Broad-spectrum insecticide based on RNA interference technology and preparation method thereof

Abstract

The invention discloses a broad-spectrum insecticide based on RNA interference technology and a preparation method thereof, and relates to the technical field of broad-spectrum insecticides, the preparation method comprises the following steps: S1, material selection; s2, preparing double-stranded siRNA (small interfering Ribonucleic Acid); s3, preparing hyaluronic acid; s4, preparing an acetamiprid solution; s5, mixing; and S6, packaging a finished product. According to the broad-spectrum insecticide based on the RNA interference technology and the preparation method of the broad-spectrum insecticide, firstly, preparation raw materials of double-stranded siRNA, hyaluronic acid and an acetamiprid solution are prepared respectively, so that three mixed materials can be formed, then the three mixed materials are put into a mixing tank to be uniformly mixed to form a uniform compound solution, and the compound solution is added into the mixing tank to prepare the broad-spectrum insecticide based on the RNA interference technology. The preparation method comprises the following steps: mixing a plurality of substances in a compound solution, then filling and packaging the solution after mixing is finished so as to facilitate subsequent selling and transportation use, and because the double-stranded siRNA in the prepared compound solution is a double-stranded RNA molecule and can specifically bind and degrade specific mRNA in cells, the expression of a target gene is inhibited, and a key gene in a pest body can be effectively silenced.

Description

Technical Field

[0001] The present invention relates to the technical field of broad-spectrum insecticides, and particularly to a broad-spectrum insecticide based on RNA interference technology and a preparation method thereof. Background Art

[0002] Aphids are a general term for the Aphidoidea of Hemiptera. They are phytophagous insects with a body length of 1-10 mm. Their body surfaces are smooth or covered with wax powder / wax filaments. They suck plant sap through their piercing-sucking mouthparts, causing leaf curling and plant dwarfing, and secreting honeydew to induce sooty mold, which affects photosynthesis. The action of insecticides is to interfere with aphid nerve conduction, leading to their paralysis and death, and can inhibit aphids to avoid excessive damage to plants.

[0003] When most insecticides are used to treat aphids, due to the much faster metabolic rate of aphids than other pests, they can quickly decompose and excrete the insecticides out of their bodies, resulting in a reduction in the efficacy of the insecticides. Moreover, aphids have the ability of a short life cycle and high reproductive capacity, enabling a new batch of aphids to be formed in a short period, making it easy for them to generate resistant populations through gene mutations. At the same time, the residual period of general insecticides is relatively short, so repeated application is required, which further increases the possibility of drug resistance, and thus the killing effect of insecticides on aphids is generally average. Summary of the Invention

[0004] The purpose of the present invention is to provide a broad-spectrum insecticide based on RNA interference technology and a preparation method thereof, so as to solve the problem that when the insecticide in the above background art is used to treat aphids, drug resistance easily appears due to the high reproductive ability of aphids, resulting in an average killing effect on aphids.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A broad-spectrum insecticide based on RNA interference technology, which is prepared by mixing double-stranded siRNA, hyaluronic acid, and acetamiprid solution in an appropriate proportion.

[0006] Preferably, the preparation raw materials of the double-stranded siRNA include: DNA extraction template of aphids and RNA polymerase. The preparation raw materials of the hyaluronic acid include: dextran or cellulose derivative and hydrolase. The preparation raw materials of the acetamiprid solution are cyanamide and ethanol.

[0007] Preferably, the dosage of the hyaluronic acid is 4-8 parts, the dosage of the acetamiprid solution is 15-75 parts, the ratio of hyaluronic acid to acetamiprid solution is 1:4, and the concentration of the double-stranded siRNA is 300 ng / μL.

[0008] A preparation method of a broad-spectrum insecticide based on RNA interference technology includes the following steps:

[0009] S1: Material selection: Prepare the raw materials for the preparation of double-stranded siRNA, hyaluronic acid, and acetamiprid solution respectively;

[0010] S2: Preparation of double-stranded siRNA: Extract the DNA template of aphids and transcribe it with RNA polymerase to generate siRNA;

[0011] S3: Preparation of hyaluronic acid: Purify it after treatment with a polysaccharide polymer and an enzyme complex solution to obtain hyaluronic acid;

[0012] S4: Preparation of acetamiprid solution: React cyanamide with ethanol and then carry out condensation to obtain a high-purity product;

[0013] S5: Mixing: Stir and mix the prepared double-stranded siRNA, hyaluronic acid, and acetamiprid solution in proportion inside the mixing roller,

[0014] S6: Finished product packaging: Can the mixed medicament to obtain the finished product.

[0015] Preferably, in S1, prepare the raw materials for double-stranded siRNA, hyaluronic acid, and acetamiprid solution and distinguish the dosages.

[0016] Preferably, in S2, prepare the DNA of aphids, design it into two 29-nt DNA oligonucleotides, transcribe them with RNA polymerase, react at 37 °C for 30 min, then add 2.5 - 3 times the volume of absolute ethanol, precipitate and recover the RNA, heat at 95 °C for five minutes, and slowly cool it to room temperature to complete annealing to form double-stranded siRNA.

[0017] Preferably, in S3, use dextran or cellulose derivative as the substrate, carry out a complex reaction with a hydrolase, carry out liquid treatment at 90 °C for 28 h, and obtain hyaluronic acid through purification.

[0018] Preferably, in S4, react cyanamide with ethanol for 4 - 6 h, control the reaction temperature at 60 - 90 °C, and carry out condensation with N-(6-chloro-3-pyridylmethyl)methylamine for 8 - 10 h after the reaction to obtain the crude acetamiprid, then add the crude product to ethanol for purification and crystallization, and the reaction time is 3 h to obtain the acetamiprid solution.

[0019] Preferably, in S5, add the prepared hyaluronic acid and acetamiprid solution to the mixing tank for mixing according to the dosage of 1:4 and the concentration of double-stranded siRNA of 300 ng / μL. Ensure that the pH value during the mixing process is between 5.3 - 6.5, and the mixing time is 3 - 4 h.

[0020] Preferably, in S6, the mixed broad-spectrum insecticide solution is filled into containers to obtain the finished product, which facilitates its transportation and sale.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: When the broad-spectrum insecticide based on RNA interference technology and its preparation method are used, first, the raw materials for the preparation of double-stranded siRNA, hyaluronic acid, and acetamiprid solution are prepared respectively, so as to form three kinds of mixed materials. Then, they are put into the mixing tank and evenly mixed to form a uniform complex solution. After that, the solution is filled and packaged after mixing, so as to facilitate subsequent sale and transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic flow chart of the broad-spectrum insecticide based on RNA interference technology and its preparation method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] Please refer to Figure 1 , the present invention provides a technical solution: A broad-spectrum insecticide based on RNA interference technology, the broad-spectrum insecticide is formed by mixing double-stranded siRNA, hyaluronic acid, and acetamiprid solution in an appropriate proportion. The raw materials for the preparation of double-stranded siRNA include: DNA extraction template of aphids and RNA polymerase. The raw materials for the preparation of hyaluronic acid include: dextran or cellulose derivatives and hydrolase. The raw materials for the preparation of acetamiprid solution are monocyanoamine and ethanol. The dosage of hyaluronic acid is 4-8 parts, and the dosage of acetamiprid solution is 15-75 parts. The ratio of hyaluronic acid to acetamiprid solution is 1:4, and the concentration of double-stranded siRNA is 300 ng / μL.

[0025] A preparation method of a broad-spectrum insecticide based on RNA interference technology, comprising the following steps:

[0026] S1: Material selection: Prepare the raw materials for the preparation of double-stranded siRNA, hyaluronic acid, and acetamiprid solution respectively;

[0027] S2: Preparation of double-stranded siRNA: Transcribe siRNA by extracting the DNA template of aphids and RNA polymerase;

[0028] S3: Preparation of hyaluronic acid: After treatment with a polysaccharide polymer and an enzyme complex solution and purification, hyaluronic acid is obtained;

[0029] S4: Preparation of acetamiprid solution: After the reaction of cyanamide with ethanol, a high-purity product is prepared through condensation;

[0030] S5: Mixing: The prepared double-stranded siRNA, hyaluronic acid, and acetamiprid solution are stirred and mixed inside a mixing roller in proportion,

[0031] S6: Finished product packaging: The mixed medicament is canned to obtain the finished product.

[0032] Preparation of double-stranded siRNA: First, the raw materials for the preparation of double-stranded siRNA, hyaluronic acid, and acetamiprid solution are prepared and their dosages are controlled. Then, aphids are selected to extract DNA, which is made into two 29-nt DNA oligonucleotides and transcribed by RNA polymerase. The reaction is carried out at 37 °C for 30 min. Subsequently, 2.5 - 3 volumes of absolute ethanol are added to the transcribed product to precipitate and recover RNA. It is heated in a heating tube at 95 °C for five minutes to ensure thorough mixing. Then, it is taken out and cooled at room temperature, maintaining the same temperature as the room temperature, and thus the preparation of double-stranded siRNA can be completed;

[0033] Preparation of hyaluronic acid: Subsequently, using dextran or cellulose derivatives as substrates, which are polysaccharide polymers, and then adding a hydrolytic enzyme, enabling the hydrolytic enzyme to undergo a complex reaction with the polysaccharide polymer with dextran or cellulose derivatives as substrates. Derivatives are synthesized through an enzyme polymerization reaction and subjected to liquid treatment at 90 °C for 28 h. Subsequently, hyaluronic acid is obtained through purification;

[0034] Preparation of acetamiprid solution: Cyanamide can react with ethanol for 4 - 6 h to generate the intermediate ethyl N-cyanoethanimidate through the catalysis of ethanol. Subsequently, it is condensed with N-(6-chloro-3-pyridylmethyl)methylamine for 8 - 10 h to obtain the crude acetamiprid. Then, the crude product is added to ethanol for purification and crystallization, with a reaction time of 3 h, and thus the acetamiprid solution is obtained.

[0035] By reacting cyanamide with ethanol for 4 - 6 h, the intermediate ethyl N-cyanoethanimidate can be generated through the catalysis of ethanol. Subsequently, it is condensed with N-(6-chloro-3-pyridylmethyl)methylamine for 8 - 10 h to obtain crude acetamiprid. Immediately, the crude product is added to ethanol for purification and crystallization, with a reaction time of 3 h, to obtain an acetamiprid solution. The obtained hyaluronic acid and the acetamiprid solution are added to a mixing tank in a ratio of 1:4 and a dosage of 300 ng / μL of double-stranded siRNA for mixing, and the mixing time is ensured to be 3 - 4 h to form a broad-spectrum insecticide solution, which is then filled and packaged.

[0036] Example

[0037]

[0038] Table 1

[0039] Since the double-stranded siRNA in the prepared complex solution is a double-stranded RNA molecule, it can specifically bind to and degrade specific mRNA in cells, thereby inhibiting the expression of target genes, effectively silencing key genes in pests. And hyaluronic acid has excellent biocompatibility and degradability, and can bind to negatively charged siRNA through electrostatic interaction to form a stable nano-complex, thereby protecting siRNA from degradation by the external environment, and can also enhance the cellular uptake efficiency of siRNA, making it easier to enter aphids. The acetamiprid solution is a commonly used insecticide that can inhibit the nervous system of aphids. Through the synergistic effect with hyaluronic acid, it can significantly improve the control effect on aphids, especially for aphids that have developed resistance, thereby forming an RNA interference effect. At the same time, the RNA interference effect can interfere with the normal physiological functions of aphids, ultimately leading to the death or decline of the reproductive ability of pests, and effectively killing and inhibiting aphids. At the same time, siRNA is designed based on the genes of aphids, which has less impact on non-target organisms and avoids affecting beneficial insects in the vicinity;

[0040] At the same time, RNA interference can significantly reduce the expression of target genes, thereby affecting the growth, development and survival of aphids, and effectively inhibiting and killing aphids.

[0041] In summary, aphids are a general term for the superfamily Aphidoidea in the Hemiptera order and belong to phytophagous insects. The action of pesticides causes paralysis and death by interfering with the nerve conduction of aphids. After preparing the raw materials for double-stranded siRNA, hyaluronic acid, and acetamiprid solution and mixing them, a complex solution is formed. The double-stranded siRNA in the complex solution is a double-stranded RNA molecule that can specifically bind to and degrade specific mRNA in cells, thereby inhibiting the expression of target genes, effectively silencing the key genes in pest bodies. At the same time, when mixed with hyaluronic acid and acetamiprid solution, it can form a stable nano-complex, avoid degradation by the external environment, and improve the control effect on aphids. Moreover, by designing siRNA based on the genes of aphids, the impact on non-target organisms is small, avoiding affecting beneficial insects in the vicinity. The content not described in detail in this specification belongs to the prior art well-known to those skilled in the art.

[0042] Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A broad-spectrum insecticide based on RNA interference technology, characterized in that: The broad-spectrum insecticide is prepared by mixing double-stranded siRNA, hyaluronic acid and acetamiprid solution in appropriate proportions.

2. The broad-spectrum insecticide based on RNA interference technology according to claim 1, characterized in that: The raw materials for preparing the double-stranded siRNA include: DNA extraction template of aphids and RNA polymerase. The raw materials for preparing the hyaluronic acid include: dextran or cellulose derivative and hydrolase. The raw materials for preparing the acetamiprid solution are cyanamide and ethanol.

3. A broad-spectrum insecticide based on RNA interference technology according to claim 1, characterized in that: The dosage of the hyaluronic acid is 4-8 parts, and the dosage of the acetamiprid solution is 15-75 parts. The ratio of hyaluronic acid to acetamiprid solution is 1:4, and the concentration of double-stranded siRNA is 300 ng / μL.

4. A preparation method of a broad-spectrum insecticide based on RNA interference technology, which applies the broad-spectrum insecticide based on RNA interference technology described in any one of claims 1-3 above, characterized in that, It includes the following steps: S1: Material selection: Prepare the raw materials for double-stranded siRNA, hyaluronic acid and acetamiprid solution respectively, and distinguish the dosages. S2: Prepare double-stranded siRNA: Transcribe siRNA by extracting the DNA template of aphids and RNA polymerase. S3: Prepare hyaluronic acid: Purify after treatment with a polysaccharide polymer and enzyme complex solution to obtain hyaluronic acid. S4: Prepare acetamiprid solution: React cyanamide with ethanol, and then condense to obtain a high-purity product. S5: Mixing: Stir and mix the prepared double-stranded siRNA, hyaluronic acid and acetamiprid solution in proportion inside the mixing roller. S6: Finished product packaging: Can the mixed medicament to obtain the finished product.

5. The preparation method of a broad-spectrum insecticide based on RNA interference technology according to claim 4, wherein: In S1, prepare the raw materials for double-stranded siRNA, hyaluronic acid and acetamiprid solution, and distinguish the dosages.

6. The preparation method of a broad-spectrum insecticide based on RNA interference technology according to claim 4, characterized in that: In S2, prepare the DNA of aphids, design it into two 29-nt DNA oligonucleotides, transcribe them with RNA polymerase, react at 37°C for 30 min, then add 2.5-3 times the volume of absolute ethanol, precipitate and recover RNA, heat at 95°C for five minutes, and let it cool slowly to room temperature to complete annealing to form double-stranded siRNA.

7. A preparation method of a broad-spectrum insecticide based on RNA interference technology according to claim 4, characterized in that: In S3, use dextran or cellulose derivative as the substrate, carry out a complex reaction with hydrolase, carry out liquid treatment at 90°C for 28 h, and obtain hyaluronic acid through purification.

8. A preparation method of a broad-spectrum insecticide based on RNA interference technology according to claim 4, characterized in that: In S4, react cyanamide with ethanol for 4-6 h, control the reaction temperature at 60-90°C, and condense with ethyl N-cyanoethanimidate for 8-10 h after the reaction to obtain the crude acetamiprid. Then add the crude product to ethanol for purification and crystallization, and the reaction time is 3 h to obtain the acetamiprid solution.

9. The preparation method of a broad-spectrum insecticide based on RNA interference technology according to claim 4, characterized in that: In S5, add the prepared hyaluronic acid and acetamiprid solution to the mixing tank for mixing according to the dosage of 1:4 and the concentration of double-stranded siRNA of 300 ng / μL. Ensure that the pH value during the mixing process is between 5.3 and 6.5, and the mixing time is 3-4 h.

10. The preparation method of a broad-spectrum insecticide based on RNA interference technology according to claim 4, characterized in that: In S6, fill the mixed broad-spectrum insecticide solution to obtain the finished product, which is convenient for transportation and sale.

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