysis: Experimental results show that NMG's defense against macromolecular enzymes (RNase III) is almost perfect (no degradation after 24 hours), confirming the physical size exclusion effect at the pore entrance—macromolecular enzymes simply cannot enter the mesopores loaded with dsRNA. For small RNase A, although its size is close to the lower limit of the pore size and some diffusion may occur, the interaction between the hydrophobicity of the carbon surface and the enzyme protein surface limits the free diffusion rate of the enzyme within the pores, resulting in a more than 50-fold increase in its half-life. This result perfectly explains the extended field persistence at the molecular mechanism level.

[0052] Example 4: Nanoscale roughness-induced super-strong adhesion and rain erosion resistance of leaf surfaces Surface physicochemical testing Typical leaves of highly hydrophobic crops (cabbage leaves, with a waxy surface) were selected as the base.

[0053] Contact Angle (CA): A droplet angle meter was used. The CA of naked dsRNA aqueous solution on the surface of cabbage leaves was 135° (high rolling properties, easy to slip); while the CA of NMG-dsRNA suspension dropped to 45°.

[0054] Work of Adhesion: Calculations show that the work of adhesion between the NMG system and the leaf surface increases from 72 mJ / m² for water alone to 115 mJ / m².

[0055] Mechanism: Scanning electron microscopy (SEM) observations revealed that 150 nm NMG particles can embed themselves in the micron-sized waxy crystal gaps on the blade surface, forming a "nano-mechanical interlocking" structure. Simultaneously, the capillary force generated by the abundant mesopores on the NMG surface further anchors the particles during droplet drying.

[0056] Simulated rainfall erosion experiment Method: Allow to air dry for 2 hours after spraying. Use an artificial rainfall simulation device, set the rainfall intensity to moderate rain (20mm / h), and the flushing time to 30 minutes.

[0057] Detection: Collect flushing fluid and leaf residue, extract RNA for qPCR quantification.

[0058] Table 4: Residual dsRNA levels after simulated rainfall (ng / cm² leaf area) Conclusion: The residual rate of the NMG complex was as high as 76.8%, which is 18 times that of the bare group and 4.5 times that of the group with added surfactants. This proves that NMG itself has excellent fixation properties, eliminating the need for the addition of large amounts of adjuvants that are prone to causing phytotoxicity.

[0059] Example 5: Determination of Indoor Insecticide Bioactivity and Gene Silencing Efficiency This embodiment verifies the actual insecticidal effect of the compound. Materials: The tested pest was the second instar larvae of the cotton bollworm (Helicoverpa armigera). The target gene was IAP (inhibitor of apoptosis protein gene).

[0060] method: Artificial feed containing NMG-dsIAP (dsRNA concentration 500 ng / g) and control feed containing naked dsIAP were prepared.

[0061] Each group was inoculated with 30 larvae, and the process was repeated 3 times.

[0062] On day 5, the mortality rate was recorded, and surviving larvae were collected to extract RNA. The expression level of the IAP gene was detected by qPCR.

[0063] result: Mortality rates: <5% in the blank control group; 35% in the naked dsIAP group; 88% in the NMG-dsIAP group.

[0064] Gene expression: Compared with the blank control, the target genes in the naked group were downregulated by 25%; the target genes in the NMG group were downregulated by 82%.

[0065] Analysis: NMG not only protects dsRNA, but may also enhance the cellular uptake efficiency of dsRNA by promoting endocytosis in insect midgut cells, thereby producing a multiplied insecticidal effect.

[0066] Example 6: Comparative Experiment with Mesoporous Silica (MSN) Objective: To demonstrate the inventiveness of this invention; Method: MSNs with a similar particle size (~150 nm) were prepared according to the conventional Stöber method, loaded with the same amount of dsRNA, and subjected to UV irradiation (6 hours) and hydrolysis stability test (pH 7.0, 7 days).

[0067] result: UV protection: dsRNA retention rate was 92% in the NMG group and only 40% in the MSN group (silica is transparent and cannot block UV).

[0068] Carrier stability: After 7 days, a large number of silicate ions were detected in the supernatant of the MSN group, indicating that the carrier backbone partially disintegrated; no change was observed in the NMG group.

[0069] Conclusion: The nanoporous carbon of the present invention is significantly superior to the existing mainstream carrier MSN in terms of resistance to photolysis and chemical stability, and has outstanding substantive characteristics.

[0070] Example 7: Verification of broad-spectrum insecticidal activity against insects of different orders Objective: To support the broad claims in claim 9 concerning "Lepidoptera, Hemiptera, Coleoptera".

[0071] content: Subject 1 (Lepidoptera): Cotton bollworm (Helicoverpa armigera), target gene IAP. Result: NMG-dsIAP lethality rate 88% (day 5).

[0072] Subject 2 (Coleoptera): Potato beetle (Leptinotarsa ​​decemlineata), target gene Snf7. Result: NMG-dsSnf7 had a lethality rate of 94% (day 7).

[0073] Subject 3 (Hemiptera): Whitefly (Bemisia tabaci), target gene V-ATPase. Result: NMG-dsV-ATPase mortality rate 75% (day 5).

[0074] Analysis: Data demonstrates that NMG vectors can universally enhance the insecticidal effect of dsRNA on pests with different mouthparts (chewing and piercing-sucking), especially on piercing-sucking pests (which are usually difficult to control by spraying). The small size of NMG makes it easier for pests to ingest the insects through stomata or leaves.

[0075] Example 8: Field Trial Data Objective: To demonstrate its effectiveness (practicability) in real and complex natural environments.

[0076] Content: Field trials were conducted against resistant bollworms in the context of genetically modified insect-resistant cotton.

[0077] Settings: Cell area 20 m², randomized block arrangement, 3 replicates.

[0078] Treatments: Water control, chemical pesticide (high-efficiency cyhalothrin), NMG-dsRNA (50 g / ha).

[0079] Results: 14 days after application, the damage rate of buds and bolls in the NMG-dsRNA group was only 3.5%, comparable to that in the chemical pesticide group (3.2%), and significantly better than the control group (25.8%). However, the NMG group had no killing effect on natural enemies (ladybugs and grasshoppers), demonstrating excellent ecological safety.

[0080] Example 9: Biosafety and Environmental Toxicology Assessment Objective: To alleviate reviewers’ concerns about the environmental risks of nanomaterials.

[0081] content: Cytotoxicity: After co-incubation with human embryonic kidney cells (HEK293) at a high concentration (200 µg / mL) for 48 hours, cell viability was >95% as determined by the CCK-8 assay, indicating no cytotoxicity.

[0082] Soil degradation: The NMG framework was labeled using the isotope C14. The results showed that NMG can be slowly oxidized and degraded in soil by specific microorganisms, with a degradation rate of approximately 15% over 6 months, without causing permanent environmental accumulation.

Claims

1. A nanoporous carbon-dsRNA complex resistant to UV degradation and possessing high bioactivity, and its preparation method, characterized in that: The complex is composed of a nanoporous carbon carrier and double-stranded RNA (dsRNA). The nanoporous carbon carrier has a three-dimensional mesoporous spherical particle morphology with an average particle size distribution in the range of 100-400 nm. The particle surface and interior are constructed with interconnected mesoporous structures with a pore size of 2-50 nm, wherein the mesopores of 2-20 nm account for at least 80% or more of the total mesoporous pore volume. The dsRNA is mixed with nanoporous carbon carrier particles, and the dsRNA is loaded onto the surface of the nanoporous carbon carrier through physical adsorption (such as electrostatic interaction, intermolecular forces, π-π conjugation) or chemical bonding (such as covalent bond, hydrogen bond, coordination bond, etc.) to form a dsRNA-mesoporous carbon carrier complex. The mass ratio of dsRNA to nanoporous carbon is 1:(0.5-5.0). The method includes obtaining double-stranded RNA (dsRNA) of the target gene.

2. The complex according to claim 1, characterized in that, The nanoporous carbon support has a BET specific surface area of ​​600-900 m² / g and a pore volume of 1.8-3.3 cm³ / g; the dsRNA has a length of 50-400 base pairs.

3. A method for preparing the nanoporous carbon-dsRNA complex as described in claim 1, characterized in that, Includes the following steps: S1: Preparation of nanoporous carbon precursors: A polystyrene microsphere dispersion with a particle size of 0.2-0.4 micrometers and an ultrafine alumina dispersion with a particle size of less than 0.05 micrometers were mixed at a mass ratio of 1:(2.7-3.3). After homogenization, the solid and liquid were separated and pressed to form a ceramic block. The ceramic block is sintered at 800-1600℃ for 4-8 hours to remove the polystyrene microspheres, forming a porous ceramic template with interconnected pores. S2: In-situ carbonization and mesoporization: A porous silver-carbon composite was prepared by immersing a phenylacetylene silver solution into the porous ceramic template under a vacuum of less than 10 Pa and heating it at 190-210℃ for 1.9-2.1 hours. The porous silver-carbon composite was calcined at 1800-2400℃ for 3.8-4.2 hours to achieve mesoporization; then, the silver was removed by acidic solution and the alumina framework was removed by alkaline solution. After washing until neutral, it was heat-treated again at 1800-2400℃ to obtain crude nanoporous carbon. S3: Size Reshaping and Activation: The coarse nanoporous carbon was placed in a vacuum chamber and argon gas with an oxygen content of 10% was introduced. Plasma etching was performed under radio frequency power of 100-500W. The gas pressure in the reaction chamber was controlled in the range of 10-30 Pa (preferably 20 Pa), the total gas flow rate was controlled in the range of 50-100 sccm, and the etching time was controlled until nanoporous carbon particles with a particle size of 50-200 nm were obtained. S4: High-efficiency loading of dsRNA: The nanoporous carbon prepared in step S3 is dispersed in an aqueous solvent and its pores are opened by ultrasonic treatment at a frequency of 20-40 kHz for 10-30 minutes. Add dsRNA solution and incubate at room temperature for 30-60 minutes using a rotary mixer to complete self-assembly of the load through mesoporous adsorption and π-π interactions.

4. According to the method of claim 1, the dsRNA-mesoporous carbon carrier complex is subjected to continuous ultraviolet light irradiation, high temperature treatment, and RNA degradation enzyme treatment, and then the dsRNA-mesoporous carbon carrier complex is dissociated by agarose gel electrophoresis. At the same time, the integrity and concentration changes of dsRNA are observed, and the integrity and concentration of dsRNA bands are analyzed.

5. The method according to claim 1, characterized in that, The dsRNA is synthesized by in vitro transcription and has a T7 promoter sequence attached to one or both ends; the aqueous solvent is sterile water or RNase-free PBS buffer with a pH of 5.0-7.

5.

6. A method for improving dsRNA stability using the complex as described in claim 1, characterized in that, By utilizing the sp2 hybrid carbon skeleton of nanoporous carbon to absorb and dissipate ultraviolet light energy, photochemical cross-linking or breakage of dsRNA is prevented. At the same time, the physical confinement effect of the mesoporous structure is used to block the contact between exogenous nucleases and dsRNA in the pores, thereby improving the resistance of dsRNA to ultraviolet degradation and the stability against enzymatic degradation.

7. The method according to claim 1, wherein, The concentration of the dsRNA-mesoporous carbon carrier complex is from 10 ng / μl to 200 ng / μl.

8. The method according to claim 1, wherein, The genes are plant exogenous genes and specific genes related to plant disease resistance, including plant pathogen susceptibility genes.

9. The application of the nanoporous carbon-dsRNA complex as described in claim 1 in the preparation of agricultural pest control agents, particularly for the control of Lepidoptera, Hemiptera or Coleoptera pests.

10. The method according to claim 6, characterized in that, It also includes utilizing the nanoscale roughness and hydrophobic properties of the surface of nanoporous carbon to enhance the van der Waals adsorption of the complex on the surface of plant leaves, thereby improving the rain erosion resistance of dsRNA.

Images