DsRNA protective agent and preparation method thereof

Abstract

The invention relates to a dsRNA protective agent and a preparation method thereof. The dsRNA protective agent comprises a calcium element, and the calcium element is selected from one or more of calcium chloride, calcium hydrophosphate and calcium sulfate. The dsRNA protective agent is combined with deinsectization dsRNA to form a micelle with a rigid structure, and the prepared biological compound composition has the advantages that the stability of the dsRNA on RNase A enzymolysis, ultraviolet radiation and high temperature is improved, and the RNAi efficiency of pests is enhanced. The biological compound composition can be prepared into a spray, and field pesticide application is facilitated. After being sprayed, the composition can be eaten by pests to enter bodies, so that target genes can be silenced by RNAi (Ribonucleic Acid Interference); meanwhile, the composition is free of chemical residues, the pesticide residues of harvested agricultural products are far lower than the national standard, the production requirements of green food and organic agricultural products are met, and the market competitiveness of the agricultural products is improved.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and biopesticides, and particularly to dsRNA protectants and their preparation methods. Background Technology

[0002] RNA interference (RNAi) technology, a highly specific gene silencing technique, is one of the few cutting-edge biotechnologies that can be simultaneously applied to human health management and the control of plant and animal diseases and pests, providing a novel technological pathway for green pest control in agriculture. RNA biopesticides developed based on RNA interference technology have demonstrated enormous application potential in the green, personalized, and intelligent control of plant diseases and pests, as well as ensuring efficient crop production. Compared with traditional chemical pesticides, RNA biopesticides have unique technological advantages: First, they possess abundant original target resources, allowing for the design of targeted double-stranded RNA (dsRNA) against specific genes of diseases and pests, achieving precise control of target diseases and pests while effectively reducing the impact on non-target organisms; second, they exhibit excellent environmental compatibility, being nucleic acid molecules that can gradually degrade in the natural environment, minimizing residual pollution and ensuring high ecological safety; third, they possess strong product compatibility and flexibility, allowing for flexible design of target sequences according to different crops and disease / pest types, and can be well integrated with existing agricultural production technology systems.

[0003] In the technical system of RNA biopesticides, the stability and bioactivity of dsRNA, the core active ingredient, directly determine the pesticide's control efficacy. However, dsRNA molecules themselves are structurally unstable and easily damaged by various external factors in agricultural applications. On the one hand, nucleases in the natural environment (such as endonucleases and exonucleases on soil and plant surfaces) can rapidly degrade dsRNA, causing it to lose activity before reaching its target site. On the other hand, environmental conditions during field application, such as ultraviolet radiation, temperature fluctuations, and pH changes, can further exacerbate dsRNA degradation and significantly reduce its bioavailability. Therefore, effectively protecting dsRNA molecules and improving their stability and persistence in agricultural applications is a key technical bottleneck in ensuring the effectiveness of RNA biopesticides.

[0004] To address the aforementioned issue of insufficient stability of dsRNA, researchers have attempted to develop various delivery materials or protective vectors to encapsulate and protect dsRNA, such as liposomes, virus-like particles, composite nanoparticles, and bio-clay. These materials can improve the stability of dsRNA and extend its duration of action to some extent under laboratory conditions. However, in practical agricultural applications, existing delivery materials and protection technologies suffer from numerous insurmountable defects, severely hindering the large-scale application of RNA biopesticides: The preparation processes of most delivery materials are complex, requiring stringent production equipment and technical conditions, resulting in high production costs and failing to meet the cost-effectiveness requirements of agricultural production; some materials themselves have poor environmental compatibility, potentially causing negative impacts on soil microbial communities and soil physicochemical properties after field application, contradicting the original intention of developing green pesticides; furthermore, the dispersibility and film-forming properties of some delivery materials are difficult to adapt to conventional agricultural application methods such as field spraying and root irrigation, further limiting their practical application value.

[0005] In summary, existing technologies for protecting dsRNA, the core active ingredient of RNA biopesticides, generally suffer from high costs, difficult preparation, and poor environmental adaptability. In particular, there is a lack of a cost-effective, easy-to-prepare, environmentally friendly dsRNA protectant that can be effectively adapted to practical agricultural applications. Developing such a protectant is of great significance for overcoming the application bottlenecks of RNA biopesticides and promoting the development of green agricultural technologies, and has become a pressing technical problem for those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to disclose a dsRNA protectant and its preparation method, so as to solve one or more technical problems existing in the existing methods and provide at least one beneficial option or create conditions.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The first aspect of this invention relates to providing the application of calcium in the preparation of dsRNA protective agents. Experimental verification of Ca... 2+ It can bind to dsRNA through electrostatic interactions to form a nucleic acid micelle complex, thereby protecting dsRNA from degradation.

[0009] A second aspect of the present invention is to provide a dsRNA protectant. The active ingredient of the dsRNA protectant is calcium.

[0010] In a further embodiment of the second aspect of the present invention, the calcium element is derived from one or more of calcium chloride (CaCl2), calcium hydrogen phosphate (CaHPO4), and calcium sulfate (CaSO4). Calcium chloride, calcium hydrogen phosphate, and calcium sulfate are commonly used compliant raw materials in the agricultural field and can be used as fast-acting calcium fertilizer, calcium phosphate compound fertilizer, and soil conditioner, respectively. They are highly safe within the prescribed application range, have no residual pollution, and are suitable for the application needs of ecological agriculture.

[0011] A third aspect of the present invention provides a biological compound composition. The biological compound composition comprises insecticidal dsRNA and the dsRNA protectant described in the second aspect of the present invention, wherein the dsRNA protectant binds to the insecticidal dsRNA to form micelles with a rigid structure. The dsRNA protectant can effectively load dsRNA, and through intercalation, exospheric coordination, and / or endospheric coordination, folded and bent crystals of the dsRNA epitopes can be detected; that is, the introduction of the dsRNA protectant causes a trend towards a reduction in the particle size of the dsRNA.

[0012] In a further embodiment of the third aspect of the invention, the dsRNA protectant contains Ca 2+ The molar ratio of the insecticidal dsRNA to the dsRNA is (1 × 10⁻⁶). 0 ~1×10 6 ): 1. Specifically, Ca 2+ The molar ratio of the insecticidal dsRNA to the dsRNA is 1:1, 10:1, 100:1, 1000:1, 10000:1, 100000:1, or 1000000:1.

[0013] In a further embodiment of the third aspect of the present invention, the bio-compound composition further includes a Tris-HCl buffer solution, trehalose, and / or mannitol. The Tris-HCl buffer solution can maintain the pH stability of the bio-compound composition (suitable for field soil pH range of 5.5~8.5), avoiding dsRNA degradation caused by pH fluctuations. Trehalose and mannitol, as freeze-drying protectants, can extend the shelf life of the protectant, meeting the needs of long-term storage in agricultural production. At the same time, the freeze-dried formulation is easy to transport, reducing logistics costs.

[0014] In a further embodiment of the third aspect of the invention, the insecticidal dsRNA targets a survival-essential gene of the pest. Preferably, the survival-essential gene is selected from... HaND3 , MdNsp1 These genes are highly conserved in the target pest population, with no risk of mutation escape, which can effectively prevent the development of pesticide resistance in pests.

[0015] In a further embodiment of the third aspect of the present invention, the pest includes at least one of the cotton bollworm and the wheat aphid. The cotton bollworm mainly damages cash crops and food crops such as cotton, corn, and soybeans, while the wheat aphid is a common pest in major wheat-producing areas. Together, the two cause annual grain losses of tens of millions of tons.

[0016] A fourth aspect of this invention is to provide a method for preparing the biological compound composition described in the third aspect of this invention. The specific steps include: A) Dissolve a calcium-containing compound in enzyme-free deionized water, heat and stir until completely dissolved, to obtain 30-40% w / v Ca. 2+ dsRNA protectant; B) Dissolve the insecticidal dsRNA lyophilized powder in nuclease-free water, remove residual inorganic salts by ultrafiltration centrifugation, and concentrate to the target concentration to obtain dsRNA stock solution; C) Dilute the dsRNA protectant with DEPC water to the required concentration, mix it with the dsRNA stock solution at a volume ratio of 1:1, and vortex to mix.

[0017] In a further embodiment of the fourth aspect of the present invention, the ultrafiltration centrifuge tube described in step B) has a specification of 30 kDa.

[0018] The fifth aspect of this invention lies in providing the application of the aforementioned biological compound composition in the control of agricultural pests and diseases. The biological compound composition can be prepared as a spray for easy field application. In the control of agricultural pests and diseases, external spraying is the core application method, suitable for mechanized operations (such as drone spraying and large sprayer spraying) of large-area crops such as wheat, corn, and cotton. After spraying, the composition can be ingested by pests and enter their bodies, achieving RNAi silencing of target genes. The control efficiency against target pests after application reaches over 80%, with a long-lasting effect of 10-15 days, reducing the number of sprays and lowering agricultural production costs. Simultaneously, the composition leaves no chemical residues, and the pesticide residue levels in harvested agricultural products are far below national standards, meeting the production needs of green food and organic agricultural products and enhancing the market competitiveness of agricultural products. Attached Figure Description

[0019] Figure 1 These are photographs observed using transmission electron microscopy and cryo-electron microscopy in Example 2; Figure 2 This is a dynamic light scattering particle size distribution diagram from Example 2; Figure 3 This is the isothermal titration thermogram from Example 2; Figure 4 This is an electrophoresis diagram of the biological compound composition resisting RNase A degradation in Example 3; Figure 5This is an electrophoresis image of naked dsRNA resisting RNase A degradation, as shown in Example 3; Figure 6 This is an electrophoresis diagram of the biological compound composition resisting degradation by strong ultraviolet light in Example 4; Figure 7 This is an electrophoresis diagram of the biological compound composition resisting high-temperature degradation in Example 5; Figure 8 This refers to the bollworm larvae in Example 6. HaND3 A bar chart of RNAi targeting genes; Figure 9 This is the wheat aphid in Example 6. MdNsp1 A bar chart of RNAi targeting genes; Figure 10 This is an electrophoresis diagram of naked dsRNA resisting RNase A degradation, determined using the second biological compound composition in Example 7; Figure 11 This is a fluorescence microscopy image taken in Example 8 showing how the third biological compound composition promotes the absorption of dsRNA by cells. Detailed Implementation

[0020] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0021] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0022] Example 1: Preparation of a biological compound composition using calcium chloride as a raw material dsRNA protectant: Dissolve CaCl2 in deenzyme-free deionized water, heat and stir until completely dissolved, to obtain 30-40% w / v CaCl2. 2+ The resulting dsRNA protectant was named BI17.

[0023] dsRNA stock solution: Take targeted eGFP The gene's dsRNA (named dseGFP) and its target HaND3The dsRNA of the gene (named dsHaND3) was prepared. Approximately 2% w / v trehalose and 2-20% mannitol were added to a 10 mg / mL dsRNA concentration, mixed well, and then lyophilized into a powder using a freeze dryer. 1 mg of the lyophilized dsRNA powder was dissolved in 10 mL of nuclease-free water, injected into a 30 kDa ultrafiltration centrifuge tube, and centrifuged for 2-5 minutes, discarding the waste liquid. An appropriate amount of fresh nuclease-free water was added to the upper chamber, mixed well, and then centrifuged again to concentrate to the target volume. This step was repeated 2-3 times to remove other inorganic salts remaining from the dsRNA synthesis and purification process, obtaining the dsRNA mother liquor.

[0024] Compounding: The dsRNA protectant was serially diluted 6 times with DEPC water at a concentration of 10 times, and then mixed with the dsRNA solution stock solution of 1 mg / mL. The mixture was vortexed to obtain biological compounding compositions with molar ratios of 1:1, 10:1, 100:1, 1000:1, 10000:1, 10000:1, and 100000:1.

[0025] Example 2: Binding of dsRNA to the dsRNA protectant TEM results showed that naked dsRNA crystallized in linear clusters that were close to each other (e.g. Figure 1 As shown in the upper left figure, after the introduction of the dsRNA protectant (BI17), the dsRNA becomes a folded, curved crystal (as shown in the figure above). Figure 1 (As shown in the lower left image); and the Cryo-EM image shows that the biocomposite composition produced linear structures at the pore edge where the complex concentration was higher, which were originally free. Figure 1 As shown in the upper right image), it transforms into a star-shaped cluster-like condensation phenomenon ( Figure 1 As shown in the lower right figure (the smaller figure is a magnified image of the agglomeration), it demonstrates that the introduction of the dsRNA protectant affects the structure of the dsRNA.

[0026] The particle size of the biological compound was found to be 438.8 ± 28.7 nm using DLS dynamic photodiffraction experiments, which is smaller than the particle size of naked dsRNA (544.3 ± 64.9 nm). Figure 2 (As shown in the left figure). Meanwhile, the PDI of the biocomposite composition increased by 0.25 compared to naked dsRNA, indicating that the biocomposite composition is less uniformly dispersed in solution than dsRNA (e.g., ...). Figure 2 (As shown in the right figure). This is because the presence of the dsRNA protectant reduces the electrostatic repulsion between phosphate groups of the dsRNA fragments, causing the dsRNA to first compress and shorten, and then tend to aggregate. Simultaneously, the smaller size of the biocomposite composition facilitates the transport of dsRNA in vivo.

[0027] ITC results showed that the dsRNA protectant could interact with dsRNA, and the dissociation constant Kd for this interaction was 3.61 × 10⁻⁶. -3 The dsRNA protectant reacts with the K of dseGFP. d This indicates that the sensitivity of the two combinations is on the order of millimoles. ΔS >0, ΔH = 2.23±1.10 kcal / mol, indicating that the process is endothermic, and that hydrogen bonds may be broken or recombined at the nucleic acid binding interface. ΔG <0 indicates that both combination processes can occur spontaneously. TΔS = -6.93 kcal / mol, indicating that the process is entropy-driven, with hydrophobic interactions as the core driving force, accompanied by polar interaction rearrangement. The dsRNA protectant intercalates between nucleic acid base pairs or binds to the major / minor grooves of nucleic acids, with the core driving force being the stacking interaction between small molecules and the hydrophobic regions of nucleic acid bases. During this process, a large number of water molecules arranged in an orderly manner on the hydrophobic surfaces of bases and small molecules are released, leading to a significant increase in the system entropy, thereby strongly driving the formation of the complex (e.g., Figure 3 (As shown).

[0028] Example 3: Improved resistance of the biocompound composition to RNase A degradation The biocomposite composition was incubated with RNase A for 30 min and obtained by agarose gel electrophoresis. The biocomposite composition was obtained at a molar ratio of 1×10⁻⁶. 3 : 1~1×10 6 : 1. The main band is significantly preserved, at 1×10 2 At time 1, the degradation rate of the main band was lower than that of naked dsRNA (e.g., Figure 4 (As shown). Therefore, it can be inferred that the "critical load" for the degradation of the main band of the biocompound is 1 × 10⁻⁶. 3 1. When naked dsRNA was mixed with RNase A and immediately added to the gel wells for electrophoresis, significant degradation of the main band was observed at 0 h, and complete degradation was achieved at 1 h (e.g., ...). Figure 5 (As shown). This demonstrates that the biological compound composition possesses extremely strong protective ability against the degradation of dsRNA under RNase A conditions.

[0029] Example 4: Improved resistance of the bio-compound composition to strong ultraviolet degradation. The ultraviolet irradiation experiment used a 254 nm low-pressure mercury lamp as the experimental light source, with an irradiance of approximately 75 μW / cm². 2 The experimental sample was placed 2 cm away from the light source, and after irradiation for 30 min, agarose gel electrophoresis was performed. The results are as follows: Figure 6 As shown, the biological compound composition is in a molar ratio of 1×10:1~1×10 6 The biocomposite exhibits significant main band retention across six orders of magnitude of 1 compared to the control group's naked dsRNA, thus demonstrating broad UV stability.

[0030] Example 5: Improved resistance of the bio-compound composition to high-temperature degradation The biological compound composition was subjected to extreme high-temperature treatment at 100°C to obtain a molar ratio of 1×10⁻⁶. 4 : 1~1×10 6 Within a ratio range of 1, nucleic acids become unstable at 100°C, but within a range of 1×10⁻⁶, the instability of nucleic acids is reduced. 3 A molar ratio of 1 has good stability (e.g., Figure 7 (As shown).

[0031] The experimental results of Examples 3 to 5 show that the biological compound composition has a yield of 1×10⁻⁶. 3 : 1~1×10 6 Within a molar ratio range of 1:1, it exhibits an effect of enhancing dsRNA stability. At 1×10⁻⁶, it also enhances dsRNA stability. 2 : 1~1×10 0 Within the range of 1, it has the effect of maintaining the stability of dsRNA.

[0032] Example 6: Improvement of RNAi efficiency in pests by biological compound composition Further investigation was conducted to determine whether the described biological compound composition affected the RNAi efficacy at the target site. Cotton bollworm larvae were selected. HaND3 RNAi targeting gene sites was performed. The results showed that using a bio-composite composition improved RNAi efficiency to varying degrees compared to naked dsRNA. The bio-composite composition with a molar ratio of 5:1 for dsHaND3 showed improved efficiency in cotton bollworm larvae compared to naked dsHaND3. HaND3 The effect was significantly reduced (p<0.01), and the target interference efficiency increased from 31.7% to 90.0%, representing a 58.3% improvement in target interference efficiency (e.g., ...). Figure 8 (As shown).

[0033] The bio-compound composition was then subjected to field testing. Three treatment groups were set up for the experiment: water treatment (blank control group), 70% imidacloprid treatment (positive control group), and the dsMdNsp1 bio-compound composition targeting MdNsp1 (experimental group, molar ratio 1×10⁻⁶). 31) Treatment. Results showed that three days after application, the control efficiency of wheat aphids in the experimental group reached 89.60%, reaching or even slightly exceeding the control efficiency of 83.73% achieved by the control group using imidacloprid. This indicates that the biological compound composition has a good pest control effect comparable to chemical pesticides (e.g., Figure 9 (As shown).

[0034] Example 7: Preparation of a biological compound composition using calcium sulfate as a raw material (1) The preparation method is the same as in Example 1, except that the raw material for dsRNA is replaced with calcium sulfate; at the same time, the mass ratio of dsRNA protectant to dsRNA solution mother liquor is set at 1×10 during the preparation of the biological compound composition. 1 : 1~1×10 6 Group 1.

[0035] (2) Following the experimental method provided in Example 3, the prepared biocomposite composition was verified to have enhanced resistance to RNase A degradation. The test results are as follows: Figure 10 As shown.

[0036] The biological compound composition is in a molar ratio of 1×10 3 : 1~1×10 6 : 1. The main band is significantly preserved, at 1×10 2 The main band was completely degraded at a molar ratio of 1×10⁻¹. Therefore, the biocomposite composition was found to be effective at a molar ratio of 1×10⁻¹⁰. 3 : 1~1×10 6 Within the range of 1, it significantly improves the stability of dsRNA by RNase A enzymatic hydrolysis.

[0037] Example 8: Preparation of a biological compound composition using dicalcium phosphate as a raw material (1) The dsRNA obtained by in vitro transcription was labeled with Cy3 using the Silencer siRNA compound labeling kit (LifeTechnologies) according to the manufacturer's instructions. The preparation method was the same as in Example 1, except that the raw material for dsRNA was replaced with calcium hydrogen phosphate; at the same time, the molar ratio of dsRNA protectant to dsRNA stock solution was set at 1×10 during the preparation of the biological compound composition. 2 Group 1.

[0038] (2) Following the experimental method provided in Example 3, the results showed that the intracellular fluorescence intensity of dsRNA-Cy3 after treatment with the biological compound composition was much greater than that after treatment with naked dsRNA. The test results are as follows: Figure 11 As shown. The prepared biological compound composition was verified to promote the absorption of dsRNA by cells.

[0039] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the invention are encompassed within the scope of the invention.

Claims

1. Application of calcium in the preparation of dsRNA protectants.

2. A dsRNA protectant, characterized in that, Includes calcium.

3. The dsRNA protectant according to claim 2, characterized in that, The calcium element is derived from one or more of calcium chloride, calcium hydrogen phosphate, or calcium sulfate.

4. A biological compound composition, characterized in that, It includes insecticidal dsRNA and the dsRNA protectant of claim 2 or 3, wherein the dsRNA protectant binds to the insecticidal dsRNA to form micelles with a rigid structure.

5. The biological compound composition according to claim 4, characterized in that, Ca in dsRNA protectant 2+ The molar ratio of the insecticidal dsRNA to the dsRNA is (1 × 10⁻⁶). 0 ~1×10 6 ) :

1.

6. The biological compound composition according to claim 5, characterized in that, It also includes Tris-HCl buffer solution, trehalose and / or mannitol.

7. The biological compound composition according to claim 4, characterized in that, The insecticidal dsRNA targets genes essential for the survival of pests.

8. The biological compound composition according to claim 7, characterized in that, The insecticidal dsRNA is synthesized using a gene with biocontrol effects as a template, and the pests include, but are not limited to, cotton bollworm and / or wheat aphid.

9. A method for preparing the biological compound composition according to any one of claims 4 to 8, characterized in that, Including the following steps: A calcium-containing compound is dissolved in enzyme-free deionized water, and the mixture is heated and stirred until completely dissolved to obtain 30-40% w / v Ca. 2+ dsRNA protectant; The insecticidal dsRNA lyophilized powder was dissolved in nuclease-free water, and residual inorganic salts were removed by ultrafiltration centrifugation in an ultrafiltration centrifuge tube. The solution was then concentrated to the target concentration to obtain the dsRNA stock solution. The dsRNA protectant was diluted with DEPC water to the required concentration and mixed with the dsRNA stock solution at a volume ratio of 1:1, and vortexed until homogeneous.

10. The use of the biological compound composition according to any one of claims 4 to 8 in the control of agricultural pests and diseases.

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