A method for interfering with expression of an insect gene by penetration of dsRNA through the insect body wall

Abstract

This invention relates to the field of nanomaterials and gene interference, specifically disclosing a method for interfering with insect gene expression by infiltrating dsRNA through the insect's body wall. The method involves complexing a nanocarrier with a functional macromolecule of dsRNA, and with the aid of a surfactant, enabling exogenous dsRNA molecules to attach, spread, penetrate the body wall, and enter various organs and tissues within the insect, thus efficiently interfering with gene expression. This invention utilizes the dual synergistic effect of the nanocarrier and the surfactant to achieve efficient and non-invasive delivery of exogenous dsRNA molecules into insect cells, ultimately promoting the correct targeting of dsRNA molecules and improving RNA interference efficiency. The method described in this invention is simple, efficient, and has low error, and has significant practical implications for scientific research on insect gene interference and the application of highly efficient, safe, and targeted RNA pesticide formulations.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials and gene interference, and more specifically, to a method for interfering with insect gene expression by infiltrating dsRNA through the insect body wall. Background Technology

[0002] RNA interference (RNA interference) refers to the highly conserved evolutionary phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA molecules). These specially designed exogenous dsRNA molecules can specifically, efficiently, and persistently reduce the expression of specific genes, which is of great significance for studying the function of unknown genes, regulating insect development and behavior, and protecting plants from viral infections. RNA interference has been widely used as an important tool in various fields of gene function identification and functional gene expression regulation. Furthermore, due to the high specificity and easy degradation of dsRNA molecules after release, it has become a genetic control strategy that specifically targets pests while remaining safe for non-target organisms.

[0003] Due to the defenses of the insect immune system and the barriers of organ basement membranes and cell membranes, exogenous dsRNA molecules have difficulty efficiently entering insect cells. Furthermore, the large variety of insect species and genes often makes injection and feeding methods for dsRNA application ineffective. Low cellular uptake of dsRNA molecules is a significant factor contributing to the poor effectiveness of RNA interference in insect genes. Injection and feeding are currently the most common methods for introducing dsRNA molecules into cells, using microinjectors to directly inject dsRNA molecules into designated sites within the insect. However, for small insects with abundant body fluids, mechanical trauma and post-operative infection are unavoidable. These methods also require sophisticated injection equipment and techniques, limiting their application to laboratory research with medium to large insects. The high operational difficulty makes them unsuitable for large-scale insect experiments and prevents widespread use in agricultural fields. Compared to injection, feeding has advantages such as ease of operation, minimal trauma, and less time consumption. However, dsRNA molecules in feed have low efficiency in penetrating the peritrophic membrane and intestinal cell membrane barrier of insects. A large number of exogenous dsRNA molecules are excreted with feces, resulting in disadvantages such as low interference efficiency of target genes, large amount of dsRNA used, and high cost. In addition, some insects lack effective artificial feed, and feeding can easily cause malnutrition and death in insects, increasing errors and affecting the accuracy of experimental results.

[0004] In recent years, nanotechnology has developed rapidly, with its applications in agriculture expanding quickly. One such application is utilizing the targeted delivery and controlled release capabilities of nanocarriers to deliver nucleic acid drugs (DNA or RNA) into insects. This breaks down organ basement membranes, cell membranes, and intestinal peritrophic membranes, promoting cellular uptake of exogenous nucleic acid molecules and enhancing their effectiveness. Successful applications have already been demonstrated in injection and feeding methods, with nanocarriers delivering dsRNA molecules significantly improving the efficiency of gene interference in insects.

[0005] Even so, microinjection can easily cause damage to the insect's internal tissues, loss of body fluids, or even death, while feeding methods cannot eliminate the problem of errors caused by malnutrition in insects.

[0006] Therefore, developing a method to efficiently interfere with insect gene expression without injection or feeding is of great significance for studying unknown gene functions, regulating insect development and behavior, and protecting plants from viral infections. Summary of the Invention

[0007] In order to solve the problems existing in the prior art and make up for the defects of injection and feeding methods, the purpose of this invention is to provide a method for interfering with insect gene expression by infiltrating dsRNA through the insect body wall.

[0008] To achieve the objectives of this invention, the technical solution is as follows:

[0009] In a first aspect, the present invention provides a method for interfering with insect gene expression by permeating dsRNA through the insect body wall. The method involves complexing a nanocarrier with a functional macromolecule of dsRNA and using a surfactant to enable exogenous dsRNA molecules to attach to, spread, permeate the body wall, and enter various organs and tissues within the insect, thereby efficiently interfering with gene expression.

[0010] The method specifically includes the following steps:

[0011] 1) The nanocarrier solution, dsRNA solution and surfactant were mixed to obtain a composite drug solution;

[0012] 2) Place the compound drug solution on the surface of the insect.

[0013] Further, in step 1), the nanocarrier in the nanocarrier solution and the dsRNA in the dsRNA solution are mixed in a charge ratio of 1:8 to 8:1 to obtain a mixture, and the surfactant is mixed with the mixture in a volume ratio of 0.01% to 10.0%.

[0014] In this mixing process, the nanocarrier and the dsRNA combine through electrostatic interactions, and / or hydrogen bonds, and / or van der Waals forces to form a stable carrier / dsRNA molecular complex. Utilizing the wetting and degreasing effects of surfactants, the waxy layer on the insect's body surface is dissolved, reducing epidermal resistance. This allows the resulting complex drug droplets to be easily adsorbed onto the insect's body surface and penetrate the body wall to enter the body cavity, tissues, and cells, thereby efficiently interfering with the expression of the target gene.

[0015] More preferably, in order to improve mixing efficiency, the nanocarrier and the dsRNA are mixed in a charge ratio of 1:1 to obtain a mixture, and the surfactant is mixed with the mixture in a volume ratio of 1%.

[0016] Furthermore, the dsRNA described in this invention is a double-stranded RNA molecule capable of specifically degrading homologous mRNA (messenger RNA), and is not limited to its fragment size and secondary structure type.

[0017] The nanocarrier described in this invention is a nanocarrier with an internal cavity structure and a positively charged surface, preferably a nanocarrier with a high density of hydrophilic functional groups (such as amino and carboxyl groups) on the periphery.

[0018] The internal cavity structure and high-density functional group structure of the nanocarrier are conducive to the loading of dsRNA molecules to form a stable complex, while protecting the dsRNA molecule from damage by acidic and alkaline environments and enzyme degradation in the body.

[0019] The delivery function of the nanocarrier promotes the evenly distributed dsRNA molecules on the insect's body surface to penetrate the insect's body wall and enter the body cavity, tissues, and cells.

[0020] The nanocarrier is functionalized with hydrophilic functional groups (including amino and carboxyl groups), making it readily compatible with the hydrophilic protein components of insect epidermis and cell membranes. This enhances the affinity between the loaded material and the carrier and the insect epidermis and cell membrane, promoting the penetration of dsRNA molecules through the insect epidermal barrier into the cells of the body cavity and various organs and tissues, thereby interfering with the expression of intracellular target genes.

[0021] The surfactants described in this invention can be selected from various natural surfactants, organic synthetic surfactants and their derivatives, such as detergents and dishwashing liquids.

[0022] The surfactant can, on the one hand, destroy the insect's protective waxy layer and increase the penetration of the drug solution, and on the other hand, reduce the surface tension of the aqueous drug solution, making it easier for the insect's body wall to adsorb and spread the drug during spraying or dripping, thus expanding the drug-receiving area.

[0023] In a specific embodiment of the present invention, commercially available goldfish detergent is used as a surfactant. However, the surfactant described in the present invention is not limited to this. As long as it has any of the properties of wetting, emulsifying and degreasing, and can reduce the surface tension of the carrier / dsRNA molecular complex solution, promote the wetting and spreading of droplets on the oily interface (protective wax layer) of the insect body wall, dissolve the lipid components in the epidermis, destroy the wax layer, destroy the defense function of the insect body surface, and promote the complex to penetrate the body wall smoothly and enter the body cavity.

[0024] Furthermore, step 2) of the method of the present invention can be specifically applied to the insect surface by means of dripping, spraying, soaking, or other application methods, including dripping the compound drug solution onto the insect surface with an appropriate size instrument (such as a pipette, microneedle, small sprayer, etc.) or directly soaking the insect.

[0025] The method described in this invention directly introduces dsRNA molecules through permeation from the insect's body surface. This method is simple and rapid, avoiding the drawbacks of traditional injection and feeding methods, such as low gene interference efficiency, instability, and high mortality. Simultaneously, the specially designed vector can carry dsRNA molecules to penetrate the insect's body wall barrier and enter various organ tissues and cells within the insect's body cavity, efficiently exerting the target and gene interference effects of dsRNA molecules to regulate the insect's growth, development, and reproductive activities. This method significantly improves gene interference efficiency, significantly reduces dsRNA molecule loss, is non-invasive, simple, and safe, providing excellent technical support for RNA interference-based insect gene function analysis experiments and the application of dsRNA formulations for pest control in farmland.

[0026] Secondly, the present invention provides a complex drug solution prepared according to the aforementioned method, and the application of the complex drug solution in interfering with gene expression.

[0027] The raw materials or reagents involved in this invention are all commercially available products, and the operations involved are all routine operations in the field unless otherwise specified.

[0028] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined to obtain specific implementation methods.

[0029] The beneficial effects of this invention are as follows:

[0030] This invention utilizes the synergistic effect of nanocarriers and surfactants to achieve efficient and non-invasive delivery of exogenous dsRNA molecules into insect cells. The internal cavity structure and external positively charged groups of the nanocarrier attract nucleic acid molecules through electrostatic interactions, and / or hydrogen bonds, and / or van der Waals forces, freely complexing to form stable complexes. The high-density amino functional groups on the carrier's periphery enhance the affinity of the complex for the insect's epidermis and cell membranes. Combined with the wetting and dispersing effects of the surfactant, this facilitates the formation of stable, uniform, and low-surface-tension droplets, promoting droplet adhesion, spread, and retention on the insect's surface. This facilitates the penetration of dsRNA molecules through the insect's body wall into the body cavity and into various tissues and cells, ultimately promoting accurate targeting of dsRNA molecules and improving RNA interference efficiency. The method described in this invention is simple, efficient, and has low error, possessing significant practical value for scientific research on insect gene interference and the application of highly efficient, safe, and targeted RNA pesticide formulations.

[0031] The advantages of the method described in this invention include at least the following:

[0032] 1. Wide range of applications. Compared with traditional feeding and injection methods, the insect body wall penetration method of this invention is not limited by the type of insect mouthparts, food source, body size, or amount of body fluid, and can be well applied in all cases.

[0033] 2. Simple and quick operation. Compared with the traditional injection method, this invention omits preoperative preparations and postoperative care such as anesthesia, site selection, injection, and disinfection. It only involves simple operations such as preparing the mixture and dripping, soaking, or spraying onto the insect's body surface. It can be completed within 1 hour, has high tolerance for experimental instruments and operating techniques, and is easy to promote and popularize.

[0034] 3. Zero mortality and minimal error. Compared to traditional injection methods, this method is non-invasive, avoiding adverse reactions such as malnutrition, mechanical damage, and postoperative infection caused by the introduction of dsRNA molecules through injection or feeding. It can guarantee a 100% survival rate in the control group, significantly reducing the impact of systematic and individual operational errors on the accuracy of experimental results, ensuring the effectiveness of each operation as much as possible, and improving work efficiency.

[0035] 4. High interference efficiency. The dual synergistic effect of the nanocarrier and detergent ensures that the dsRNA molecules in the drug solution can quickly penetrate the insect body wall and enter the target cells for correct targeting, effectively regulating gene expression, causing abnormal insect growth and development, reduced egg production, or even death. It has good application value for both RNA interference-based insect functional gene analysis and genetic control of pests. Detailed Implementation

[0036] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] Example 1

[0040] This embodiment illustrates the preparation method of the composite drug solution of the present invention, as follows:

[0041] 1. Raw materials:

[0042] (1) Nanocarrier: This embodiment takes the fluorescent nanocarrier molecule disclosed in patent number 201310119837.3 as an example. The fluorescent core inside the carrier has a high fluorescence quantum yield, good light, heat and chemical stability, which is beneficial for tracing in organisms. The formation of a good cavity structure between each side chain and the presence of a large number of specific functional groups are conducive to the binding and assembly of dsRNA molecules and the formation of a stable dsRNA molecule / carrier complex. The entire complex has good insect cell affinity, which is conducive to the rapid entry of dsRNA molecules into cells to target genes.

[0043] (2) dsRNA: dsRNA that interferes with genes synthesized in the fat body.

[0044] Following the standard method for in vitro synthesis of dsRNA provided by Promega, dsRNA was synthesized using the T7RiboMAXExpress RNAi System kit provided by the company. The nucleotide sequence of the dsRNA is shown in SEQ ID NO.1.

[0045] (3) Sterile water, generally commercially available products.

[0046] (4) Surfactant: Goldfish brand detergent, which is available commercially, is used.

[0047] 2. Method:

[0048] (1) Prepare a nanocarrier solution with a concentration of 1.0 mg / mL using sterile water.

[0049] (2) Prepare a dsRNA solution with a concentration of 600.0 ng / μL.

[0050] (3) Take 1.5 μg of nanocarrier solution and 1.0 μg of dsRNA solution and mix them in a centrifuge tube. Vortex the two substances to make them fully contact each other. The two molecules combine under the action of electrostatic interaction, hydrogen bonding and van der Waals forces to form a stable carrier / dsRNA molecular complex drug solution.

[0051] (4) Add 1% by volume of surfactant and mix it evenly with a pipette tip to reduce the surface tension of subsequent drug droplets and form small-volume drug droplets to be added to the insect body surface.

[0052] Example 2

[0053] This embodiment illustrates how to use the complex drug solution described in Example 1 to deliver dsRNA into the small piercing-sucking insect, the soybean aphid, to interfere with the expression of key genes and control the aphid's oviposition.

[0054] This embodiment uses the soybean aphid as a representative test insect. Its abdomen is filled with body fluid; microinjection of dsRNA molecules causes internal tissue damage and loss of most body fluids, leading to death. Herbivorous soybean aphids lack nutritionally complete artificial feed, making it difficult to eliminate errors caused by malnutrition when feeding them dsRNA molecules. Furthermore, their small size (approximately 2 μm in length) makes them susceptible to drowning by insufficiently atomized large volumes of pesticide solution during application. This embodiment uses the soybean aphid, a species where RNA interference is relatively difficult to manipulate, as a representative test insect to elucidate the key technologies of this invention.

[0055] The specific steps are as follows:

[0056] 1. The composite drug solution prepared in Example 1 was repeatedly blown and agitated with a pipette to promote emulsification. Small droplets of 10nL to 100nL were formed using a microsyringe with an appropriate volume and adhered to and dropped onto the surface of the soybean aphid. The surfactant molecules in the detergent were adsorbed on the surface of the droplets, replacing the water molecules at the droplet-air interface, which reduced the uneven force on the droplet molecules and lowered the surface tension of the droplets. The droplets spread rapidly at the oil interface on the surface of the soybean aphid. The hydrophobic part of the surfactant distributed on the insect's body surface inserted into the lipid components of the body wall, while the hydrophilic part faced outward, changing the hydrophobicity of the insect's body surface and further promoting its adsorption and absorption of the water-soluble carrier droplets. To better demonstrate the role of the detergent in this invention, two control treatments were introduced: Treatment 1 (carrier / sterile water) and Treatment 2 (carrier / sterile water / detergent). These treatments were used to penetrate the soybean aphids. In Treatment 1, the detergent was difficult to adsorb onto the oily epidermal interface due to surface tension, making it difficult to absorb. In Treatment 2, the detergent was used to evenly distribute the detergent on the aphids, and the detergent was quickly absorbed by the soybean aphids.

[0057] To demonstrate the penetration effect of the carrier in this method, the carrier location on the surface and inside the aphid was examined under a light microscope one hour later. Before this, the entire soybean aphid was washed with sterile water 3-4 times to remove residual substances on the surface of the insect.

[0058] Under a microscope, it was clearly observed that the fluorescent nanocarriers in treatment 2 (carrier / sterile water / detergent) were distributed throughout the entire aphid body, while no carrier fluorescence was detected on the entire surface of the aphid in control treatment 1 (carrier / sterile water).

[0059] The carrier localization in the internal tissues of aphids under two different treatments was observed by dissection under a fluorescence microscope: no carrier fluorescence signal was detected in the tissue of control treatment 1 (carrier / sterile water), while in treatment 2 (carrier / sterile water / detergent), carrier localization was detected in various important tissues of the soybean aphid: the egg chamber and embryo related to reproduction and oviposition, the fat body related to energy metabolism, storage and transport, and the intestine related to digestion and absorption. This indicates that the surfactant in the method described in this invention can promote the adsorption, spread and penetration of the carrier aqueous solution at the lipid interface of the insect body wall.

[0060] 2. Insect cell wall permeation method for delivery of dsRNA molecules and efficient interference with insect gene expression: To better demonstrate the role of detergent and carrier in promoting the efficient and non-invasive introduction of exogenous dsRNA molecules into insect cells to interfere with gene expression, gene interference experiments were conducted using dsRNA molecules. Control treatment 1: carrier / sterile water / dsRNA; Control treatment 2: detergent / sterile water / dsRNA; Treatment 3: carrier / detergent / sterile water / dsRNA. dsRNA molecules designed with genes synthesized in the fat body as target genes were delivered to the fat bodies of soybean aphids using the cell wall permeation method described above. Gene interference efficiency was detected after 48 hours, and the phenotype of soybean aphids was observed. In soybean aphids treated with the composite drug solution described in this invention, the target gene was inhibited at the transcriptional level. In treatment 3, the target gene was inhibited at the transcriptional level, with a relative expression decrease of over 95%. In contrast, the expression levels of the interfered target genes in control treatments 1 and 2 did not show significant changes.

[0061] Meanwhile, compared to control treatment 1, control treatment 2, and treatment 3, the normal growth and development of soybean aphids were hindered, aphid production decreased or ceased, and the population density of soybean aphids in soybean seedlings significantly decreased. This indicates that, under the dual synergistic effect of detergent and nanocarrier, exogenous dsRNA molecules can penetrate the insect's body wall barrier, organ basement membrane barrier, and cell membrane barrier, rapidly enter the cell to exert gene interference effects, regulate insect life activities, hinder insect growth and development, and reduce reproductive capacity.

[0062] It should be understood that the technical solutions obtained by proportionally increasing or decreasing the amount of reagents or raw materials used in the above embodiments are essentially the same as those in the above embodiments.

[0063] The nanomaterial described in Example 1 of this invention is merely an example and can be replaced by the water-soluble fluorescent dendritic macromolecule disclosed in Chinese Patent No. 201310095194.3, the water-soluble hyperbranched fluorescent polymer disclosed in Chinese Patent No. 201310456288.9, or the fluorescent dendritic nanomacromolecule disclosed in Chinese Patent No. 201410258179.0, etc.

[0064] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> China Agricultural University <120> A method for interfering with insect gene expression by infiltrating dsRNA through the insect body wall <141> 2017-12-15 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 493 <212> DNA / RNA <213> Artificial Sequence <400> 1 tacgtcattt atttgttcca agtccgagtt caatgaattt aatatcttac cggcgtacca 60 cacgtgtccg aatatcggcg actgtccgga aaacagaatc tattacgacg gatgttgcga 120 acagtgcaac aacacgggaa tcattaaaac agaggaccat agtctttgcg ctccagagag 180 cttacccata aatcaaacag tcggtctagt aacagaagac aatccattcc atgatgcgtg 240 cacgaatatc gaaggcatag ttgggttcac ggagtgtcgg ggtctttgcg attcatacac 300 gtatttcaat aaaaaaacaa taaaacacga ttcgaaatgt caatgttgtc agccaatccg 360 tttcgataaa ttgtctgtgg tgttacagtg caaagatggt tatcagtata acaagtctgt 420 tccagtgccg tcggcttgtt cttgtacggc gtgtggcgga gagagctttt cgataaaaaa 480 atccagaaaa ctt 493

Claims

1. A method for interfering with insect gene expression by infiltrating dsRNA through the insect body wall, characterized in that, The method includes the following steps: 1) The nanocarrier solution, dsRNA solution, and surfactant were mixed to obtain a composite drug solution; The nanocarrier is a nanocarrier with an internal cavity structure and a positively charged surface, and has a high density of hydrophilic functional groups on its periphery; the nanocarrier in the nanocarrier solution and the dsRNA in the dsRNA solution are mixed in a charge ratio of 1:8 to 8:1 to obtain a mixture, and the surfactant is mixed with the mixture in a volume ratio of 0.01% to 10.0%; 2) Place the compound drug solution on the surface of the insect's body. The surfactant dissolves the waxy layer on the insect's body surface. The high-density hydrophilic functional groups on the periphery of the nanocarrier are used to enhance the affinity between the hydrophilic protein components of the insect epidermis. Force enables the composite drug solution to penetrate the insect's body wall and enter its body. .

2. The method according to claim 1, characterized in that, The nanocarrier and the dsRNA are mixed in a charge ratio of 1:1 to obtain a mixture, and the surfactant is mixed with the mixture at a volume ratio of 1%.

3. The method according to claim 1 or 2, characterized in that, Step 2) can be specifically applied to the insect's body surface by dripping or spraying.