Method for producing sterile lepidopteran insects and composition for sterilizing lepidopteran insects

Abstract

To provide methods for producing an infertile lepidopteran insect by a sterilization method that can simply, stably, and efficiently sterilize lepidopteran insects and cause abnormalities only in germline cells, and to provide sterilizing compositions capable of performing the method.SOLUTION: The expression of the nanosO gene and the nanosP gene in a host lepidopteran insect is suppressed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing sterile lepidopteran insects and a composition for sterilizing lepidopteran insects to be used in said method. [Background technology]

[0002] Genetic modification techniques, such as genetic recombination, are essential for the functional analysis of genes and proteins, as well as for the production of proteins and other substances. Traditionally, Escherichia coli and yeast have been used primarily as host organisms for genetic recombination. However, these organisms are not considered suitable for use as mass production systems for proteins and other substances.

[0003] Therefore, in recent years, silkworms (Bombyx mori) have been attracting attention as a host for mass production of proteins. Silkworms are insects that have long been used industrially to produce silk, and because they spin cocoons during the pre-pupal stage, they can produce large amounts of silk thread in a short period of time. This is due to the high protein production capacity of the silkworm's silk gland. Utilizing this production capacity to create genetically modified silkworms (transgenic silkworms) and mass-produce useful proteins other than silk thread has attracted attention.

[0004] On the other hand, substance production systems using genetic engineering technology pose the problem of genetic environmental contamination due to the release of genetically modified organisms into the wild. Because silkworms have completely lost their ability to fly, they are approved for Type 1 use under the Cartagena Protocol, meaning that they can be used without preventing their spread into the environment. However, the possibility of hybridization between male silkworms and female silkworms cannot be ruled out; this is because the closely related species, the mandarin silkworm (Bombyx mandarina), has the ability to fly and can invade from the outside. Therefore, the development of a sterilization technique for silkworms that does not produce offspring is industrially important, both for maintaining useful strains and preventing the spread of genetically modified organisms into the environment.

[0005] Methods for sterilizing insects have been developed and implemented mainly for producing sterile insects in the Sterile Insect Technique (SIT), including, for example, irradiation.

[0006] The radiation irradiation method is a method of sterilizing target insects by irradiating the germ cells of the target insects with radiation such as X-rays or gamma rays, thereby inactivating sperm, causing egg loss, reducing the number of eggs laid, and preventing mating (Non-Patent Documents 1 and 2). However, this method has safety concerns, such as the need for an irradiation facility, and concerns that radiation exposure may reduce the vitality of the insects. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Agriculture, Forestry and Fisheries Journal (1980), Vol. 3, No. 2, pp. 32-34 [Non-patent document 2] Chemistry and Biology (1993), vol.31, No.2, pp.137-139 Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present invention is to develop a new sterilization method that can sterilize Lepidoptera insects simply, stably, and efficiently and that causes abnormalities only in germline cells, and to develop and provide a method for producing sterile Lepidoptera insects using the same, and a sterilization composition that can be used to carry out the method. [Means for solving the problem]

[0009] The present inventors conducted extensive research aimed at solving the above-mentioned problems and found that when the nanosO and nanosP genes, out of the four nanos genes present in Lepidoptera insects (nanosM gene, nanosN gene, nanosO gene, and nanosP gene), were double suppressed during embryonic development, significant abnormalities thought to be due to germline dysgenesis, such as a decrease in the number of mature eggs in the adult ovaries or egg disappearance, and testicular dwarfism, occurred, resulting in sterility. However, it was also revealed that all cells except the germline remained normal. By applying this phenomenon, it is possible to genetically sterilize Lepidoptera insects. The present invention is based on these findings and provides the following.

[0010] (1) A method for producing a sterile Lepidoptera insect, comprising a step of suppressing the expression of the nanosO gene and the nanosP gene. (2) The production method described in (1), wherein the expression of the gene is suppressed using a gene knockdown method. (3) The production method according to (2), wherein the gene knockdown method is the RNAi method or the antisense oligonucleotide method. (4) The method according to any one of (1) to (3), wherein the nanosO gene comprises a nucleotide sequence encoding a nanosO protein having the amino acid sequence shown in the following (a) to (c): (a) the amino acid sequence shown in SEQ ID NO: 1; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1; or (c) an amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 1 (5) The production method according to (4), wherein the nanosO gene consists of the base sequence shown in SEQ ID NO: 2. (6) The method according to any one of (1) to (5), wherein the nanosP gene comprises a nucleotide sequence encoding a nanosP protein having the amino acid sequence shown in the following (d) to (f): (d) the amino acid sequence shown in SEQ ID NO: 3; (e) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 3; or (f) an amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 3 (7) The method according to (6), wherein the nanosP gene consists of the base sequence shown in SEQ ID NO: 4. (8) Sterile lepidopteran insects in which the expression of the nanosO and nanosP genes is suppressed. (9) The sterile Lepidoptera insect according to (8), wherein the expression is suppressed by gene knockdown of each gene. (10) The sterile Lepidoptera insect according to (9), wherein the gene knockdown method is an RNAi method or an antisense oligonucleotide method. (11) A composition for sterilizing lepidopteran insects, comprising as an active ingredient a gene expression inhibitor that suppresses the expression of the nanosO gene and the nanosP gene. (12) The sterilization composition according to (11), wherein the gene expression inhibitor is a transcription product inhibitor that targets the transcription product of each of the genes. (13) The sterilization composition according to (12), wherein the transcription inhibitor is an RNAi agent or an antisense oligonucleotide. (14) The sterilization composition according to (13), wherein the RNAi agent is an expression vector operably carrying a nucleic acid encoding an shRNA for each of the genes. (15) The sterilization composition according to (11), wherein the gene expression inhibitor is a translation product inhibitor that targets the translation product of each of the genes. [Effects of the Invention]

[0011] According to the method for producing sterile lepidopteran insects of the present invention, sterile lepidopteran insects having abnormalities only in germline cells can be produced simply, stably, and efficiently.

[0012] Furthermore, the composition for sterilizing lepidopteran insects of the present invention can easily, stably, and efficiently sterilize any desired lepidopteran insect. [Brief explanation of the drawings]

[0013] [Figure 1]FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosO-RNAi, and the number of individuals with that number of mature eggs. [Figure 2] FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosP-RNAi, and the number of individuals with that number of mature eggs. [Figure 3] FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosM / O-RNAi, and the number of individuals with that number of mature eggs. [Figure 4] FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosN / O-RNAi, and the number of individuals with that number of mature eggs. [Figure 5] FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosO / P-RNAi, and the number of individuals with that number of mature eggs. [Figure 6] These are photographs of the morphology of testes excised from males treated with nanos-RNAi. A shows the testes of a wild-type adult male, B shows the testes of a nanosO-RNAi-treated male, and C shows the testes of a nanosO / P-RNAi-treated male. In C, the arrowhead indicates the dwarfed testes, and the arrow indicates the transparent area. [Figure 7] FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosM-RNAi, and the number of individuals with that number of mature eggs. [Figure 8] FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosN-RNAi, and the number of individuals with that number of mature eggs. [Figure 9] FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosM / P-RNAi, and the number of individuals with that number of mature eggs. [Figure 10] FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosN / P-RNAi, and the number of individuals with that number of mature eggs. [Figure 11] FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanossM / N / O-RNAi, and the number of individuals with that number of mature eggs. [Figure 12]FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosM / N / P-RNAi, and the number of individuals with that number of mature eggs. [Figure 13] FIG. 1 shows the number of mature eggs in the ovaries of female silkworms treated with nanosM / N / O / P-RNAi, and the number of individuals with that number of mature eggs. DETAILED DESCRIPTION OF THE INVENTION

[0014] 1. Method for producing sterile lepidopteran insects 1-1. Overview A first aspect of the present invention is a method for producing sterile Lepidopteran insects. The method of the present invention comprises a step of suppressing the expression of two nanos genes in a target Lepidopteran insect, disrupting the function of these genes to induce abnormalities in the insect's germline cells and produce a sterile Lepidopteran insect.

[0015] 1-2.Definition of Terms The definitions of terms frequently used in this specification are explained below. "Lepidoptera insects" refers to insects that belong to the taxonomic order Lepidoptera, and include butterflies and moths. Butterflies include insects that belong to the families Nymphalidae, Papilionidae, Pieridae, Lycaenidae, and Hesperiidae. Moths include insects that belong to the families Saturniidae, Bombycidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Geometridae, Archtiidae, Noctuidae, Pyralidae, and Sphingidae. For example, moths include species belonging to the genera Bombyx, Samia, Antheraea, Saturnia, Attacus, and Rhodinia, specifically silkworms, Bombyx mandarina, Samia cynthia (including Samia cynthia ricini and hybrids of Samia cynthia and Samia ricini), Antheraea yamamai, Antheraea pernyi, Saturnia japonica, and Actias gnoma, but the Lepidoptera insects of the present invention are not limited to these. Silkworms are preferred.

[0016] As used herein, the term "target Lepidopteran insect" refers to a Lepidopteran insect to which the method for producing a sterile Lepidopteran insect of the present invention is applied, or a Lepidopteran insect to which the composition for sterilizing Lepidopteran insects of the present invention is administered.

[0017] As used herein, "infertility" refers to the loss or significant reduction of fertility, resulting in the loss or reduction of the reproductive ability to produce individuals of the next generation. Infertility as used herein can be caused primarily by, but is not limited to, abnormalities in germ line cells.

[0018] As used herein, the term "sterilization" refers to changing an individual in a normal state to an infertile state.

[0019] As used herein, "infertility" refers to a state of infertility or having the characteristics of infertility.

[0020] As used herein, the term "germline cells" refers to eggs or ova and sperm that are directly involved in reproduction, and cells that will become eggs and sperm in the future. Specifically, it includes eggs (ova) and sperm, oogonia, oocytes, spermatogonia, spermatocytes, sperm cells, and primordial germ cells (PGCs) that will differentiate into the above cells in the future.

[0021] As used herein, the term "germline cell abnormality" refers to aplasia such as loss of germline cells, as well as abnormalities in morphology and properties.

[0022] As used herein, "suppressing gene expression" or "suppressing gene expression" is also referred to as gene knockdown, and refers to suppressing the expression and function of the protein encoded by that gene. Specifically, this includes suppressing the function of the transcription product (mRNA) or translation product (protein) of the target gene at the transcription stage, post-transcription, translation stage, or post-translation stage in the expression of the target gene. Gene knockdown is distinct from gene knockout, which destroys the target gene and completely eliminates the function of the protein encoded by that gene.

[0023] As used herein, the term "expression vector" refers to a vector that contains an operably encoded nucleic acid molecule encoding a protein or functional nucleic acid and that can control the expression of that nucleic acid molecule. Examples include plasmids. Furthermore, as used herein, "operably" refers to placing a nucleic acid molecule of interest under the control of a promoter within the expression vector. This results in a state in which expression of the nucleic acid molecule of interest is initiated by the activity of the promoter.

[0024] As used herein, the term "nucleic acid molecule" refers to a gene encoding a protein, or a polynucleotide or oligonucleotide encoding a functional nucleic acid. While not limited thereto, nucleic acid molecules are primarily composed of natural nucleic acids, such as DNA and / or RNA. However, they may also include artificial nucleic acids.

[0025] As used herein, the term "functional nucleic acid" refers to a nucleic acid molecule that has a specific biological function in a living organism or cell, such as an enzymatic function, a catalytic function, or a biological inhibitory or enhancing function (e.g., inhibition or enhancement of transcription or translation). Specific examples include RNAi agents, nucleic acid aptamers (such as DNA aptamers or RNA aptamers), antisense oligonucleotides, and nucleic acid enzymes.

[0026] As used herein, "genetic modification" refers to the artificial modification of the natural genetic information of a host organism. Modification of genetic information here includes addition, deletion, substitution, etc. of genetic information. Artificial modification of genetic information includes gene recombination and genome editing.

[0027] "Genetic recombination" includes methods of adding foreign genetic information not possessed by the host organism into the genome, etc., using vectors such as plasmids or transposons, or methods of modifying or destroying genetic information possessed by the host organism.

[0028] "Genome editing" is a gene targeting technology that utilizes DNA repair mechanisms associated with double strand breaks (DSBs) caused by DNA-cleaving enzymes to insert foreign genes (knock-in) or destroy target genes (knock-out) at any location in the genome.

[0029] 1-3. Method The method for producing sterile Lepidoptera insects of the present invention includes a gene expression suppression step as an essential step, which will be described in detail below.

[0030] 1-3-1. Gene expression suppression process The "gene expression suppression step" is a step of suppressing the expression of the nanosO gene and the nanosP gene (herein, these genes are often collectively referred to as "nanosO / P genes") in a target lepidopteran insect. Research results by the present inventors have revealed that disrupting the function of the two proteins, the nanosO protein and the nanosP protein (herein, these proteins are often collectively referred to as "nanosO / P proteins"), at the embryonic development stage in lepidopteran insects inhibits the formation of germline cells, resulting in sterility in both males and females. This step utilizes this phenomenon to suppress the expression of the nanosO / P genes in a target lepidopteran insect, thereby sterilizing the insect.

[0031] The "nanos gene" (or nos gene) encodes the nanos protein, an evolutionarily conserved protein with a zinc finger motif. Studies using fruit flies (Drosophila melanogaster) suggest that the nanos protein plays a role in the survival and proliferation of primordial germ cells (PGCs) (Keuckelaere ED et al., 2018, Cell Mol Life Sci., 75:1929-1946). Unlike other insects, lepidopterans possess four nanos paralogs (nanosM, nanosN, nanosO, and nanosP) (Nakao H., et al., 2008, Evolution & Development, 10(5): 548-554; Carter JM, et al., 2015, PLoS ONE, 10: e0144471). Similarly, four paralogous genes (Bm-nosM, Bm-nosN, Bm-nosO, and Bm-nosP) have been identified in silkworms, and tissue expression studies suggest that the Bm-nosO protein is important for the formation of primordial germ cells. The phenotype of Bm-nosO gene knockout silkworms, generated using genome editing technology, showed abnormal oogenesis and, in rare cases, a reduced number of mature eggs, suggesting that the Bm-nosO protein is involved in the egg (germ cell) formation process (Nakao H. and Takasu Y., 2019, Developmental Biology, 445:209-36). However, the specific functions of other nanos paralogs in lepidopteran insects remain unknown.

[0032] The nanosO / P gene to be targeted for expression suppression in this specification is not particularly limited as long as it is the nanosO / P gene of the target Lepidoptera insect for which sterilization is desired. The nanosO gene ortholog and nanosP gene ortholog of each species can be targeted. Their nucleotide sequences may be those that encode the respective nanos proteins.

[0033] For example, in the case of the nanosO gene, if the target Lepidoptera insect is a silkworm, examples of such genes include a wild-type Bm-nosO gene encoding a wild-type Bm-nosO protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a mutant Bm-nosO gene encoding a mutant Bm-nosO protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, or added to the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity to the amino acid sequence set forth in SEQ ID NO: 1. A specific example of the wild-type Bm-nosO gene is a Bm-nosO gene consisting of the nucleotide sequence set forth in SEQ ID NO: 2. As used herein, "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Furthermore, "(amino acid) substitution" refers to substitution within a conservative amino acid group that has similar properties, such as charge, side chain, polarity, and aromaticity, among the 20 amino acids that constitute natural proteins. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to be less likely to cause changes in the properties of polypeptides. Furthermore, "amino acid identity" refers to the percentage (%) of identical amino acid residues in one amino acid sequence relative to the total number of amino acid residues in the other amino acid sequence when two amino acid sequences are aligned and gaps are introduced into one or both amino acid sequences as necessary to maximize the degree of amino acid identity between the two.

[0034] In the case of the nanosP gene, if the target Lepidoptera insect is a silkworm, examples of such genes include a wild-type Bm-nosP gene encoding a wild-type Bm-nosP protein consisting of the amino acid sequence set forth in SEQ ID NO: 3, or a mutant Bm-nosP gene encoding a mutant Bm-nosP protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity to the amino acid sequence set forth in SEQ ID NO: 3. A specific example of the wild-type Bm-nosP gene is a Bm-nosP gene consisting of the nucleotide sequence set forth in SEQ ID NO: 4.

[0035] In this step, the expression of the two nanos genes is suppressed in the target Lepidoptera insect to be sterilized. The method for suppressing gene expression will be specifically described below.

[0036] A. Methods for suppressing gene expression As used herein, the term "method for suppressing gene expression" refers to a method for specifically suppressing the expression of a target gene. Methods for suppressing the expression of a specific gene in lepidopteran insects may be any method known in the art, and are not particularly limited. Examples include gene knockdown methods and methods in which low-molecular-weight compounds that specifically suppress the functions of two target proteins, nanosO / P proteins, are administered to the target lepidopteran insect. Gene knockdown methods are particularly preferred because they are technically established, simple, and expected to be highly effective.

[0037] "Gene knockdown" is a method for depleting target gene products in cells via (poly)nucleotides or (poly)peptides. It can target gene products after transcription but before translation of the target gene, i.e., gene transcription products such as mRNA, or it can target translation products after translation, i.e., proteins. Specific examples of gene knockdown methods include (1) the RNAi method, (2) the antisense oligonucleotide method, (3) the nucleic acid enzyme method, and (4) the aptamer method. Each method is explained below.

[0038] (1)RNAi method The "RNAi method (RNA interference method)" is a method in which an RNAi agent is administered to a host and the expression of a target gene is suppressed at the post-transcriptional, pre-translational, or transcriptional level by utilizing the RNA interference (RNAi) of the RNAi agent. RNA interference is sequence-specific gene silencing that suppresses the expression of a target gene through, for example, degradation of the transcript of that gene. In this specification, this method is performed using an RNAi agent that induces specific gene silencing for each transcript of the nanosO / P genes. The RNAi agent in this specification is assumed to be a mechanism that suppresses the expression of the nanosO gene or nanosP gene of Lepidoptera insects at the post-transcriptional, pre-translational, level.

[0039] "RNAi agent" refers to a substance that induces RNA interference (RNAi) in vivo and suppresses (silences) the expression of a target gene through degradation of the gene's transcription product. Examples include artificially synthesized dsRNA, siRNA, or shRNA, and endogenous miRNA (microRNA) (including pri-miRNA and pre-miRNA). RNA interference is described in detail in, for example, Bass BL, 2000, Cell, 101, 235-238; Sharp PA, 2001, Genes Dev., 15, 485-490; Zamore PD, 2002, Science, 296, 1265-1269; Dernburg AF & Karpen, GH, 2002, Cell, 111, 159-162. In the present invention, the RNAi agents used are primarily dsRNA, siRNA, or shRNA, which can be arbitrarily designed to target the nanosO and nanosP genes. However, if miRNAs targeting the nanosO or nanosP genes exist, they can also be used. Each RNA agent is described in detail below.

[0040] (i) dsRNA (composition) "dsRNA" (double-stranded RNA) is a double-stranded RNA consisting of 50 to 1000 bases containing a desired RNA sense strand sequence selected from the base sequence of the sense strand of a target gene, and has been shown to be effective as an RNAi agent in insects. The administered dsRNA is ultimately processed into siRNA, which will be described later, within the cell, causing RNA interference. dsRNA may be designed by known methods based on the base sequence of the target gene.

[0041] (How to install) To achieve gene expression-suppressing effects, dsRNA can be introduced by administering in vitro prepared dsRNA to a host. While methods known in the art, such as microinjection or immersion in a dsRNA solution, are available for administration, in the present invention, where the host is a Lepidoptera insect, injection administration is preferred. The concentration of the dsRNA solution used for introduction should be such that a certain concentration or higher is present in the insect body (e.g., eggs) after introduction. More specifically, although not limited thereto, a solution with a concentration of several μg / μL to several tens of μg / μL can typically be injected in a volume of, for example, 2 nL to 40 nL, 5 nL to 30 nL, 8 nL to 25 nL, or 10 nL to 20 nL.

[0042] Microinjection is most effective when performed on eggs 2 to 8 hours after oviposition, before cleavage, when the nuclei are incorporated into the cell membrane. Alternatively, to achieve the same effect as egg injection, dsRNA can be introduced into mature eggs from females before oviposition, followed by fertilization and oviposition. A marker gene can also be introduced as a selectable marker at the same time as dsRNA introduction, making it easier to select individuals into which dsRNA has been introduced.

[0043] (Selected) After the introduction of dsRNA, the introduced individuals can be selected as necessary. The selection method is not limited. For example, a portion of the dsRNA-introduced individuals can be collected and the reduction in the expression level of the target gene compared to control individuals can be confirmed by RT-PCR or the like. Alternatively, as described above, when a marker gene is simultaneously introduced as a selection marker during the injection of dsRNA, the introduced individuals can be selected based on the activity of the marker gene.

[0044] As used herein, a "marker gene" refers to a gene that encodes a marker protein. A marker protein refers to a polypeptide whose activity can be used to determine whether or not a marker gene is expressed. "Based on activity" means "based on the results of activity detection." Activity may be detected directly by detecting the activity of the marker protein itself, or indirectly via a metabolite such as a pigment produced by the activity of the marker protein. Detection may be chemical detection (including enzyme reaction detection), physical detection (including behavioral analysis detection), or sensory detection by the detector (including detection by sight, touch, smell, hearing, or taste).

[0045] The type of labeled protein is not particularly limited, as long as its activity can be detected by a method known in the art. A labeled protein that is less invasive to the host lepidopteran insect upon detection is preferred. Examples include fluorescent proteins, pigment-synthesizing proteins, luminescent proteins, exocrine proteins, and proteins that control external morphology. Fluorescent proteins, pigment-synthesizing proteins, luminescent proteins, and exocrine proteins are particularly preferred because they can be visually detected under specific conditions, are less invasive to lepidopteran insects, and are easy to distinguish and select.

[0046] The fluorescent protein refers to a protein that emits fluorescence of a specific wavelength when a lepidopteran insect is irradiated with excitation light of a specific wavelength. It may be either natural or non-natural. The excitation wavelength and fluorescence wavelength are not particularly limited. Specific examples include CFP, AmCyan, RFP, DsRed (including derivatives such as DsRed monomer and DsRed2), YFP, GFP (including derivatives such as EGFP and EYFP), etc.

[0047] The pigment synthesis protein is a protein involved in pigment biosynthesis and is usually an enzyme. The "pigment" referred to here refers to a low molecular weight compound or peptide capable of imparting a pigment to a transformant, regardless of type. Preferably, the pigment is one that appears as the external color of the individual. Examples include melanin-based pigments (including dopamine melanin), ommochrome-based pigments, and pteridine-based pigments.

[0048] The photoprotein refers to a substrate protein that can emit light without requiring excitation light or an enzyme that catalyzes the luminescence of the substrate, such as aequorin and the enzyme luciferase.

[0049] (passage) In principle, the suppression effect of dsRNA on target genes lasts for only one generation.

[0050] (ii) siRNA (composition) "siRNA" (small interference RNA) is a small double-stranded RNA consisting of a sense strand (passenger strand) whose base sequence corresponds to a portion of the sense strand of a target gene, and an antisense strand (guide strand) that is its antisense strand. siRNA can induce RNA interference by introducing it into eukaryotic cells (Fire A. et al., 1998, Nature, 391, 806-811).

[0051] siRNAs can be designed based on the nucleotide sequence of a target gene by known methods, such as those described by Ui-Tei et al. (Nucleic Acids Res., 2004, 32:936-948), Reynolds et al. (Nat. Biotechnol., 2004, 22:326-330), or Amarzguioui et al. (Biochem. Biophys. Res. Commun., 2004, 316:1050-1058).

[0052] As a specific example of siRNA design, when the nanosO gene is used as the target gene, a contiguous base sequence of 15 to 35 bases, preferably 15 to 30 bases or 18 to 25 bases, is selected as the base sequence of the RNAi sense strand (passenger strand) from the base sequence shown in SEQ ID NO: 2. Care must be taken to ensure that the base sequence of the selected region completely matches the base sequence of the target gene. Therefore, it is preferable to design the selected region so that it does not include known mutation sites (e.g., SNPs) in the target gene. The base sequence of the RNAi antisense strand (guide strand) may be a base sequence complementary to the base sequence of the selected RNAi sense strand. Note that, when preparing siRNA, T (thymine) bases in the selected region of both the sense strand and the antisense strand are converted to U (uracil) bases.

[0053] The selected region of the RNAi sense strand is not particularly limited as long as it has a sequence specific to the target gene. It is preferably at least 50 bases downstream from the start codon, more preferably 70 to 100 bases downstream. Furthermore, it is preferable to select a base sequence region having AA (adenine-adenine) on the 5' side within the candidate region of the RNAi sense strand. The GC (guanine-cytosine) content within the selected region is preferably 20 to 80%, more preferably 30 to 70% or 40 to 60%. Numerous siRNA designs are available online, and effective and appropriate siRNAs can be designed online by inputting the base sequence of the target gene. Representative siRNA design websites include siDirect (http: / / sidirect2.rnai.jp / ) and siDESIGN Center (https: / / horizondiscovery.com / en / ordering-and-calculation-tools / sidesign-center).

[0054] One or both ends of the siRNA may contain a base sequence consisting of one or more DNAs, RNAs, and / or nucleic acid analogs that are unrelated to the base sequence of the target gene or a base sequence complementary thereto. The number of bases present at the ends of such siRNA is not particularly limited, but is preferably within the range of 1 to 20. Specific examples include adding TT (thymine-thymine) or UU (uracil-uracil) to the 3'-end of each base strand (Tuschl T et al., 1999, Genes Dev, 13(24):3191-7).

[0055] (How to install) The method for introducing siRNA is similar to the method for introducing dsRNA, and therefore, a detailed explanation is omitted here.

[0056] (Selected) After the introduction of siRNA, the introduced individuals can be selected as needed. The selection method is similar to the selection method for dsRNA described above, and therefore will not be explained here.

[0057] (passage) As with dsRNA, the suppression effect of siRNA on target genes is, in principle, only one generation long.

[0058] (iii) shRNA (composition) "shRNA" (short hairpin RNA) refers to a single-stranded RNA in which the two RNA strands (RNAi sense strand and RNAi antisense strand) that make up the siRNA are linked by a spacer sequence consisting of an appropriate base sequence. In other words, shRNA contains an RNAi sense region as the RNAi sense strand and an RNAi antisense region as the RNAi antisense strand within a single molecule, and these regions form a stem structure through base pairing with each other, and the spacer sequence forms a loop structure, so that the entire molecule has a hairpin stem-loop structure.

[0059] When shRNA is introduced into cells, the loop structure is cleaved to generate double-stranded RNA molecules, or siRNAs, which can suppress the expression of target genes through the same RNA interference mechanism as siRNAs described in the previous section.

[0060] For example, shRNA is designed by linking the 3' end of the sense region of the siRNA to the 5' end of the antisense strand with a spacer sequence. The spacer sequence may generally be 3 to 24 bases, preferably 4 to 15 bases. There are no particular limitations on the spacer sequence, as long as it is a sequence that allows base pairing with siRNA.

[0061] The shRNA can also be inserted so that the DNA encoding it is operably under the control of a promoter in an expression vector. When such an shRNA expression vector is introduced into a target organism or target cell, the promoter activates the shRNA to express the shRNA. After expression, the shRNA is processed into siRNA through self-folding within the host cell or the activity of Dicer, thereby exerting its effects. Furthermore, using this shRNA expression vector, the DNA encoding the shRNA can be inserted (knocked in) into the genome of a lepidopteran insect using the transposon method or genome editing method described below.

[0062] (How to install) The basic method for introducing shRNA is similar to the method for introducing dsRNA described above. Therefore, we will not discuss the common introduction methods for dsRNA, and will only discuss the unique introduction methods for shRNA.

[0063] As mentioned above, shRNA can also be introduced in the form of DNA as an shRNA expression vector, allowing the shRNA to be expressed in host cells. In this case, the method for introducing the shRNA expression vector can be similar to that for dsRNA introduction. When introducing the shRNA into eggs via mature female eggs, the shRNA expression vector can be injected into the abdominal cavity of the pupae and transferred to mature eggs, or the shRNA expression vector can be inserted (knocked in) into the genome of the lepidopteran insect. However, when introducing the shRNA expression vector, care must be taken to select a developmental stage-specific promoter so that the shRNA is not expressed in germline cells at developmental stages prior to egg maturation. This is because expression of shRNA in early germline cells may prevent the formation of eggs.

[0064] Methods for introducing shRNA expression vectors into the genome of lepidopteran insects include the transposon method and genome editing.

[0065] The "transposon method" utilizes the inverted terminal repeat sequence of a transposon (Handler AM. et al., 1998, Proc. Natl. Acad. Sci. USA 95:7520-5) and the activity of a transposon transferase to insert a foreign gene into a genome. Transposons that can be used in Lepidoptera include piggyBac, mariner, and minos, and any of these can be used (Shimizu, K. et al., 2000, Insect Mol. Biol., 9, 277-281; ​​Wang W. et al., 2000, Insect Mol. Biol. 9(2):145-55).

[0066] An exogenous gene can be introduced into a host genome by methods known in the art using an expression vector containing two transposon inverted terminal repeats and an exogenous gene located between them. For example, if the host is a silkworm, the method of Tamura et al. can be used (Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84). Briefly, an expression vector containing an exogenous gene and a helper vector containing a transposon transferase gene are injected into early silkworm embryos. An example of a helper vector is pHA3PIG. The activity of the transposon transferase produced by the helper vector allows the exogenous gene to be inserted into the silkworm genome by homologous recombination via the transposon inverted terminal repeats.

[0067] The "genome editing method" refers to a method using the aforementioned genome editing technology. Known genome editing methods include, for example, the zinc finger nuclease (ZFN) method, the TALEN method, and the CRISPR / Cas9 method, and any of these methods may be used herein. Gene targeting techniques using these methods are all well known in the art, and kits and support tools for efficiently knocking out target genes are commercially available from various life science manufacturers for the genome editing method used herein, and these may also be used. Regarding a method for inserting a foreign gene using genome editing in silkworms, a knock-in technique utilizing microhomology of TALEN cleavage sites has been published as the TAL-PITCH method, and this method may also be used (Nakade, S. et al., 2014, Nature Communications, 5:5560).

[0068] (Selected) Selection of shRNA-introduced individuals can be performed in the same manner as for siRNA. When introducing an shRNA expression vector, by inserting a marker gene into the vector, introduced individuals can be selected based on the activity of the marker gene.

[0069] (passage) As with siRNA, the suppression effect of shRNA on target genes generally lasts for only one generation. However, when shRNA is inserted into the genome of a host lepidopteran insect, the target gene can be suppressed by inducing expression of the shRNA in progeny, even if the lineage is passed down.

[0070] As used herein, the term "progeny" refers to a descendant of the first-generation genetically modified individuals that retain DNA encoding the shRNA of the present invention, as well as the asRNA, ribozyme, or RNA aptamer described below, in their genomes. The number of generations of the progeny is not important, as long as they retain the shRNA.

[0071] (2) Antisense oligonucleotide method The "antisense oligonucleotide (ASO) method" refers to a method in which an antisense oligonucleotide is administered to a host to suppress the expression of a target gene post-transcriptionally but before translationally, through the activity of the antisense oligonucleotide. The antisense oligonucleotide herein suppresses the expression of the nanosO gene or nanosP gene of a lepidopteran insect post-transcriptionally but before translationally.

[0072] (structure) An "antisense oligonucleotide (often referred to herein as "ASO")" is a single-stranded nucleic acid molecule that is composed of a base sequence complementary to all or part of the base sequence of mRNA, which is the transcription product of a target gene, and that hybridizes to the target mRNA to suppress its translation.

[0073] ASOs are known to be of DNA and RNA types. DNA-type ASOs are a nuclease-mediated gene suppression method that hybridizes to RNA molecules such as mRNA, which are transcription products of target genes, to form heteroduplex structures, and then cleave and degrade the target RNA molecules using intracellular RNase H activity to suppress target gene expression. On the other hand, RNA-type ASOs hybridize to RNA molecules such as mRNA, which are transcription products of target genes, to form double-stranded RNA, and then, after undergoing a process, can ultimately exert the same gene expression suppression effect through RNAi as siRNA, etc. Although not limited thereto, DNA-type ASOs are usually widely used due to their intracellular stability and ease of synthesis.

[0074] DNA-type ASOs are primarily composed of natural DNA, but may also contain natural RNA as well as nucleic acid analogs such as LNA / BNA (Locked Nucleic Acid / Bridged Nucleic Acid) and PMO (Phosphorodiamidate Morpholino Oligomers), and / or modified nucleic acids such as 2'-OMe-RNA, 2'-F-RNA, 2'-MOE-RNA, and phosphorothioates. They also include ASOs with special structures, such as gapmers, which are RNA-degrading ASOs with wing regions composed of nucleic acid analogs or modified nucleic acids at the 5' and 3' ends, and mixmers, which are splicing-controlling ASOs composed of nucleic acid analogs or modified nucleic acids.

[0075] Furthermore, the heteroduplex oligonucleotide (HDO) method, which is an application of the DNA-based ASO method, can also be used. A heteroduplex oligonucleotide (often abbreviated as "HDO" herein) is a double-stranded nucleic acid composed of a main strand (DNA strand) with ASO function and a cRNA (complementarily RNA) strand with a base sequence complementary to the main strand. The cRNA strand may also contain nucleic acid analogs or modified nucleic acids, but at least all or part of the central portion of the HDO is a heteroduplex nucleic acid composed of DNA and RNA. Therefore, the complementary cRNA strand can be cleaved and degraded by intracellular RNase H, and the isolated main strand can function as an ASO.

[0076] RNA ASOs consist of a base sequence similar to that of an antisense strand that can hybridize to a portion of mRNA, a transcription product of a target gene, and are generally composed of natural RNA. Therefore, to distinguish them from DNA ASOs, they are often referred to as "antisense RNA (asRNA)" in this specification.

[0077] The asRNA can also be inserted so that the DNA encoding it is operably under the control of a promoter in an expression vector. Similar to the shRNA expression vector described above, such an "antisense RNA expression vector (asRNA expression vector)" expresses the asRNA through the activity of the promoter when introduced into a target organism or target cell. After expression, the asRNA exerts its effects by hybridizing with the transcription product of the target gene within the host cell. Furthermore, similar to the shRNA expression vector, DNA encoding the asRNA can also be knocked into the genome of lepidopteran insects using the transposon method or genome editing method described below.

[0078] As a specific example of the base sequence design of an ASO, regardless of whether it is DNA or RNA, when the nanosO gene is used as the target gene, a base sequence complementary to a contiguous base sequence of 10 to 30 bases, preferably 12 to 25 bases, or 13 to 20 bases, from the base sequence of the mRNA strand shown in SEQ ID NO: 2 can be selected as the selected region. In this case, for example, a region encompassing the start codon or a region that can form a single-stranded structure in the predicted secondary structure of the mRNA strand can be selected as the target region.

[0079] (How to install) The method for introducing ASO is similar to the method for introducing siRNA. Therefore, a detailed explanation will be omitted here. As mentioned above, asRNA can be introduced in the form of DNA as an asRNA expression vector, similar to the shRNA expression vector, and the asRNA can be expressed in host cells. The method for introducing the asRNA expression vector may also be similar to the method for introducing the shRNA expression vector.

[0080] (Selected) Selection of ASO-introduced individuals can be performed in the same manner as for siRNA. When introducing an asRNA expression vector, a marker gene can be inserted into the vector, as with shRNA expression vectors, and introduced individuals can be selected based on the activity of the marker gene.

[0081] (passage) Like siRNA, the target gene suppression effect of ASOs generally lasts for only one generation. However, if DNA encoding asRNA is inserted into the genome of a host lepidopteran insect, the target gene can be suppressed by inducing asRNA expression in progeny, even if the lineage is passed down.

[0082] (3) Nucleic acid enzyme method The "nucleic acid enzyme method" refers to a method in which a nucleic acid enzyme is administered to a host, and the expression of a target gene is suppressed after transcription but before translation by the activity of the nucleic acid enzyme. The nucleic acid enzyme used herein suppresses the expression of the nanosO gene or nanosP gene of a lepidopteran insect after transcription but before translation.

[0083] (composition) A "nucleic acid enzyme" is a nucleic acid molecule with catalytic activity that specifically binds to a target mRNA as a substrate, cleaves a specific site in the target mRNA, and can suppress the expression of a target gene after transcription but before translation. The nucleic acid enzyme used herein suppresses the expression of the nanosO gene or nanosP gene of a lepidopteran insect after transcription but before translation.

[0084] Known nucleic acid enzymes include deoxyribozymes, which are composed of DNA, and ribozymes, which are also called riboenzymes and are composed of RNA, but the nucleic acid enzymes referred to herein may be either. Both deoxyribozymes and ribozymes may contain chemically modified nucleic acids, artificial nucleic acids, and / or nucleic acid analogs as part of their constituent bases.

[0085] For ribozymes composed of RNA, DNA encoding the ribozyme can be inserted into an expression vector so that it is operably under the control of a promoter. Similar to the shRNA expression vector described above, when such a "ribozyme expression vector" is introduced into a target organism or target cell, the ribozyme is expressed by the activity of the promoter.

[0086] (How to install) The method for introducing the nucleic acid enzyme is similar to the method for introducing the dsRNA. Therefore, detailed explanations will be omitted here. As mentioned above, the ribozyme can also be introduced in the form of DNA as a ribozyme expression vector, similar to the shRNA expression vector, to express the ribozyme in the host cell. The method for introducing the ribozyme expression vector can also be similar to the method for introducing the shRNA expression vector.

[0087] (Selected) Selection of individuals into which the nucleic acid enzyme has been introduced can be performed in the same manner as for siRNA. When a ribozyme expression vector is introduced, a marker gene can be inserted into the vector in the same way as for shRNA expression vectors, and introduced individuals can be selected based on the activity of the marker gene.

[0088] (passage) Like siRNA, the target gene suppression effect of nucleic acid enzymes generally lasts for only one generation. However, if DNA encoding a ribozyme is inserted into the genome of a host lepidopteran insect, the target gene can be suppressed by inducing ribozyme expression in progeny, even if the lineage is passed down.

[0089] (4) Aptamer method The "aptamer method" is a method for post-translationally suppressing target gene expression by administering an aptamer to a host and inhibiting the function of a target protein through its target binding activity. The aptamer herein binds to the nanosO protein or nanosP protein and post-translationally suppresses the expression of the nanosO gene or nanosP gene in lepidopteran insects.

[0090] (structure) An "aptamer" is a ligand molecule that binds tightly and specifically to a target substance due to its three-dimensional molecular structure, thereby inhibiting the function of the target substance. Aptamers have the same functional effects as antibodies, but generally have higher specificity and affinity for the target substance than antibodies. Furthermore, aptamers are superior to antibodies in that they can distinguish between closely related molecules because fewer amino acid residues of the target are required for binding compared to antibodies. Furthermore, aptamers have lower immunogenicity and toxicity than antibodies, and can be produced in a short period of time (approximately 3-4 weeks), and can also be mass-produced by chemical synthesis.

[0091] Aptamers can be broadly classified into nucleic acid aptamers and peptide aptamers depending on the type of constituent molecule. The aptamer in this specification may be either type of aptamer, but is preferably a nucleic acid aptamer.

[0092] As used herein, a "nucleic acid aptamer" refers to an aptamer composed of nucleic acid, which binds strongly and specifically to a target substance through a three-dimensional structure formed based on the secondary structure and further tertiary structure of a single-stranded nucleic acid molecule mediated by hydrogen bonds, etc.

[0093] Although nucleic acid aptamers are generally known as RNA aptamers composed of RNA and DNA aptamers composed of DNA, the nucleic acids constituting the nucleic acid aptamers herein are not particularly limited. Examples include DNA aptamers, RNA aptamers, and aptamers composed of a combination of DNA and RNA. While typically composed solely of natural nucleic acids (DNA or RNA), they may also contain non-natural artificial nucleic acids or modified nucleic acids. The base length of the aptamer of the present invention is not limited, but is preferably within the range of 10 to 100 bases, and more preferably within the range of 15 to 80 bases. Aptamers are a well-known technology, and for details, see, for example, Janasena, Clin. Chem. 45:1628-1650 (1999).

[0094] RNA aptamers can be prepared by in vitro selection using, for example, the SELEX (systematic evolution of ligands by exponential enrichment) method. The SELEX method involves selecting RNA molecules that bind to a target molecule (in this case, the nanosO protein or the nanosP protein) from an RNA pool composed of RNA molecules with a random sequence region and primer binding regions at both ends. After recovery, the RNA molecules are amplified by RT-PCR, and then transcribed using the resulting cDNA molecules as templates to create an RNA pool for the next round. This cycle is repeated several to several dozen times to select RNAs with stronger binding affinity to the target molecule. The lengths of the random sequence region and primer binding region are not particularly limited. Typically, the random sequence region is 20 to 80 bases long, and the primer binding region is 15 to 40 bases long, respectively. To enhance specificity for a target molecule, a molecule similar to the target molecule can be mixed with the RNA pool, and the pool of RNA molecules that did not bind to the target molecule can be used. The RNA molecules finally obtained by this method are used as RNA aptamers. The SELEX method is a known method, and the specific method may be carried out, for example, according to Pan et al. (Proc. Natl. Acad. Sci. USA, (1995) 92: 11509-11513).

[0095] When the nucleic acid aptamer is composed of natural RNA, the DNA encoding it can be inserted into an expression vector so that it is operable under the control of a promoter. Similar to the shRNA expression vector described above, this "RNA aptamer expression vector" is introduced into a target organism or target cell, where it expresses the RNA aptamer through the activity of the promoter. After expression, it binds to the target protein in the host cell and suppresses its function. Furthermore, similar to the shRNA expression vector, DNA encoding the RNA aptamer can be inserted (knocked in) into the genome of a lepidopteran insect using the transposon method or genome editing method described below.

[0096] (How to install) The method for introducing the aptamer is similar to the method for introducing the siRNA. Therefore, detailed explanations will be omitted here. As mentioned above, the ribozyme can be introduced in the form of DNA as an RNA aptamer expression vector, similar to the shRNA expression vector, to express the RNA aptamer in the host cell. The method for introducing the RNA aptamer expression vector may also be similar to the method for introducing the shRNA expression vector.

[0097] (Selected) Selection of aptamer-introduced individuals can be performed in the same manner as for siRNA. When introducing an RNA aptamer expression vector, a marker gene can be inserted into the vector, as with shRNA expression vectors, and introduced individuals can be selected based on the activity of the marker gene.

[0098] (passage) As with siRNA, the target gene suppression effect of aptamers generally lasts for only one generation. However, when DNA encoding an RNA aptamer is inserted into the genome of a host lepidopteran insect, the target gene can be suppressed by inducing expression of the RNA aptamer in progeny, even if the lineage is passed on.

[0099] 2. Sterile Lepidoptera 2-1. Overview A second aspect of the present invention is a sterile Lepidopteran insect. The sterile Lepidopteran insect of the present invention is characterized in that expression of the nanosO / P gene is suppressed. As a result, fertility is reduced or lost in both males and females. The sterile Lepidopteran insect of the present invention can be produced by the method for producing a sterile Lepidopteran insect described in the first aspect.

[0100] 2-2.Configuration Sterile lepidopteran insects are characterized by the suppression of nanosO / P gene expression in germline cells during early embryonic development. This can be achieved by suppressing the expression of both nanosO / P genes (double knockdown).

[0101] The sterile Lepidoptera insect in which gene expression is suppressed may be, but is not limited to, an individual obtained through a gene knockdown method in the method for producing a sterile Lepidoptera insect described in the first aspect, or an individual in which gene expression suppression has been induced in progeny containing a gene expression inhibitor in the genome.

[0102] The type of sterile Lepidoptera insect is not limited, but preferably includes industrially useful insects, such as species used for silk production, such as Bombyx mori, Manchuria cocoon, Cynthia striola (Eri silkworm), Saturniidae moth, Scutellaria japonica, and Scutellaria nigra. Bombyx mori is preferred.

[0103] The developmental stage of the sterile Lepidoptera insect is not limited, and may be any of egg, larva, pupa, and adult. However, for the purpose of sterility, it is preferably an adult which is naturally capable of reproduction.

[0104] Sterile Lepidoptera insects of the present invention can be distinguished from fertile Lepidoptera insects based on molecular genetic techniques and / or phenotypic characteristics.

[0105] Distinction based on molecular genetic techniques can be achieved, for example, by measuring or detecting the expression level of the nanosO / P gene using a fertile Lepidoptera insect of the same species with normal nanosO / P gene expression as a control. Methods known in the art, such as RT-PCR and Northern hybridization, can be used to measure or detect gene expression levels. In the sterile Lepidoptera insects of the present invention, the expression level of the nanosO / P gene is significantly reduced (e.g., p<0.05, p<0.01, p<0.001) compared to that of the control individuals.

[0106] Discrimination based on phenotypic characteristics can be confirmed by observing morphological abnormalities in reproductive organs or germ cells. In the case of sterile Lepidoptera insects of the present invention, adult females lay significantly fewer eggs than control individuals, or do not lay eggs at all. Adult males also exhibit dwarfed and transparent testes and reduced fertility.

[0107] 3. Lepidoptera insect sterilization composition Overview A third aspect of the present invention is a composition for sterilizing lepidopteran insects. The sterilizing composition of the present invention contains, as an active ingredient, a gene expression inhibitor that specifically suppresses the expression of the nanosO gene and the nanosP gene. By using the sterilizing composition of the present invention, any type of lepidopteran insect can be sterilized easily and efficiently.

[0108] 3-2.Configuration The components of the lepidopteran insect sterilization composition of the present invention will now be described.

[0109] 3-2-1. Active ingredients The sterilizing composition for lepidopteran insects of the present invention contains as active ingredients at least two gene expression inhibitors that inhibit the expression of each of the nanosO / P genes. A "gene expression inhibitor" is an agent that specifically inhibits the expression of either the nanosO gene or the nanosP gene, which are target genes. Gene expression inhibitors are divided into transcription product inhibitors and translation product inhibitors based on their inhibitory effect on gene expression.

[0110] (1) Transcription inhibitors As used herein, a "transcription inhibitor" refers to an agent that targets mRNA, a transcription product of the target gene nanosO or nanosP, and degrades or inactivates it to inhibit the expression of that gene. Specific examples of transcription inhibitors include RNAi agents, antisense oligonucleotides (ASOs), and nucleic acid enzymes. The specific configurations of these agents are similar to those of the RNAi agents, ASOs, and nucleic acid enzymes described in detail in the first embodiment (1) RNAi method, (2) antisense oligonucleotide method, and (3) nucleic acid enzyme method, respectively, and therefore will only be briefly described here.

[0111] Examples of the transcript inhibitors that are RNAi agents include nanosO-dsRNA, nanosO-siRNA, or nanosO-shRNA directed against the nanosO gene, and nanosP-dsRNA, nanosP-siRNA, or nanosP-shRNA directed against the nanosP gene. When the transcript inhibitor is an shRNA, it may be in the form of an expression vector containing an operably encoded nucleic acid. The effect of the RNAi agent as a transcript inhibitor generally lasts for only one generation. However, by inserting an expression vector encoding nanosO-shRNA or nanosP-shRNA into the genome of a host lepidopteran insect, the effect of the RNAi agent can be obtained by inducing expression of the nanosO-shRNA or nanosP-shRNA in progeny carrying the expression vector, even over successive generations of the lineage.

[0112] Examples of ASOs that can be used as transcription inhibitors include nanosO-ASOs targeting the nanosO gene and nanosP-ASOs targeting the nanosP gene. Furthermore, when the ASO is RNA-type, it may be in the form of a nanosO-asRNA expression vector or a nanosP-asRNA expression vector that operably harbors the nucleic acid encoding it. While the effect of the ASO as a transcription inhibitor generally lasts for only one generation, the as expression vector can be inserted into the genome of a host lepidopteran insect, allowing the effect of the ASO to be obtained by inducing expression of the nanosO-ASO or nanosP-ASO in progeny carrying the expression vector, even over successive generations of the lineage.

[0113] If the transcription product inhibitor is a nucleic acid enzyme, examples include a nanosO-(deoxy)ribozyme for the nanosO gene and a nanosP-(deoxy)ribozyme for the nanosP gene. If the nucleic acid enzyme is a ribozyme, it may be in the form of a nanosO-ribozyme expression vector or a nanosP-ribozyme expression vector that operably contains a nucleic acid encoding it.

[0114] (2) Translation product inhibitors As used herein, a "translation product inhibitor" refers to an agent that targets a protein that is a translation product of the target gene, the nanosO gene or the nanosP gene, and inhibits the expression of that gene by degrading or inactivating the protein. Specific examples of translation product inhibitors include an anti-nanosO aptamer for the nanosO protein and an anti-nanosP aptamer for the nanosP protein. The specific structure of the aptamer is similar to the structure of the aptamer detailed in "(4) Aptamer" of the first aspect, and therefore will only be briefly described here.

[0115] When the transcript inhibitor is an RNA aptamer, it may be in the form of an expression vector containing an operably encoded nucleic acid. In principle, the effect of an aptamer as a transcript inhibitor lasts for only one generation, but if the expression vector encodes a nanosO-RNA aptamer or nanosP-RNA aptamer, it can be inserted into the genome of a host lepidopteran insect, and even if the lineage is passed on, the effect of the RNA aptamer can be obtained by inducing expression of the nanosO-RNA aptamer or nanosP-RNA aptamer in progeny carrying the expression vector.

[0116] The sterilization composition of the present invention uses a pair of gene expression inhibitors, at least one for the nanosO gene and the other for the nanosP gene.

[0117] In addition to the above reasons, the sterilization composition of the present invention may also contain two or more gene expression inhibitors for either the nanosO gene or the nanosP gene. For example, the sterilization composition of the present invention may contain a total of three active ingredients: two types of gene expression inhibitors for the nanosO gene, ie, siRNA and shRNA, and one type of gene expression inhibitor for the nanosP gene, ie, siRNA.

[0118] 3-2-2. Other ingredients The sterilizing composition for lepidopteran insects of the present invention may contain, in addition to the active ingredient, a solvent or carrier acceptable in the field of entomology, as necessary. "Acceptable in the field of entomology" means that the solvent or carrier is commonly used in the field of entomology and is harmless or has little effect on the lepidopteran insects to which it is administered.

[0119] Examples of solvents include water or aqueous solutions, and organic solvents acceptable to lepidopteran insects. Examples of aqueous solutions include buffers (such as phosphate buffer and sodium acetate buffer), saline, and isotonic solutions.

[0120] Examples of the carrier include glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, as well as low-concentration nonionic surfactants and polyoxyethylene sorbitan fatty acid esters.

[0121] 3-3.Administration method The sterilizing composition of the present invention may be administered to lepidopteran insects in accordance with the method for introducing each gene expression inhibitor described in the gene expression inhibitor method of the first embodiment. [Example]

[0122] Example 1 (the purpose) The sterility and sterilization efficiency of sterile Lepidoptera insects obtained by the method for producing sterile Lepidoptera insects of the present invention will be verified.

[0123] (method) (1) Material The host lepidopteran insect used was the silkworm (pnd strain) serially reared at the National Agriculture and Food Research Organization (NARO, Japan).

[0124] (2) Preparation of gene expression inhibitors In this example, RNAi agents for the target genes, nanosO gene and nanosP gene, were prepared as gene expression inhibitors to be used in the gene expression inhibitory step.

[0125] First, the nucleotide sequence shown in SEQ ID NO: 5 was amplified using the silkworm nanosO gene consisting of the nucleotide sequence shown in SEQ ID NO: 2 as a template and the primer pair shown in SEQ ID NOs: 6 and 7 (O-Fw and O-Rv, respectively). PCR was performed in two steps: (98°C, 10 s; 55°C, 15 s; 68°C, 60 s) × 15, followed by (98°C, 10 s; 68°C, 60 s) × 25 using GXL polymerase (Takara Bio Inc.).

[0126] Similarly, the silkworm nanosP gene consisting of the nucleotide sequence shown in SEQ ID NO: 4 was used as a template to amplify the nucleotide sequence shown in SEQ ID NO: 8 using the primer pairs shown in SEQ ID NOs: 9 and 10 (P-Fw and P-Rv, respectively). Each Fw primer contained a T7 promoter sequence at its 5' end. The PCR reaction conditions were the same as those described above.

[0127] Furthermore, dsRNA was similarly prepared for the nanos paralogous genes, the nanosM gene and the nanosN gene. The nanosM gene was amplified using the silkworm nanosM gene consisting of the nucleotide sequence shown in SEQ ID NO: 11 as a template and the primer pair shown in SEQ ID NO: 13 and 14 (M-Fw and M-Rv, respectively) to amplify the nucleotide sequence shown in SEQ ID NO: 12. The nanosN gene was amplified using the silkworm nanosN gene consisting of the nucleotide sequence shown in SEQ ID NO: 15 as a template and the primer pair shown in SEQ ID NO: 17 and 18 (N-Fw and N-Rv, respectively) to amplify the nucleotide sequence shown in SEQ ID NO: 16. The PCR reaction conditions were as described above.

[0128] Next, double-stranded RNA (dsRNA) derived from each nanos gene was prepared using the resulting PCR amplification products with the Megascript RNAi kit (Ambion) according to the protocol attached to the kit.

[0129] For injection, dsRNA for each nanos gene was prepared in distilled water at a concentration of 3 μg / μL. To simultaneously suppress the expression of two target genes, the dsRNAs for the two nanos genes were mixed in equal proportions at a concentration of 3 μg / μL. Five types of nanos-RNAi agents were prepared: (1) nanosO-dsRNA alone, (2) nanosP-dsRNA alone, (3) a mixture of nanosM-dsRNA and nanosO-dsRNA (nanosM / O-dsRNA), (4) a mixture of nanosN-dsRNA and nanosO-dsRNA (nanosN / O-dsRNA), and (5) a mixture of nanosO-dsRNA and nanosP-dsRNA (nanosO / P-dsRNA).

[0130] (3) Injection of dsRNA into silkworm eggs Next, mated female moths were placed on an egg-laying mat and allowed to lay eggs for 1 hour. Between 2 and 4 hours after egg laying, 10-20 nL of each nanos-RNAi agent was microinjected into each egg using a glass micropipette. After injection, the injection hole was sealed with instant adhesive (Turilon Instant Adhesive, Multi-Purpose, Fast-Curing Type, Alpha Shoji). Eggs laid at the same time but without injection were used as negative controls. The egg-laying mat was then quickly transferred to a petri dish and the lid was placed on top. To maintain humidity, a water-soaked kitchen towel was placed inside the tight box, and the petri dish was placed on top of it. The tight box was then closed. The tight box was then incubated at 25-28°C for approximately 10 days until the eggs hatched.

[0131] (4) Rearing silkworms and confirming the phenotype of adult gonads After hatching, the larvae were reared in a rearing room at 28°C on an artificial diet (Silkmate original species 1st to 3rd instar S, Nippon Nosan Kogyo) for all stages. The artificial diet was changed as necessary.

[0132] After emergence, the ovaries were removed from virgin female adults and the number of mature eggs in the ovaries was counted. Note that in silkworms, most of the eggs in the ovaries are usually mature by the time the female emerges.

[0133] In addition, testes were removed from virgin male adults after emergence and the morphology of the testes was observed. Furthermore, male adults obtained after injection were mated with wild-type female adults and the normal mating rate was calculated. The normal mating rate was calculated from the number of normal matings relative to the total number of matings. Mating was considered normal when the number of fertilized eggs laid by female individuals after mating was 100 or more. Fertilized eggs were counted as pigmented eggs (colored eggs that develop several days after egg laying).

[0134] Furthermore, we also examined phenotypes other than those of germline cells (behavior, growth, morphology, etc.) for each individual after nanos-RNAi injection.

[0135] (result) Figures 1–5 show the results for the number of females and the number of mature eggs in their ovaries after injection, and Figure 6 shows the morphology of the excised testes. Figure 1 shows the results for nanosO-RNAi, in which nanosO-dsRNA was administered alone; Figure 2 shows the results for nanosO-RNAi, in which nanosP-dsRNA was administered alone; Figure 3 shows the results for nanosM / O-RNAi, in which nanosM / O-dsRNA was administered; Figure 4 shows the results for nanosN / O-RNAi, in which nanosN / O-dsRNA was administered; and Figure 5 shows the results for nanosO / P-RNAi, in which nanosO / P-dsRNA was administered. In Figure 6, A shows the testes of wild-type adult males, B shows the testes of males treated with nanosO-RNAi, and C shows the testes of males treated with nanosO / P-RNAi.

[0136] (oogenesis) The results in Figures 1-5 show that none of the individuals treated with nanosO-RNAi, nanosP-RNAi, nanosM / O-RNAi, or nanosN / O-RNAi had a mature egg count of 150 or less. These results suggest that the nanosO gene, either alone or in combination with other nanos paralogs (nanosM or nanosN), does not affect oogenesis. Nakao H. and Takasu Y. (2019, cited above) have reported that a rare decrease in the number of mature eggs was observed in Bm-nosO gene knockout silkworms. In this example, no abnormalities in the number of mature eggs were observed when nanosO gene expression was suppressed with nanosO-RNAi, which is largely consistent with the results of Nakao H. and Takasu Y. (2019), who disrupted the nanosO gene. This suggests that suppressing or inhibiting nanosO gene expression alone is insufficient to induce stable sterility in lepidopteran insects. Furthermore, as shown in Figure 2, the same results as with the nanosO gene were confirmed when nanosP gene expression was suppressed alone. This indicates that suppressing the expression of the nanosP gene alone is insufficient to induce stable sterility in lepidopteran insects.

[0137] In contrast, the results in Figure 5 demonstrate that dual suppression of the nanosO and nanosP genes significantly reduced the number of mature eggs, resulting in clear abnormalities in oogenesis. Approximately 70% of injected females had fewer than 150 mature eggs. These results demonstrate that mature egg formation is inhibited only when expression of the nanosO / P genes is dually suppressed.

[0138] (Testicular morphology) No morphological differences were observed between the testes of wild-type males and those of nanosO-RNAi-treated males, as shown in Figure 6, A and B. However, in the testes of nanosO / P-RNAi-treated males shown in C, partially transparent testes were observed (indicated by arrows), as well as multiple significantly dwarfed testes (indicated by arrowheads).

[0139] (mating rate) The results are shown in Table 1.

[0140] [Table 1]

[0141] The normal mating rate was significantly reduced only when nanosO / P expression was double-silenced by RNAi. On the other hand, the normal mating rate was similar when either nanosO or nanosP expression was silenced by RNAi, and when either nanosM / O or nanosN / O expression was double-silenced by RNAi. This result suggests that functional spermatogenesis is inhibited in males with nanosO / P double-silenced by RNAi, resulting in reduced or lost fertility.

[0142] These results revealed that only when the expression of the nanosO / P genes was double suppressed did male individuals not only develop abnormal testicular morphology but also lose the fertilizing ability of sperm.

[0143] (Other phenotypes) Throughout the rearing process, observations of phenotypes other than germline cells (behavior, growth, morphology, etc.) revealed no notable phenotypic differences other than sterility. That is, they grew, molted, metamorphosed, produced cocoons normally, and matured into adults, with no changes in mating behavior. Furthermore, no differences were observed in the size of larvae or adults compared to wild-type.

[0144] (Conclusion) These results suggest that the nanosO and nanosP genes function redundantly in the formation of germline cells such as primordial germ cells, that their function cannot be complemented by other nanos paralogs, and that dual suppression of the nanosO / P genes during early embryogenesis leads to stable abnormalities in germline cell formation and sterility. These results suggest that stable sterility induction in Lepidoptera requires the suppression or inhibition of nanosO / P gene expression during early embryogenesis.

[0145] <Comparative Example 1> (the purpose) We will prepare expression inhibitors for the nanos genes, both individually and in combination, which were not verified in Example 1, and confirm that the fertility suppression effect in lepidopteran insects of the present invention can only be obtained when the expression of the nanosO / P genes is doubly suppressed or inhibited.

[0146] (method) The basic operations are similar to those of the first embodiment, so only the differences from the first embodiment will be explained here.

[0147] (1) Preparation of gene expression inhibitors In this comparative example, RNAi agents were prepared for the nanosM gene and nanosN gene, which are the nanos paralogous genes used in Example 1, and the nanosO gene and nanosP gene, which are the target genes of the present invention. Specific preparation methods were as described in Example 1.

[0148] For injection, the dsRNA of each nanos gene was adjusted to a concentration of 3 μg / μL with distilled water. Two to four types of RNAi agents were prepared by mixing equal ratios of dsRNA of each nanos gene at a concentration of 3 μg / μL.

[0149] In this comparative example, in addition to the control groups not tested in Example 1, namely (1) nanosM-dsRNA alone (nanosM-dsRNA), (2) nanosN-dsRNA alone (nanosN-dsRNA), (3) a mixture of nanosM-dsRNA and nanosP-dsRNA (nanosM / P-dsRNA), and (4) a mixture of nanosN-dsRNA and nanosP-dsRNA (nanosN / P-dsRNA), (5) a mixture of nanosM-dsRNA, nanosN- Seven nanos-RNAi agents were prepared: (1) a mixture of dsRNA and nanosO-dsRNA (nanosM / N / O-dsRNA), (2) a mixture of nanosM-dsRNA, nanosN-dsRNA, and nanosP-dsRNA (nanosM / N / P-dsRNA), and (3) a mixture of nanosM-dsRNA, nanosN-dsRNA, nanosO-dsRNA, and nanosP-dsRNA (nanosM / N / O / P-dsRNA). Injection of dsRNA into silkworm eggs, rearing of silkworms, and confirmation of adult gonad phenotypes were performed according to the method described in Example 1.

[0150] (result) The number of females after injection and the number of mature eggs in their ovaries are shown in Figures 7 to 13. Figure 7 shows nanosM-dsRNA, Figure 8 shows nanosN-dsRNA, Figure 9 shows nanosM / P-dsRNA, Figure 10 shows nanosN / P-dsRNA, Figure 11 shows nanosM / N / O-dsRNA, Figure 12 shows nanosM / N / P-dsRNA, and Figure 13 shows nanosM / N / O / P-dsRNA.

[0151] The results in Figures 7 to 13 show that, except for the quadruple suppression of the nanos gene (nanosM / N / O / P-dsRNA) in Figure 13, which includes double suppression of the nanosO and nanosP genes, no individuals treated with any RNAi had fewer than 150 mature eggs. These results suggest that single suppression of the nanosM and nanosN genes, or double or triple suppression with other nanos genes, does not affect oogenesis in Lepidoptera, as long as it does not include double suppression of the nanosO and nanosP genes.

[0152] Taking the results of Example 1 and this Comparative Example together, it was demonstrated that a stable sterilization effect in Lepidoptera insects is induced only when the nanosO gene and the nanosP gene are double-suppressed, and that double suppression of other nanos genes or multiple suppression excluding the nanosO gene and the nanosP gene is insufficient.

Claims

1. A method for producing a sterile lepidopteran insect, comprising a step of suppressing expression of the nanosO gene and the nanosP gene, the method for producing a nanosO gene, wherein the nanosO gene comprises a nucleotide sequence encoding a nanosO protein having the amino acid sequence shown in (a) or (b) below, and the nanosP gene comprises a nucleotide sequence encoding a nanosP protein having the amino acid sequence shown in (c) or (d) below; (a) the amino acid sequence shown in SEQ ID NO: 1; or (b) an amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 1; (c) the amino acid sequence shown in SEQ ID NO: 3; (d) an amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 3;

2. The method of claim 1 , wherein the gene expression is suppressed using a gene knockdown method.

3. The production method according to claim 2, wherein the gene knockdown method is the RNAi method or the antisense oligonucleotide method.

4. The method of claim 1, wherein the nanosO gene consists of the base sequence shown in SEQ ID NO:

2.

5. The method according to claim 1 or 4, wherein the nanosP gene consists of the base sequence shown in SEQ ID NO:

4.

6. A sterile lepidopteran insect in which expression of the nanosO gene and the nanosP gene is suppressed, the sterile Lepidoptera insect, wherein the nanosO gene comprises a nucleotide sequence encoding a nanosO protein having the amino acid sequence shown in (a) or (b) below, and the nanosP gene comprises a nucleotide sequence encoding a nanosP protein having the amino acid sequence shown in (c) or (d) below; (a) the amino acid sequence shown in SEQ ID NO: 1; or (b) an amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 1; (c) the amino acid sequence shown in SEQ ID NO: 3; (d) an amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 3;

7. The sterile Lepidoptera insect according to claim 6 , wherein the expression is suppressed by gene knockdown of each gene.

8. The sterile Lepidoptera insect according to claim 7, wherein the gene knockdown method is an RNAi method or an antisense oligonucleotide method.

9. A sterilization composition for lepidopteran insects containing, as an active ingredient, a gene expression inhibitor that suppresses the expression of the nanosO gene and the nanosP gene, the infertility composition, wherein the nanosO gene comprises a nucleotide sequence encoding a nanosO protein consisting of the amino acid sequence shown in (a) or (b) below, and the nanosP gene comprises a nucleotide sequence encoding a nanosP protein consisting of the amino acid sequence shown in (c) or (d) below; (a) the amino acid sequence shown in SEQ ID NO: 1; or (b) an amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 1; (c) the amino acid sequence shown in SEQ ID NO: 3; (d) an amino acid sequence having 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 3;

10. The sterilization composition according to claim 9, wherein the gene expression inhibitor is a transcript inhibitor that targets the transcript of each of the genes.

11. The sterilization composition of claim 10, wherein the transcript inhibitor is an RNAi agent or an antisense oligonucleotide.

12. The sterilization composition of claim 11, wherein the RNAi agent is an expression vector operably carrying a nucleic acid encoding an shRNA for each of the genes.

13. The sterilization composition according to claim 9, wherein the gene expression inhibitor is a translation product inhibitor that targets the translation product of each of the genes.

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