Beta-tubulin protein-related dsRNA (double-stranded ribonucleic acid) and application thereof in pest killing

Abstract

The invention discloses dsRNA (double-stranded ribonucleic acid) related to beta-tubulin protein and application of the dsRNA in insect killing, and belongs to the technical field of biology. The dsRNA is a double-stranded RNA, and the dsRNA targets mRNA of a coding gene of a protein with an amino acid sequence of SEQ ID NO: 7. Experiments prove that the dsRNA provided by the invention has high efficiency and specificity, and can effectively kill corresponding mosquitoes from the dsRNA.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to dsRNA associated with β-tubulin protein and its application in insecticidal applications. Background Technology

[0002] Mosquitoes transmit pathogens of many human infectious diseases, leading to a high incidence of vector-borne diseases globally. Due to their widespread distribution in tropical and subtropical regions, these areas suffer significant morbidity and mortality rates. Many serious infectious diseases, such as dengue fever, yellow fever, chikungunya, West Nile virus, and malaria, are transmitted by mosquitoes. Humans are infected with vector-borne diseases primarily through three routes: direct mosquito bites, transmission from mosquitoes to humans through the bite of domestic animals, and the transmission of viruses through a human-mosquito-human cycle. Aedes aegypti is a major vector for vector-borne diseases, preferring to feed on human blood and primarily transmitting infectious diseases such as Zika virus, dengue virus, and yellow fever. They are mainly distributed in tropical and subtropical regions and are continuously expanding their distribution to other areas. With the continued growth of the global population and human-induced global climate change, temperatures are rising, and the spread of many vector-borne diseases has expanded, posing an increasing threat to public health. Aedes mosquitoes are widely active, both indoors and outdoors, biting both day and night. With the widespread use of chemical pesticides, many mosquitoes, which transmit viruses, have developed pesticide resistance. Currently, mosquito control still relies primarily on chemical methods. The long-term, large-scale, and widespread use of chemical pesticides has led to increasingly severe mosquito resistance, increasing the difficulty of control and necessitating the development of mosquito control agents that target new pathogens and are safe for higher animals. The development of RNA drugs can not only open up new areas of mosquito control but is also relatively safe for higher animals. To achieve sustainable development, some international organizations have proposed measures to limit the use of chemical pesticides. Mosquitoes have frequent contact with humans in daily life, relying on blood-sucking behavior to reproduce, and various viruses can also enter the bloodstream for transmission. Therefore, research on new mosquito control technologies is of great value. Currently, controlling the spread of vector-borne diseases mainly targets the larval and egg stages of mosquitoes. With the continuous development of science and technology, scientists have investigated the distribution of mosquito species in different regions, providing important reference data for vector-borne disease control. The research results have important theoretical basis and practical value for developing new mosquito control agents that specifically target mosquito genes.

[0003] With the rapid development of biotechnology, researchers both domestically and internationally have begun to focus on the application of engineered bacteria in mosquito RNAi control. A series of highly specific and targeted mosquito-resistant dsRNA molecules have been developed for design and preparation. Compared to traditional chemical insecticides, mosquito RNAi technology is environmentally friendly and highly specific (no resistance, no residue, and targets only mosquitoes without affecting other insects). Regarding engineered bacteria production and expression systems, researchers have successfully constructed various highly efficient genetically engineered bacterial systems for producing dsRNA molecules with mosquito-resistant activity. In terms of efficacy evaluation within mosquitoes, various detection methods have been established to assess the mosquito-resistant activity of engineered bacteria synthesis and nanodelivery systems. In recent years, the habitats of mosquito larvae, namely various outdoor water storage containers, have received increasing attention in research. Compared to adult mosquitoes, mosquito larvae exhibit aggregation and limited mobility, making them the easiest stage of development to capture. Internationally, efforts have begun to develop yeast-based larvicides to control mosquito larvae in outdoor water containers. Research focuses on mosquito odor receptors, reproductive capacity, and developmental stages. Furthermore, scientists are exploring the use of bioengineering techniques, such as gene editing, to modify mosquito larvae's genes, preventing them from developing into adult mosquitoes and thus controlling mosquito populations. In practical applications, some regions have begun to experiment with these novel control methods. For example, in some tropical areas, where mosquito-borne diseases such as dengue fever and malaria pose a serious threat to local residents' health, local governments are collaborating with research institutions to apply these new larvicides to outdoor water containers. The effectiveness of these methods is assessed by regularly monitoring the number and types of mosquito larvae, and the application strategy is adjusted according to the actual situation. Besides direct chemical and genetic control methods, environmental management measures have also proven to be effective in controlling mosquito populations. For example, regularly cleaning and maintaining water containers to prevent water accumulation can significantly reduce mosquito larval habitat. In addition, introducing natural enemies such as fish and certain microorganisms can also inhibit mosquito reproduction to some extent. The implementation of these comprehensive measures will not only help reduce the threat of mosquitoes to human health, but also help protect the balance of the ecological environment. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide new applications for β-tubulin protein and related biomaterials. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention also claims protection for dsRNA, said dsRNA targeting the mRNA of a gene encoding a protein, said protein being any of the following: A1) a protein whose amino acid sequence is SEQ ID NO:7; A2) a protein having more than 80% identity and the same function as the protein shown, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1); A3) a fusion protein having the same function, obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

[0006] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0007] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0008] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0009] The above-mentioned dsRNA is a double-stranded RNA consisting of one strand and its reverse complementary sequence, wherein the nucleotide sequence of the one strand is SEQ ID NO:5 or has 40% or more identity with SEQ ID NO:5.

[0010] The nucleotide sequence of one strand of the dsRNA described above is SEQ ID NO:4 or SEQ ID NO:6.

[0011] This invention also claims protection for biological materials, said biological materials being any of the following: G1) The gene encoding the aforementioned RNA molecule; G2) An expression cassette containing the gene described in G1); G3) A recombinant vector containing the gene described in G1) or a recombinant vector containing the expression cassette described in G2); G4) A recombinant microorganism containing the gene described in G1) or a recombinant microorganism containing the expression cassette described in G2) or a recombinant microorganism containing the recombinant vector described in G3).

[0012] The expression cassette containing nucleic acid molecules, as described in G2, refers to DNA capable of expressing the aforementioned coding genes in host cells. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules containing all regulatory sequences necessary for expressing the DNA of any of the aforementioned proteins or RNA molecules. These regulatory sequences, under compatible conditions, guide the expression of the coding sequence in a suitable host cell for the DNA of any of the aforementioned proteins or RNA molecules. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule, adapter-equipped regulatory sequences may be provided. The regulatory sequences may be suitable promoter sequences, i.e., nucleic acid sequences recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences mediating the DNA expression of the protein or the RNA molecule. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively linked to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively linked to the 5' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a protein, capable of guiding the DNA encoding the protein or the RNA molecule into the cellular secretion pathway. Signal peptide coding regions that can guide the expressed protein or the DNA of the RNA molecule into the secretion pathway of the host cell used can be used in this invention. Adding regulatory sequences that can modulate the expression of proteins or RNA molecules according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are systems that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that enable gene amplification.

[0013] The vector may be a plasmid, granule, bacteriophage, or viral vector. The microorganism may be yeast, bacteria, algae, or fungi.

[0014] The present invention also claims protection for dsRNA compositions, said compositions being any of the following: D1) the composition comprises two dsRNAs, wherein the nucleotide sequence of one strand of one dsRNA is SEQ ID NO:4, and the nucleotide sequence of one strand of the other dsRNA is SEQ ID NO:5; D2) the composition comprises two dsRNAs, wherein the nucleotide sequence of one strand of one dsRNA is SEQ ID NO:4, and the nucleotide sequence of one strand of the other dsRNA is SEQ ID NO:6; D3) the composition comprises two dsRNAs, wherein the nucleotide sequence of one strand of one dsRNA is SEQ ID NO:5, and the nucleotide sequence of one strand of the other dsRNA is SEQ ID NO:6; D4) the composition comprises three dsRNAs, wherein the nucleotide sequence of one strand of one dsRNA is SEQ ID NO:4, the nucleotide sequence of one strand of the other dsRNA is SEQ ID NO:5, and the nucleotide sequence of one strand of the other dsRNA is SEQ ID NO:6.

[0015] The present invention also claims protection for insecticides for killing mosquitoes, said insecticide comprising the aforementioned dsRNA or containing the aforementioned biological material or a composition containing the aforementioned dsRNA.

[0016] The insecticide is applied by soaking.

[0017] The mosquitoes mentioned include Aedes aegypti, Culex quinquefasciatus, and Aedes albopictus.

[0018] This invention claims protection for the use of a protein or a substance that inhibits, reduces, or downregulates the expression of the gene encoding the protein or the content and / or activity of the protein in any of the following: C1) Increase the mortality rate of mosquitoes; C2) Prepare products that increase the mortality rate of mosquitoes; C3) Prepare insecticides that kill mosquitoes; The protein is any one of the following: A1) The amino acid sequence of this protein is SEQ ID NO:7; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

[0019] The mosquitoes mentioned include Aedes aegypti, Culex quinquefasciatus, and Aedes albopictus.

[0020] In the above applications, the substance is any of the following biomaterials: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the aforementioned protein, or RNA molecules that knock out the gene encoding the aforementioned protein; B2) The gene encoding the RNA molecule described in B1); B3) An expression cassette containing the gene described in B2); B4) A recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) A recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

[0021] In the above applications, the RNA molecule is dsRNA.

[0022] In the above applications, the dsRNA is a double-stranded RNA consisting of one strand and its reverse complementary sequence, wherein the nucleotide sequence of the one strand is SEQ ID NO:5 or has 40% or more identity with SEQ ID NO:5.

[0023] The advantage of this invention lies in the fact that, based on the characteristics and specificity of the dsRNA sequence, the synthesis patterns of numerous engineered bacteria representing other key genes affecting life activities in mosquitoes can be replicated. The dsRNA provided by this invention is highly efficient and specific, effectively killing the corresponding mosquitoes from which the dsRNA originates. Attached Figure Description

[0024] Figure 1 This is an electrophoresis diagram of the target gene synthesis. 1 and 2 represent Aedes aegypti; 3 and 4 represent Aedes albopictus; and 5 and 6 represent Culex quinquefolius. M1 is a 2000m marker, and M2 is a 5000mArker.

[0025] Figure 2 A schematic diagram of the construction of the dsRNA β-tub L4440 vector and its gel electrophoresis image.

[0026] Figure 3 The study investigated the use of biosynthesized dsRNA to interfere with the survival rate of first-instar larvae induced by Aedes aegypti. At a specific optimal concentration, the 7-day mortality rate of mosquito larvae reached over 90%.

[0027] Figure 4 To analyze the relative gene expression levels of first-instar larvae of Aedes aegypti mosquitoes by using biosynthesized dsRNA. a. After 24 hours of soaking, the results showed that at a concentration of 500 ng / µl, there was no statistically significant difference between the eGFP and water groups (n=15), but a statistically significant difference between the eGFP and β-tub groups (**). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*). P<0.05)(n=15). At a concentration of 750 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15). At a concentration of 1000 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (**). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15). b. After 48 hours of soaking, the results showed that at a concentration of 500 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15). At a concentration of 750 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (**). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*). P <0.05)(n=15). At a concentration of 1000 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (**). P <0.05)(n=15). c. After 72 hours of soaking, the results showed that at a concentration of 500 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (**). P <0.05)(n=15). At a concentration of 750 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but a statistically significant difference between the eGFP and β-tub groups (***). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (***P<0.05)(n=15). At a concentration of 1000 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (***P<0.05)(n=15).P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15).

[0028] Figure 5 The study investigated the use of biosynthesized dsRNA to interfere with the survival rate of first-instar larvae induced by Aedes albopictus. At a specific optimal concentration, the 7-day mortality rate of mosquito larvae in the β-tub group exceeded 90%.

[0029] Figure 6 To analyze the relative gene expression levels of first-instar larvae of Aedes albopictus mosquitoes by using biosynthesized dsRNA. a. After 24 hours of soaking, the results showed that at a concentration of 500 ng / µl, there was no statistically significant difference between the eGFP and water groups (n=15), but a statistically significant difference between the eGFP and β-tub groups (****). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (**). P <0.05)(n=15). At a concentration of 750 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (**). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (**). P <0.05)(n=15). At a concentration of 1000 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (***). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (**). P <0.05)(n=15). b. After 48 hours of soaking, the results showed that at a concentration of 500 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*). P <0.05)(n=15). At a concentration of 750 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but a statistically significant difference between the eGFP and β-tub groups (***). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15). At a concentration of 1000 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P<0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (**). P <0.05)(n=15). c. After 72 hours of soaking, the results showed that at a concentration of 500 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (****). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (**** P <0.05)(n=15). At a concentration of 750 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (****). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15). At a concentration of 1000 ng / ul, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15).

[0030] Figure 7 The study investigated the use of biosynthesized dsRNA to interfere with the survival rate of first-instar larvae induced by Aedes aegypti. At a specific optimal concentration, the 7-day mortality rate of mosquito larvae reached over 90%.

[0031] Figure 8 Analysis of relative gene expression levels in first-instar Culex pipiens quinquefolia larvae induced by biosynthetic dsRNA interference. A: 24 h after soaking, the results showed that at a concentration of 500 ng / μL, there was no statistically significant difference between the eGFP and water groups (n=15), but a statistically significant difference between the eGFP and β-tub groups (**). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*). P <0.05)(n=15). At a concentration of 750 ng / μL, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15). At a concentration of 1000 ng / μL, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (**). P<0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15). B: After 48 hours of soaking, the results showed that at a concentration of 500 ng / μL, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15). At a concentration of 750 ng / μL, there was no statistically significant difference between the eGFP and water groups (n=15), but a statistically significant difference between the eGFP and β-tub groups (**). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*). P <0.05)(n=15). At a concentration of 1000 ng / μL, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (**). P <0.05)(n=15). C: After 72 hours of soaking, the results showed that at a concentration of 500 ng / μL, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (*). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (**). P <0.05)(n=15). At a concentration of 750 ng / μL, there was no statistically significant difference between the eGFP and water groups (n=15), but a statistically significant difference between the eGFP and β-tub groups (***). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15). At a concentration of 1000 ng / μL, there was no statistically significant difference between the eGFP and water groups (n=15), but there was a statistically significant difference between the eGFP and β-tub groups (***). P <0.05)(n=15), there was a statistically significant difference between the water and β-tub groups (*** P <0.05)(n=15). Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0035] The following examples used GraphPad Prism 8 statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and one-way ANOVA was used. P <0.05 (*) indicates a significant difference.

[0036] Example 1: Construction of Recombinant Expression Vector 1. Target gene amplification (1) Primer design Searching for β-tubulin sequences (encoding the protein's amino acid sequence SEQ ID NO:7, totaling 447 amino acids) on NCBI included Aedes aegypti mosquitoes. Aedes aegypti (XM_001655975.2), Culex pipiens quinquefasciatus Culex quinquefasciatus (XM_038251688.1), Aedes albopictus Aedes albopictus (XM_019696866.4). Primers were designed using SnapGene and suitable restriction enzyme sites were added to both ends of the primers. The specific primers are as follows: The upstream and downstream primers for Aedes aegypti β-tubulin are: F1: 5'-acctcgagggGAAATCATCTCCG-3'; R1: 5'-ACGAGCTCGGCACGGTACTGTTG-3'.

[0037] F1 and R1 were used to amplify nucleotides 62-850 of Aedes aegypti β-tubulin (SEQ ID NO:1, total 2075bp, of which nucleotides 270-1613 are the CDS region).

[0038] Primers for inducing Culex quinquefolia β-tubulin: F2: 5'-ACCTCGAGGGACAACTTTGTGTTCGGACA-3'; R2: 5'-ACGAGCTCACAGGGCTTCATTGTCGAT-3'.

[0039] F2 and R2 were used to amplify nucleotides 527-888 of Culex pipiens quinquefolius β-tubulin (SEQ ID NO:2, total 2004bp, of which nucleotides 267-1610 are the CDS region).

[0040] Aedes albopictus β-tubulin upstream and downstream primer sequences: F3: 5'-ACCTCGAGTCGAAAACACCGACGAGACGT-3'; R3: 5'-ACGAGCTCGCAACTGTCAGGTAACGTCCATGT-3'; F3 and R3 were used to amplify nucleotides 578-941 of Aedes albopictus β-tubulin (SEQ ID NO:3, total 2139bp, of which nucleotides 321-1664 are the CDS region).

[0041] (2) Laboratory culture of mosquitoes Aedes aegypti and Aedes albopictus: described in the non-patent literature “Minghui Zhao, Xin Ran, DanXing, Yun Liao, Wei Liu, Yu Bai, Qiang Zhang, Kan Chen, Lan Liu, Mingyu Wu, Zu Ma, Jian Gao, Hengduan Zhang, Tongyan Zhao. Evolution of knockdownresistance (kdr) mutations of Aedes aegypti and Aedes albopictus in Hainan Island and Leizhou Peninsula, China. Front Cell Infect Microbiol. 2023 Sep21:13:1265873. doi: 10.3389 / fcimb.2023.1265873. eCollection 2023.”, which is available to the public from the Academy of Military Medical Sciences of the Chinese People's Liberation Army. This biological material is only for repeating the relevant experiments of this invention and shall not be used for other purposes.

[0042] Culex quinquefasciatus: described in the non-patent literature "Gao Heting, Gu Zhenyu, Xing Dan, Yang Qiaojiang, Li Jianhang, Zhou Xinyu, Zhao Teng, Li Chunxiao. Identification of differentially expressed genes based on antennae RNA-seq analyses in Culex quinquefasciatus and Culex pipiens molestus. Parasit Vectors. 2022 Oct 1;15(1):353. doi: 10.1186 / s13071-022-05482-6.", is available to the public from the Academy of Military Medical Sciences of the Chinese People's Liberation Army. This biological material is only for repeating the relevant experiments of this invention and shall not be used for other purposes.

[0043] The author's affiliation in the two non-patent documents mentioned above, "State Key Laboratory of Pathogen and Biosecurity," belongs to the Academy of Military Medical Sciences of the Chinese People's Liberation Army.

[0044] Aedes aegypti, Aedes albopictus, and Culex quinquefolius were identified by the Academy of Military Medical Sciences and then reared in a vector breeding room for a long period. The environment was maintained at 26℃±1°C, relative humidity of approximately 75%±5%, with a 14-hour dark / 10-hour light cycle, and fed with 8% sugar water.

[0045] (3) Mosquito RNA extraction Trizol method: Place 1-2 mosquitoes and 5-6 magnetic beads in each 1.5 mL EP tube; add 1 mL of RNAiso (TAKARA) to the tube, homogenize at 60 Hz for 300 s, remove and let stand at room temperature for 5 min to allow complete lysis. Transfer the liquid to a new 1.5 mL EP tube, add 0.2 mL of chloroform to each tube, shake to mix, let stand at room temperature for 3 min, 12000 g, 4℃, 15 min; transfer the supernatant to a new 1.5 mL EP tube, add an equal volume of pre-frozen isopropanol solution, shake vigorously to allow the reaction to complete, let stand for 10 min, 12000 g, 4℃, 10 min; at this point, RNA forms a white precipitate at the bottom of the tube; if the amount is small, there will be no white precipitate. Aspirate the supernatant, add 1 mL of 75% ethanol, shake to suspend and wash the RNA, incubate at 13000g for 5 min, remove and discard the supernatant, place a piece of kitchen paper on the tube, invert the EP tube to allow the ethanol to drain completely, and add 30 μL of RNA-free water to dissolve the RNA. Analyze the concentration of the extracted RNA using a Nanodrop instrument.

[0046] (4) Mosquito cDNA synthesis Use the TAKARA PrimeScript kit. TM II 1 st To prevent reagent degradation or premature reaction, the entire process of thawing reagents and performing operations is conducted on an ice pack using the Strand cDNA Synthesis Kit. Perform the reverse transcription reaction according to the instructions.

[0047] (5) Amplification of the target fragment Using TAKARA's Premix Taq TM The designed upstream and downstream primers and cDNA were added. A 25 μL reaction system was prepared with RNA-free water. The PCR reaction program was as follows: pre-denaturation at 98℃ for 5 min, denaturation at 98℃ for 30 s, annealing (temperature varies depending on the primers) for 40 s, extension at 72℃ for 1 min, and final extension at 72℃ for 10 min, for 30 cycles. The PCR products were subjected to agarose gel electrophoresis. The correctly banded PCR products were excised under UV light, weighed, and transferred to a new 1.5 mL EP tube. The gel was extracted using the TATAKAMiniBEST Agarose Gel DNA Extraction Kit Ver4.0. The DNA concentration of the collected solution was determined using a nanometer.

[0048] 2. Carrier Construction (1) Ligation of the target fragment with the cloning vector Using the Vazyme 5 min TA / Blunt-Zero Cloning Kit, the amplified fragments of Aedes aegypti, Aedes albopictus, and Culex quinquefolius were ligated with the plasmids in the aforementioned kit. The reaction mixture was prepared according to the instructions, the bottom of the tube was gently tapped to mix, and all liquid was collected at the bottom of the tube by slow, brief centrifugation. The tubes were incubated at 37°C for 20 min in a PCR instrument. After the reaction was complete, the tubes were transferred to ice to obtain the ligation products.

[0049] (2) Construction of T1 vector Take 50 μL of competent cells thawed on ice, add the ligation product from "(1) Ligation of the target fragment and cloning vector", mix gently, and operate according to the instructions on ice. Add 500 μL of sterile SOC or LB medium (without antibiotics) to a centrifuge tube, mix well, and incubate at 37°C and 200 rpm for 1 h to allow the bacteria to recover. Take 200 μL of the recovered bacterial solution and spread it on LB solid medium containing antibiotics, and incubate overnight at 37°C. Select single colonies and add them to 10 μL of ddH2O. Prepare the solution and set the PCR reaction program according to the instructions of the selected enzyme. Perform electrophoresis on the PCR products. Send the correctly detected bacterial solution to the company for sequencing to further confirm the correct insertion of the target fragment.

[0050] 3. Construction and transformation of L4440 expression vector (1) Plasmid extraction Using the Novizan plasmid extraction kit, take 1-5 mL of bacterial culture that has been correctly sequenced in “(2) Construction of T1 vector” and perform the experimental operation according to the instructions. To avoid contamination, the operation should be carried out in a biosafety cabinet. The collected plasmid DNA can be used for subsequent experiments or stored at -20℃.

[0051] (2) Linearization of L4440 vector and double enzyme digestion of target gene L4440 vector sequence:

[0052] The plasmid extracted in "(1) Plasmid Extraction" and the L4440 vector were double-digested using the restriction enzymes selected in the primer design. The restriction enzyme system was added according to the instructions, and the cells were placed in a PCR instrument at 37°C for 1 hour. The digested fragments were then subjected to agarose gel electrophoresis. The PCR products with the correct bands were excised under UV light, weighed, and placed in a new 1.5 mL EP tube. The gel was recovered using the TATAKA MiniBEST Agarose Gel DNA Extraction Kit Ver4.0. The DNA concentration of the collected solution was detected using NANO. The following fragments were obtained: L4440 vector digestion fragment, Aedes aegypti amplified fragment digestion product, Aedes albopictus amplified fragment digestion product, and Culex quinquefasciatus amplified fragment digestion product.

[0053] (3) The target fragment is ligated to the L4440 vector. HT115 (defective) competent cells: Beijing Zhuangmeng International Biotechnology Co., Ltd., catalog number ZK269.

[0054] The digested fragments of Aedes aegypti, Aedes albopictus, and Culex quinquefolius were ligated with L4440 vector digested fragments using NEB's T4 DNA ligase. The T4 DNA ligase ligation system was prepared according to the product instructions. The ligation products were incubated overnight at 25°C in a PCR instrument to obtain the ligated products.

[0055] Take 50 μL of thawed HT115 (deficient) competent cells from an ice bath, add the ligation product from the previous step, mix gently, and follow the manufacturer's instructions while still on ice. Add 500 μL of sterile SOC or LB medium (antibiotic-free) to a centrifuge tube, mix well, and incubate at 37°C and 200 rpm for 1 h to allow the bacteria to recover. Take 200 μL of the recovered bacterial culture and spread it on LB solid medium containing antibiotics, incubate overnight at 37°C. Select single colonies and add them to 10 μL of ddH2O. Prepare the solution and set the PCR reaction program according to the instructions of the selected enzyme. Perform electrophoresis on the PCR products. Perform double digestion of the correctly detected plasmid with the corresponding restriction enzyme for verification. Send the correctly sequenced bacterial culture to the sequencing company for further confirmation of the correct insertion of the target fragment. Transfer the correctly sequenced bacterial culture to 5 mL of liquid medium containing ampicillin, incubate overnight at 37°C with shaking at 240 rpm, and store the bacterial culture at -80°C.

[0056] PCR product electrophoresis results are as follows Figure 1As shown in the image. Lanes 1 and 2 show the electrophoretic bands of the Aedes aegypti β-tubulin PCR amplification fragment, which match the length of the Aedes aegypti β-tubulin fragment, indicating successful construction of the Aedes aegypti β-tubulin L4440 recombinant expression vector. Lanes 3 and 4 show the electrophoretic bands of the Culex quinquefolius PCR amplification fragment, which match the length of the Culex quinquefolius β-tubulin fragment, indicating successful construction of the Culex quinquefolius β-tubulin L4440 recombinant expression vector. Lanes 5 and 6 show the electrophoretic bands of the Aedes albopictus β-tubulin PCR amplification fragment, which match the length of the Aedes albopictus β-tubulin fragment, indicating successful construction of the Aedes albopictus β-tubulin L4440 recombinant expression vector.

[0057] The successfully constructed recombinant expression vectors are as follows: The recombinant expression vector L4440-A-β-tub is obtained by replacing the small fragment between nucleotides 2024 and 2817 of the L4440 vector with nucleotides 331-1118 of SEQ ID NO:1 while keeping the other nucleotide sequences of the L4440 vector unchanged. The expressed nucleotide sequence is the dsRNA of sequence 4 (SEQ ID NO:4) in the sequence listing (U is replaced by T in the sequence listing to meet the nucleotide sequence listing standards).

[0058] The recombinant expression vector L4440-B-β-tub is obtained by replacing the small fragment between nucleotides 2024 and 2817 of the L4440 vector with nucleotides 527-888 of SEQ ID NO:2 while keeping the other nucleotide sequences of the L4440 vector unchanged. The expressed nucleotide sequence is the dsRNA of sequence 5 (SEQ ID NO:5) in the sequence listing (U is replaced by T in the sequence listing to meet the nucleotide sequence listing standards).

[0059] The recombinant expression vector L4440-C-β-tub is obtained by replacing the small fragment between nucleotides 2024 and 2817 of the L4440 vector with nucleotides 578-941 of SEQ ID NO:3 while keeping the other nucleotide sequences of the L4440 vector unchanged. The expressed nucleotide sequence is the dsRNA of sequence 6 (SEQ ID NO:6) in the sequence listing (U is replaced by T in the sequence listing to meet the nucleotide sequence listing standards).

[0060] Example 2: dsRNA-induced expression The frozen bacterial cultures containing recombinant expression vectors L4440-A-β-tub, L4440-B-β-tub, and L4440-C-β-tub were thawed at room temperature and inoculated into fresh LB broth containing antibiotics at a 1:100 volume ratio. The cultures were then incubated at 37°C and 240 rpm for 3-4 hours until the OD value was reached. 600nm When the pH value was 0.4-0.6, IPTG was added, with a final IPTG concentration of 1 mmol / L. The culture was continued with shaking for 4-5 h to induce dsRNA synthesis. The bacterial culture was then collected in a 50 mL centrifuge tube, centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was carefully aspirated. 1 mL of Trizol reagent was added to the tube, and the precipitate was dissolved by vortexing before being transferred to a new 1.5 mL EP tube. The experimental procedure was performed according to the mosquito RNA Trizol extraction method. The concentration of the extracted RNA was determined using a nanometer.

[0061] The HT115 strain could not produce enzymes that degrade dsRNA, so DNase I and RNase A were used to degrade the extracted RNA. The gene fragments from *Aedes aegypti* were approximately 788 bp in length, those from *Aedes albopictus* were approximately 364 bp, and those from *Culex quinquefasciatus* were approximately 362 bp. The extracted RNA and the degraded RNA were detected by 2% agarose gel electrophoresis.

[0062] The results are as follows Figure 2As shown, the M1 Maker2000 bands, from top to bottom, are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp; the M2 Maker5000 bands are 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. Lane 1 contains uninduced RNA from the *Aedes aegypti* expression strain; lane 2 contains the product of uninduced RNA from the *Aedes aegypti* expression strain after digestion with DNase I and RNase A; lane 3 contains induced RNA from the *Aedes aegypti* expression strain; lane 4 contains the product of induced RNA from the *Aedes aegypti* expression strain after digestion with DNase I and RNase A; lane 5 contains uninduced RNA from the *Aedes albopictus* expression strain; and lane 6 contains the product of uninduced RNA from the *Aedes albopictus* expression strain after digestion with DNase I and RNase A. Lane 7 contains the RNA from the Aedes albopictus expression strain after induction, and lane 8 contains the product of the inducible RNA from the Aedes albopictus expression strain after digestion with DNase I and RNase A. As can be seen, bright target bands appeared at approximately 750 bp and 360 bp after digestion with DNase I and RNase A, respectively, indicating successful dsβ-tub induction and the production of large quantities of dsRNA after IPTG induction.

[0063] The aforementioned "uninduced RNA" refers to electrophoresis performed directly on RNA extracted from the bacterial culture without the addition of IPTG for induction during bacterial culture. The aforementioned "products of uninduced RNA digested with DNase I and RNase A" refers to electrophoresis performed on the sample after digestion of RNA extracted from the bacterial culture with DNase I and RNase A without the addition of IPTG for induction during bacterial culture. The aforementioned "induced RNA" refers to electrophoresis performed directly on RNA extracted from the bacterial culture after the addition of IPTG for induction during bacterial culture. The aforementioned "products of induced RNA digested with DNase I and RNase A" refers to electrophoresis performed on the sample after digestion of RNA extracted from the bacterial culture with DNase I and RNase A after the addition of IPTG for induction during bacterial culture.

[0064] The aforementioned degraded dsRNA solution targeting β-tubulin in Aedes aegypti is named A-β-tub dsRNA solution, the dsRNA solution targeting β-tubulin in Aedes albopictus is named B-β-tub dsRNA solution, and the dsRNA solution targeting β-tubulin in Culex quinquefolius is named C-β-tub dsRNA solution.

[0065] Example 3: Effects of different concentrations of dsRNA on mosquito survival A-β-tub 1000 ng / μL group: The A-β-tub dsRNA solution extracted in "Example 2, dsRNA-induced expression" was diluted to 1000 ng / μL. The diluted dsRNA solution was added to each well of a 12-well cell culture plate, with 1 mL added to each well. Three replicate wells were set up. 100 first-instar larvae (<24 hours) of Aedes aegypti (or Aedes albopictus, or Culex quinquefasciatus) were added to each well for immersion. Larval survival was observed at 24, 48, and 72 hours. After the observation period, larval growth cycle, pupation, and emergence were continuously observed. Larval mortality rate was calculated as: Larval mortality rate = (Number of dead larvae / Total number of larvae) * 100%. Simultaneously, surviving larvae were collected at 24h, 48h, and 72h, one larva per tube, and RNA was extracted. The gene expression level of β-tubulin in the larvae was detected using a full-gold real-time quantitative PCR kit. The detection method was as follows: Full-gold one-step qRT-PCR kit was used. Reaction components: SuperMix, 10μL; EnzymeMix, 0.4μL; forward primer (10μM), 0.4μL; reverse primer (10μM), 0.4μL; Passive Reference Dye (50×), 0.4μL; RNA sample, 2μL; water added to a final volume of 20μL. Reaction program: 45℃, 5min; 94℃, 30s; 94℃, 5s, 60℃, 15s, 72℃, 10s, 40 cycles; Dissociation Stage. The relative expression level of the positive control group eGFP was used as a control. -△△CT The relative expression levels of the target gene were calculated. Three replicates were set up for each mosquito species in this group.

[0066] Aedes aegypti β-tubulin qRT-PCR upstream and downstream primer sequences: Upstream: 5'-ATGAGAGAAATCGTCCACATCCAAG-3'; Downstream: 5'-GAATATCACAGACGGCGGTC-3'; Primer sequences for Culex quinquefolia β-tubulin qRT-PCR: Upstream: 5'-AGCGCATCAACGTGTACTAC-3'; Downstream: 5'-GCATGAAGAAGTGCAGACGT-3'; Primer sequences for Aedes albopictus β-tubulin qRT-PCR: Upstream: 5'-ATGGGCACACTGTTGATCTCC-3'; Downstream: 5'-GGAGATACGCTTGAACAGTTCCT-3'.

[0067] A-β-tub-750 ng / μL group: The difference between this group and the A-β-tub-1000 ng / μL group is that the A-β-tub dsRNA solution is diluted to 750 ng / μL, and the rest of the operation is the same as the A-β-tub-1000 ng / μL group.

[0068] A-β-tub-500 ng / μL group: The difference between this group and the A-β-tub-1000 ng / μL group is that the A-β-tub dsRNA solution is diluted to 500 ng / μL, and the rest of the operation is the same as the A-β-tub-1000 ng / μL group.

[0069] B-β-tub-1000 ng / μL group: The difference between this group and the A-β-tub-1000 ng / μL group is that the B-β-tub dsRNA solution is used instead of the A-β-tub dsRNA solution, and the rest of the operation is the same as the A-β-tub-1000 ng / μL group.

[0070] B-β-tub-750 ng / μL group: The difference between this group and the B-β-tub-1000 ng / μL group is that the B-β-tub dsRNA solution is diluted to 750 ng / μL, and the rest of the operation is the same as the B-β-tub-1000 ng / μL group.

[0071] B-β-tub-500 ng / μL group: The difference between this group and the B-β-tub-1000 ng / μL group is that the B-β-tub dsRNA solution is diluted to 500 ng / μL, and the rest of the operation is the same as the B-β-tub-1000 ng / μL group.

[0072] C-β-tub-1000 ng / μL group: The difference between this group and the A-β-tub-1000 ng / μL group is that the C-β-tub dsRNA solution is used instead of the A-β-tub dsRNA solution, and the rest of the operation is the same as the A-β-tub-1000 ng / μL group.

[0073] C-β-tub-750 ng / μL group: The difference between this group and the C-β-tub-1000 ng / μL group is that the C-β-tub dsRNA solution is diluted to 750 ng / μL, and the rest of the operation is the same as the C-β-tub-1000 ng / μL group.

[0074] C-β-tub-500 ng / μL group: The difference between this group and the C-β-tub-1000 ng / μL group is that the C-β-tub dsRNA solution is diluted to 500 ng / μL, and the rest of the operation is the same as the C-β-tub-1000 ng / μL group.

[0075] eGFP-1000 ng / μL group: The difference between this group and the A-β-tub-1000 ng / μL group is that 1000 ng / mL eGFP dsRNA solution (Shanghai Bolang Biotechnology Co., Ltd., DS001-01) is used instead of 1000 ng / mL A-β-tub dsRNA solution. The rest of the operation is the same as the A-β-tub-1000 ng / μL group.

[0076] The sequence of eGFP dsRNA is as follows:

[0077] Target the 300-500 bp region of the gene encoding the eGFP protein. Preparation method for 1000 ng / mL eGFP dsRNA solution: Dilute 1000 ng of powder with 1 ml of double-distilled water.

[0078] eGFP-750 ng / μL group: The difference between this group and the eGFP-1000 ng / μL group is that the 750 ng / mL eGFP dsRNA solution is used instead of the 1000 ng / mL eGFP dsRNA solution. The eGFP dsRNA content in the 750 ng / mL eGFP dsRNA solution is 750 ng / mL. The rest of the operation is the same as the eGFP-1000 ng / μL group.

[0079] eGFP-500 ng / μL group: The difference between this group and the eGFP-1000 ng / μL group is that the 500 ng / mL eGFP dsRNA solution is used instead of the 1000 ng / mL eGFP dsRNA solution. The eGFP dsRNA content in the 500 ng / mL eGFP dsRNA solution is 500 ng / mL. The rest of the operation is the same as the eGFP-1000 ng / μL group.

[0080] Water1, Water2, or Water3: This group differs from the A-β-tub-1000 ng / μL group in that it uses sterile, enzyme-free water at a concentration of 1000 ng / mL for A-β-tub dsRNA solution. All other procedures are the same as in the A-β-tub-1000 ng / μL group.

[0081] The survival curve of Aedes aegypti ( Figure 3 ), Aedes albopictus survival curve ( Figure 5 ), survival curve of Culex pipiens quinquefolius ( Figure 7It was found that soaking larvae in dsRNA solutions targeting β-tubulin in *Aedes aegypti*, *Aedes albopictus*, and *Culex quinquefolius* significantly reduced larval survival rates, with mortality rates exceeding 80%. From day one to day ten, the survival rate of *Aedes aegypti* at a concentration of 1000 ng / μL decreased from 100% to 12%; at 750 ng / μL, it decreased from 100% to 15%; and at 500 ng / μL, it decreased from 100% to 23%. The survival rates of *Culex quinquefolius* at 1000 ng / μL decreased from 100% to 23%; at 750 ng / μL, it decreased from 100% to 18%; and at 500 ng / μL, it decreased from 100% to 28%. The survival rates of *Culex quinquefolius* at 1000 ng / μL, it decreased from 100% to 23%; at 750 ng / μL, it decreased from 100% to 18%; and at 500 ng / μL, it decreased from 100% to 28%. The survival rates of *Culex quinquefolius* at 1000 ng / μL, it decreased from 100% to 23%; and at 500 ng / μL, it decreased from 100% to 28%. The survival rates of *Culex quinquefolius* at 100% to 28% remained above 93%. The survival rate of Aedes albopictus mosquitoes decreased from 100% to 15% at a concentration of 1000 ng / μL; from 100% to 13% at 750 ng / μL; and from 100% to 17% at 500 ng / μL. The survival rate remained above 93% at all concentrations in both the eGFP and control groups. The 7-day mortality rate of mosquito larvae in the β-tub group at specific concentrations reached over 90%. This indicates that immersion allows dsRNA to penetrate the body wall and enter the mosquito larvae.

[0082] Quantitative fluorescence results of Aedes aegypti mosquitoes ( Figure 4 ), quantitative fluorescence results of Aedes albopictus ( Figure 6 ), quantitative fluorescence results of Culex pipiens broodstock ( Figure 8 It was found that soaking larvae in dsRNA solutions targeting β-tubulin in Aedes aegypti, Aedes albopictus, and Culex quinquefolius significantly reduced the expression level of β-tubulin in the larvae.

[0083] Because RNAi is highly specific, for example, the dsRNA sequence of SEQ ID NO:4 targeting Aedes aegypti can only kill Aedes aegypti and has no effect on other mosquitoes. Therefore, this application does not present data on the killing effect of the same dsRNA on other mosquitoes.

[0084] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. dsRNA, characterized by, The dsRNA targets the mRNA of the gene encoding the protein, wherein the protein is any one of the following: A1) The amino acid sequence of this protein is SEQ ID NO:7; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

2. The dsRNA according to claim 1, characterized in that, It is a double-stranded RNA consisting of a single strand and its inverse complementary sequence, wherein the nucleotide sequence of the single strand is SEQ ID NO:5 or has 40% or more identity with SEQ ID NO:

5.

3. The dsRNA according to claim 1 or 2, characterized in that, The nucleotide sequence of one of the chains is SEQ ID NO:4 or SEQ ID NO:

6.

4. dsRNA composition, wherein the composition is any one of the following: D1) The composition comprises two dsRNAs, wherein the nucleotide sequence of one strand of one dsRNA is SEQ ID NO:4, and the nucleotide sequence of one strand of the other dsRNA is SEQ ID NO:5; D2) The composition comprises two dsRNAs, one of which has a nucleotide sequence of one strand of dsRNA as SEQ ID NO:4 and the other has a nucleotide sequence of one strand of dsRNA as SEQ ID NO:

6. D3) The composition comprises two dsRNAs, one of which has a nucleotide sequence of one strand of dsRNA as SEQ ID NO:5 and the other has a nucleotide sequence of one strand of dsRNA as SEQ ID NO:

6. D4) The composition comprises three dsRNAs, wherein the nucleotide sequence of one strand of one dsRNA is SEQ ID NO:4, the nucleotide sequence of one strand of another dsRNA is SEQ ID NO:5, and the nucleotide sequence of one strand of yet another dsRNA is SEQ ID NO:

6.

5. A biomaterial, characterized in that, The biomaterial is any one of the following: G1) Expressing the gene encoding the RNA molecule according to any one of claims 1-3; G2), an expression cassette containing the gene described in G1); G3), a recombinant vector containing the gene described in G1), or a recombinant vector containing the expression cassette described in G2; G4) recombinant microorganisms containing the gene described in G1), or recombinant microorganisms containing the expression cassette described in G2), or recombinant microorganisms containing the recombinant vector described in G3).

6. An insecticide for killing mosquitoes, characterized in that, The insecticide comprises the dsRNA according to any one of claims 1-3, or contains the biological material according to claim 5, or contains the dsRNA composition according to claim 4.

7. Application, characterized in that, The application includes the use of the protein of claim 1 or a substance that inhibits, reduces, or downregulates the expression of the gene encoding the protein or the content and / or activity of the protein in any of the following: C1) Increases mosquito mortality; C2) Prepare products that increase mosquito mortality rates; C3) Prepare insecticides to kill mosquitoes.

8. The application according to claim 7, characterized in that, The substance is any of the following biological materials: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein of claim 1, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the protein, or RNA molecules that knock out the gene encoding the protein of claim 7. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4).

9. The application according to claim 8, characterized in that, The RNA molecule is dsRNA.

10. The application according to claim 9, characterized in that, The dsRNA is a double-stranded RNA consisting of one strand and its reverse complementary sequence, wherein the nucleotide sequence of the one strand is SEQ ID NO:5 or has 40% or more identity with SEQ ID NO:5.

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