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 molybdenum element, and the molybdenum element is selected from one or more of ammonium molybdate, ammonium tetramolybdate or sodium molybdate. 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 a dsRNA protectant and its preparation method. 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 molybdenum in the preparation of dsRNA protective agents. Experimental verification of Mo... 6+ 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 molybdenum.

[0010] In a further embodiment of the second aspect of the present invention, the molybdenum element is derived from ammonium molybdate ((NH4)6Mo7O). 24 Ammonium tetramolybdate ((NH4)2Mo4O) 13Ammonium molybdate, sodium molybdate (Na2MoO4), and sodium molybdate are micronutrient fertilizer raw materials approved for use by the agricultural sector and have no toxic side effects on crops, soil microorganisms, or humans.

[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 present invention, the mass ratio of molybdenum salt to insecticidal dsRNA in the dsRNA protectant is (0.005~5000):1. Specifically, the mass ratio of molybdenum salt to insecticidal dsRNA is 5000:1, 500:1, 50:1, 5:1, 0.5:1, 0.05:1, or 0.005:1.

[0013] In a further embodiment of the third aspect of the present invention, the biological compound composition further includes trehalose and mannitol as freeze-drying protectants, which can extend the shelf life of the protectants and meet the needs of long-term storage in agricultural production. At the same time, the freeze-dried formulation is easy to transport and reduces 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. The survival-essential gene is selected from... HaND3 , HaNur-4 , Sfshd , SaZFP One or more of 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 pests include, but are not limited to, at least one of the following: cotton bollworm, fall armyworm, and wheat aphid. Cotton bollworm mainly damages cash crops and food crops such as cotton, corn, and soybeans; fall armyworm is a globally invasive pest; and wheat aphid is a common pest in major wheat-producing areas. The combined annual grain loss caused by these three pests reaches 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 molybdenum-containing compound in enzyme-free deionized water, heat and stir until completely dissolved to obtain a dsRNA protectant with 40-50% w / v molybdenum. 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 in Example 2; Figure 2 This is the high-performance liquid chromatogram from Example 2; Figure 3 This is a combined diagram of isothermal titration calorimetry and micro-thermophoresis techniques used in Example 2; Figure 4 This is an electrophoresis diagram of the biological compound composition resisting RNase A degradation in Example 3; Figure 5 This is an electrophoresis diagram of the biological compound composition resisting degradation by strong ultraviolet light in Example 4; Figure 6 This is an electrophoresis diagram of the biological compound composition resisting high-temperature degradation in Example 5; Figure 7 This refers to the bollworm larvae in Example 6. HaND3 A bar chart of RNAi targeting genes; Figure 8 This refers to the bollworm larvae in Example 6. HaNur-4A bar chart of RNAi targeting genes; Figure 9 This is the treatment of fall armyworm in Example 6. Sfshd A bar chart of RNAi targeting genes; Figure 10 This is the control of wheat aphids in Example 6. SaZFP A bar chart of RNAi targeting genes; Figure 11 Example 7 uses the second biological compound composition to target cotton bollworm larvae. HaND3 A bar chart of RNAi targeting genes; Figure 12 This is an electrophoresis image of the third biocomposite composition used in Example 8 to resist RNase A degradation; Figure 13 This is an electrophoresis diagram of the third biological compound composition used in Example 8 to resist the degradation of midgut fluid in cotton bollworm larvae. 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 sodium molybdate as a raw material dsRNA protectant: Dissolve Na2MoO4 in deenzyme-free deionized water, heat and stir until completely dissolved to obtain a 40-50% w / v molybdenum elemental solution.

[0023] dsRNA stock solution: Take eGFP The gene's dsRNA (named dseGFP) and its target HaNur-4 The dsRNA of the gene (named dsHaNur-4) 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-fold, and then mixed with the dsRNA solution stock solution at a concentration of 1 mg / mL. The mixture was vortexed to obtain biological compounding compositions with mass ratios of 5000:1, 500:1, 50:1, 5:1, 0.5:1, 0.05:1, and 0.005:1.

[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 on the left side of the middle section); after the introduction of the dsRNA protectant, the dsRNA becomes a folded, curved crystal (as shown on the left side of the middle section). Figure 1 As shown on the right side of the image, this indicates that the introduction of the dsRNA protectant affects the structure of the dsRNA.

[0026] Furthermore, HPLC was used to identify the biological compound composition. The results are as follows: Figure 2 As shown, with increasing concentration of the dsRNA protectant, the peak elution time shifts to the right, indicating that the introduction of the dsRNA protectant leads to a decrease in the particle size of the dsRNA.

[0027] ITC results showed that the dsRNA protectant could interact with dsRNA, and the dissociation constant K of their interaction was... d It is 2.69 × 10 -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. ΔH >0, ΔS >0, ΔH = 276 kcal / mol, a relatively large value, indicating that the binding process is highly endothermic. This is due to the energy consumption of desolvation enthalpy penalty, phosphate group dehydration, and conformational adjustment. Long-chain dsRNA may undergo local bending or compression to adapt to ion binding, consuming a large amount of energy. TΔS = -280 kcal / mol, a large value. This is because the dsRNA protectant is a multivalent cation, which generates a stronger entropy increase when it binds to dsRNA. The interface between the cation and dsRNA releases a large number of ordered water molecules (such as...). Figure 3 (As shown at points a and b). Combined with the MST experiment, the K of the biological compound composition can be determined. d Approximately 2.62 × 10 -8 The interaction between the dsRNA protectant and dsRNA was verified using both methods (e.g., Figure 3 (As shown at point c). This ultimately demonstrates that the process is significantly endothermic and exhibits a significant increase in entropy. ΔG A value less than 0 indicates that both combination processes can occur spontaneously. -TΔS The value is <0 and the absolute value is relatively large, indicating that the formation process of the biological compound composition is dominated by electrostatic interaction and involves a desolvation entropy increase process of large-scale solvent recombination.

[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 showed significant retention of the main band at mass ratios of 5000:1 to 500:1, significant degradation of the main band at 50:1, and complete degradation at 5:1 (e.g., ...). Figure 4 (As shown at point a). Therefore, it can be inferred that the "critical load" for the degradation of the main band of the biological compound composition is 500:1.

[0029] Further testing was conducted on the long-term RNase A enzymatic stability of the biocompound composition under critical loading. The results showed that no major band degradation occurred in the biocompound composition within 0–9 days. The biocompound composition exhibited RNase A enzymatic stability for nearly 15 days (e.g., ...). Figure 4 (As shown at point b). 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 time 0, and complete degradation was achieved at 1 hour (e.g., ...). Figure 4 (As shown at point c). This demonstrates that the biological compound composition possesses a strong protective ability against the degradation of dsRNA under RNase A conditions.

[0030] 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 5 As shown, the biological compound composition significantly retained the major band of naked dsRNA within three orders of magnitude range of mass ratios from 5000:1 to 50:1; at a mass ratio of 5:1, there was no significant difference compared to naked dsRNA. This indicates that the described dsRNA protectant has the ability to effectively stabilize dsRNA under UV conditions.

[0031] 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. Within a mass ratio range of 5000:1 to 50:1, the biological compound composition showed significantly better retention of the major band compared to naked dsRNA, exhibiting high-temperature stability consistent with its UV stability range (see Figure 1). Figure 6 (As shown).

[0032] The experimental results of Examples 3 to 5 show that the biological compound composition enhances dsRNA stability within a mass ratio range of 5000:1 to 50:1, and maintains dsRNA stability within a mass ratio range of 5:1 to 0.005:1.

[0033] 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. [Selection / Option] HaND3 , HaNur-4 RNAi was performed on gene targets in two cotton bollworm larvae. The results showed that the RNAi efficiency was improved to varying degrees compared to naked dsRNA after using the bio-composite composition. Compared to naked dsRNA, the dsHaND3 bio-composite composition at a mass ratio of 5:1 significantly improved the RNAi efficiency in cotton bollworm larvae. HaND3 The expression level decreased significantly (p<0.01), with the relative expression level decreasing from 0.48 to 0.19, and the target interference efficiency increased by 29.0% (e.g., Figure 7 (As shown). Compared with the naked dsHaNur-4 group, the dsHaNur-4 biocompound with a critical mass ratio of 500:1... HaNur-4 The expression level decreased, and the efficiency of target interference increased from 42.0% to 65.3% (e.g., Figure 8 (As shown). This illustrates a relatively wide range of BI6 / dsRNA quality ratios, indicating that BI6@dsRNA can achieve good RNAi effects in pests.

[0034] At the same time, another option was chosen Sfshd , SaZFP They are fall armyworm ( Spodoptera frugiperda ) and wheat aphid ( Sitobion avenae RNAi was performed on the gene target of ) . The results showed that the dsSfshd biopolymer composition at a mass ratio of 5:1 was significantly better than naked dsSfshd. Sfshd The relative expression level of the gene decreased, from 0.41 to 0.24, and the target interference efficiency increased by 22.7% (e.g. Figure 9 (As shown). Compared with the naked dsSaZFP group, the dsSaZFP biocompound composition with a mass ratio of 5:1... SaZFP The expression level was significantly reduced ( p<0.01), the target interference efficiency increased from 48.7% to 97.7% (e.g., ...). Figure 10 (As shown). The results showed that the RNAi efficiency was improved to varying degrees compared to naked dsRNA after using the biological compound composition. This indicates that the biological compound composition has a relatively broad-spectrum RNAi pest control effect.

[0035] Example 7: Preparation of a biological compound composition using ammonium molybdate 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 (NH4)6Mo7O 24 Meanwhile, during the preparation of the biological compound composition, only groups with a mass ratio of dsRNA protectant to dsRNA solution stock solution of 5000:1, 500:1, 50:1, and 5:1 were set.

[0036] (2) Following the experimental method provided in Example 3, the improved resistance of the prepared biocompound composition to RNase A degradation was verified. The test results are as follows: Figure 9 and Figure 10 As shown.

[0037] The biocomposite composition showed significant retention of the main band at mass ratios of 5000:1 to 500:1, but complete degradation of the main band at a mass ratio of 50:1. Therefore, the "critical load" for main band degradation in the biocomposite composition was determined to be 500:1 (e.g., ...). Figure 9 (As shown).

[0038] No major degradation occurred in the biocomposite composition within 12 hours (e.g. Figure 10 (As shown). This demonstrates that the biological complex prepared with ammonium molybdate also possesses a strong protective ability against the degradation of dsRNA under RNase A conditions.

[0039] (3) The experimental method provided in Example 6 was used to verify the improvement of the efficiency of the prepared biological compound composition on the RNAi of pests.

[0040] cotton bollworm larvae HaND3 RNAi was performed targeting the gene. The results showed that the RNAi efficiency was improved compared to naked dsRNA when using the described biological complex combination. Compared to dseGFP, the biological complex combination with a mass ratio of 500:1... HaND3 The expression level was significantly reduced ( p <0.001) (e.g. Figure 11 (As shown). This demonstrates that the described biological compound combination can achieve a good RNAi effect on the cotton bollworm pest.

[0041] Example 8: Preparation of a biological compound composition using ammonium tetramolybdate 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 (NH4)2Mo4O 13 Meanwhile, during the preparation of the biological compound composition, groups were set up with the following mass ratios of dsRNA protectant to dsRNA solution stock solution: 5000:1, 500:1, 50:1, 5:1, 0.5:1, 0.05:1, and 0.005:1.

[0042] (2) Following the experimental method provided in Example 3, the improved resistance of the prepared biocompound composition to RNase A degradation was verified. The test results are as follows: Figure 12 As shown, the "critical load" for the degradation of the main band of the biological compound composition is 500:1.

[0043] (3) Following the experimental method provided in Example 3, the biological compound composition was incubated with the midgut fluid of *Helicoverpa armigera* for 30 min to verify that the prepared biological compound composition exhibited enhanced resistance to degradation by complex enzymes in the midgut fluid of *Helicoverpa armigera*. Furthermore, by reducing the amount of dsRNA protectant in the compound composition system, the results showed that dsRNA could still be cleaved by nucleases in the insect (including DICER), laying the foundation for the successful occurrence of RNAi mechanisms in insects (e.g., ...). Figure 13 (As shown).

[0044] 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 in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

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

2. A dsRNA protectant, characterized in that, Including molybdenum.

3. The dsRNA protectant according to claim 2, characterized in that, The molybdenum element is derived from one or more of ammonium molybdate, tetraammonium molybdate, or sodium molybdate.

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, The mass ratio of molybdenum salt to insecticidal dsRNA in the dsRNA protectant is (0.005~5000):

1.

6. The biological compound composition according to claim 5, characterized in that, It also includes 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 effect as a template, and the pests include, but are not limited to, cotton bollworm, fall armyworm, and 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 molybdenum-containing compound was dissolved in enzyme-free deionized water and heated and stirred until completely dissolved to obtain a dsRNA protectant with 40-50% w / v molybdenum. 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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