AGENT FOR ENHANCING RNAi EFFICIENCY IN LEPIDOPTERAN INSECTS, AND AGENT FOR CONTROLLING LEPIDOPTERAN INSECTS

Abstract

To provide an agent for enhancing RNAi efficiency in lepidopteran insects and an agent for controlling lepidopteran insects that make it possible to exert efficacy via transdermal application.SOLUTION: There is provided an agent for enhancing RNAi efficiency in lepidopteran insects for a target gene, the agent comprising a complex of a double-stranded RNA that suppresses expression of a predetermined gene, nanoparticles, and a surfactant, wherein the predetermined gene includes a target gene of the lepidopteran insects, at least two genes encoding a double-stranded RNA degrading enzyme, at least one gene encoding a nuclease having a PIN domain, and / or at least two genes encoding enzymes involved in chitin synthesis of skin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for improving RNAi efficiency in lepidopteran insects, and an agent for controlling lepidopteran insects. [Background technology]

[0002] Lepidoptera are important agricultural pests, accounting for approximately 70% of all insect pests, and in recent years, populations have developed resistance to chemical pesticides. The development of RNA pesticides, which are more specific to target species than chemical pesticides, is anticipated as a new pest control method that can simultaneously improve productivity and be environmentally safe. However, RNA interference (RNAi) is significantly less effective against Lepidoptera pests than against Coleoptera and Hemiptera pests, and methods other than oral administration, such as transdermal application of double-stranded RNA to the skin, have yet to be developed.

[0003] For herbivorous lepidopteran pests, attempts have been made to orally ingest double-stranded RNA by mixing it with food, but this is inhibited by strong RNA-degrading activity within the digestive tract or cells, making it difficult to achieve a sufficient lethal effect. As a means of avoiding degradation within the digestive tract, double-stranded RNA can be administered transdermally. Because sap-sucking pests will not ingest RNA simply by spraying it on the surface of crops, methods are currently being developed to apply dsRNA directly to the skin of pests using nanoparticles or surfactants, and success has been reported for some Hemiptera insects (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] ZhangYH, et al., “Nanocarrier-delivered dsRNA suppresseswing development of green peach aphids.” Insect Sci.2022 Jun;29(3):669-682. Epub2021 Oct 13. Summary of the Invention [Problem to be solved by the invention]

[0005] On the other hand, applying double-stranded RNA to the skin of lepidopteran pests using the method described in Non-Patent Document 1 does not produce an RNAi effect. Furthermore, 90% of crop damage caused by lepidopteran pests is caused by final-stage larvae, and therefore, killing the larvae before the final stage is important for pest control. However, while oral ingestion of RNAi has been reported to delay the development of lepidopteran larvae and inhibit pupation, its effectiveness in killing young larvae is low, making it an insufficient pest control method for reducing pest damage.

[0006] An object of the present invention is to provide a novel agent for improving RNAi efficiency against a target gene in lepidopteran insects, which is effective even when administered transdermally, and a novel agent for controlling lepidopteran insects. [Means for solving the problem]

[0007] The present inventors formed a complex of double-stranded RNA that suppresses the expression of specific genes, including genes involved in the development of Lepidoptera insects, with nanoparticles and a surfactant, and found that when the complex was applied dropwise to the skin of Spodoptera exigua (Japanese name: beet armyworm), the mortality rate of Spodoptera exigua was improved compared to when only double-stranded RNA that suppresses the expression of genes involved in the development of Lepidoptera insects was introduced. The present invention is based on this novel finding.

[0008] That is, the present invention relates to, for example, the following inventions. [1] An agent for improving RNAi efficiency against target genes in lepidopteran insects, The present invention relates to a method for producing a pharmaceutical composition comprising: a complex of double-stranded RNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant; The target gene, At least two genes encoding double-stranded RNA degrading enzymes, and At least one gene encoding a nuclease having a PIN domain, and / or The enhancer comprises at least two genes encoding enzymes involved in chitin synthesis in the skin. [2] The predetermined gene is The target gene, at least two genes encoding the double-stranded RNase, and at least one gene encoding the nuclease having a PIN domain, or The agent according to [1], comprising the target gene, at least two genes encoding the double-stranded RNA-degrading enzyme, at least one gene encoding the nuclease having the PIN domain, and at least two genes encoding enzymes involved in chitin synthesis in the skin. [3] The agent according to [1] or [2], wherein the at least one gene encoding the double-stranded RNase is of three or more types. [4] The improving agent according to any one of [1] to [3], wherein the double-stranded RNA degrading enzyme includes dsRNase. [5] The enhancer according to any one of [1] to [4], wherein the nuclease having a PIN domain includes a REase and / or a PIN domain and transmembrane domain-containing protein (PTDCP). [6] The improver according to any one of [1] to [5], wherein the enzymes involved in chitin synthesis in the skin include chitin synthase A and UDP-N-acetylglucosamine pyrophosphorylase. [7] The improver according to any one of [1] to [6], wherein the nanoparticles comprise carbon-based nanoparticles. [8] The improver according to any one of [1] to [7], wherein the nanoparticles comprise chitosan-based nanoparticles. [9] The improver according to any one of [1] to [8], wherein the surfactant comprises a cationic surfactant.

[10] In the improver according to any one of [1] to [9], The agent for controlling lepidopteran insects, wherein the target gene is at least one gene involved in the growth, survival, development and / or reproduction of lepidopteran insects.

[11] A method for improving RNAi efficiency against a target gene in a lepidopteran insect, The method comprises introducing into a lepidopteran insect a composition containing a complex of double-stranded RNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant, wherein the predetermined gene is The target gene, At least two genes encoding double-stranded RNA degrading enzymes, and At least one gene encoding a nuclease having a PIN domain, and / or A method for improving the efficiency of RNAi against the target genes, which are at least two genes encoding enzymes involved in chitin synthesis in the skin.

[12] A method for controlling lepidopteran insects in a plant, comprising: The method comprises introducing into a lepidopteran insect a composition containing a complex of double-stranded RNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant, wherein the predetermined gene is at least one gene involved in the growth, survival, development and / or reproduction of a lepidopteran insect; At least two genes encoding double-stranded RNA degrading enzymes, and At least one gene encoding a nuclease having a PIN domain, and / or A method for controlling lepidopteran insects in a plant, comprising at least two genes encoding enzymes involved in chitin synthesis in the skin. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a novel agent for improving RNAi efficiency in lepidopteran insects and a novel agent for controlling lepidopteran insects, which are effective even when administered transdermally. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a graph showing the results of evaluating the mortality rate of beet armyworm when a solution containing a complex of double-stranded RNA, nanoparticles, and a surfactant is applied. [Figure 2] 1 is a graph showing the results of evaluating the mortality rate of beet armyworm when a solution containing a complex of double-stranded RNA, nanoparticles, and a surfactant is applied. [Figure 3] 1 is a graph showing the results of evaluating the mortality rate of beet armyworm when a solution containing a complex of double-stranded RNA, nanoparticles, and a surfactant is applied. [Figure 4] 1 is a graph showing the results of evaluating the mortality rate of beet armyworm when a solution containing a complex of double-stranded RNA, nanoparticles, and a surfactant is applied. [Figure 5] 1 is a graph showing the results of evaluating the mortality rate of Spodoptera litura when a solution containing a complex of double-stranded RNA, nanoparticles, and a surfactant was applied. [Figure 6] 1 is a graph showing the results of evaluating the mortality rate of beet armyworm when a solution containing a complex of double-stranded RNA, nanoparticles, and a surfactant is applied. [Figure 7] 1 is a graph showing the results of evaluating the mortality rate of beet armyworm when a solution containing a complex of double-stranded RNA, nanoparticles, and a surfactant is applied. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0012] [RNAi efficiency enhancer] According to this embodiment, an agent for improving RNAi efficiency of a target gene in a lepidopteran insect (hereinafter simply referred to as "improver") contains a complex of double-stranded RNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant (hereinafter also referred to as "dsRNA-nanoparticle-surfactant complex"). Here, the predetermined gene includes the target gene, at least two genes encoding a double-stranded RNA-degrading enzyme, and at least one gene encoding a nuclease having a PIN domain, and / or at least two genes encoding enzymes involved in chitin synthesis in the skin.

[0013] The improver according to this embodiment is used for lepidopteran insects. In this specification, "lepidopteran insects" means insects that belong to the taxonomic order Lepidoptera (scientific name: Lepidoptera), and includes butterflies, moths, etc. Lepidopteran insects may be in the form of adults, pupae, or larvae, preferably larvae, and more preferably 1st to 2nd instar larvae.

[0014] Examples of Lepidoptera insects include, but are not limited to, insects belonging to the Tortricidae, Hesperiidae, Pieridae, Lycaenidae, or Noctuidae families, with cutworms (genus Spodoptera) being preferred.

[0015] More specific examples of lepidopteran insects include Hedya salicella (Tortricidae), Epinotia nisella (Tortricidae), Acleris literana (Tortricidae), Adoxophyes orana (Tortricidae; the smaller apple tortrix), Adoxophyes honmai (Tortricidae; the smaller tea tortrix), Archips xylosteana (Tortricidae; the common orchid tortrix), Pyrgus malvae (Hesperiidae), Carterocephalus palaemon (Hesperiidae), and Parnara guttata. guttata (Hesperiidae; Common Skipper), Ochlodes venatus (Hesperiidae; Common Skipper), Thymelicus lineola (Hesperiidae; Scarlet Skipper), Pieris brassicae (Pieridae), Leptidea sinapis (Pieridae), Colias croceus (Pieridae), Pieris melete (Pieridae; Striped White), Colias erate (Pieridae; Colias), Pieris rapae (Pieridae; Cabbage White), Lycaena flaeas phlaeas (Lycaenidae), Aricia agestis (Lycaenidae), Cyaniris semiargus (Lycaenidae), Lampides boeticus (Lycaenidae), Chilades pandava (Lycaenidae), Everes argiades (Lycaenidae), Mythimna ferrago (Noctuidae), Atethmia centrago (Noctuidae), Xestia c-nigrum (Noctuidae; Black cutworm), Mythimna separata (Noctuidae; Armyworm), Mythimna loreyi (Noctuidae; African armyworm), Sarcopolia illoba (Noctuidae; Large yellow underwing), Spodoptera litura (Noctuidae; Tobacco cutworm), Agrotis segetum (Noctuidae; Turnip moth), Agrotis ipsilon (Noctuidae; Black cutworm), Xanthodes transversa (Noctuidae; Square-spot dart), Eudocima tyrannus (Noctuidae; Akebia leafroller), Arcte coerula (Noctuidae; Yellow ermine), Acronicta rumicis (Noctuidae; Dark dagger), or Spodoptera exigua (Noctuidae; Beet armyworm), etc. can be mentioned.;

[0016] <dsRNA-nanoparticle-surfactant complex> The enhancer according to this embodiment contains a dsRNA-nanoparticle-surfactant complex, thereby improving the RNAi efficiency against the target gene in Lepidoptera insects. That is, when the enhancer according to this embodiment is administered (introduced) to the above-mentioned Lepidoptera insects, the effect of suppressing the expression of the target gene by double-stranded RNA that can suppress the expression of the target gene is improved as compared with the case where only the above-mentioned double-stranded RNA is administered (introduced) to the above-mentioned Lepidoptera insects.

[0017] The dsRNA-nanoparticle-surfactant complex can be prepared by known methods. For example, the complex can be prepared by mixing dsRNA and nanoparticles, centrifuging to remove the supernatant, redissolving the precipitate in water, adding a surfactant, and mixing thoroughly. A stirrer or homogenizer may be used to redissolve the precipitate and mix with the surfactant.

[0018] (double-stranded RNA that suppresses the expression of a specific gene) The predetermined genes include a gene of interest, at least two genes encoding double-stranded RNases, and at least one gene encoding a nuclease having a PIN domain, and / or at least two genes encoding enzymes involved in chitin synthesis in the skin.

[0019] As used herein, "double-stranded RNA that suppresses the expression of a specific gene" refers to double-stranded RNA that can specifically induce degradation of the transcription product (mRNA) of the specific gene via the RNAi mechanism. In the improving agent of this embodiment, double-stranded RNA that suppresses the expression of a specific gene includes double-stranded RNA that suppresses the expression of a target gene, double-stranded RNA that suppresses the expression of at least two genes encoding double-stranded RNA-degrading enzymes, and double-stranded RNA that suppresses the expression of at least one gene encoding a nuclease having a PIN domain, and / or double-stranded RNA that suppresses the expression of at least two genes encoding enzymes involved in chitin synthesis in the skin.

[0020] The length of the double-stranded RNA that suppresses the expression of a predetermined gene may be, for example, 20 or more bases, 30 or more bases, 50 or more bases, 70 or more bases, 100 or more bases, 150 or more bases, 180 or more bases, or 200 or more bases, or 1000 or less bases, 900 or less bases, 800 or less bases, 700 or less bases, or 650 or less bases, or may be 20 to 2000 bases, 30 to 2800 bases, 50 to 1500 bases, 100 to 1200 bases, 150 to 1000 bases, 200 to 1000 bases, 300 to 800 bases, 300 to 700 bases, 200 to 650 bases, or 300 to 650 bases. The double-stranded RNA that suppresses the expression of a predetermined gene may be double-stranded RNA consisting of a base sequence identical to the sequence of the gene whose expression is suppressed or a partial sequence thereof.

[0021] The target gene is a gene possessed by a lepidopteran insect, and is not particularly limited as long as it is any gene whose expression is to be suppressed as a target of RNAi. As used herein, "target gene" refers to a DNA sequence capable of expressing a target protein. The target gene may include, for example, a region (transcription region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell, an expression regulatory region located upstream and / or downstream of the transcription region, and an untranslated region (5'UTR, 3'UTR). The transcription region may be an open reading frame (ORF) containing not only exons but also introns, or may be a CDS. Note that "target gene" encompasses all genes targeted by RNAi, including the target gene.

[0022] The expression regulatory region may be, for example, a transcription regulatory region or a translation regulatory region. Examples of the transcription regulatory region include a promoter, an enhancer, and a silencer. Examples of the translation regulatory region include a ribosome binding region.

[0023] The gene of interest is not particularly limited, but may be, for example, at least one gene involved in the growth, survival, development and / or reproduction of the lepidopteran insect, from the viewpoint of enabling control of the lepidopteran insect. The at least one gene involved in the growth, survival, development and / or reproduction of the lepidopteran insect will be described later.

[0024] The target gene in the improving agent according to this embodiment may be one type or two or more types.

[0025] The gene encoding the double-stranded RNase in the enhancer according to this embodiment is a gene possessed by lepidopteran insects, and the type of gene is not particularly limited. Those skilled in the art can search for genes encoding double-stranded RNase in each lepidopteran insect. For example, a method can be used to determine whether a protein expressed by a certain gene has a DNA / RNA non-sp endonuclease domain or the like, using a database or tertiary structure prediction software. An example of tertiary structure prediction software is InterProScan version 5.65-97.0 (https: / / www.ebi.ac.uk / interpro / search / sequence / ).

[0026] The gene encoding the double-stranded RNase in the improver according to this embodiment may be, for example, a gene encoding dsRNase 1 to 5 (dsRNase 1 to 5 genes).

[0027] Whether or not a target gene is a dsRNase gene can be determined, for example, by determining whether the expressed protein is a dsRNase. A specific method for this determination can be, for example, the following procedure. First, InterProScan version 5.65-97.0 is used to search for domains contained in the amino acid sequence of the target protein. The parameters are not changed, and the search is run with default parameters. If the analysis results in a hit for a protein having a DNA / RNA_non-sp endonuclease domain, the target protein is determined to be a dsRNase.

[0028] In InterProScan version 5.65-97.0, the only parameter that can be changed by the user is the domain database to be searched. That is, when determining whether a protein is a PTDCP ​​using InterProScan version 5.65-97.0, the following databases are used: NCBIfam, SFLD, PANTHER, HAMAP, PROSITE profiles, PROSITE patterns, SMART, CDD, PRINTS, Pfam, PIRSF, SUPERFAMILY, CATH-Gene3D, Phobius, SignalP, Coils, MobiDBLite, TMHMM, SignalP_EUK, SignalP_GRAM_POSITIVE, SignalP_GRAM_NEGATIVE, AntiFam, FunFam, and PIRSR. By default, InterProScan version 5.65-97.0 searches for protein domains using all of these databases.

[0029] Examples of the nucleotide sequence of the dsRNase1 gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 1 to 2, and a nucleotide sequence that expresses mRNA shown in SEQ ID NOs: 3 to 4. The protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is the dsRNase1 protein of the beet armyworm, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is the dsRNase1 protein of the common cutworm. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 is the mRNA of dsRNase1 of the beet armyworm, and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4 is the mRNA of dsRNase1 of the common cutworm.

[0030] As used herein, "sequence identity" refers to the percentage (%) of identical DNA bases or amino acid residues relative to the total overlapping DNA base sequences or total amino acid sequences in an optimal alignment of two DNA base sequences or amino acid sequences, which are achieved using a mathematical algorithm known in the art. For example, Multiple Sequence Alignment (Clustal Omega: https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) can be used to create the alignment, and Percent Identity Matrix (Clustal2.1) can be used to calculate sequence identity.

[0031] Examples of the partial sequence of the dsRNase 1 gene used to synthesize double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 5 and 6. The nucleotide sequence shown in SEQ ID NO: 5 is a partial sequence of the dsRNase 1 gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 6 is a partial sequence of the dsRNase 1 gene of the common cutworm. The double-stranded RNA that suppresses the expression of the dsRNase1 gene is not particularly limited as long as it can suppress the expression of the dsRNase1 gene, and examples thereof include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequences shown in SEQ ID NOs: 7 and 8. The nucleotide sequence shown in SEQ ID NO: 7 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the dsRNase1 gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 8 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the dsRNase1 gene of the common cutworm.

[0032] Examples of the nucleotide sequence of the dsRNase2 gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 9 to 10, and a nucleotide sequence that expresses mRNA shown in SEQ ID NOs: 11 to 12. The protein consisting of the amino acid sequence shown in SEQ ID NO: 9 is the dsRNase2 protein of the beet armyworm, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 10 is the dsRNase2 protein of the common cutworm. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 11 is the mRNA of dsRNase2 of the beet armyworm, and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 12 is the mRNA of dsRNase2 of the common cutworm.

[0033] Examples of the partial sequence of the dsRNase2 gene used to synthesize double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 13 to 14. The nucleotide sequence shown in SEQ ID NO: 13 is a partial sequence of the dsRNase2 gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 14 is a partial sequence of the dsRNase2 gene of the common cutworm. The double-stranded RNA that suppresses the expression of the dsRNase2 gene is not particularly limited as long as it can suppress the expression of the dsRNase2 gene, and examples thereof include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 15 to 16. The nucleotide sequence shown in SEQ ID NO: 15 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the dsRNase2 gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 16 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the dsRNase2 gene of the common cutworm.

[0034] Examples of the nucleotide sequence of the dsRNase3 gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, and a nucleotide sequence that expresses the mRNA shown in SEQ ID NO: 18. The protein consisting of the amino acid sequence shown in SEQ ID NO: 17 is the dsRNase3 protein of beet armyworm. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 18 is the mRNA of dsRNase3 of beet armyworm.

[0035] Examples of the partial sequence of the dsRNase3 gene used to synthesize double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 19. The nucleotide sequence shown in SEQ ID NO: 19 is a partial sequence of the dsRNase3 gene of the beet armyworm. The double-stranded RNA that suppresses the expression of the dsRNase3 gene is not particularly limited as long as it can suppress the expression of the dsRNase3 gene, and examples thereof include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 20. The base sequence shown in SEQ ID NO: 20 is the base sequence of a double-stranded RNA that suppresses the expression of the dsRNase3 gene of the beet armyworm.

[0036] Examples of the nucleotide sequence of the dsRNase4 gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 21 and 22, and a nucleotide sequence that expresses mRNA shown in SEQ ID NOs: 23 and 24. The protein consisting of the amino acid sequence shown in SEQ ID NO: 21 is the dsRNase4 protein of the beet armyworm, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 22 is the dsRNase4 protein of the common cutworm. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 23 is the mRNA of dsRNase4 of the beet armyworm, and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 24 is the mRNA of dsRNase4 of the common cutworm.

[0037] Examples of the partial sequence of the dsRNase4 gene used to synthesize double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 25 to 26. The nucleotide sequence shown in SEQ ID NO: 25 is a partial sequence of the dsRNase4 gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 26 is a partial sequence of the dsRNase4 gene of the common cutworm. The double-stranded RNA that suppresses the expression of the dsRNase4 gene is not particularly limited as long as it can suppress the expression of the dsRNase4 gene, and examples thereof include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 27 to 28. The nucleotide sequence shown in SEQ ID NO: 27 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the dsRNase4 gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 28 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the dsRNase4 gene of the common cutworm.

[0038] Examples of the nucleotide sequence of the dsRNase5 gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 29 to 30, and a nucleotide sequence that expresses mRNA shown in SEQ ID NOs: 31 to 32. The protein consisting of the amino acid sequence shown in SEQ ID NO: 29 is the dsRNase5 protein of the beet armyworm, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 30 is the dsRNase5 protein of the common cutworm. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 31 is the mRNA of dsRNase5 of the beet armyworm, and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 32 is the mRNA of dsRNase5 of the common cutworm.

[0039] Examples of the partial sequence of the dsRNase5 gene used to synthesize double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 33 to 34. The nucleotide sequence shown in SEQ ID NO: 33 is a partial sequence of the dsRNase5 gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 34 is a partial sequence of the dsRNase5 gene of the common cutworm. The double-stranded RNA that suppresses the expression of the dsRNase5 gene is not particularly limited as long as it can suppress the expression of the dsRNase5 gene, and examples thereof include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequences shown in SEQ ID NOs: 35 to 36. The nucleotide sequence shown in SEQ ID NO: 35 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the dsRNase5 gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 36 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the dsRNase5 gene of the common cutworm.

[0040] The gene encoding the double-stranded RNase in the improver of this embodiment may be two or more types, or three or more types, preferably three or more types, more preferably four or more types, and even more preferably five or more types.

[0041] Genes encoding nucleases having a PIN domain are genes found in lepidopteran insects, and the type is not particularly limited. The PIN domain is a protein domain that functions as a nuclease that cleaves single-stranded RNA. Those skilled in the art can search for genes encoding nucleases having a PIN domain in each lepidopteran insect. For example, a method can be used to determine whether a protein expressed by a certain gene has a PIN domain using a database or tertiary structure prediction software. An example of tertiary structure prediction software is InterProScan version 5.65-97.0 (https: / / www.ebi.ac.uk / interpro / search / sequence / ).

[0042] Examples of genes encoding nucleases having a PIN domain include genes encoding REase (REase genes) and genes encoding PTDCP ​​(PTDCP ​​genes), and it is preferable that the nucleases having a PIN domain include REase and / or PTDCP.

[0043] REase (RNAi efficiency-related nuclease) is a lepidopteran-specific RNA degrading enzyme containing a PIN domain. PTDCP ​​(PIN / TMD domain containing protein) is a protein that contains a PIN domain and a transmembrane domain as its protein domain.

[0044] A transmembrane domain is a protein domain consisting of a transmembrane region that penetrates the cell membrane. Whether a protein contains a transmembrane domain can be predicted, for example, using protein three-dimensional structure prediction software commonly used by those skilled in the art. Examples of such software include InterProScan version 5.65-97.0 (https: / / www.ebi.ac.uk / interpro / search / sequence / ).

[0045] Whether a target gene is a REase gene or a PTDCP ​​gene can be determined, for example, by determining whether the expressed protein is a REase or a PTDCP. A specific method for this determination can be, for example, the following procedure. First, InterProScan version 5.65-97.0 is used to search for domains contained in the amino acid sequence of the target protein. The parameters are not changed, and the program is run with default parameters. If the analysis results show a hit for the PIN-like domain superfamily, the target protein is determined to have a PIN domain. If the analysis results show a hit for TRANSMEMBRANE, the target protein is determined to have a transmembrane domain. If the target protein is determined to have only a PIN domain, the target protein is determined to be a REase. If the target protein is determined to have both a PIN domain and a transmembrane domain, the target protein is determined to be a PTDCP. More detailed operating procedures are as follows. 1. To use the browser version of InterProScan, go to the website https: / / www.ebi.ac.uk / interpro / search / sequence / . 2. Enter the amino acid sequence of the protein of interest in the box and press the “search” button. 3. Based on the analysis results, if the PIN-like domain superfamily is found, the protein of interest is determined to have a PIN domain. If the TRANSMEMBRANE is found, the protein of interest is determined to have a transmembrane domain.

[0046] Examples of the nucleotide sequence of the REase gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 37 to 38, and a nucleotide sequence that expresses mRNA shown in SEQ ID NOs: 39 to 40. The protein consisting of the amino acid sequence shown in SEQ ID NO: 37 is the REase protein of Spodoptera exigua, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 38 is the REase protein of Spodoptera litura. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 39 is the mRNA of REase from Spodoptera exigua, and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 40 is the mRNA of REase from Spodoptera litura.

[0047] Examples of partial sequences of the REase gene used to synthesize double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequences shown in SEQ ID NOs: 41 to 42. The nucleotide sequence shown in SEQ ID NO: 41 is a partial sequence of the REase gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 42 is a partial sequence of the REase gene of the common cutworm. The double-stranded RNA that suppresses expression of the REase gene is not particularly limited as long as it can suppress expression of the REase gene, and examples include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequences shown in SEQ ID NOs: 43 to 44. The nucleotide sequence shown in SEQ ID NO: 43 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the REase gene of the beet armyworm.The nucleotide sequence shown in SEQ ID NO: 44 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the REase gene of the common cutworm.

[0048] Examples of the nucleotide sequence of the PTDCP ​​gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 45 to 46, and a nucleotide sequence that expresses mRNA shown in SEQ ID NOs: 47 to 48. The protein consisting of the amino acid sequence shown in SEQ ID NO: 45 is the PTDCP ​​protein of beet armyworm, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 46 is the PTDCP ​​protein of common cutworm. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 47 is the PTDCP ​​mRNA of beet armyworm, and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 48 is the PTDCP ​​mRNA of common cutworm.

[0049] Examples of partial sequences of the PTDCP ​​gene used to synthesize double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequences shown in SEQ ID NOs: 49 to 50. The nucleotide sequence shown in SEQ ID NO: 49 is a partial sequence of the PTDCP ​​gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 50 is a partial sequence of the PTDCP ​​gene of the common cutworm. The double-stranded RNA that suppresses expression of the PTDCP ​​gene is not particularly limited as long as it can suppress expression of the PTDCP ​​gene, and examples thereof include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequences shown in SEQ ID NOs: 51 to 52. The nucleotide sequence shown in SEQ ID NO: 51 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the PTDCP ​​gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 52 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the PTDCP ​​gene of the common cutworm.

[0050] The gene encoding the nuclease having a PIN domain in the improver according to this embodiment may be one type or two or more types.

[0051] The at least two genes encoding enzymes involved in skin chitin synthesis are genes possessed by Lepidoptera insects, and the types thereof are not particularly limited. Those skilled in the art can search for enzymes involved in skin chitin synthesis in each Lepidoptera insect. For example, a method can be used to search for sequence identity using a database by using the amino acid sequence of a protein expressed by a certain gene and a known enzyme involved in skin chitin synthesis.

[0052] Examples of genes encoding enzymes involved in chitin synthesis in the skin include chitin synthase A (CHA), UDP-N-acetylglucosamine pyrophosphorylase (UAP), glucose phosphate isomerase (GPI), and 3,4-dihydroxyphenylacetaldehyde (DopaL) synthase. Preferably, the enzymes involved in chitin synthesis in the skin include chitin synthase A (CHA) and UDP-N-acetylglucosamine pyrophosphorylase (UAP).

[0053] Examples of the nucleotide sequence of the CHA gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 53-54, and a nucleotide sequence that expresses mRNA shown in SEQ ID NOs: 55-56. The protein consisting of the amino acid sequence shown in SEQ ID NO: 53 is the CHA protein of Spodoptera exigua, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 54 is the CHA protein of Spodoptera litura. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 55 is the CHA mRNA of Spodoptera exigua, and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 56 is the CHA mRNA of Spodoptera litura.

[0054] Examples of the partial sequence of the CHA gene used to synthesize the double-stranded RNA include double-stranded RNAs containing a nucleotide sequence with 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NOs: 57-58. The nucleotide sequence shown in SEQ ID NO: 57 is a partial sequence of the CHA gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 58 is a partial sequence of the CHA gene of the common cutworm. The double-stranded RNA that suppresses the expression of the CHA gene is not particularly limited as long as it can suppress the expression of the CHA gene, and examples include double-stranded RNAs containing a nucleotide sequence with 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NOs: 59-60. The nucleotide sequence shown in SEQ ID NO: 59 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the CHA gene in the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 60 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the CHA gene in the common cutworm.

[0055] Examples of the nucleotide sequence of the UAP gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 61 to 62, and a nucleotide sequence that expresses mRNA shown in SEQ ID NOs: 63 to 64. The protein consisting of the amino acid sequence shown in SEQ ID NO: 61 is the UAP protein of Spodoptera exigua, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 62 is the UAP protein of Spodoptera litura. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 63 is the UAP mRNA of Spodoptera exigua, and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 64 is the UAP mRNA of Spodoptera litura.

[0056] Examples of the partial sequence of the UAP gene used for synthesizing the double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NOs: 65 to 66. The nucleotide sequence shown in SEQ ID NO: 65 is a partial sequence of the UAP gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 66 is a partial sequence of the UAP gene of the common cutworm. The double-stranded RNA that suppresses the expression of the UAP gene is not particularly limited as long as it can suppress the expression of the UAP gene, and examples thereof include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NOs: 67 to 68. The nucleotide sequence shown in SEQ ID NO: 67 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the UAP gene in Spodoptera exigua.The nucleotide sequence shown in SEQ ID NO: 68 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the UAP gene in Spodoptera litura.

[0057] In the enhancer of this embodiment, the ratio of the content of double-stranded RNA that suppresses the expression of a target gene in a lepidopteran insect, the content of double-stranded RNA that suppresses the expression of at least one gene encoding a double-stranded RNA degrading enzyme, the content of double-stranded RNA that suppresses the expression of at least one gene encoding a nuclease having a PIN domain, and the content of double-stranded RNA that suppresses the expression of at least one gene encoding an enzyme involved in chitin synthesis in the skin may be, for example, 1-10:1-10:1-10:1-10, 1-5:1-5:1-5:1-5, 1-3:1-3:1-3:1-3, 1-2:1-2:1-2:1-2, or approximately 1:1:1:1.

[0058] The gene encoding the enzyme involved in chitin synthesis in the skin in the improver according to this embodiment may be of two or more types, or may be of three or more types.

[0059] The dsRNA-nanoparticle-surfactant complex in the enhancer of this embodiment may also contain double-stranded RNA that suppresses the expression of any gene that is thought to improve RNAi efficiency. Examples of such genes include genes encoding enzymes with nuclease activity, such as the asteroid gene and single-stranded RNase (ssRNase) genes.

[0060] Double-stranded RNA can be synthesized by selecting all or part of the base sequence of any gene and synthesizing it based on the nucleotide sequence of the target gene using known methods, such as the phosphite triester method, the phosphoramidite method, and in vitro enzymatic transcription methods.

[0061] (nanoparticles) Nanoparticles are particles with diameters on the order of nanometers. The nanoparticles are not particularly limited, but may be, for example, carbon-based nanoparticles, polymer-based nanoparticles, chitosan-based nanoparticles, or lipid-based nanoparticles, and are preferably carbon-based nanoparticles and / or chitosan-based nanoparticles.

[0062] Examples of carbon-based nanoparticles include carbon quantum dots (CQDs), fullerenes, carbon nanotubes, graphene, and carbon black, with carbon quantum dots being preferred.

[0063] Examples of polymer nanoparticles include polyethyleneimine nanoparticles, polylysine nanoparticles, star-shaped polycation nanoparticles, and poly(β-amino ester) nanoparticles.

[0064] Chitosan nanoparticles include, for example, nanoparticles of low molecular weight chitosan, nanoparticles of high molecular weight chitosan, and nanoparticles of chemically modified chitosan. Low molecular weight chitosan nanoparticles are preferred, and nanoparticles of low molecular weight chitosan with a deacetylation degree of 75% or more are more preferred. Chitosan is produced by N-deacetylation of chitin and is generally characterized by its degree of deacetylation (DDA).

[0065] Examples of lipid-based nanoparticles include phosphatidylcholine nanoparticles, phosphatidylethanolamine nanoparticles, phosphatidylserine nanoparticles, phosphatidylglycerol nanoparticles, sphingomyelin nanoparticles, and cholesterol nanoparticles.

[0066] The diameter of the nanoparticles may be, for example, 1 nm to 100 nm, 1 nm to 80 nm, 1 nm to 60 nm, 1 nm to 50 nm, 1 nm to 40 nm, 1 nm to 20 nm, or 1 nm to 10 nm.

[0067] The nanoparticles may be used alone or in combination of two or more types.

[0068] (surfactant) The surfactant is not particularly limited, and may be an ionic surfactant (anionic surfactant, cationic surfactant), a nonionic surfactant, or an amphoteric surfactant.

[0069] Examples of anionic surfactants include higher fatty acid salts, linear alkylbenzene sulfonates, α-sulfofatty acid methyl ester salts, α-olefin sulfonates, alkyl sulfate ester salts, and polyoxyethylene alkyl sulfate ester salts.

[0070] Examples of cationic surfactants include alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkyldimethylbenzylammonium salts, and benzyltrimethylammonium salts, and may also include benzyldimethyldodecylammonium chloride (BDDAC).

[0071] Examples of nonionic surfactants include sorbitan fatty acid esters, sucrose fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, polyoxyalkylphenyl ethers, and the like, and may also include TRITON (registered trademark)-X100, Tween (registered trademark) 40, etc.

[0072] Examples of amphoteric surfactants include alkylamino fatty acid salts, alkylbetaines, alkylamine oxides, and the like, and may also be 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).

[0073] The content of the dsRNA-nanoparticle-surfactant complex in the improving agent according to this embodiment can be adjusted as appropriate depending on the type of Lepidoptera insect, the type of dosage form, the administration method, etc. The content of the dsRNA-nanoparticle-surfactant complex in the improving agent according to this embodiment may be, for example, 0.01% by mass to 100% by mass, 0.1% by mass to 100% by mass, 1% by mass to 100% by mass, or 10% by mass to 100% by mass, based on the total amount of the improving agent according to this embodiment.

[0074] Furthermore, the content of the dsRNA-nanoparticle-surfactant complex in the improver of this embodiment may be, for example, 1 μg / μl or more, 2 μg / μl or more, 4 μg / μl or more, 6 μg / μl or more, 8 μg / μl or more, 10 μg / μl or more, 12 μg / μl or more, 14 μg / μl or more, 16 μg / μl or more, 18 μg / μl or more, or 20 μg / μl or more, or 200 μg / μl or less, 150 μg / μl or less, 100 μg / μl or less, or 50 μg / μl or less, calculated as the dsRNA content when the recovery rate during complex synthesis is estimated to be 100% based on the total amount of the improver of this embodiment.

[0075] The improver according to this embodiment is a composition, and may be in the form of a liquid, gel, paste, or the like, which can be appropriately selected depending on the method of use, etc.

[0076] The improver according to this embodiment may contain additives such as excipients, thickeners, binders, stabilizers, preservatives, pH adjusters, colorants, flavoring agents, and spreaders. The various additives are not particularly limited, and materials known in the technical field of biopesticides can be used, and the amounts of these additives can be adjusted appropriately based on the techniques known to those skilled in the art. A spreader is an agent that promotes the adsorption and penetration of pesticides onto plant surfaces.

[0077] An improvement in RNAi efficiency may mean that the expression level of a target gene is statistically significantly reduced when the enhancer according to this embodiment is administered to a lepidopteran insect, compared to when only double-stranded RNA that suppresses the expression of the target gene is administered. An improvement in RNAi efficiency may mean that the expression level of a target gene is reduced by 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, or 40% or more when the enhancer according to this embodiment and double-stranded RNA that suppresses the expression of the target gene are simultaneously administered to the lepidopteran insect, compared to when only double-stranded RNA that suppresses the expression of the target gene is administered. An improvement in RNAi efficiency can be evaluated, for example, by measuring the expression level of a target gene using quantitative real-time PCR or the like.

[0078] The method of administering the improver according to the present embodiment is not particularly limited, and for example, the improver according to the present embodiment may be administered by adhering it to the skin of a lepidopteran insect (transdermal administration), specifically, the improver according to the present embodiment may be administered by applying or spraying it on the skin of a lepidopteran insect, or the improver according to the present embodiment may be applied or sprayed on the surface of a plant body that the lepidopteran insect feeds on and then brought into contact with the lepidopteran insect, but it is preferable to administer the improver according to the present embodiment by applying or spraying it on the skin of a lepidopteran insect. That is, the improver according to the present embodiment may be for transdermal administration.

[0079] In this specification, unless otherwise specified, the term "plant body" refers to any organ of a plant, such as flowers (including buds), buds, leaves, stems, roots, rhizomes, tubers, and underground stems.

[0080] Examples of methods for applying or spraying the improver according to this embodiment include applying it to the skin of a lepidopteran insect or the surface of a plant using a brush or spraying it with a spray.

[0081] The dosage of the enhancer according to this embodiment to the Lepidoptera insects may be, for example, 0.01 μg to 50 μg of the double-stranded RNA per Lepidoptera insect.

[0082] [A kit for improving RNAi efficiency of target genes in lepidopteran insects] The present invention can also be understood as a kit for improving RNAi efficiency against a target gene in a lepidopteran insect (hereinafter also referred to as "kit 1 according to this embodiment"). The kit includes double-stranded RNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant. Here, the predetermined gene includes double-stranded RNA that suppresses the expression of at least two genes encoding double-stranded RNA-degrading enzymes, and at least one gene encoding a nuclease having a PIN domain, and / or at least two genes encoding enzymes involved in chitin synthesis in the skin.

[0083] The double-stranded RNA, nanoparticles, and surfactant that suppress the expression of a specific gene in Kit 1 of this embodiment, as well as the double-stranded RNA that suppresses the expression of a target gene, can be used to prepare a dsRNA-nanoparticle-surfactant complex, which can be used as an enhancer of this embodiment.

[0084] The specific aspects of the kit according to this embodiment, such as the dsRNA that suppresses the expression of a predetermined gene, the nanoparticles, and the surfactant, can be applied without limitation to the specific aspects described above.

[0085] The kit according to this embodiment may contain, in addition to the dsRNA that suppresses the expression of a specific gene, nanoparticles, and surfactant, reagents necessary for carrying out RNAi, such as a buffer solution, RNA polymerase for synthesizing double-stranded RNA, nucleotide triphosphates (NTPs), double-stranded DNA used for synthesizing double-stranded DNA, single-stranded DNA, and single-stranded RNA.

[0086] [Method for improving RNAi efficiency of target genes in Lepidoptera insects] The method for improving RNAi efficiency for a target gene in a lepidopteran insect according to the present embodiment (hereinafter also referred to simply as the "improvement method according to the present embodiment") comprises introducing into the lepidopteran insect a composition containing a complex of double-stranded RNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant (dsRNA-nanoparticle-surfactant). Here, the predetermined gene includes the target gene of the lepidopteran insect, at least two genes encoding a double-stranded RNA-degrading enzyme, and at least one gene encoding a nuclease having a PIN domain, and / or at least two genes encoding enzymes involved in chitin synthesis in the skin.

[0087] The dsRNA-nanoparticle-surfactant complex is as described above. The composition containing the dsRNA-nanoparticle-surfactant is the enhancer according to this embodiment and is as described above. The method for introducing the composition containing the dsRNA-nanoparticle-surfactant into a lepidopteran insect is as described in the administration method above.

[0088] The method for improving the dsRNA-nano-particle-surfactant composition according to this embodiment may include preparing a dsRNA-nano-particle-surfactant mixture, which can be prepared as described above, for example by mixing dsRNA, nanoparticles, and a surfactant.

[0089] [Lepidoptera insect control agent] The lepidopteran insect control agent of this embodiment is an enhancer according to this embodiment, in which the target gene is at least one gene involved in the growth, survival, development, and / or reproduction of lepidopteran insects. That is, the control agent according to this embodiment contains a complex (dsRNA-nanoparticle-surfactant complex) of double-stranded RNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant. Here, the predetermined gene includes at least one gene involved in the growth, survival, development, and / or reproduction of lepidopteran insects, at least two genes encoding a double-stranded RNA-degrading enzyme, and at least one gene encoding a nuclease having a PIN domain, and / or at least two genes encoding an enzyme involved in chitin synthesis in the skin.

[0090] The control agent for lepidopteran insects of this embodiment can be applied in the same manner as described for the improver of this embodiment, except that the target gene of lepidopteran insects is at least one gene involved in the growth, survival, development and / or reproduction of lepidopteran insects.

[0091] <At least one gene involved in the growth, survival, development and / or reproduction of Lepidoptera insects> Examples of genes involved in the growth, survival, development, and / or reproduction of Lepidoptera include genes whose inhibition induces growth inhibition or lethality or suppresses the proliferation of Lepidoptera. Examples of such genes include apoptosis inhibitor genes, genes encoding proteins that are structural subunits of complexes required for the autophagy pathway, detoxification enzyme genes, genes encoding neuropeptides and their receptors, genes encoding vacuolar ATPase (V-ATPase) subunits, genes encoding proteins that are structural subunits of ribosomal proteins, genes encoding proteins that are subunits of multimeric complexes that form membrane vesicles, and genes encoding proteins required for insect morphogenesis. The at least one gene involved in the growth, survival, development, and / or reproduction of Lepidoptera may be one or more genes selected from the group consisting of apoptosis inhibitor genes, genes encoding proteins that are structural subunits of complexes required for the autophagy pathway, detoxification enzyme genes, genes encoding neuropeptides and their receptors, genes encoding vacuolar ATPase subunits, genes encoding proteins that are structural subunits of ribosomal proteins, and genes encoding proteins that are subunits of multimeric complexes that form membrane vesicles.

[0092] An apoptosis inhibitor gene is a gene that inhibits cell death, and suppressing its expression causes cell death, leading to growth inhibition or lethality. Examples of apoptosis inhibitor genes include genes encoding proteins containing baculovirus IAP (inhibitor of apoptosis) repeats (IAP family proteins). Examples of IAP family proteins include IAP1, IAP2, IAP3, IAP4, IAP5, and IAP6. In the control agent of this embodiment, the apoptosis inhibitor gene may include, for example, a gene encoding IAP1 (hereinafter also referred to as "IAP1 gene").

[0093] Genes encoding proteins that are structural subunits of complexes required for the autophagy pathway are responsible for cellular waste recycling, which involves the uptake, transport, sorting, and degradation of unwanted proteins through endosomes, thereby maintaining cellular homeostasis. Specific examples include CHMP4B, a structural subunit of the ESCRTIII complex, and Atg13, which is responsible for initiating autophagy.

[0094] Detoxification enzyme genes are responsible for the detoxification metabolism of xenobiotics. Examples include genes for enzymes that detoxify plant defense substances and chemicals such as pesticides. Specific examples include cytochrome P450 (CYP) genes. For example, the detoxification enzyme CYP6k1 is known to be involved in chemical resistance in onion thrips.

[0095] Neuropeptides and their receptors are small proteins produced by neurons that act on G protein-coupled receptors and are responsible for delayed and long-term regulation of synaptic transmission. Examples of neuropeptides and their receptors include the genes for the neuropeptide CAPA receptor (capaR) and the ecdysis triggering hormone (ETH) and its receptor (ETHR). capaR is a gene involved in water regulation in insects; its suppression causes the insect to dry out and die. Furthermore, ETH and ETHR are genes that induce molting and metamorphosis; their suppression inhibits molting and metamorphosis, resulting in stunted development or even lethality in the insect. In fact, it has been reported that suppressing the expression of ETH and ETHR by RNA interference resulted in lethality in western flower thrips (Patent Publication No. 2017-131201).

[0096] An example of a vacuolar ATPase (V-ATPase) subunit is V-ATPase-B (vascuolar ATP synthase subunit B). In fact, it has been reported that suppressing the expression of this gene by RNAi is lethal to western flower thrips.

[0097] Ribosomal proteins, which are structural components involved in protein synthesis, include, for example, S4 (RpS4) and S9 (RpS9), which are components of the cytoplasmic ribosomal small subunit, and L9 and L19, which are localized in the ribosome.

[0098] An example of a gene encoding a protein that is a subunit of a multimeric complex that forms a membrane of a membrane vesicle is the Coatomer subunit beta (COPB) gene.

[0099] Examples of genes encoding proteins necessary for insect morphogenesis include the imaginal disc growth factor (IDGF) gene, the forkhead (foxo) gene, which is involved in cell growth, proliferation, and differentiation, and the chitinase gene, a chitin-degrading enzyme in the skin.

[0100] The at least one gene involved in the growth, survival, development, and / or reproduction of the Lepidoptera insect may be selected from the group consisting of, for example, a gene encoding IAP1, a gene encoding cytochrome P450 (CYP), a gene encoding P-450 monooxygenase, a gene encoding capaR, a gene encoding ETH, a gene encoding ETHR, a gene encoding a V-ATPase subunit, a gene encoding RpS4, a gene encoding RpS9, a gene encoding COPB, a gene encoding nitroferrin 2, a gene encoding a carboxylesterase, a gene encoding a pheromone binding protein, a gene encoding TsetseEP, and a gene encoding IDGF, or may be selected from the group consisting of a gene encoding IAP1 and a gene encoding a cytochrome P450 (CYP).

[0101] Examples of the nucleotide sequence of the IAP1 gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequences set forth in SEQ ID NOs: 69 to 70, and a nucleotide sequence that expresses mRNA set forth in SEQ ID NOs: 71 to 72. The protein consisting of the amino acid sequence set forth in SEQ ID NO: 69 is the IAP1 protein of Spodoptera exigua, and the protein consisting of the amino acid sequence set forth in SEQ ID NO: 70 is the IAP1 protein of Spodoptera litura. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 71 is the mRNA of IAP1 of Spodoptera exigua, and a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 72 is the mRNA of IAP1 of Spodoptera litura.

[0102] Examples of the partial sequence of the IAP1 gene used for synthesizing the double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequences shown in SEQ ID NOs: 73 to 74. The nucleotide sequence shown in SEQ ID NO: 73 is a partial sequence of the IAP1 gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 74 is a partial sequence of the IAP1 gene of the common cutworm. The double-stranded RNA that suppresses the expression of the IAP1 gene is not particularly limited as long as it can suppress the expression of the IAP1 gene, and examples thereof include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequences shown in SEQ ID NOs: 75 to 76. The nucleotide sequence shown in SEQ ID NO: 75 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the IAP1 gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 76 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the IAP1 gene of the common cutworm.

[0103] Examples of the nucleotide sequence of the CHMP4B gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 77 to 78, and a nucleotide sequence that expresses mRNA shown in SEQ ID NOs: 79 to 80. The protein consisting of the amino acid sequence shown in SEQ ID NO: 77 is the CHMP4B protein of Spodoptera exigua, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 78 is the CHMP4B protein of Spodoptera litura. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 79 is the CHMP4B mRNA of Spodoptera exigua, and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 80 is the CHMP4B mRNA of Spodoptera litura.

[0104] The partial sequence of the CHMP4B gene used to synthesize the double-stranded RNA includes, for example, a double-stranded RNA containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 81 to 82. The nucleotide sequence shown in SEQ ID NO: 81 is a partial sequence of the CHMP4B gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 82 is a partial sequence of the CHMP4B gene of the common cutworm. The double-stranded RNA that suppresses the expression of the CHMP4B gene is not particularly limited as long as it can suppress the expression of the CHMP4B gene, and examples include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequences shown in SEQ ID NOs: 83 to 84. The nucleotide sequence shown in SEQ ID NO: 83 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the CHMP4B gene of the beet armyworm. The nucleotide sequence shown in SEQ ID NO: 84 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the CHMP4B gene of the common cutworm.

[0105] Examples of the nucleotide sequence of the ETH gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 85, and a nucleotide sequence that expresses the mRNA shown in SEQ ID NO: 86. The protein consisting of the amino acid sequence shown in SEQ ID NO: 85 is the ETH protein of beet armyworm. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 86 is the ETH mRNA of beet armyworm.

[0106] Examples of the partial sequence of the ETH gene used to synthesize the double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 87. The nucleotide sequence shown in SEQ ID NO: 87 is a partial sequence of the ETH gene of the beet armyworm. The double-stranded RNA that suppresses the expression of the ETH gene is not particularly limited as long as it can suppress the expression of the ETH gene, and examples include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 88. The nucleotide sequence shown in SEQ ID NO: 88 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the ETH gene of the beet armyworm.

[0107] Examples of the nucleotide sequence of the ETHR gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 89, and a nucleotide sequence that expresses the mRNA shown in SEQ ID NO: 90. The protein consisting of the amino acid sequence shown in SEQ ID NO: 89 is the ETHR protein of beet armyworm. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 90 is the ETHR mRNA of beet armyworm.

[0108] Examples of the partial sequence of the ETHR gene used to synthesize the double-stranded RNA include double-stranded RNAs containing a nucleotide sequence with 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 91. The nucleotide sequence shown in SEQ ID NO: 91 is a partial sequence of the ETHR gene of the beet armyworm. The double-stranded RNA that suppresses the expression of the ETHR gene is not particularly limited as long as it can suppress the expression of the ETHR gene, and examples include double-stranded RNAs containing a nucleotide sequence with 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 92. The nucleotide sequence shown in SEQ ID NO: 92 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the ETHR gene of the beet armyworm.

[0109] Examples of the nucleotide sequence of the COPB gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 98, and a nucleotide sequence that expresses the mRNA shown in SEQ ID NO: 99. The protein consisting of the amino acid sequence shown in SEQ ID NO: 98 is the COPB protein of beet armyworm. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 99 is the COPB mRNA of beet armyworm.

[0110] Examples of partial sequences of the COPB gene used to synthesize double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 94. The nucleotide sequence shown in SEQ ID NO: 94 is a partial sequence of the COPB gene of the beet armyworm. The double-stranded RNA that suppresses the expression of the COPB gene is not particularly limited as long as it can suppress the expression of the COPB gene, and examples include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 96. The nucleotide sequence shown in SEQ ID NO: 96 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the COPB gene of the beet armyworm.

[0111] Examples of the nucleotide sequence of the IDGF gene include a nucleotide sequence that expresses a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 100, and a nucleotide sequence that expresses the mRNA shown in SEQ ID NO: 101. The protein consisting of the amino acid sequence shown in SEQ ID NO: 100 is the IDGF protein of beet armyworm. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. Furthermore, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 101 is the IDGF mRNA of beet armyworm.

[0112] Examples of the partial sequence of the IDGF gene used to synthesize the double-stranded RNA include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 95. The nucleotide sequence shown in SEQ ID NO: 95 is a partial sequence of the IDGF gene of the beet armyworm. The double-stranded RNA that suppresses the expression of the IDGF gene is not particularly limited as long as it can suppress the expression of the IDGF gene, and examples include double-stranded RNAs containing a nucleotide sequence having 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 97. The nucleotide sequence shown in SEQ ID NO: 97 is the nucleotide sequence of a double-stranded RNA that suppresses the expression of the IDGF gene of the beet armyworm.

[0113] The at least one gene involved in the growth, survival, development, and / or reproduction of Lepidopteran insects may be a plurality of genes, or may be at least two genes. In the case of a plurality of genes, the double-stranded RNA that suppresses the expression of at least one gene involved in the growth, survival, development, and / or reproduction of Lepidopteran insects may be a double-stranded RNA that simultaneously suppresses a plurality of genes, or may be a double-stranded RNA that suppresses each gene individually. The at least one gene involved in the growth, survival, development, and / or reproduction of Lepidopteran insects may include one or more selected from the IAP1 gene, CHMP4B gene, ETH gene, ETHR gene, COPB gene, and IDGF gene, and may include the IAP1 gene and CHMP4B gene, the ETH gene and ETHR gene, and / or the COPB gene and IDGF gene.

[0114] [Kit for controlling lepidopteran insects] The present invention can also be understood as a kit for controlling lepidopteran insects (hereinafter also referred to as "kit 2 of this embodiment"). The kit comprises dsRNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant. Here, the predetermined gene includes at least one gene involved in the growth, survival, development, and / or reproduction of lepidopteran insects, at least two genes encoding a double-stranded RNA degrading enzyme, and at least one gene encoding a nuclease having a PIN domain, and / or at least two genes encoding an enzyme involved in chitin synthesis in the skin.

[0115] The same aspects as those of Kit 1 of this embodiment can be applied, except that the dsRNA used to suppress the expression of a specific gene is a dsRNA that suppresses the expression of at least one gene involved in the growth, survival, development, and / or reproduction of Lepidoptera insects.

[0116] In addition to the dsRNA that suppresses the expression of a specific gene, the nanoparticles, and the surfactant, the kit according to this embodiment may also contain reagents necessary for controlling lepidopteran insects, such as a buffer solution, an RNA polymerase for synthesizing double-stranded RNA, and nucleotide triphosphates (NTPs).

[0117] [Method for controlling lepidopteran insects in plants] According to this embodiment, a method for controlling lepidopteran insects in a plant comprises introducing into the lepidopteran insect a composition containing a complex of double-stranded RNA (dsRNA) that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant, wherein the predetermined gene includes a target gene of the lepidopteran insect, at least one gene involved in the growth, survival, development, and / or reproduction of the lepidopteran insect, at least two genes encoding a double-stranded RNA degrading enzyme, and at least one gene encoding a nuclease having a PIN domain, and / or at least two genes encoding an enzyme involved in chitin synthesis in the skin.

[0118] The method for controlling lepidopteran insects according to this embodiment can be applied in the same manner as described for the improvement method according to this embodiment, except that the target gene of lepidopteran insects is at least one gene involved in the growth, survival, development and / or reproduction of lepidopteran insects. [Example]

[0119] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0120] (Example 1: Evaluation of mortality of beet armyworm 1) A solution containing a complex of double-stranded RNA (dsRNA) that suppresses the expression of a specific target gene, nanoparticles, and a surfactant (dsRNA-nanoparticle-surfactant complex) was dropped onto the skin of the beet armyworm, and the mortality rate of the beet armyworm was evaluated.

[0121] <Synthesis of dsRNA for use against beet armyworm> For each gene sequence registered in the database (GenBank), dsRNases 1 to 5, REase, PTDCP, CHA, UAP, IAP1, CHMP4B, ETH, and ETHR, 400 bp partial sequences (dsRNases 1 to 5 partial sequences: SEQ ID NOs: 5, 13, 19, 25, 33; REase partial sequence: SEQ ID NO: 41; PTDCP ​​partial sequence: SEQ ID NO: 49; CHA partial sequence: SEQ ID NO: 57; UAP partial sequence: SEQ ID NO: 65; IAP1 partial sequence: SEQ ID NO: 73; CHA partial sequence: SEQ ID NO: 81; ETH partial sequence: SEQ ID NO: 87; ETHR partial sequence: SEQ ID NO: 91) for which primers for amplifying 200 bp to 400 bp DNA could be designed were selected and artificially synthesized, and the partial sequences (artificial genes) were cloned into the pUCFa vector (ampicillin resistant) (FASMAC). These artificial genes were used as templates for dsRNA.

[0122] Using these artificial genes as templates and primers with T7 promoter sequences attached to the 5' ends, DNA fragments with T7 promoter sequences attached to both ends of the target gene partial sequence were amplified by PCR using GoTaq® Green Master MIX (Promega). Using the PCR products as templates, dsRNA of the target genes (dsRNases 1 to 5 (SEQ ID NOs: 7, 15, 20, 27, 35), dsREase (SEQ ID NO: 43), dsPTDCP ​​(SEQ ID NO: 51), dsCHA (SEQ ID NO: 59), dsUAP (SEQ ID NO: 67), dsIAP1 (SEQ ID NO: 75), dsCHMP4B (SEQ ID NO: 83), dsETH (SEQ ID NO: 88), dsETHR (SEQ ID NO: 92)) was synthesized using the T7 RiboMAX Express Large Scale RNA Production System.

[0123] Also, using pGFPuv (Takara Bio Inc.) as a template and primers with a T7 promoter sequence added to the 5'-end, a DNA fragment with T7 promoter sequences added to both ends of a partial sequence of the GFP gene was amplified. Using this PCR product as a template, dsRNA of GFP (dsGFP, SEQ ID NO: 93) was synthesized.

[0124] The target genes in this example are shown in Table 1. A group of genes encoding ribonucleases involved in the degradation of double-stranded RNA (dsRNase3-5 genes), a group of genes encoding nucleases having a PIN domain (REase gene and PTDCP gene), and a group of genes encoding enzymes involved in chitin synthesis in the skin (chitin synthase A (CHA) gene and UDP-N-acetylglucosamine pyrophosphorylase (UAP) gene) were used as target genes. And each group of genes was designated as Group A - C and used as a basic target gene set. Also, the IAP1 gene and CHMP4B gene, which are genes important for insect development, were also used as target genes.

[0125]

Table 1

[0126] <Preparation of dsRNA-Nanoparticle-Surfactant Composite Solution> As the dsRNA, those synthesized above were used in a predetermined combination. As the nanoparticles, carbon quantum dots (CQD) prepared from polyethylene glycol #200 and ethyleneimine (polymer) (both from Nacalai Tesque Inc.) were used. As the surfactant, benzyl dimethyl dodecyl ammonium chloride (Sigma-Aldrich) was used to form a complex of dsRNA, nanoparticles, and surfactant, and a dsRNA-nanoparticle-surfactant composite solution was prepared.

[0127] First, ethyleneimine (polymer, approximately 30% aqueous solution), polyethylene glycol #200, and distilled water were mixed in a volume ratio of 2:9:3. The mixture was then heated in a microwave oven (800W, approximately 3 minutes) until golden brown. After heating, the mixture was cooled to room temperature. The dsRNA was then dissolved in 50 mM sodium sulfate solution at a ratio of 40 μg / 100 μL, and an equal volume of CQDs was added. The mixture was then vortexed for 1 minute. After stirring, the mixture was stored overnight at 4°C. The mixture was then centrifuged at 15,000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was then washed three times with distilled water and dried at room temperature. Distilled water was added to the precipitate, and the mixture was stirred with an ultrasonic homogenizer until the precipitate was completely dissolved. Next, benzyldimethyldodecylammonium chloride was added to the solution containing the nanoparticle-dsRNA complexes at a concentration of 0.1%, and the mixture was stirred for approximately 30 seconds. The mixture was then left at room temperature for 30 minutes to form a dsRNA-nanoparticle-surfactant complex, preparing a dsRNA-nanoparticle-surfactant complex solution. To ensure that the total concentration of double-stranded RNA was equal in each test group, dsGFP was used appropriately to prepare the dsRNA-nanoparticle-surfactant complex solution. As a control, a dsRNA-nanoparticle-surfactant complex solution was prepared using the above-mentioned double-stranded RNAs that suppress the expression of the IAP1 gene, CHMP4B gene, and GFP gene.

[0128] <Administration of the complex solution to beet armyworm> First-instar larvae of the beet armyworm were reared at 25°C. A few hours before the larvae began head capsule slippage (HCS), 1 μL of the dsRNA-nanoparticle-surfactant complex solution was added dropwise to the larvae at a rate of 1 μL per individual (so that the administration concentrations of each double-stranded RNA were as shown in Table 1 above, and the double-stranded RNA concentration was 11 μg / μL). The larvae were then immersed for 10 minutes. After that, excess liquid was absorbed with Kimwipes or similar. The food was placed in a petri dish, and the mortality rate of the beet armyworms (molting rate to the second instar) was observed.

[0129] The results are shown in Figure 1. As shown in Figure 1, in the control group, a dsRNA-nanoparticle-surfactant complex solution was prepared using double-stranded RNAs that suppress the expression of the IAP1 gene, CHMP4B gene, and GFP gene. When the control dsRNA-nanoparticle-surfactant complex solution was applied dropwise to the skin of S. exigua, the mortality rate of the moths was approximately 20% (first from the left in Figure 1). On the other hand, when dsRNA-nanoparticle-surfactant complex solutions prepared using the genes of Groups A and B, Groups A and C, or Groups A, B, and C and double-stranded RNAs that suppress the expression of the IAP1 gene and CHMP4B gene were applied dropwise to the skin of S. exigua (third and fifth to sixth from the left in Figure 1), the mortality rate of the moths was significantly higher than when the control dsRNA-nanoparticle-surfactant complex solution was applied dropwise to the skin of S. exigua.

[0130] The results of Example 1 suggest that the RNAi efficiency against the IAP1 gene and CHMP4B gene was improved by applying a dsRNA-nanoparticle-surfactant complex solution prepared using double-stranded RNA that suppresses the expression of dsRNase3 to 5 genes, REase gene, PTDCP ​​gene, CHA gene, UAP gene, and IAP1 gene and CHMP4B gene to the skin of the beet armyworm.

[0131] Therefore, it is believed that a solution containing a complex of double-stranded RNA that suppresses the expression of a target gene in a lepidopteran insect, a gene encoding a double-stranded RNA degrading enzyme, a gene encoding a nuclease having a PIN domain, and / or a gene encoding an enzyme involved in chitin synthesis in the skin, nanoparticles, and a surfactant can improve the RNAi efficiency of a target gene (in this example, the IAP1 gene and the CHMP4B gene) in a lepidopteran insect, even by transdermal administration.

[0132] (Example 2: Evaluation of mortality of beet armyworm 2) Except for using dsRNases 1 to 5, REase or PTDCP, and IAP1 and CHMP4B as target genes, mortality of beet armyworm was evaluated in the same manner as in Example 1. The dsRNAs used to suppress the expression of each gene were those prepared in <Synthesis of dsRNA to be used against beet armyworm>, and the double-stranded RNAs suppressing the expression of dsRNases 1 to 5 genes, REase gene, and PTDCP ​​gene were administered at a concentration of 1 μg / μl each, and the double-stranded RNAs suppressing the expression of IAP1 gene and CHMP4B gene were administered at a concentration of 2 μg / μl each.

[0133] The dsRNA-nanoparticle-surfactant complex solution was prepared using dsGFP appropriately so that the total concentration of double-stranded RNA was 7 μg / μl.

[0134] The results are shown in Figure 2. As shown in Figure 2, even when a dsRNA-nanoparticle-surfactant complex solution prepared using a combination of any two of the dsRNase 1 to 5 genes, the REase gene or the PTDCP ​​gene, and double-stranded RNA that suppresses the expression of the IAP1 gene and the CHMP4B gene was applied dropwise to the skin of beet armyworms, the mortality rate of the beet armyworms significantly increased compared to the control dsRNA-nanoparticle-surfactant complex solution.

[0135] (Example 3: Evaluation of mortality of beet armyworm 3) The mortality rate of beet armyworm was evaluated in the same manner as in Example 2, except that the CHA gene and UAP gene were used as target genes instead of the REase gene or PTDCP ​​gene. The dsRNA used to suppress the expression of each gene was prepared as described in <Synthesis of dsRNA used against beet armyworm>. The dsRNA-nanoparticle-surfactant complex solution was prepared using dsGFP appropriately so that the total concentration of double-stranded RNA was 9 μg / μl.

[0136] The results are shown in Figure 3. As shown in Figure 3, even the dsRNA-nanoparticle-surfactant complex solutions prepared using three combinations of dsRNase 1 to 5 genes and double-stranded RNAs that suppress the expression of the CHA gene, UAP gene, and IAP1 gene, and CHMP4B gene significantly increased the mortality rate of the beet armyworm when applied to the skin of the moth, compared to the control dsRNA-nanoparticle-surfactant complex solution.

[0137] (Example 4: Evaluation of mortality of beet armyworm 4) The mortality rate of beet armyworms was evaluated in the same manner as in Example 1, except that in addition to IAP1 and CHMP4B, the target genes were the molting hormone (ETH) gene and the ETH receptor (ETHR) gene, which are important genes for insect development.

[0138] The dsRNA used to suppress the expression of each gene was prepared as described in "Synthesis of dsRNA for use against beet armyworm." The dsRNA-nanoparticle-surfactant complex solution was prepared using dsGFP appropriately to achieve a total double-stranded RNA concentration of 11 μg / μL. When using only one type of double-stranded RNA to suppress the expression of a target gene important for development, the concentration of the double-stranded RNA was adjusted to 4 μg / μL. When using two types of double-stranded RNA, the concentration of each double-stranded RNA was adjusted to 2 μg / μL.

[0139] The results are shown in Figure 4. As shown in Figure 4, regardless of whether the IAP1 gene, CHMP4B gene, ETH gene, or ETHR gene was used as a gene important for insect development, the mortality rate of beet armyworms was increased when a dsRNA-nanoparticle-surfactant complex solution was formed in combination with double-stranded RNA that suppresses the expression of genes in groups A to C, compared to the control dsRNA-nanoparticle-surfactant complex solution.

[0140] Example 5: Evaluation of mortality of Spodoptera litura The mortality rate of the common cutworm was evaluated in the same manner as in Example 1, except that the common cutworm was used instead of the beet cutworm, the basic target gene set in the common cutworm shown in Table 2 was used instead of the basic target gene set in the beet cutworm shown in Table 1, and benzyldimethyldodecylammonium chloride was dropped onto the skin of the common cutworm as a control.

[0141] [Table 2]

[0142] dsRNA synthesis was performed in the same manner as for beet armyworm, and 400 bp artificial genes were synthesized (artificial genes for dsRNase 1, 2, 4, and 5: sequence numbers 6, 14, 26, and 34; artificial gene for REase: sequence number 42; artificial gene for PTDCP: sequence number 50; artificial gene for CHA: sequence number 58; artificial gene for UAP: sequence number 66; artificial gene for IAP1: sequence number 74; artificial gene for CHMP4B: sequence number 82), which were used as templates for dsRNA synthesis.

[0143] Using these artificial genes as templates and primers with T7 promoter sequences added to the 5' ends, DNA fragments with T7 promoter sequences added to both ends of partial sequences of target genes were amplified in the same manner as in Example 1. Using the PCR products as templates, dsRNAs of the target genes (dsRNases 1 to 2 and 4 to 5 (SEQ ID NOs: 8, 16, 28, 36), dsREase (SEQ ID NO: 44), dsPTDCP ​​(SEQ ID NO: 52), dsCHA (SEQ ID NO: 60), dsUAP (SEQ ID NO: 68), dsIAP1 (SEQ ID NO: 76), and dsCHMP4B (SEQ ID NO: 84)) were synthesized in the same manner as in Example 1.

[0144] The results are shown in Figure 5. When a dsRNA-nanoparticle-surfactant complex solution prepared using double-stranded RNA that suppresses the expression of genes in groups A to C was applied to the skin of Spodoptera litura, mortality was significantly increased compared to when a control dsRNA-nanoparticle-surfactant complex solution was applied to the skin of Spodoptera litura.

[0145] (Example 6: Evaluation of mortality of beet armyworm 5) The mortality rate of beet armyworms was evaluated in the same manner as in Example 1, except that double-stranded RNA that suppresses the expression of a specific gene was used as the target gene, a dsRNA-nanoparticle-surfactant complex solution was prepared and administered using compositions A to C shown in Table 3, and the dsRNA-nanoparticle-surfactant complex solution was applied to both first-instar beet armyworm larvae (day 2) and second-instar beet armyworm larvae (day 1). The values ​​in Table 3 are in μg / μL. As a control, double-stranded RNA that suppresses the expression of GFP was administered at a concentration of 16 μg / μL. The dsRNA that suppresses the expression of each gene was prepared as described in <Synthesis of dsRNA used against beet armyworm>.

[0146] [Table 3]

[0147] The results are shown in Figure 6. As shown in Figure 6, when the dsRNA-nanoparticle-surfactant complex solution of composition C, which was prepared using double-stranded RNA that suppresses the expression of dsRNase 1 to 5 genes, the REase gene, the PTDCP ​​gene, and the ETH gene and the ETHR gene, was applied dropwise to the skin of S. exigua, the mortality rate of first-instar larvae (day 2) of S. exigua was significantly increased compared to the control dsRNA-nanoparticle-surfactant complex solution. Furthermore, the mortality rate of second-instar larvae (day 1) of S. exigua was similar to that of first-instar larvae (day 2).

[0148] Even when the administration concentration of double-stranded RNA that suppresses the expression of the ETH gene and ETHR gene was changed, the effect of double-stranded RNA that suppresses the expression of the dsRNases 1 to 5 genes and the REase and PTDCP ​​genes on improving RNA efficiency was confirmed. Furthermore, the dsRNA-nanoparticle-surfactant complex solution prepared using double-stranded RNA that suppresses the expression of the dsRNases 1 to 5 genes and the REase and PTDCP ​​genes was effective against not only first-instar larvae of the beet armyworm, but also second-instar larvae of the beet armyworm.

[0149] Furthermore, when applied to the skin of S. exigua, both the dsRNA-nanoparticle-surfactant complex solution of Composition A, prepared using double-stranded RNA that suppresses the expression of dsRNases 3 to 5, REase and PTDCP, CHA and UAP, and ETH and ETHR genes, and the dsRNA-nanoparticle-surfactant complex solution of Composition B, prepared using double-stranded RNA that suppresses the expression of dsRNases 1 to 5, REase and PTDCP, CHA and UAP, and ETH and ETHR genes, significantly increased the mortality rate of first-instar (day 2) S. exigua larvae compared with the control dsRNA-nanoparticle-surfactant complex solution. The dsRNA-nanoparticle-surfactant complex solutions of Compositions A and B showed no significant effect on second-instar S. exigua larvae (results for Composition B are not shown).

[0150] As described above, the dsRNA-nanoparticle-surfactant complex solution of composition C was highly effective not only against first-instar larvae of S. exigua, but also against second-instar larvae of S. exigua. The dsRNA-nanoparticle-surfactant complex solution of composition C contained the same total amount of double-stranded RNA as the dsRNA-nanoparticle-surfactant complex solutions of compositions A and B, but had a higher total amount of double-stranded RNA that suppressed the expression of target genes important for development. These results suggest that a higher RNAi effect can be achieved even in older lepidopteran insects by simply changing the administration concentration of double-stranded RNA that suppresses the expression of target genes, such as those important for development, in the dsRNA-nanoparticle-surfactant complex solution.

[0151] (Example 7: Evaluation of mortality of beet armyworm 6) The mortality rate of S. exigua was evaluated in the same manner as in Example 1, except that the target genes were dsRNases 1 to 5, REase and PTDCP, and COPB and IDGF, COPB and CHMP4B, or ETH and ETHR; chitosan-based nanoparticles were used instead of carbon-based nanoparticles; the dsRNA-nanoparticle-surfactant complex solution was applied to first-instar larvae (day 1) or first-instar larvae (day 2) of S. exigua; and the double-stranded RNAs inhibiting the expression of dsRNases 1 to 5, REase, and PTDCP ​​were each administered at a concentration of 0.19 μg / μl; the double-stranded RNAs inhibiting the expression of COPB, IDGF, CHMP4B, ETH, and ETHR were each administered at a concentration of 0.84 μg / μl; and the double-stranded RNA concentration was 3 μg / μl. As a control, double-stranded RNA suppressing GFP expression was administered at a concentration of 3 μg / μl. The dsRNA suppressing the expression of dsRNases 1-5, REase, and PTDCP, CHMP4B, ETH, and ETHR genes was prepared as described in <Synthesis of dsRNA for use against S. exigua>. The dsRNA suppressing the expression of COPB and IDGF genes was prepared as described below in the same manner as described in <Synthesis of dsRNA for use against S. exigua>.

[0152] For each of the COPB and IDGF gene sequences registered in the database (GenBank), a 400-bp partial sequence (COPB partial sequence: SEQ ID NO: 94; IDGF partial sequence: SEQ ID NO: 95) for which primers for amplifying 200-400 bp DNA could be designed was selected and artificially synthesized using the Primer Design Tool website (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The partial sequence (artificial gene) was then cloned into the pUCFa vector (ampicillin resistant) (Fasmac Co., Ltd.). These artificial genes were used as templates for dsRNA.

[0153] Using these artificial genes as templates and primers with T7 promoter sequences attached to the 5' ends, DNA fragments with T7 promoter sequences attached to both ends of the partial sequences of the target genes were amplified by PCR using GoTaq® Green Master MIX (Promega). Using the PCR products as templates, dsRNAs of the target genes (dsCOPB (SEQ ID NO: 96) and dsIDGF (SEQ ID NO: 97)) were synthesized using the T7 RiboMAX Express Large Scale RNA Production System.

[0154] The results are shown in Figure 7. As shown in Figure 7, even when a dsRNA-nanoparticle-surfactant complex solution prepared using double-stranded RNA that suppresses the expression of genes targeting dsRNase 1 to 5, REase and PTDCP, COPB and IDGF, COPB and CHMP4B, or ETH and ETHR, and chitosan-based nanoparticles as nanoparticles was used, the mortality rate of beet armyworms was significantly increased when applied to the skin of beet armyworms compared to the control dsRNA-nanoparticle-surfactant complex solution. Furthermore, compared to the results of Example 6, the mortality rate of beet armyworms was higher when a dsRNA-nanoparticle-surfactant complex solution prepared using chitosan-based nanoparticles as nanoparticles was used compared to a dsRNA-nanoparticle-surfactant complex solution prepared using carbon-based nanoparticles as nanoparticles.

Claims

1. An agent for improving RNAi efficiency against a target gene in a lepidopteran insect, The composition contains a complex of double-stranded RNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant, and the predetermined gene is the target gene, At least two genes encoding double-stranded RNases, and At least one gene encoding a nuclease having a PIN domain, and / or An enhancer comprising at least two genes encoding enzymes involved in chitin synthesis in the skin.

2. The predetermined gene is the target gene, at least two genes encoding the double-stranded RNase, and at least one gene encoding the nuclease having a PIN domain, or The agent according to claim 1, comprising the target gene, at least two genes encoding the double-stranded RNase, at least one gene encoding the nuclease having a PIN domain, and at least two genes encoding enzymes involved in chitin synthesis in the skin.

3. The agent according to claim 1 , wherein the at least one gene encoding the double-stranded RNase is of three or more types.

4. The agent according to claim 1 , wherein the double-stranded RNA degrading enzyme comprises dsRNase.

5. The agent according to claim 1 , wherein the nuclease having a PIN domain comprises a REase and / or a PIN domain and transmembrane domain-containing protein (PTDCP).

6. The agent according to claim 1, wherein the enzymes involved in chitin synthesis in the skin include chitin synthase A and UDP-N-acetylglucosamine pyrophosphorylase.

7. The enhancer of claim 1 , wherein the nanoparticles comprise carbon-based nanoparticles.

8. The enhancer of claim 1 , wherein the nanoparticles comprise chitosan-based nanoparticles.

9. The enhancer of claim 1 , wherein the surfactant comprises a cationic surfactant.

10. The improver according to any one of claims 1 to 9, The agent for controlling lepidopteran insects, wherein the target gene is at least one gene involved in the growth, survival, development and / or reproduction of lepidopteran insects.

11. A method for improving RNAi efficiency for a target gene in a lepidopteran insect, The method comprises introducing into a lepidopteran insect a composition containing a complex of double-stranded RNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant, wherein the predetermined gene is the target gene, At least two genes encoding double-stranded RNases, and At least one gene encoding a nuclease having a PIN domain, and / or at least two genes encoding enzymes involved in chitin synthesis in the skin.

12. A method for controlling lepidopteran insects in a plant, comprising: The method comprises introducing into a lepidopteran insect a composition containing a complex of double-stranded RNA that suppresses the expression of a predetermined gene, nanoparticles, and a surfactant, wherein the predetermined gene is At least one gene involved in the growth, survival, development and / or reproduction of a lepidopteran insect; At least two genes encoding double-stranded RNases, and At least one gene encoding a nuclease having a PIN domain, and / or at least two genes encoding enzymes involved in chitin synthesis in the skin.

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