JP2023067775A - Method for producing infertile lepidopteran insects and composition for sterilizing lepidopteran insects - Google Patents

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

The present invention relates to a method for producing sterile lepidopteran insects and a composition for sterilizing lepidopteran insects used in the method.

Gene modification technology represented by gene recombination technology is an indispensable technology in the functional analysis of genes and proteins and in the production of substances such as proteins. Conventionally, Escherichia coli and yeast have been mainly used as host organisms used in gene recombination technology. However, these organisms are not suitable for mass production of substances such as proteins.

Therefore, in recent years, silkworms (Bombyx mori) have attracted attention as hosts for mass production of proteins. Silkworms are insects that have been used industrially for the production of silk for a long time, and because they form cocoons in the prepupal stage, they can produce a large amount of silk thread in a short period of time. This is based on the high ability of protein production in silkworm silk glands. Techniques for producing genetically modified silkworms (transgenic silkworms) using this production capacity and mass-producing useful proteins other than silk thread are attracting attention.

On the other hand, substance production systems using genetic recombination technology are accompanied by the problem of genetic environmental contamination due to outflow of genetically modified organisms into the wild. Since silkworms have completely lost the ability to fly, they are approved for type 1 use based on the Cartagena Law, that is, use without preventing their spread into the environment. However, it cannot be ruled out that male and female silkworms, which are related species that can fly and can be invaded from the outside, interbreed. Therefore, it is industrially important to develop techniques for sterilizing silkworms from which the next generation cannot be obtained, in order to maintain useful strains and prevent the spread of genetically modified organisms into the environment.

Methods of sterilizing insects have heretofore been developed and practiced primarily for producing sterile insects in the Sterile Insect Technique (SIT). For example, a radiation irradiation method is known as a sterilization method.

The radiation irradiation method is to sterilize the target insect by irradiating the reproductive cells of the target insect with radiation such as X-rays and γ-rays to induce inactivation of sperm, loss of egg production, reduction in the number of eggs laid, and inability to mate. method (Non-Patent Documents 1 and 2). However, in addition to concerns about safety, etc., that requires an irradiation facility, this method has problems such as concerns that radiation exposure may reduce the vitality of the worms.

Journal of Agriculture, Forestry and Fisheries (1980), Vol. 3, No. 2, p.32-34 Chemistry and Biology (1993), vol.31, No.2, p.137-139

An object of the present invention is to develop a new sterilization method that can simply, stably, and efficiently sterilize lepidopteran insects and to cause abnormalities only in germ line cells, and to develop sterilized butterflies using the method. To develop and provide a method for producing insects of the order Insects and a sterilizing composition capable of implementing the method.

As a result of intensive studies aimed at solving the above problems, the present inventors found that among the four nanos genes (nanosM gene, nanosN gene, nanosO gene, and nanosP gene) present in lepidopteran insects, When the nanosO gene and nanosP gene are double-suppressed in adult ovaries, remarkable abnormalities such as a decrease in the number of mature eggs in the adult ovary, egg disappearance, and testicular dwarfing, which are thought to be caused by germline hypoplasia, appear, leading to infertility. became. However, they were also found to be normal except for the germline cells. By applying this phenomenon, it becomes possible to genetically sterilize lepidopteran insects. The present invention is based on this finding and provides the following.

(1) A method for producing a sterile lepidopteran insect, comprising a step of suppressing the expression of the nanosO gene and the nanosP gene.
(2) The production method according to (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 an RNAi method or an antisense oligonucleotide method.
(4) The production method according to any one of (1) to (3), wherein the nanosO gene comprises a base sequence encoding a nanosO protein consisting of the amino acid sequences shown in (a) to (c) below. (a) the amino acid sequence shown in SEQ ID NO: 1,
(b) an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, or (c) 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 1 (5) The production method according to (4), wherein the nanosO gene consists of the nucleotide sequence shown in SEQ ID NO:2.
(6) The production method according to any one of (1) to (5), wherein the nanosP gene comprises a base sequence encoding a nanosP protein consisting of the amino acid sequences shown in (d) to (f) below. (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) 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 3 (7) The production method according to (6), wherein the nanosP gene consists of the nucleotide sequence shown in SEQ ID NO:4.
(8) A sterile lepidopteran insect in which the expression of the nanosO gene and the nanosP gene is suppressed.
(9) The sterile lepidopteran insect according to (8), wherein the expression is suppressed by a gene knockdown method for each gene.
(10) The sterile lepidopteran 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, which contains, as an active ingredient, a gene expression suppressing agent 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 targeting the transcript of each gene.
(13) The sterilizing composition according to (12), wherein the transcript inhibitor is an RNAi agent or an antisense oligonucleotide.
(14) The sterilizing composition according to (13), wherein the RNAi agent is an expression vector operably containing a nucleic acid encoding an shRNA for each gene.
(15) The sterilization composition according to (11), wherein the gene expression inhibitor is a translation product inhibitor targeting the translation product of each gene.

INDUSTRIAL APPLICABILITY 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.

In addition, according to the composition for sterilizing lepidopteran insects of the present invention, any desired lepidopteran insect can be sterilized easily, stably, and efficiently.

FIG. 3 is a diagram showing the number of mature eggs in the ovaries of nanosO-RNAi-treated female silkworm individuals and the number of mature eggs. FIG. 3 is a diagram showing the number of mature eggs in the ovaries of nanosP-RNAi-treated female silkworm individuals and the number of mature eggs. FIG. 3 is a diagram showing the number of mature eggs in the ovaries of nanosM/O-RNAi-treated female silkworm individuals and the number of mature eggs. FIG. 4 is a diagram showing the number of mature eggs in the ovaries of nanosN/O-RNAi-treated female silkworm individuals and the number of mature eggs. FIG. 3 is a diagram showing the number of mature eggs in the ovaries of nanosO/P-RNAi-treated female silkworm individuals and the number of mature eggs. Fig. 10 shows the morphology of testis excised from each nanos-RNAi-treated male. A, wild-type male adult testes, B, nanosO-RNAi-treated adult male testes, and C, nanosO/P-RNAi-treated adult male testes. In panel C, arrowheads indicate dwarfed testes and arrows indicate clearing. FIG. 3 is a diagram showing the number of mature eggs in the ovaries of nanosM-RNAi-treated female silkworm individuals and the number of mature eggs. FIG. 3 is a diagram showing the number of mature eggs in the ovaries of nanosN-RNAi-treated female silkworm individuals and the number of mature eggs. FIG. 4 is a diagram showing the number of mature eggs in the ovaries of nanosM/P-RNAi-treated female silkworm individuals and the number of mature eggs. FIG. 3 is a diagram showing the number of mature eggs in the ovaries of nanosN/P-RNAi-treated female silkworms and the number of mature eggs. FIG. 4 is a diagram showing the number of mature eggs in the ovaries of nanoss M/N/O-RNAi-treated female silkworm individuals and the number of mature eggs. FIG. 4 is a diagram showing the number of mature eggs in the ovaries of nanosM/N/P-RNAi-treated female silkworm individuals and the number of mature eggs. FIG. 4 is a diagram showing the number of mature eggs in the ovaries of nanosM/N/O/P-RNAi-treated female silkworm individuals and the number of mature eggs.

1. Method for Producing Sterile Lepidoptera 1-1. SUMMARY A first aspect of the present invention is a method for producing sterile lepidopteran insects. The production method of the present invention includes a step of suppressing the expression of two nanos genes in a target lepidopteran insect, and disrupting the functions of these genes induces germline cell abnormalities in the insect, resulting in infertility. characterized by producing lepidopteran insects.

1-2. DEFINITIONS OF TERMS Definitions of terms frequently used in this specification are explained below.
"Lepidoptera" refers to insects belonging to the taxonomic order Lepidoptera, which are butterflies or moths. Butterflies include insects belonging to the families Nymphalidae, Papilionidae, Pieridae, Lycaenidae, and Hesperiidae. Moths include Saturniidae, Bombycidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Geometridae, and Archtiidae. , Noctuidae, Pyralidae, Sphingidae, and the like. For example, in the case of moths, species belonging to the genus Bombyx, Samia, Antheraea, Saturnia, Attacus, and Rhodinia, specifically silkworms, Bombyx mandarina, Samia cynthia; ricini and hybrids of Shinjusan and Erisan), Antheraea yamamai, Antheraea pernyi, Saturnia japonica, and Actias gnoma. It is not limited to these. Silkworms are preferred.

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. refers to insects.

As used herein, "infertility" refers to loss or reduction in fertility to form the next generation of individuals due to loss or significant decline in fertility. Infertility herein can result primarily, but not exclusively, from germline cell abnormalities.

As used herein, the term "sterilization" refers to changing a normal individual into a sterile state.

As used herein, the term “infertility” refers to being infertile or having the characteristics of infertility.

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

As used herein, "abnormality of germline cells" refers to hypoplasia such as disappearance of germline cells, as well as morphological and dispositional abnormalities.

As used herein, “suppression of gene expression” or “suppression of gene expression” is also referred to as gene knockdown, and refers to suppression of the expression and function of a protein encoded by the gene. Specifically, in the expression of the target gene, suppression of 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 can be mentioned. Gene knockdown is distinguished from gene knockout, which disrupts a target gene and results in complete loss of function of the protein encoded by that gene.

As used herein, the term "expression vector" refers to a vector that operably contains a nucleic acid molecule encoding a protein or functional nucleic acid and that is capable of controlling the expression of that nucleic acid molecule. Examples thereof include plasmids and the like. As used herein, the term "operable" refers to placement of a nucleic acid molecule of interest under the control of a promoter within an expression vector. As a result, the expression of the nucleic acid molecule of interest is initiated by the activity of the promoter.

As used herein, the term "nucleic acid molecule" refers to a gene that encodes a protein, or a polynucleotide or oligonucleotide that encodes a functional nucleic acid. Without limitation, nucleic acid molecules are composed principally of naturally occurring nucleic acids, DNA and/or RNA. However, it may contain an artificial nucleic acid.

As used herein, the term "functional nucleic acid" refers to a specific biological function, such as an enzymatic function, a catalytic function, or a biological inhibitory or enhancing function (e.g., transcription, translation inhibition or It refers to a nucleic acid molecule having an enhancement). Specific examples include RNAi agents, nucleic acid aptamers (DNA aptamers, RNA aptamers, etc.), antisense oligonucleotides, nucleic acid enzymes, and the like.

As used herein, the term "genetic modification" refers to artificial modification of the natural genetic information of the host organism. Modification of genetic information as used herein includes addition, deletion, substitution, and the like of genetic information. Artificial modification of genetic information includes genetic recombination and genome editing.

"Genetic recombination" means a method of adding foreign genetic information not possessed by the host organism to the genome, etc., using a vector such as a plasmid or a transposon, or a method of modifying or destroying the genetic information possessed by the host organism. is mentioned.

"Genome editing" refers to the insertion (knock-in) of a foreign gene at any position on the genome and the target gene using the DNA repair mechanism associated with double strand break (DSB) by a DNA cutting enzyme It is a gene targeting technology that knocks out the

1-3. Method The method for producing sterile lepidopteran insects of the present invention includes a gene expression suppression step as an essential step. This step will be specifically described below.

1-3-1. Gene Expression Suppression Step The “gene expression suppression step” is to suppress the gene expression of the nanosO gene and nanosP gene (herein, these genes are often collectively referred to as “nanosO/P gene”) in the target lepidopteran insect. It is a step of suppressing. In lepidopteran insects, the functions of nanosO protein and nanosP protein (herein, these proteins are often collectively referred to as "nanosO/P protein") are discontinued at the stage of embryogenesis, resulting in germline cell formation. The research results of the present inventors have revealed that formation is inhibited and both males and females become infertile. In this step, this phenomenon is used to suppress the expression of the nanosO/P gene in the target lepidopteran insect, thereby rendering the insect sterile.

A "nanos gene" (or nos gene) is a gene that encodes a nanos protein. A "nanos protein" is an evolutionarily conserved protein with a zinc finger motif. Studies using fruit flies (Drosophila melanogaster) suggest that nanos proteins function in the survival and proliferation of primordial germ cells (PGCs) (Keuckelaere E.D. et al., 2018, Cell Mol Life Sci., 75:1929- 1946). Unlike other insects, lepidopteran insects have four nanos paralogs (nanosM, nanosN, nanosO, and nanosP) genes (Nakao H., et al., 2008, Evolution & Development, 10(5): 548 -554; Carter J-M, et al., 2015, PLoS ONE, 10: e0144471). Similarly, four paralogous genes (Bm-nosM, Bm-nosN, Bm-nosO, and Bm-nosP, respectively) have been identified in silkworms. important has been suggested. From the phenotype of Bm-nosO gene knockout silkworms using genome editing technology, in addition to oogenesis abnormalities, a decrease in the number of mature eggs was observed in rare cases, and the Bm-nosO protein is involved in the egg (germ cell) formation process. (Nakao H. and Takasu Y., 2019, Developmental Biology, 445:209-36). On the other hand, the specific functions of other nanos paralogs in lepidopteran insects are unknown.

In the present specification, the nanosO/P gene whose expression is to be suppressed is not particularly limited as long as it is the nanosO/P gene of the target lepidopteran insect for which sterilization is intended. NanosO gene orthologs and nanosP gene orthologs of each species can be targeted. Those base sequences should just be the base sequence which codes each nanos protein.

For example, in the case of the nanosO gene, if the target lepidopteran insect is a silkworm, the wild-type Bm-nosO gene encoding the wild-type Bm-nosO protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 1 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% of the amino acid sequence in which one or more amino acids are deleted, substituted or added, or the amino acid sequence shown in SEQ ID NO: 1 A mutant Bm-nosO gene encoding a mutant Bm-nosO protein consisting of an amino acid sequence having an amino acid identity of 99% or more. A specific example of the wild-type Bm-nosO gene is the Bm-nosO gene having the nucleotide sequence shown in SEQ ID NO:2. In this specification, "plurality" means, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4 or 2 to 3 say. In addition, "(amino acid) substitution" refers to substitution within a group of conservative amino acids with similar properties such as charge, side chain, polarity, and aromaticity among the 20 types of amino acids that make up natural proteins. . For example, 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 substitutions within the aromatic amino acid group (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 the polypeptide. Furthermore, "amino acid identity" means that two amino acid sequences are aligned (aligned) and, if necessary, gaps are introduced into either or both amino acid sequences so that the degree of amino acid identity between the two is maximized. It refers to the ratio (%) of identical amino acid residues in one amino acid sequence to the total number of amino acid residues in the other amino acid sequence.

In the case of the nanosP gene, if the target lepidopteran insect is a silkworm, the wild-type Bm-nosP gene encoding the wild-type Bm-nosP protein consisting of the amino acid sequence shown in SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 3 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% of the amino acid sequence in which one or more amino acids are deleted, substituted or added, or the amino acid sequence shown in SEQ ID NO: 3 A mutant Bm-nosP gene encoding a mutant Bm-nosP protein consisting of an amino acid sequence having an amino acid identity of 99% or more. A specific example of the wild-type Bm-nosP gene is the Bm-nosP gene having the nucleotide sequence shown in SEQ ID NO:4.

In this step, an operation for suppressing the expression of the two nanos genes is performed in the target lepidopteran insect for the purpose of sterilization. The method for suppressing gene expression will be specifically described below.

A. Gene Expression Suppression Method As used herein, the term “gene expression suppression method” refers to a method for specifically suppressing the expression of a target gene. Methods for suppressing the expression of specific genes in lepidopteran insects may be methods known in the art, and are not particularly limited. Examples include a method of administering a low-molecular-weight compound that specifically suppresses the functions of nanosO/P proteins, which are two types of target proteins, to a target lepidopteran insect, and a gene knockdown method. A gene knockdown method that is technically established, simple, and expected to be highly effective is particularly preferable.

A "gene knockdown method" is a method for depleting a target gene product in a cell via (poly)nucleotides or (poly)peptides. There are cases where the target gene is a gene product after transcription and before translation, that is, a gene transcription product such as mRNA, and where there is a case where a translation product after translation, that is, a protein is targeted. Specific examples of gene knockdown methods include (1) RNAi method, (2) antisense oligonucleotide method, (3) nucleic acid enzyme method, and (4) aptamer method. Each method will be described below.

(1) RNAi method “RNAi method (RNA interference method)” is a method in which an RNAi agent is administered to the host, and the RNA interference (RNAi) possessed by the RNAi agent is used to control the expression of the target gene after transcription and before translation. Alternatively, it is a method of suppressing at the transcription level. RNA interference is sequence-specific gene silencing that suppresses the expression of a target gene through degradation or the like of the gene transcript. Here, this is done using an RNAi agent that induces gene silencing specific to each transcript of the nanosO/P gene. The RNAi agent used herein assumes a mechanism that suppresses the expression of the lepidopteran nanosO gene or nanosP gene after transcription and before translation.

"RNAi agent (RNA interference agent)" induces RNA interference (RNAi) in the body and suppresses the expression of the target gene through degradation of the transcript (silencing). A substance that Examples thereof include artificially synthesized dsRNA, siRNA, or shRNA and endogenous miRNA (microRNA) (including pri-miRNA and pre-miRNA). For RNA interference see, for example, Bass B.L., 2000, Cell, 101, 235-238; Sharp P.A., 2001, Genes Dev., 15, 485-490; Zamore P.D., 2002, Science, 296, 1265-1269; A.F. & Karpen, G.H., 2002, Cell, 111, 159-162. In the present invention, dsRNA, siRNA, or shRNA, which can be arbitrarily designed, is used as a main RNAi agent against the nanosO gene and nanosP gene, which are target genes. However, if miRNAs targeting the nanosO gene or nanosP gene exist, they can also be used. Each RNA agent is described in detail below.

(i) dsRNA
(composition)
"dsRNA" (double-stranded RNA: double-stranded RNA) is a double-stranded RNA consisting of 50 to 1000 bases containing a desired RNA sense strand sequence selected from the sense strand nucleotide sequence of a target gene. It has been shown to be effective against insects as an agent. The administered dsRNA is finally processed intracellularly into siRNA, which will be described later, and causes RNA interference. A dsRNA may be designed by a known method based on the base sequence of the target gene.

(Introduction method)
A method of introducing dsRNA for obtaining a gene expression-suppressing effect is to administer dsRNA prepared in vitro to a host. As the administration method, methods known in the art, such as injection administration by microinjection and the like, and immersion in a dsRNA solution, can be used. In the present invention, in which the host is a lepidopteran insect, injection administration is preferred. The concentration of the dsRNA solution used for introduction may be such that a certain concentration or more exists in the worm body (eg, egg) after introduction. More specifically, but not limited to, a solution with a concentration of several μg/μL to several tens of μg/μL, for example, in an amount of 2 nL to 40 nL, 5 nL to 30 nL, 8 nL to 25 nL, or 10 nL to 20 nL should be injected with

It is effective to perform microinjection on eggs 2 to 8 hours after spawning, before the nucleus is incorporated into the cell membrane after spawning. Alternatively, in order to obtain the same effect as in the case of injection into eggs, a method of introducing dsRNA into mature eggs of females before oviposition, followed by fertilization and oviposition may also be considered. A labeling gene may be co-introduced as a selection marker when the dsRNA is introduced. This facilitates selection of individuals into which the dsRNA has been introduced.

(Selected)
After the introduction of dsRNA, the introduced individuals can be selected if necessary. The selection method is not limited. For example, there is a method of collecting a part of the dsRNA-introduced individual and confirming a decrease in the expression level of the target gene compared to the control individual by RT-PCR or the like. Alternatively, as described above, when a marker gene is co-introduced as a selection marker at the time of dsRNA injection, introduced individuals can be selected based on the activity of the marker gene.

As used herein, a "marker gene" is a gene that encodes a marker protein. A labeling protein refers to a polypeptide that can determine the presence or absence of expression of a labeling gene based on its activity. "Based on activity" means "based on activity detection results." Activity may be detected directly by detecting the activity of the labeled protein itself, or may be indirectly detected via metabolites such as dyes produced by the activity of the labeled protein. Detection may be by chemical detection (including enzymatic detection), physical detection (including behavioral detection), or sensory detection of the detector (including detection by sight, touch, smell, hearing, taste). Either can be used.

The type of labeling protein is not particularly limited as long as its activity can be detected by a method known in the art. Preferably, the labeling protein is less invasive to the host lepidopteran insect upon detection. Examples thereof include fluorescent proteins, chromosynthetic proteins, luminescent proteins, exosecretory proteins, proteins that control external morphology, and the like. Fluorescent proteins, chromosynthetic proteins, luminescent proteins, and exocrine proteins are particularly preferred because they are visually detectable under certain conditions, are less invasive to lepidopteran insects, and are easier to distinguish and select. .

The fluorescent protein is a protein that emits fluorescence of a specific wavelength when the lepidopteran insect is irradiated with excitation light of a specific wavelength. It may be either natural or non-natural. Also, 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).

The pigment synthesis protein is a protein involved in pigment biosynthesis and is usually an enzyme. The term “pigment” as used herein refers to a low-molecular-weight compound or peptide capable of imparting a pigment to a transformant, regardless of the type thereof. Preferred are pigments that appear as the external color of the solid. Examples thereof include melanin pigments (including dopamine melanin), ommochrome pigments, and pteridine pigments.

The luminescent protein refers to a substrate protein capable of emitting light without requiring excitation light or an enzyme that catalyzes the luminescence of a substrate. Examples include aequorin and luciferase as an enzyme.

(Passage)
In principle, the dsRNA suppresses the target gene only for one generation.

(ii) siRNAs
(composition)
“siRNA” (small interference RNA) consists of an RNA sense strand (passenger strand) composed of base sequences corresponding to part of the sense strand of the target gene, and its antisense strand RNA antisense. It is a small double-stranded RNA consisting of a strand (guide strand). siRNA can induce RNA interference by introducing it into a subject's cells (eukaryotic cells) (Fire A. et al., 1998, Nature, 391, 806-811).

siRNA may be designed by a known method based on the base sequence of the target gene. For example, Ui-Tei et al. (Nucleic Acids Res., 2004, 32:936-948), Reynolds et al. (Nat. Biotechnol., 2004, 22:326-330), Amarzguioui et al. Res. Commun., 2004, 316: 1050-1058).

To give a specific example of siRNA design, when the nanosO gene is the target gene, for example, the base sequence of the RNAi sense strand (passenger strand) from the base sequence shown in SEQ ID NO: 2 is 15 bases or more and 35 bases or less, preferably 15 bases. A continuous base sequence of 30 bases or less, or 18 bases or more and 25 bases or less, is selected as a selection region. Care should be taken that the nucleotide sequence of the selected region at this time completely matches the nucleotide sequence of the target gene. Therefore, it is preferable to design the selected region so as not to include known mutation sites (including SNPs, for example) of the target gene. The nucleotide sequence of the RNAi antisense strand (guide strand) may be a nucleotide sequence complementary to the selected nucleotide sequence of the RNAi sense strand. When preparing siRNA, T (thymine) bases in the selected region are converted to U (uracil) bases in both the sense strand and the antisense strand.

The selection region of the RNAi sense strand is not particularly limited as long as it is a sequence specific to the target gene. It is preferably at least 50 bases, more preferably 70 to 100 bases downstream from the initiation codon. Furthermore, it is preferable to select a nucleotide 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 20-80%, more preferably 30-70% or 40-60%. Many designs of siRNA are published on the website, and effective and appropriate siRNA can be designed on the web by inputting the nucleotide sequence of the target gene. Typical siRNA design websites include siDirect (http://sidirect2.rnai.jp/) and siDESIGN Center (https://horizondiscovery.com/en/ordering-and-calculation-tools/sidesign-center). mentioned.

One or both ends of the siRNA may contain a base sequence consisting of one or more DNAs, RNAs, and/or nucleic acid analogues unrelated to the base sequence of the target gene or its complementary base sequence. good. Although the number of bases present at the end of such siRNA is not particularly limited, it is preferably in the range of 1-20. Specifically, for example, TT (thymine-thymine) or UU (uracil-uracil) is added to the 3' end of each base chain (Tuschl T et al., 1999, Genes Dev, 13(24):3191-7).

(Introduction method)
Since the method for introducing siRNA conforms to the above-mentioned method for introducing dsRNA, the explanation is omitted here.

(Selected)
After the introduction of siRNA, the introduced individuals can be selected if necessary. Since the selection method conforms to the above-mentioned dsRNA selection method, the description is omitted here.

(Passage)
The effect of suppressing target genes by siRNA is, in principle, only for one generation, similar to dsRNA.

(iii) shRNAs
(composition)
“shRNA” (short hairpin RNA) refers to a single-stranded RNA in which two RNA strands (RNAi sense strand and RNAi antisense strand) constituting the siRNA are linked by a spacer sequence consisting of an appropriate base sequence. . In other words, shRNA contains an RNAi sense region as an RNAi sense strand and an RNAi antisense region as an RNAi antisense strand within one molecule, and these regions base-pair with each other to form a stem structure, and a spacer sequence. By adopting a loop structure, the molecule as a whole is configured to have a hairpin-shaped stem-loop structure.

When shRNA is introduced into cells, the loop structure portion is cleaved to generate a double-stranded RNA molecule, ie siRNA. The resulting siRNA can suppress target gene expression by the same RNA interference mechanism as the siRNA described above.

The shRNA is designed, for example, by connecting the 3' end of the sense region in the siRNA and the 5' end of the antisense strand with a spacer sequence. The spacer sequence is usually 3 to 24 bases, preferably 4 to 15 bases. The spacer sequence is not particularly limited as long as it is a sequence that allows siRNA to base-pair.

The shRNA can also be operably inserted under the control of the promoter of the expression vector with the DNA encoding it. When such an shRNA expression vector is introduced into a target organism or target cell, the shRNA is expressed by promoter activity. After expression, it is processed into siRNA through self-folding in host cells and activity such as Dicer, and can exhibit its effects. Furthermore, using this shRNA expression vector, it is also possible to insert (knock-in) a DNA encoding shRNA into the genome of a lepidopteran insect by the transposon method or genome editing method described below.

(Introduction method)
The basic introduction method of shRNA conforms to the aforementioned dsRNA introduction method. Therefore, the description of the introduction method common to dsRNA is omitted, and only the characteristic introduction method of shRNA is explained here.

As described above, shRNA can be introduced in the form of DNA as an shRNA expression vector to express shRNA in host cells. In this case, the shRNA expression vector can be introduced according to the dsRNA introduction method. When introducing shRNA expression vectors into the laid eggs via mature female eggs, the shRNA expression vector is injected into the peritoneal cavity of the pupa and transferred to the mature eggs, or the shRNA expression vector is inserted into the genome of the lepidopteran insect (knock-in). It is also possible to keep However, when introducing an shRNA expression vector, care must be taken, such as selecting a developmental stage-specific promoter so that shRNA is not expressed in germline cells at a developmental stage prior to egg maturation. This is because the expression of shRNA in early germline cells may prevent the eggs themselves from forming.

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

The "transposon method" uses the inverted terminal repeat sequence of a transposon (Handler AM. et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:7520-5) and the activity of a transposon transferase. is used to insert a foreign gene into the genome. PiggyBac, mariner, minos, and the like are known transposons that can be used in lepidopteran insects, and any of them 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).

The exogenous gene can be introduced into the host genome by a method known in the art using an expression vector containing two transposon inverted terminal repeats and the exogenous gene interposed therebetween. For example, if the host to be introduced 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 may be injected into early silkworm embryos. Helper vectors include, for example, pHA3PIG. The activity of the transposon transferase produced by the helper vector allows the insertion of exogenous genes into the silkworm genome by homologous recombination via the transposon inverted terminal repeats.

"Genome editing method" is a method using the aforementioned genome editing technology. Genome editing methods include, for example, the zinc finger nuclease (ZFN) method, the TALEN method, and the CRISPR/Cas9 method, and any method may be used herein. Gene targeting techniques using these methods are all known techniques in the art, and kits and support tools for efficiently knocking out the target gene for the genome editing method used herein are available in each life science field. They are commercially available from manufacturers and can also be used. As for the foreign gene insertion method using the genome editing method in silkworms, the TAL-PITCH method has been published as a knock-in technology using the microhomology of the cleaved surface by TALEN, and this method may be used (Nakade , S. et al., 2014, Nature Communications, 5:5560).

(Selected)
Selection of shRNA-introduced individuals may be performed according to the siRNA selection method. When an shRNA expression vector is introduced, by inserting a marker gene into the vector, the introduced individuals can be selected based on the activity of the marker gene.

(Passage)
In principle, shRNA suppresses target genes for only one generation, similar to siRNA. However, when the shRNA is inserted into the genome of the host lepidopteran insect, the target gene can be suppressed by inducing the expression of the SHRNA in the progeny even if the strain is passaged.

As used herein, the term “progeny” refers to genetically modified first-generation offspring individuals that retain in their genome the shRNA of the present invention or DNA encoding the asRNA, ribozyme, or RNA aptamer described below. It refers to an individual who has As long as the progeny retains this shRNA, the number of generations does not matter.

(2) Antisense oligonucleotide method "Antisense oligonucleotide (ASO) method" is a method in which antisense oligonucleotides are administered to the host, and the activity of the antisense oligonucleotides suppresses the expression of the target gene after transcription and before translation. It is a way to The antisense oligonucleotides herein suppress the expression of the lepidopteran nanosO gene or nanosP gene post-transcriptionally and pre-translationally.

(structure)
“AntiSense Oligonucleotide (herein often referred to as “ASO”)” refers to a base sequence complementary to all or part of the base sequence of mRNA, which is the transcription product of a target gene. It is a single-stranded nucleic acid molecule that is organized and hybridizes to a target mRNA to inhibit its translation.

ASO is known to have a DNA type and an RNA type. The DNA form hybridizes to RNA molecules such as mRNA, which is the transcription product of the target gene, and forms a heteroduplex structure. A nuclease-mediated gene silencing method for silencing gene expression. On the other hand, the RNA type hybridizes to RNA molecules such as mRNA, which is the transcription product of the target gene, to form a double-stranded RNA, and then undergoes a process that ultimately suppresses gene expression by RNAi, similar to siRNA. can be effective. Although not limited, DNA-type ASOs are commonly used due to their stability in cells, ease of synthesis, and the like.

DNA-type ASOs are mainly composed of natural DNA, but in addition to natural RNA, nucleic acid analogues 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 may also be included. In addition, Gapmer, an RNA-degrading ASO having wing regions composed of nucleic acid analogues, modified nucleic acids, etc. at the 5' and 3' ends, and Mix, a splicing-regulated ASO composed of nucleic acid analogues, modified nucleic acids, etc. ASOs with special structures such as mers are also included.

Furthermore, a heteroduplex nucleic acid (HDO) method, which is an application of the DNA-type ASO method, can also be used. “HeteroDuplex Oligonucleotide (HeteroDuplex Oligonucleotide: In this specification, often referred to as “HDO”) refers to a main chain (DNA chain) having an ASO function and cRNA (complementary RNA) having a base sequence complementary to the main chain. ) strand is a double-stranded nucleic acid. Although the cRNA strand may also contain nucleic acid analogues, modified nucleic acids, etc., all or part of at least the central part of HDO is a heterogeneous nucleic acid composed of DNA-RNA, so the cRNA strand is a complementary strand by intracellular RNase H. is cleaved and degraded, and the single main chain can function as ASO.

An RNA-type ASO consists of a nucleotide sequence similar to that of an antisense strand that can hybridize to a part of mRNA, etc., which is a transcription product of a target gene, and in principle consists of natural RNA. Therefore, in the present specification, it is often referred to as "antisense RNA (asRNA)" in order to distinguish it from DNA-type ASO.

The asRNA can also be operably inserted under the control of the promoter of the expression vector with the DNA encoding it. Such an "antisense RNA expression vector (asRNA expression vector)" expresses asRNA by promoter activity when introduced into a target organism or target cell, like the shRNA expression vector described above. After expression, it exhibits its effect by, for example, hybridizing with the transcription product of the target gene in the host cell. Furthermore, like shRNA expression vectors, DNA encoding asRNA can be knocked into the genome of lepidopteran insects by the transposon method or genome editing method described below.

As a specific example of ASO base sequence design, regardless of DNA type or RNA type, if the nanosO gene is the target gene, for example, 10 bases or more and 30 bases or less from the base sequence of the mRNA chain shown in SEQ ID NO: 2 Preferably, a continuous base sequence of 12 to 25 bases, or 13 to 20 bases is used as a selection region, and a complementary base sequence may be selected. At this time, for example, a region including an initiation codon or a region capable of forming a single-stranded structure in the expected secondary structure of the mRNA chain can be selected as the target region.

(Introduction method)
The method for introducing ASO conforms to the above-mentioned method for introducing siRNA. Therefore, detailed description is omitted here. In addition, as described above, asRNA can be introduced in the form of DNA as an asRNA expression vector in the same manner as shRNA expression vectors, and asRNA can be expressed in host cells. The method for introducing the asRNA expression vector may also be according to the method for introducing the shRNA expression vector.

(Selected)
ASO-introduced individuals may also be selected according to the siRNA selection method. When an asRNA expression vector is introduced, by inserting a marker gene into the vector in the same manner as for the shRNA expression vector, the introduced individuals can be selected based on the activity of the marker gene.

(Passage)
The effect of ASO on target gene suppression is, in principle, only for one generation, similar to siRNA. However, when DNA encoding asRNA is inserted into the genome of the host lepidopteran, target genes can be suppressed by inducing the expression of asRNA in progeny even if the strain is passaged. .

(3) Nucleic Acid Enzyme Method “Nucleic acid enzyme method” is a method of administering a nucleic acid enzyme to a host and suppressing post-transcriptional and pre-translational expression of a target gene by the activity of the nucleic acid enzyme. The nucleic acid enzyme herein suppresses the expression of the lepidopteran nanosO gene or nanosP gene post-transcriptionally and pre-translationally.

(composition)
“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 suppresses the expression of the target gene after transcription and before translation. can. The nucleic acid enzyme herein suppresses the expression of the lepidopteran nanosO gene or nanosP gene post-transcriptionally and pre-translationally.

Deoxyribozymes composed of DNA and ribozymes composed of RNA, also known as ribozymes, are known as nucleic acid enzymes, but any of them may be used as the nucleic acid enzyme in the present specification. Both deoxyribozymes and ribozymes can partially contain chemically modified nucleic acids, artificial nucleic acids and/or nucleic acid analogues in their constituent bases.

A ribozyme composed of RNA can also be operably inserted under the control of the promoter of the expression vector with the DNA encoding it. Such a "ribozyme expression vector" expresses a ribozyme by promoter activity when introduced into a target organism or target cell, like the shRNA expression vector described above.

(Introduction method)
The method for introducing the nucleic acid enzyme conforms to the above-mentioned method for introducing dsRNA. Therefore, detailed description is omitted here. In addition, as described above, ribozymes can be introduced in the form of DNA as ribozyme expression vectors in the same manner as shRNA expression vectors, and the ribozymes can be expressed in host cells. The method for introducing the ribozyme expression vector may also be according to the method for introducing the shRNA expression vector.

(Selected)
Nucleic acid enzyme-introduced individuals may be selected according to the siRNA selection method. When a ribozyme expression vector is introduced, by inserting a marker gene into the vector in the same manner as the shRNA expression vector, the introduced individuals can be selected based on the activity of the marker gene.

(Passage)
In principle, the effect of suppressing a target gene by a nucleic acid enzyme is only for one generation, similar to siRNA. However, when the DNA encoding the ribozyme is inserted into the genome of the host lepidopteran insect, the target gene can be suppressed by inducing the expression of the ribozyme in the progeny even if the strain is passaged. .

(4) Aptamer method The “aptamer method” is a method of administering an aptamer to a host and suppressing target gene expression post-translationally by suppressing target protein function by 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 of Lepidoptera insects.

(structure)
An “aptamer” is a ligand molecule that strongly and specifically binds to a target substance due to its steric structure and suppresses the function of the target substance. Aptamers have effects similar to those of antibodies, but generally have higher specificity and affinity for target substances than antibodies, and even if the number of target amino acid residues required for binding is smaller than that of antibodies, The good news is that they are superior to antibodies in that they can distinguish between closely related molecules. Furthermore, they have the advantage that they are lower in immunogenicity and toxicity than antibodies, can be produced in a short period of about 3 to 4 weeks, and can be produced in large amounts by chemical synthesis.

Aptamers can be broadly classified into nucleic acid aptamers and peptide aptamers according to the types of constituent molecules. Aptamers herein may be any aptamers, but are preferably nucleic acid aptamers.

As used herein, the term “nucleic acid aptamer” refers to an aptamer composed of nucleic acids, which is targeted by a three-dimensional structure formed based on the secondary structure and tertiary structure of a single-stranded nucleic acid molecule via hydrogen bonding and the like. Binds strongly and specifically to substances.

As nucleic acid aptamers, RNA aptamers composed of RNA and DNA aptamers composed of DNA are generally known, but the nucleic acids that constitute the nucleic acid aptamers in the present specification are not particularly limited. Examples include DNA aptamers, RNA aptamers, aptamers composed of a combination of DNA and RNA, and the like. It is usually composed only of natural nucleic acids (DNA or RNA), but may partially contain non-natural artificial nucleic acids or modified nucleic acids. Although the base length of the aptamer of the present invention is not limited, it is preferably within the range of 10 to 100 bases. More preferably, it is within the range of 15-80 bases. Aptamers are a well known technology and for details see, for example, Janasena, Clin. Chem. 45:1628-1650 (1999).

RNA aptamers can be produced by in vitro selection using, for example, the SELEX (systematic evolution of ligands by exponential enrichment) method. The SELEX method selects and recovers RNA molecules bound to a target molecule (nanosO protein or nanosP protein in the present invention) from an RNA pool composed of RNA molecules having a random sequence region and primer binding regions at both ends thereof. After amplification by RT-PCR reaction, transcription is performed using the resulting cDNA molecule as a template, and the next round of RNA pool is repeated for several to several tens of rounds to bind more to the target molecule. This is a method for selecting strong RNAs. The base sequence lengths of the random sequence region and the primer binding region are not particularly limited. Generally, the random sequence region ranges from 20-80 bases and the primer binding region ranges from 15-40 bases each. In order to increase the specificity to the target molecule, a molecule similar to the target molecule is mixed with an RNA pool, and a pool consisting of RNA molecules that do not bind to the molecule may be used. The RNA molecules finally obtained by such methods are used as RNA aptamers. The SELEX method is a known method, and a specific method may be performed, for example, according to Pan et al. (Proc. Natl. Acad. Sci. U.S.A., (1995) 92: 11509-11513).

When the nucleic acid aptamer is composed of natural RNA, the DNA encoding it can also be operably inserted under the control of the promoter of the expression vector. Such an "RNA aptamer expression vector" expresses an RNA aptamer by promoter activity when introduced into a target organism or target cell, like the shRNA expression vector described above. After expression, it binds to the target protein in the host cell and suppresses its function. Furthermore, it is also possible to insert (knock-in) a DNA encoding an RNA aptamer into the genome of a lepidopteran insect by the transposon method or the genome editing method described below, similarly to the shRNA expression vector.

(Introduction method)
The aptamer introduction method conforms to the siRNA introduction method described above. Therefore, detailed description is omitted here. In addition, as described above, the ribozyme can be introduced in the form of DNA as an RNA aptamer expression vector in the same manner as the shRNA expression vector, and the RNA aptamer can be expressed in the host cell. The method for introducing the RNA aptamer expression vector may also be according to the method for introducing the shRNA expression vector.

(Selected)
Selection of aptamer-introduced individuals may also be performed according to the siRNA selection method. When an RNA aptamer expression vector is introduced, by inserting a marker gene into the vector in the same manner as the shRNA expression vector, the introduced individuals can be selected based on the activity of the marker gene.

(Passage)
In principle, aptamers suppress target genes for only one generation, similar to siRNA. However, when the DNA encoding the RNA aptamer is inserted into the genome of the host lepidopteran insect, even if the strain is passaged, the target gene is suppressed by inducing the expression of the RNA aptamer in the progeny. be able to.

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 the 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 according to the first aspect.

2-2. Structure Sterile lepidopteran insects are characterized by repression of the nanosO/P gene expression in germline cells during early embryogenesis. It is sufficient that the expression of both nanosO/P genes is suppressed (double knocked down).

The gene expression-suppressed sterile lepidopteran insect is, but not limited to, an individual obtained through the gene knockdown method in the method for producing a sterile lepidopteran insect according to the first aspect, or a gene expression inhibitor. is an individual that induces gene silencing in progeny that contain .

The types of sterile lepidopteran insects are not limited, but are preferably industrially useful insects, for example, types for the purpose of collecting silk threads, such as silkworms, mulberries, sycamores (Erisan), wild tiger moths, sakusans, cypresses, etc. Silkworms are preferred.

The stage of development of sterile lepidopteran insects is not limited. Any of eggs, larvae, pupae and adults may be used. However, for purposes of sterility, the worms are preferably naturally fertile adults.

The sterile lepidopteran insects of the present invention can be distinguished from fertile lepidopteran insects based on molecular genetic techniques and/or phenotypic characteristics.

Distinction based on molecular genetic techniques may be achieved by, for example, measuring or detecting the expression level of the nanosO/P gene using a conspecific lepidopteran insect with normal nanosO/P gene expression and fertility 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 lepidopteran insect of the present invention, the expression level of the nanosO/P gene is significantly reduced (for example, p<0.05, p<0.01, p<0.001) compared to that of the control individual.

Distinction based on phenotypic characteristics may be confirmed by observing morphological abnormalities in reproductive organs or germ cells. In the case of sterile lepidopteran insects of the present invention, adult females lay significantly fewer or no eggs compared to control individuals. In the case of male worms, testes become dwarfed and transparent, and fertility is reduced.

3. Sterilizing composition for lepidopteran insects 3-1. SUMMARY A third aspect of the present invention is a composition for sterilizing lepidopteran insects. The sterilization composition of the present invention contains, as an active ingredient, a gene expression suppressor that specifically suppresses the expression of each of the nanosO gene and the nanosP gene. Any type of lepidopteran insect can be easily and efficiently sterilized using the sterilizing composition of the present invention.

3-2. Configuration The components of the composition for sterilizing lepidopteran insects of the present invention will be described.

3-2-1. Active Ingredients The composition for sterilizing 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 the target gene, the nanosO gene or the nanosP gene.
Gene expression inhibitors are classified into transcription product inhibitors and translation product inhibitors based on their gene expression inhibitory action.

(1) Transcript suppressor As used herein, the term “transcript suppressor” refers to targeting mRNA, which is the transcription product of the nanosO gene or nanosP gene, which is a target gene, and degrading or inactivating it. It is an agent that has the effect of suppressing gene expression. Specific examples of transcript inhibitors include RNAi agents, antisense oligonucleotides (ASOs), and nucleic acid enzymes. As for specific configurations of these agents, the RNAi agent, ASO, and Since it follows the construction of nucleic acid enzymes, only a brief description will be given here.

If the transcript inhibitor is an RNAi agent, examples include nanosO-dsRNA, nanosO-siRNA or nanosO-shRNA against the nanosO gene and nanosP-dsRNA, nanosP-siRNA or nanosP-shRNA against the nanosP gene. When the transcript repressing agent is an shRNA, it may be in the form of an expression vector operably containing a nucleic acid encoding it. The effect of RNAi agents as transcription product inhibitors is, in principle, limited to one generation. Even if the line is subcultured, the effect of the RNAi agent can be obtained by inducing the expression of nanosO-shRNA or nanosP-shRNA in progeny having the expression vector.

If the transcript repressor is an ASO, examples include nanosO-ASO for the nanosO gene and nanosP-ASO for the nanosP gene. In addition, when the ASO is of RNA type, it may be in the form of a nanosO-asRNA expression vector or a nanosP-asRNA expression vector containing a nucleic acid encoding it in an operable state. In principle, the effect of ASO as a transcription product suppressor is limited to one generation. , the effect of ASO can be obtained by inducing the expression of nanosO-ASO or nanosP-ASO in progeny carrying the expression vector.

If the transcription product inhibitor is a nucleic acid enzyme, examples include nanosO-(deoxy)ribozyme against the nanosO gene and nanosP-(deoxy)ribozyme against the nanosP gene. When 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 containing nucleic acid encoding it in an operable state.

(2) Translation product inhibitors As used herein, the term "translation product inhibitor" refers to targeting a protein that is the translation product of the nanosO gene or nanosP gene, which is a target gene, and degrading or inactivating it. It is an agent that has the effect of suppressing gene expression. Specific examples of translation product inhibitors include anti-nanosO aptamers against nanosO proteins and anti-nanosP aptamers against nanosP proteins. Since the specific structure of the aptamer conforms to the structure of the aptamer described in detail in "(4) Aptamer" of the first aspect, only a brief description will be given here.

When the transcription product repressing agent is an RNA aptamer, it may be in the form of an expression vector operably containing a nucleic acid encoding it. The effect of aptamers as transcription product inhibitors is, in principle, limited to one generation, but if an expression vector encoding nanosO-RNA aptamer or nanosP-RNA aptamer is inserted into the genome of the host lepidopteran insect, Therefore, even if the line is subcultured, the effect of the RNA aptamer can be obtained by inducing the expression of the nanosO-RNA aptamer or the nanosP-RNA aptamer in the offspring having the expression vector.

The sterilization composition of the present invention uses at least a gene expression inhibitor for the nanosO gene and a gene expression inhibitor for the nanosP gene in pairs.

For reasons other than those mentioned above, the sterilization composition of the present invention can also contain two or more gene expression inhibitors for either the nanosO gene or the nanosP gene. For example, the sterilizing composition of the present invention contains a total of three active ingredients, consisting of two gene expression inhibitors for the nanosO gene, siRNA and shRNA, and one gene expression inhibitor for the nanosP gene, siRNA. You can stay.

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

Solvents include, for example, water or aqueous solutions, or organic solvents acceptable to lepidopteran insects. Aqueous solutions include, for example, buffers (phosphate buffer, sodium acetate buffer, etc.), physiological saline, and isotonic solutions.

Carriers include glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, low concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like.

3-3. Administration Method The method for administering the sterilizing composition of the present invention to lepidopteran insects may be carried out according to the method for introducing each gene expression suppressing agent described in the method for suppressing gene expression of the first aspect.

<Example 1>
(the purpose)
The sterility and sterilization efficiency of sterile lepidopteran insects obtained by the method for producing sterile lepidopteran insects of the present invention are verified.

(Method)
(1) Materials As a host lepidopteran insect, silkworms (pnd strain) subcultured at the National Agriculture and Food Research Organization (NARO, Japan) were used.

(2) Preparation of Gene Expression Suppressant In this Example, RNAi agents against each of the target gene nanosO gene and nanosP gene were prepared as gene expression inhibitors used in the gene expression suppression step.

First, the silkworm nanosO gene consisting of the nucleotide sequence shown in SEQ ID NO: 2 was used as a template, and the primer pair shown in SEQ ID NOS: 6 and 7 (O-Fw and O-Rv, respectively) was used to amplify the nucleotide sequence shown in SEQ ID NO: 5. . The reaction conditions for PCR were (98°C, 10s; 55°C, 15s; 68°C, 60s) x 15, and then (98°C, 10s; 68°C, 60s) x 25. GXL polymerase (Takara Bio company).

Similarly, the silkworm nanosP gene consisting of the nucleotide sequence shown in SEQ ID NO: 4 was used as a template, and the primer pair shown in SEQ ID NOS: 9 and 10 (P-Fw and P-Rv, respectively) was used to amplify the nucleotide sequence shown in SEQ ID NO: 8. . The 5' end of each Fw primer contains a T7 promoter sequence. The PCR reaction conditions were the same as those described above.

Furthermore, dsRNA was similarly prepared for the nanos paralogue genes nanosM gene and nanosN gene. The nanosM gene was obtained by using the silkworm nanosM gene having the nucleotide sequence shown in SEQ ID NO: 11 as a template, and the primer pair shown in SEQ ID NOS: 13 and 14 (M-Fw and M-Rv, respectively). and the nanosN gene is shown in SEQ ID NO: 16 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 NOS: 17 and 18 (N-Fw and N-Rv, respectively). Each base sequence was amplified. The PCR reaction conditions were the same as those described above.

Next, using each obtained PCR amplification product, a double-stranded RNA (dsRNA) derived from each nanos gene was prepared with Megascript RNAi kit (Ambion). A specific preparation method followed the protocol attached to the kit.

For injection, the concentration of dsRNA of each nanos gene was adjusted to 3 μg/μL with distilled water. When simultaneously suppressing the expression of two types of target genes, dsRNAs of two types of nanos genes in the mixed solution were prepared at equal ratios so that the concentration of each was 3 μg/μL. Here, (1) nanosO-dsRNA alone, (2) nanosP-dsRNA alone, (3) a mixture of nanosM-dsRNA and nanosO-dsRNA (nanosM/O-dsRNA), (4) nanosN-dsRNA and nanosO-dsRNA A mixture (nanosN/O-dsRNA) and (5) a mixture of nanosO-dsRNA and nanosP-dsRNA (nanosO/P-dsRNA) were prepared as nanos-RNAi agents.

(3) Injection of dsRNA into Silkworm Eggs Subsequently, mated female moths were placed on an egg-laying board and allowed to lay eggs for 1 hour. Between 2 and 4 hours after egg laying, 10 nL to 20 nL of each prepared nanos-RNAi agent was microinjected into each egg using a glass micropipettor. After the injection, the injection hole was closed with an instant adhesive (Turiron instant adhesive, multi-purpose, fast-hardening type: Alpha Shoji Co., Ltd.). Eggs laid at the same time without injection were used as negative controls. After that, the egg-laying mount was quickly transferred to a petri dish and the lid was closed. Subsequently, in order to create a humidified state, a kitchen towel moistened with water was laid inside the tight box, the petri dish was placed thereon, and the tight box was covered with a lid. The tight boxes were incubated at 25-28°C for about 10 days until the eggs hatched.

(4) Breeding of silkworms and confirmation of phenotypes of adult gonads Larvae after hatching are reared in a rearing room at 28°C with artificial feed (silkmate original species 1-3rd instar S, Nosan Kogyo) for all ages. bottom. The artificial diet was changed as needed.

After eclosion, ovaries were removed from unmated female adults, and the number of mature eggs in the ovaries was counted. In silkworms, most of the eggs in the ovaries are usually mature at the time of eclosion of females.

In addition, testes were removed from unmated male adults after eclosion, 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 to the total number of matings. When the number of fertilized eggs laid by the female after mating was 100 or more, it was regarded as normal mating. Fertilized eggs were counted as cyanoocytes (colored eggs associated with egg development several days after spawning).

Furthermore, phenotypes other than germline cells (behavior, growth, morphology, etc.) were also examined for each individual after nanos-RNAi injection.

(result)
Figures 1 to 5 show the number of females after injection and the number of mature eggs in their ovaries, and Figure 6 shows the morphology of excised testes. Figure 1 shows the results of nanosO-RNAi administered with nanosO-dsRNA alone, Figure 2 shows the results of nanosO-RNAi administered with nanosP-dsRNA alone, and Figure 3 shows the results of nanosM administered with nanosM/O-dsRNA. FIG. 4 shows the results of nanosN/O-RNAi administered with nanosN/O-dsRNA, and FIG. 5 shows the results of nanosO/P-RNAi administered with nanosO/P-dsRNA. be. In FIG. 6, A is the testis of a wild-type male adult, B is the testis of a nanosO-RNAi-treated adult male, and C is the testis of a nanosO/P-RNAi-treated adult male.

(oogenesis)
From the results of FIGS. 1 to 5, among the individuals treated with nanosO-RNAi, nanosP-RNAi, nanosM/O-RNAi, and nanosN/O-RNAi, no individuals with 150 or less mature eggs were found. These results suggest that the nanosO gene alone or double suppression with other nanos paralogues, the nanosM or nanosN genes, does not affect oogenesis. Nakao H. and Takasu Y. (2019, supra) disclose that Bm-nosO gene knockout silkworms rarely show a decrease in the number of mature eggs. Even in this example, when the expression of the nanosO gene was suppressed by nanosO-RNAi, no abnormality in the number of mature eggs could be confirmed. not inconsistent. In other words, it is suggested that suppression or inhibition of nanosO gene expression alone is insufficient to induce stable sterilization of lepidopteran insects. Furthermore, from FIG. 2, the same results as those of the nanosO gene were confirmed in the single suppression of the nanosP gene expression. In other words, it was shown that even if the expression of the nanosP gene is suppressed alone, it is insufficient to induce stable sterilization of lepidopteran insects.

On the other hand, the results of FIG. 5 revealed that double suppression of the nanosO gene and the nanosP gene significantly decreased the number of mature eggs, resulting in clear oogenesis abnormalities. Females with less than 150 mature eggs accounted for about 70% of the injected individuals. These results demonstrate that mature egg formation is inhibited only when the expression of the nanosO/P gene is double-suppressed.

(testis morphology)
No morphological difference was observed between the wild-type male testis and the nanosO-RNAi-treated male testis from FIGS. 6A and 6B. However, in the nanosO/P-RNAi-treated male testes shown in C, partially cleared testes were observed as indicated by arrows, and multiple remarkably dwarfed testes were observed as indicated by arrowheads. .

(mating rate)
Table 1 shows the results.

The normal mating rate was significantly reduced only when the expression of nanosO/P was double suppressed by RNAi. On the other hand, when only the expression of nanosO or nanosP was suppressed by RNAi, the normal mating rate was similar when the expression of nanosM/O or nanosN/O was double suppressed by RNAi. This result suggests that spermatogenesis with normal function is inhibited in males in which nanosO/P expression is double-suppressed by RNAi, and fertility is reduced or lost.

From these results, it was clarified that only when the expression of the nanosO/P gene was double-suppressed, males not only showed abnormal testicular morphology, but also lost the fertilizing ability of sperm.

(other phenotypes)
Observation of phenotypes other than germline cells (behavior, growth, morphology, etc.) throughout the breeding process revealed no noteworthy phenotypic differences other than infertility. In other words, they grew, molted and metamorphosed as usual, formed cocoons normally, became adults, and had no change in their mating behavior. In addition, no difference from the wild type was observed in the sizes of larvae and adults.

(Conclusion)
From the above, it was concluded that the nanosO and nanosP genes function redundantly in germ line cell formation such as primordial germ cells, that their functions cannot be complemented by other nanos paralogs, etc. Double suppression has been shown to cause stable abnormalities in germline cell formation and infertility. In other words, it is suggested that stable induction of sterilization in lepidopterous insects requires suppression or inhibition of nanosO/P gene expression during early embryogenesis.

<Comparative Example 1>
(the purpose)
The fertility-suppressing effect on the lepidopterous insects of the present invention was obtained by double-suppressing or inhibiting the expression of the nanosO/P genes by preparing expression-suppressing agents for the nanos genes alone and in combination, which were not verified in Example 1. to be obtained only in

(Method)
Since the basic operation conforms to the first embodiment, only the differences from the first embodiment will be explained here.

(1) Preparation of gene expression inhibitor In this comparative example, RNAi agents for each of the nanos paralogue genes nanosM gene and nanosN gene used in Example 1 and the target genes nanosO gene and nanosP gene of the present invention were prepared. bottom. The specific preparation method followed the method described in Example 1.

For injection, the concentration of dsRNA of each nanos gene was adjusted to 3 μg/μL with distilled water. 2 to 4 types of RNAi agents were prepared by equal ratio of dsRNA of each nanos gene, and each concentration was adjusted to 3 μg/μL.

In this comparative example, (1) nanosM-dsRNA alone (nanosM-dsRNA), (2) nanosN-dsRNA alone (nanosN-dsRNA), (3) nanosM-dsRNA, which are control groups that were not verified in Example 1 and nanosP-dsRNA (nanosM / P-dsRNA), and (4) a mixture of nanosN-dsRNA and nanosP-dsRNA (nanosN / P-dsRNA), (5) nanosM-dsRNA, nanosN-dsRNA and nanosO- 3 kinds of mixture of dsRNA (nanosM/N/O-dsRNA), (6) 3 kinds of mixture of nanosM-dsRNA, nanosN-dsRNA and nanosP-dsRNA (nanosM/N/P-dsRNA) and (7) nanosM-dsRNA, Seven types of nanos-RNAi agents were prepared, including a mixture of nanosN-dsRNA, nanosO-dsRNA and nanosP-dsRNA (nanosM/N/O/P-dsRNA). The method described in Example 1 was followed for injection of dsRNA into silkworm eggs, rearing of silkworms, and confirmation of the phenotype of adult gonads.

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

From the results of FIGS. 7 to 13, individuals treated with any RNAi, except for nanos gene quadruple suppression (nanosM/N/O/P-dsRNA) in FIG. Individuals with less than 150 mature eggs were 0. These results show that even if the nanosM gene and the nanosN gene are singly suppressed, or double-suppressed or triple-suppressed with other nanos genes, the eggs of lepidopteran insects do not include the double-silenced nanosO gene and nanosP gene. suggesting that it does not affect formation.

Summarizing the results of Example 1 and this comparative example, the stable sterilization effect of lepidopteran insects was induced only in the case of double suppression of the nanosO gene and the nanosP gene. Double suppression and multiple suppression without the nanosO and nanosP genes proved inadequate.

Claims (15)

A method for producing a sterile lepidopteran insect, comprising a step of suppressing the expression of nanosO gene and nanosP gene. 2. The production method according to claim 1, wherein the suppression of gene expression uses a gene knockdown method. 3. The production method according to claim 2, wherein the gene knockdown method is an RNAi method or an antisense oligonucleotide method. The production method according to any one of claims 1 to 3, wherein the nanosO gene comprises a base sequence encoding a nanosO protein consisting of the amino acid sequences shown in (a) to (c) below.
(a) the amino acid sequence shown in SEQ ID NO: 1,
(b) an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, or (c) 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 1 amino acid sequence having 5. The production method according to claim 4, wherein the nanosO gene consists of the nucleotide sequence shown in SEQ ID NO:2. 4. The production method according to any one of claims 1 to 3, wherein the nanosP gene comprises a nucleotide sequence encoding a nanosP protein consisting of the following amino acid sequences (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) 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 3 amino acid sequence having 7. The production method according to claim 6, wherein the nanosP gene consists of the base sequence shown in SEQ ID NO:4. A sterile lepidopteran insect in which the expression of the nanosO gene and nanosP gene is suppressed. 9. The sterile lepidopteran insect according to claim 8, wherein said expression suppression is by a gene knockdown method for each gene. 10. The sterile lepidopteran insect of claim 9, wherein said gene knockdown method is RNAi method or antisense oligonucleotide method. A composition for sterilizing lepidopteran insects, containing 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 claim 11, wherein the gene expression inhibitor is a transcription product inhibitor targeting the transcript of each gene. 13. The sterilizing composition of Claim 12, wherein said transcript inhibitor is an RNAi agent or an antisense oligonucleotide. 14. The sterilizing composition of claim 13, wherein said RNAi agent is an expression vector operably containing a nucleic acid encoding an shRNA for each said gene. 12. The sterilization composition according to claim 11, wherein the gene expression inhibitor is a translation product inhibitor targeting the translation product of each gene.

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