Method for producing sterile lepidopteran insect and sterilizing composition of lepidopteran insect

Abstract

To provide a method for producing a sterile lepidopteran insect by a sterilization method which can simply, stably and efficiently sterilize a lepidopteran insect and causes abnormality only in germ line cells, and a sterilizing composition capable of executing the method.SOLUTION: The expression of nanosO and nanosP genes 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 method for sterilizing lepidopteran insects. It relates to a composition. [Background technology]

[0002] Genetic modification technology, typified by genetic recombination technology, is used to analyze the functions of genes and proteins, It is an essential technology for the production of substances such as bacteria and proteins. The host organisms used for this purpose have mainly been Escherichia coli and yeast. It was difficult to say that this system was suitable as a material production system for the mass production of proteins, etc.

[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 been used industrially for silk production for a long time. Because they make cocoons during the pupal stage, they can produce a large amount of silk thread in a short period of time. This production ability is utilized to produce recombinant corn. Creating transgenic silkworms and mass-producing useful proteins other than silk thread Technology is in the spotlight.

[0004] On the other hand, in substance production systems using genetic engineering technology, there is a risk of genetically modified organisms leaking into the wild. This involves the problem of genetic contamination of the environment. Silkworms have completely lost their ability to fly, so they are not subject to the Cartagena Protocol. It is approved for Type 1 use based on the REACH Act, i.e., use without preventing its spread into the environment. However, males of the closely related species, the mandarin silkworm (Bombyx mandarina), have the ability to fly and can invade from outside. The possibility of crossbreeding between female silkworms cannot be ruled out. Therefore, it is necessary to maintain useful strains and to carry out genetic recombination. The development of sterilization techniques for silkworms that cannot produce new generations is essential to prevent the spread of the virus into the environment. It is important for business purposes.

[0005] Conventionally, the main method for sterilizing insects has been the Sterile Insect Technique (SIT). Sterilization methods have been developed and implemented to produce sterile insects in the Radiation irradiation methods are known.

[0006] The radiation irradiation method involves irradiating the germ cells of the target insect with radiation such as X-rays or gamma rays, resulting in sperm A method of sterilizing target insects by inducing offspring inactivation, egg loss, reduced egg count, and mating inability. However, this method requires an irradiation facility and raises safety concerns. In addition, there were concerns that radiation exposure would 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 provide a method for sterilizing lepidopteran insects simply, stably, and efficiently, and We have developed a new sterilization method that induces abnormalities only in germline cells, and are conducting sterilization choriocarcinoma using this method. By developing and providing a method for producing cormorant insects and a sterilization composition that can be used to carry out the method, be. [Means for solving the problem]

[0009] As a result of intensive research aimed at solving the above problems, the present inventors have discovered a novel lepidopteran phenotype present in lepidopteran insects. The four nanos genes present (nanosM, nanosN, nanosO, and nanosP) When the nanosO and nanosP genes were double suppressed during embryonic development, This is due to a decrease in the number of mature eggs or the disappearance of eggs, as well as germ cell line agenesis such as testicular dwarfism. However, the cells other than the germline cells were normal. It was also revealed that this phenomenon can be applied to genetically sterilize lepidopteran insects. The present invention is based on this finding and provides the following.

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

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

[0012] Furthermore, the sterilization composition for lepidopteran insects of the present invention can be used to sterilize any desired lepidopteran insects. Sterilization can be achieved easily, stably, and efficiently. [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 the steps of: The method includes a step of suppressing the expression of two nanos genes in the target lepidopteran insect, and By disrupting the function of offspring, it induces abnormalities in the germline cells of the insect, resulting in infertility in lepidopterans. It is characterized by producing insects.

[0015] 1-2.Definition of Terms The definitions of terms frequently used in this specification are explained below. "Lepidoptera insects" are insects that belong to the taxonomic order Lepidoptera, It refers to butterflies or moths. Butterflies include the Nymphalidae, Papilionidae, and apilionidae, Pieridae, Lycaenidae, and Xenopus The family includes insects belonging to the Hesperiidae family. ae), Bombycidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Geometridae, Archtiidae, Noctuidae, Pyralidae, Sphingidae This includes insects belonging to the Sphingidae family. For example, moths include the Bombyx and Samia genera, and Ant Species belonging to the genera Heraea, Saturnia, Attacus, and Rhodinia, specifically silkworms and mulberry Bombyx mandarina, Samia cynthia, Samia cynthia ricinus including hybrids of the Japanese silk moth and the Eri silk moth), the Japanese stag moth (Antheraea yamamai), Antheraea pernyi, Saturnia japonica, Act. However, the lepidopteran insects of the present invention are not limited to these. Silkworms are particularly popular.

[0016] As used herein, the term "target Lepidoptera insect" refers to a sterile Lepidoptera insect produced by the method of the present invention. or the insects to which the sterilizing composition for lepidopteran insects of the present invention is administered. This refers to insects of the order Lepidoptera.

[0017] As used herein, "infertility" refers to the inability to form the next generation due to loss or significant reduction of fertility. Infertility as used herein includes, but is not limited to, the following: It can be caused primarily by abnormalities in germline cells.

[0018] As used herein, "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. This refers to

[0020] As used herein, "germline cells" refers to eggs or ova and sperm that are directly involved in reproduction. These are the cells that will become eggs and sperm in the future. Specifically, eggs (ovum), sperm, oogonia, and oocytes Primordial germ cells (PGCs) that differentiate into the following cells: spermatogonia, spermatocytes, spermatids, and spermatids. These cells contain progenitor germ cells (PGCs).

[0021] As used herein, "germline cell abnormality" refers to a malformation such as loss of germline cells. It also refers to abnormalities in shape and characteristics.

[0022] As used herein, "suppressing gene expression" or "suppressing gene expression" refers to the act of inhibiting the expression of a gene. Also known as knockdown, this suppresses the expression and function of the protein encoded by the gene. Specifically, in the expression of a target gene, it refers to the transcriptional, post-transcriptional, translational, or suppresses the function of the transcription product (mRNA) or translation product (protein) of target genes after translation Gene knockdown involves disrupting a target gene and increasing the amount of the protein it encodes. This is distinct from gene knockout, which results in the complete loss of function of a protein.

[0023] As used herein, the term "expression vector" refers to a vector that carries a nucleic acid molecule that encodes a protein or a functional nucleic acid. A vector that contains a nucleic acid molecule in an operative state and can control the expression of the nucleic acid molecule. In addition, in this specification, "operable state" means that the target gene is in an expression vector. This refers to placing a target nucleic acid molecule under the control of a promoter. The activity of the ATP-dependent ATPase activates the expression of the nucleic acid molecule of interest.

[0024] As used herein, the term "nucleic acid molecule" refers to a gene encoding a protein or a gene encoding a functional nucleic acid. It refers to a polynucleotide or oligonucleotide that encodes a nucleic acid molecule, including but not limited to a nucleic acid molecule. In principle, they are composed of natural nucleic acids, such as DNA and / or RNA. However, they also contain artificial nucleic acids. That's fine.

[0025] As used herein, the term "functional nucleic acid" refers to a nucleic acid that performs a specific biological function in a living body or cell. functions, such as enzymatic functions, catalytic functions, or biological inhibitory or enhancing functions (e.g., transcription, translation, etc.) Specifically, for example, RNAi agents, nucleic acid aptamers, etc. aptamers (DNA aptamers, RNA aptamers, etc.), antisense oligonucleotides, nucleic acids Enzymes and the like are included.

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

[0027] "Genetic recombination" refers to the use of vectors such as plasmids or transposons to transform a host organism. a method of adding foreign genetic information into the genome, etc., that the host organism does not possess, or a method of adding foreign genetic information into the genome, etc., that the host organism does not possess, or Examples of methods include altering or destroying information.

[0028] "Genome editing" refers to the process of creating double strand breaks (DSBs) caused by DNA cleavage enzymes. Using DNA repair mechanisms, etc., foreign genes can be inserted (knocked in) at any position on the genome. It is a gene targeting technology that destroys (knocks out) target genes.

[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. This step will be explained in detail below.

[0030] 1-3-1. Gene expression suppression process The "gene expression suppression process" is a process for suppressing the expression of the nanosO gene and the nanosP gene in the target lepidopteran insect. (Herein, these genes are often collectively referred to as "nanosO / P genes.") In lepidopteran insects, the expression of the nanosO protein and nanosP protein is suppressed. Proteins (herein, these proteins are often collectively referred to as "nanosO / P proteins" By disrupting the function of these two genes during embryonic development, the formation of germline cells is inhibited. The results of the research by the present inventors have revealed that the process damages the ovaries, resulting in infertility in both males and females. So, we will use this phenomenon to suppress the expression of the nanosO / P gene in the target lepidopteran insect, sterilize the insects.

[0031] The "nanos gene" (or nos gene) is a gene that encodes the nanos protein. "nanos proteins" are evolutionarily conserved proteins with zinc finger motifs. Research using fruit flies (Drosophila melanogaster) has revealed that nanos Proteins are thought to play a role in the survival and proliferation of primordial germ cells (PGCs) (Keuckelae re ED et al., 2018, Cell Mol Life Sci., 75:1929-1946). Unlike insects, there are four nanos paralogs (nanosM, nanosN, nanosO, and nanosP) (Nakao H., et al., 2008, Evolution & Development, 10(5): 548-554; Carter J- M, et al., 2015, PLoS ONE, 10: e0144471). In silkworms, four paralogous genes ( Bm-nosM, Bm-nosN, Bm-nosO, and Bm-nosP have been identified, and the results of tissue expression It has been suggested that the Bm-nosO protein is important for the formation of primordial germ cells. The phenotype of Bm-nosO gene knockout silkworms using the collection technique showed abnormal egg formation and rare A decrease in the number of mature eggs was observed, and the Bm-nosO protein is involved in the egg (germ cell) formation process. It has been suggested that this is the case (Nakao H. and Takasu Y., 2019, Developmental Biology, 445:20 9-36) On the other hand, the specific functions of other nanos paralogs in lepidopteran insects are unknown. be.

[0032] The nanosO / P gene to be suppressed in this specification is a gene encoding the nanosO / P gene of the target lepidopteran organism to be sterilized. There are no particular limitations as long as it is an insect nanosO / P gene. The target genes are nanosP and nanosP orthologs. Any nucleotide sequence encoding the nos protein will suffice.

[0033] For example, in the case of the nanosO gene, if the target Lepidoptera insect is a silkworm, the A wild-type Bm-nosO gene encoding a wild-type Bm-nosO protein consisting of the amino acid sequence or one or more amino acids are deleted, substituted or deleted in the amino acid sequence shown in SEQ ID NO: 1. is 90% or more of the added amino acid sequence or the amino acid sequence shown in SEQ ID NO: 1, preferably or have 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity The mutant Bm-nosO gene encoding the mutant Bm-nosO protein consisting of the amino acid sequence A specific example of the wild-type Bm-nosO gene is a gene comprising the base sequence shown in SEQ ID NO: 2. In this specification, "plurality" 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 amino acids. ) substitutions are substitutions that change the charge, side chain, and This refers to substitutions within a conservative amino acid group that have similar properties such as polarity and aromaticity. For example, Uncharged polar amino acids with side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched chain amino acids amino acid group (Leu, Val, Ile), neutral amino acid group (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), aromatic amino acids (Phe, Tyr, Trp Amino acid substitutions within these groups may alter the properties of the polypeptide. It is preferred because it is known that the quality is less likely to change. The two amino acid sequences are aligned, and one or both of the sequences are aligned as needed. When gaps are introduced into the amino acid sequence to maximize the amino acid identity between the two. The number 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. The percentage of amino acid residues.

[0034] In the case of the nanosP gene, if the target Lepidoptera insect is a silkworm, the sequence shown in SEQ ID NO: 3 a wild-type Bm-nosP gene encoding a wild-type Bm-nosP protein consisting of the amino acid sequence In the amino acid sequence shown in SEQ ID NO: 3, one or more amino acids are deleted, substituted, or 90% or more of the added amino acid sequence or the amino acid sequence shown in SEQ ID NO: 3, preferably or an amino acid sequence having 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity. The mutant Bm-nosP gene encodes a mutant Bm-nosP protein consisting of the amino acid sequence A specific example of the wild-type Bm-nosP gene is the Bm-nosP gene having the base sequence shown in SEQ ID NO: 4. An example is the m-nosP gene.

[0035] In this step, the two types of nanos genes are expressed in the target lepidopteran insect to be sterilized. The following describes the gene expression suppression method in detail. do.

[0036] A. Methods for suppressing gene expression As used herein, the term "method for inhibiting gene expression" refers to a method for specifically inhibiting the expression of a target gene. The method for suppressing the expression of a specific gene in a lepidopteran insect is known in the art. For example, the method for detecting two target proteins, nanosO / Small molecules that specifically inhibit the function of each P protein are administered to target lepidopteran insects. These include the gene knockdown method and the established technique, which are simple and highly effective. Promising gene knockdown methods are particularly preferred.

[0037] The "gene knockdown method" is a method of detecting target gene products in cells using (poly)nucleotides. or (poly)peptide-mediated depletion of target genes. They target gene products, i.e., gene transcription products such as mRNA, and post-translational translation products. A specific example of gene knockdown is (1) RNAi method, (2) antisense oligonucleotide method, (3) nucleic acid enzyme method, and (4) Aptamer method, etc. Each method will be explained below.

[0038] (1)RNAi method "RNAi method (RNA interference method)" refers to a method in which an RNAi agent is administered to a host, and the RNA interference (RNA RNA interference (RNAi) is used to suppress target gene expression at the post-transcriptional, pre-translational, or transcriptional level. RNA interference is a method of inhibiting the target gene by, for example, degrading the transcript of the gene. This is sequence-specific gene silencing that suppresses the expression of the nanosO / P gene. RNAi agents are used to induce specific gene silencing for each transcript of a gene. The RNAi agent herein is a gene encoding the nanosO gene or the nanosP gene of a lepidopteran insect. We hypothesize a mechanism that suppresses the expression of the gene after transcription and before translation.

[0039] "RNAi agents" are agents that induce RNA interference (RNAi) in vivo. It induces the expression of the target gene by degrading the transcript of the gene (silencing). For example, artificially synthesized dsRNA, siRNA, shRNA, or endogenous These include miRNAs (microRNAs) (including pri-miRNAs and pre-miRNAs) that are involved in RNA interference. For details, see, for example, Bass BL, 2000, Cell, 101, 235-238; Sharp PA, 2001, Genes D ev., 15 ,485-490;Zamore PD, 2002, Science, 296, 1265-1269;Dernburg,AF & K The target gene of the present invention is described in detail in Arpen, GH, 2002, Cell, 111, 159-162. dsRNA or siRNA that can be arbitrarily designed against the nanosO gene and the nanosP gene, or The primary RNAi agent is shRNA, but miRNA targeting the nanosO gene or nanosP gene is not included. If there is a specific RNA agent, it can be used. do.

[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) The method of introducing dsRNA to obtain the gene expression suppression effect is to introduce in vitro prepared dsRNA into the host. The administration method can be injection by microinjection or dsRNA dissolution. However, if the host is a lepidopteran insect, it is preferable to use a method known in the art, such as immersion in a solution. In some embodiments of the present invention, injection administration is preferred. The concentration of the dsRNA solution used for introduction is determined based on the concentration of the dsRNA in the insect after introduction. It is sufficient to ensure that a certain concentration or more exists in the body (for example, eggs). More specifically, the limitation is However, usually, solutions with concentrations of several μg / μL to several tens of μg / μL are prepared, for example, in volumes of 2 nL to 40 nL, 5 nL to The amount of injection may be 30 nL, 8 nL to 25 nL, or 10 nL to 20 nL.

[0042] Microinjection was performed 2-8 hours after egg laying, before the nucleus was integrated into the cell membrane. It is effective to do this to eggs for a while. Or, it has the same effect as if it were injected into eggs. To obtain this information, we have also considered a method in which dsRNA is introduced into mature eggs of females before spawning, followed by fertilization and spawning. When dsRNA is introduced, a marker gene may be introduced at the same time as the dsRNA. This makes 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 needed. For example, a portion of the dsRNA-introduced individuals can be collected and analyzed by RT-PCR to check the expression level of the target gene. Alternatively, as mentioned above, the reduction in the number of dsRNAs can be confirmed by measuring the number of dsRNAs in the control individuals. When a marker gene is introduced as a selectable marker at the same time as the transfection, The introduced individuals can be selected based on the activity.

[0044] As used herein, a "marker gene" is a gene that encodes a marker protein. The recognition protein is a protein that can distinguish the presence or absence of expression of a marker gene based on its activity. "Based on activity" means "based on the results of activity detection." The activity may be detected directly by detecting the activity of the labeled protein itself, or by detecting the activity of the labeled protein. It may also be detected indirectly via metabolites such as pigments produced by protein activity. Detection can be chemical detection (including enzyme reaction detection), physical detection (including behavioral analysis detection), etc. ), or the sensory detection of the detector (including detection by sight, touch, smell, hearing, or taste) This may also be the case.

[0045] The type of labeled protein may be any of the following, as long as its activity can be detected by a method known in the art: There are no particular limitations. Preferably, the pathogen is less invasive to the host insect of the order Lepidoptera upon detection. These are useful labeled proteins. For example, fluorescent proteins, pigment-synthesizing proteins, and luminescent proteins are Proteins that control external morphology, exocytic proteins, and fluorescent proteins. Proteins, pigment synthesis proteins, photoproteins, and exocrine proteins are secreted under certain conditions. They are visually detectable, minimally invasive to lepidopteran insects, and easy to identify and select. Therefore, it is particularly suitable.

[0046] The fluorescent protein emits a specific wavelength when excitation light of a specific wavelength is irradiated onto a lepidopteran insect. It refers to a protein that emits fluorescence. It may be either a natural or non-natural type. There are no particular limitations on the excitation wavelength and fluorescence wavelength. Specific examples include CFP, AmCyan, RFP, and DsRed. (including derivatives such as DsRed monomer and DsRed2), YFP, GFP (including derivatives such as EGFP and EYFP) (including) etc.

[0047] The pigment synthesis protein is a protein involved in the biosynthesis of pigments, and is usually an enzyme. The term "pigment" used here refers to a low molecular weight compound that can impart a pigment to a transformant. or peptides, of any type. Preferably, they are pigments that appear as external colors on the individual. For example, melanin pigments (including dopamine melanin), ommochrome pigments, or Examples include pteridine dyes.

[0048] The photoprotein is a substrate protein that can emit light without the need for excitation light. An enzyme that catalyzes the luminescence of a protein or substrate. For example, aequorin, enzymatic luciferase, Examples include enzymes.

[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 part of the sense strand of a target gene. The RNA sense strand (passenger strand) consists of a base sequence corresponding to the siRNA is a small double-stranded RNA consisting of a sense strand and an antisense (guide) strand of RNA. can induce RNA interference by introducing it into a subject's cell (eukaryotic cell). (Fire A. et al., 1998, Nature, 391, 806-811).

[0051] siRNA may be designed by known methods based on the base sequence of the target gene. , the method of Ui-Tei et al. (Nucleic Acids Res., 2004, 32:936-948), the method of Reynolds et al. (Nat. Biotechnol., 2004, 22:326-330), the method of Amarzguioui et al. (Biochem. Biophys. Res. Co mmun.,2004, 316: 1050-1058).

[0052] As a specific example of siRNA design, when the nanosO gene is used as the target gene, for example, the sequence number The base sequence of the RNAi sense strand (passenger strand) is 15 bases or more from the base sequence shown in No. 2. A sequence of 35 bases or less, preferably 15 to 30 bases or 18 to 25 bases A base sequence is selected as the selection region. The base sequence of the selected region is the target sequence. Therefore, the selected region must contain the target gene. It is preferable to design the gene so as not to include known mutation sites (e.g., SNPs). The base sequence of the RNAi antisense strand (guide strand) is selected from the base sequence of the RNAi sense strand. When preparing siRNA, the base sequence should be complementary to the sense strand. In both the sense and antisense strands, T (thymine) bases in the selected region are converted to U (uracil) bases.

[0053] The selected region of the RNAi sense strand is not particularly limited as long as it is a sequence specific to the target gene. Preferably, at least 50 bases from the start codon, more preferably 70 to 100 bases. Furthermore, within the candidate region of the RNAi sense strand, there is a region with AA (adenine-adenine It is preferable to select a base sequence region having GC (guaia) on the 5' side. The amino acid-cytosine content is preferably 20 to 80%, more preferably 30 to 70% or 40 to 60%. Many siRNA designs are available on the web, and the base sequence of the target gene can be easily identified. By inputting the columns, effective and appropriate siRNAs can be designed online. NA design websites include siDirect (http: / / sidirect2.rnai.jp / ) and siDESIGN Cent er(https: / / horizondiscovery.com / en / ordering-and-calculation-tools / sidesign-cent er), etc.

[0054] At one or both ends of the siRNA, the base sequence of the target gene or a base sequence complementary thereto is inserted. There are sequences of one or more DNA, RNA, and / or nucleic acid analogs that are not related to the sequence. The number of bases present at the end of such siRNA is not particularly limited, but may be 1 to 20. Specifically, for example, a T is preferably added to the 3' end of each base strand. Examples include the addition of T (thymine-thymine) or UU (uracil-uracil) (Tus chl 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 method is similar to the sRNA selection method, so the explanation will be omitted 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 the two RNA strands (RNAi sense) that make up the siRNA. A single-stranded RNA (RNAi strand and RNAi antisense strand) is connected by a spacer sequence consisting of an appropriate base sequence. In other words, shRNA consists of an RNAi sense region and an RNAi amplifier region within a single molecule. The RNAi sequence contains an RNAi antisense region as the antisense strand, and these regions base pair with each other to form a sequence. The spacer sequence forms a loop structure, and the entire molecule It is constructed to have a hairpin stem-loop structure.

[0059] When shRNA is introduced into cells, the loop structure is cleaved to form a double-stranded RNA molecule, i.e. The resulting siRNA is targeted by the same RNA interference mechanism as the siRNA described in the previous section. The expression of target genes can be suppressed.

[0060] The shRNA is designed, for example, by dividing the 3' end of the sense region of the siRNA into the 3' end of the antisense region of the siRNA. The 5' end of the strand is linked to the 5' end of the strand by a spacer sequence. The spacer sequence is usually 3 to 24 bases, preferably The spacer sequence may be 4 to 15 bases long. There are no particular restrictions on columns.

[0061] shRNA is a gene that encodes an shRNA and is placed under the control of a promoter in an expression vector. Such shRNA expression vectors can be inserted into target organisms or target cells. By introducing the shRNA into the host cell, the promoter activates the shRNA, and after expression, siRNA processing occurs through the self-folding of ribosomal RNA and the activity of Dicer. Furthermore, this shRNA expression vector can be used to The DNA encoding hRNA is then transformed into lepidopteran insects using the transposon method or genome editing method described below. It is also possible to insert (knock in) the gene into the genome.

[0062] (How to install) The basic method for introducing shRNA is similar to the method for introducing dsRNA. We will omit the explanation of the transfection method used and only explain the characteristic transfection method of shRNA. Reveal.

[0063] As mentioned above, shRNA is introduced in the form of DNA as an shRNA expression vector, and then expressed as shRNA in host cells. In this case, the shRNA expression vector can be introduced by dsRNA introduction. When introducing the shRNA expression vector into the eggs of the female through mature eggs, The shRNA expression vector is injected into the abdominal cavity of the pupa and allowed to develop into a mature egg. However, it is possible to insert (knock in) the shRNA into the genome. The shRNA is expressed in germline cells at the developmental stage before oocyte maturation. Care must be taken to select a developmental stage-specific promoter so that This is because the expression of shRNA in early germline cells may prevent the formation of eggs. do.

[0064] The shRNA expression vector can be introduced into the genome of lepidopteran insects using transposon or Examples include genome editing methods.

[0065] The "transposon method" is a method in which the inverted terminal repeats of transposons are used to eat sequence)(Handler AM. et al., 1998, Proc. Natl. Acad. Sci. USA 95:7520-5 ) and a method of inserting a foreign gene into the genome using the activity of a transposon transferase. Transposons that can be used in lepidopteran insects include piggyBac, mariner, and minos. are known, and any of them can be used (Shimizu, K. et al., 2000, Insect Mo l. Biol., 9, 277-281; ​​Wang W. et al., 2000, Insect Mol Biol 9(2):145-55).

[0066] The method for introducing exogenous genes into the host genome is to use two transposon inverted terminal repeats and Using an expression vector containing an exogenous gene located therebetween, the vector can be expressed by methods known in the art. For example, if the host to be introduced is a silkworm, the method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84). Specifically, the gene for a transposon transferase was inserted into an expression vector containing an exogenous gene. The helper vector containing the gene can be injected into early silkworm embryos. An example of a transposon produced by a helper vector is pHA3PIG. The activity of the transposon transferase leads to homologous recombination via the transposon inverted terminal repeats. In this way, exogenous genes can be inserted into the silkworm genome.

[0067] "Genome editing" is a method that uses the aforementioned genome editing technology. For example, zinc finger nuclease (ZFN) method, TALEN method, and CRISPR / Cas9 method are known. In the present specification, either method may be used. The targeting techniques are all well known in the art, and are used herein as the genomic targeting techniques. Kits and support tools for editing methods and efficient knockout of target genes are available at each laboratory. These are commercially available from bioscience manufacturers, and can be used. Regarding the method of inserting foreign genes using genome editing in the TAL-PITCH method, A paper has been published on knock-in technology that utilizes microhomology of the cut surface. , you can use this method (Nakade, S. et al., 2014, Nature Communications, 5:55 60).

[0068] (Selected) Selection of shRNA-introduced individuals can be performed in the same manner as for siRNA. When inserting a marker gene into a vector, it is possible to detect the activity of the marker gene. This allows for the selection of introduced individuals.

[0069] (passage) As with siRNA, the suppression effect of shRNA on target genes is generally only one generation. When shRNA is inserted into the genome of a host lepidopteran insect, the line can be subcultured to In addition, target genes can be suppressed by inducing expression of SHRNA in progeny.

[0070] In this specification, the term "progeny" refers to the descendants of the first generation of genetically modified individuals. , DNA encoding the shRNA of the present invention, asRNA, ribozyme, or RNA aptamer described below. The offspring will continue to carry this shRNA for as long as it is present in the genome. Regardless.

[0071] (2) Antisense oligonucleotide method The "antisense oligonucleotide (ASO) method" refers to the administration of antisense oligonucleotides to a host. The antisense oligonucleotide is administered to induce target gene expression through its activity. The antisense oligonucleotides used herein are It inhibits the expression of the nanosO or nanosP gene in lepidopteran insects post-transcriptionally and before translation.

[0072] (structure) "Antisense Oligonucleotide" ("Antisense Oligonucleotide" as used herein) ASO (often referred to as "ASO") is a gene that encodes the entire or partial base sequence of mRNA, which is the transcription product of a target gene. consists of a partially complementary base sequence, hybridizes to the target mRNA, and initiates its translation. It is a single-stranded nucleic acid molecule that inhibits

[0073] ASOs are known to be of DNA and RNA types. DNA types are those that target genes, such as mRNA, which is a transcription product of the target gene. After hybridizing to the RNA molecule to form a heteroduplex structure, it inhibits RNase H activity in the cell. Nuclease-mediated suppression of target gene expression by cleaving and degrading target RNA molecules. On the other hand, the RNA type is a method for suppressing genes by silencing RNA molecules such as mRNA, which is the transcription product of the target gene. After hybridizing with the molecule to form double-stranded RNA, it undergoes a process and finally becomes the same as siRNA, etc. This can have the effect of suppressing gene expression by RNAi, similar to that of RNAi. DNA-type ASOs are often used due to their ease of synthesis and efficacy.

[0074] DNA-type ASOs are mainly composed of natural DNA, but also contain natural RNA and LNA / BNA (Loc ked Nucleic Acid / Bridged Nucleic Acid) and PMO (Phosphorodiamidate Morpholino Olig) Nucleic acid analogs such as 2'-OMe-RNA, 2'-F-RNA, 2'-MOE-RNA and phosphorothioates The wing region may also comprise a nucleic acid analog or a modified nucleic acid. Gapmers, which are RNA-degrading ASOs with 5'- and 3'-terminals, nucleic acid analogs, modified nucleic acids, etc. ASOs with special structures such as mixmers, which are splicing-controlling ASOs composed of Contains.

[0075] Furthermore, the heteroduplex oligonucleotide (HDO) method, which is an application of the DNA-type ASO method, can also be used. Heteroduplex oligonucleotide (often referred to as "HDO" herein) The term "ASO" refers to a cRNA ( cRNA strands are double-stranded nucleic acids composed of complementary RNA strands. cRNA strands can also be used as nucleic acid analogs or modified nucleic acids. However, at least the whole or part of the central part of the HDO is a heterogeneous DNA-RNA structure. Because it is a nucleic acid, the complementary strand, cRNA, is cut and degraded by RNase H in the cell, leaving it alone The backbone can function as an ASO.

[0076] RNA-type ASOs are antibodies that can hybridize to parts of mRNA, which is the transcription product of a target gene. It consists of the same base sequence as the sense strand and is essentially made up of natural RNA. In the specification, the term "antisense RNA (asRNA)" is often used to distinguish it from DNA-type ASOs. It is written as A).

[0077] The asRNA is designed so that the DNA encoding it can operate under the control of a promoter in an expression vector. Such an "antisense RNA expression vector (asRNA expression vector)" can also be inserted into Like the shRNA expression vectors mentioned above, the vectors are introduced into target organisms or target cells. Once expressed, the asRNA is transduced into the target gene in the host cell. Furthermore, the shRNA expression vector As with the target, DNA encoding asRNA can be created by transposon or genome editing, which will be described later. It is also possible to knock-in the gene into the genome of a lepidopteran insect.

[0078] As a specific example of ASO base sequence design, regardless of whether it is DNA or RNA, for example, the nanosO gene In the case of a target gene, for example, 10 or more bases from the base sequence of the mRNA strand shown in SEQ ID NO: 2 A sequence of 30 or less bases, preferably 12 to 25 bases, or 13 to 20 bases The base sequence is used as a selection region, and a base sequence complementary to it is selected. , the region encompassing the initiation codon, or the predicted secondary structure of the mRNA strand, forming a single-stranded structure. Any region where this is possible can be selected as the target region.

[0079] (How to install) The method for introducing ASO is similar to the method for introducing siRNA, and therefore, detailed explanations are omitted here. As mentioned above, asRNA can be used as an asRNA expression vector in the same way as shRNA expression vectors. It is also possible to introduce asRNA in the form of DNA and express asRNA in host cells. The method for introducing the vector may 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. In this case, inserting a marker gene into the vector in the same way as with the shRNA expression vector will Transgenic individuals can be selected based on the activity of the marker gene.

[0081] (passage) As with siRNA, the suppression effect of ASO on target genes is, in principle, only for one generation. However, when the DNA encoding the asRNA is inserted into the genome of the host lepidopteran insect, Even after subculture, the target gene is suppressed by inducing asRNA expression in the progeny. can be done.

[0082] (3) Nucleic acid enzyme method The "nucleic acid enzyme method" refers to administering a nucleic acid enzyme to a host and using the activity of the nucleic acid enzyme to transfect a target gene. The nucleic acid enzyme in this specification is a method for inhibiting expression after transcription and before translation. It represses the expression of the nanosO gene or nanosP gene after transcription and before translation.

[0083] (composition) "Nucleic acid enzymes" are nucleic acid molecules with catalytic activity that specifically bind to target mRNA as a substrate. It cleaves specific sites in target mRNA and suppresses the expression of target genes post-transcriptionally and pre-translationally. The nucleic acid enzyme in the present specification can be derived from the nanosO gene or nanosP gene of lepidopteran insects. It suppresses gene expression post-transcriptionally but before translation.

[0084] Nucleic acid enzymes include deoxyribozymes, which are made up of DNA, and ribozymes, which are made up of RNA. Ribozymes that are synthesized are known, but the nucleic acid enzymes used herein are any of Both deoxyribozymes and ribozymes have chemically modified nucleic acids and artificial nucleic acids in their constituent bases. Acid and / or nucleic acid analogs may be included in the composition.

[0085] Ribozymes, which are composed of RNA, are expressed by inserting the DNA encoding them under the control of a promoter in an expression vector. Such a "ribozyme expression vector" can be operably inserted into the In the same way as the shRNA expression vectors mentioned above, they can be introduced into target organisms or target cells. 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 carried out in the same manner as for selection of siRNA. When introducing a target gene, insert the target gene into the vector in the same way as the shRNA expression vector. By doing so, introduced individuals can be selected based on the activity of the marker gene.

[0088] (passage) The suppression effect of nucleic acid enzymes on target genes is, in principle, only one generation, similar to siRNA. However, the DNA encoding the ribozyme is inserted into the genome of the host lepidopteran insect. In this case, even if the line is subcultured, the target gene is induced by inducing ribozyme expression in the progeny. can be suppressed.

[0089] (4) Aptamer method The "aptamer method" is a method in which an aptamer is administered to a host and its target binding activity is utilized to bind to the target. This method inhibits the function of proteins, thereby suppressing the expression of target genes after translation. The aptamer in the present specification binds to the nanosO protein or the nanosP protein and It post-translationally suppresses the expression of the nanosO gene or nanosP gene in Coraca insects.

[0090] (structure) An "aptamer" is a molecule that binds strongly and specifically to a target substance due to its three-dimensional structure. Aptamers are ligand molecules that inhibit the function of target substances. However, they generally have higher specificity and affinity for target substances than antibodies, and also have a higher binding affinity. The number of target amino acid residues required for targeting is smaller than that of antibodies. They are superior to antibodies in that they can distinguish between molecules. Furthermore, they have lower immunogenicity and toxicity than antibodies. Furthermore, it can be produced in a short period of time, about 3 to 4 weeks, and can also be mass-produced by chemical synthesis. It has the advantage of being able to

[0091] Aptamers are broadly classified into nucleic acid aptamers and peptide aptamers depending on the type of constituent molecule. The aptamer in this specification may be any aptamer, Preferably, it is a nucleic acid aptamer.

[0092] As used herein, the term "nucleic acid aptamer" refers to an aptamer composed of nucleic acid, The three-dimensional structure formed based on the secondary structure and tertiary structure of single-stranded nucleic acid molecules via elementary bonds, etc. Its structure allows it to bind strongly and specifically to target substances.

[0093] Nucleic acid aptamers are generally classified into RNA aptamers composed of RNA and DNA aptamers composed of DNA. Although nucleic acids constituting the nucleic acid aptamers in this specification are known, there are no particular limitations on the nucleic acids constituting the nucleic acid aptamers. For example, DNA aptamers, RNA aptamers, and combinations of DNA and RNA It includes aptamers, etc. Usually, it is composed only of natural nucleic acids (DNA or RNA), but it is also possible to use non-natural nucleic acids. The aptamer of the present invention may partially contain an artificial nucleic acid or modified nucleic acid. However, it is preferable that the length is in the range of 10 to 100 bases, and more preferably in the range of 15 to 80 bases. Aptamers are a well-known technology, and for details, see, for example, Janasena, Cl See, e.g., in. Chem. 45:1628-1650 (1999).

[0094] RNA aptamers can be synthesized using, for example, SELEX (systematic evolution of ligands by exponential The SELEX method can be used to generate genes by random selection in vitro. The RNA pool consists of RNA molecules with a primer binding region at both ends. RNA molecules bound to a target molecule (in the present invention, the nanosO protein or the nanosP protein) After selection, the cDNA fragments are amplified by RT-PCR and transcribed using the resulting cDNA as a template. This cycle is repeated several to several tens of times to create an RNA pool for the next round. In other words, it is a method to select RNA with stronger binding strength to the target molecule. The length of the base sequence of the random sequence region and the primer binding region is not particularly limited. The length of the primer binding region is in the range of 15 to 40 bases. To enhance the affinity, a molecule similar to the target molecule is mixed with the RNA pool and the target molecule is then bound to the RNA. The pool of RNA molecules that were not purified by this method can be used. The RNA molecules thus obtained are used as RNA aptamers. Such a method is described, for example, in Pan et al. (Proc. Natl. Acad. Sci. USA, (1995) 92: 11509-11513) It should be carried out in accordance with the following.

[0095] When the nucleic acid aptamer is composed of natural RNA, the DNA encoding it is inserted into an expression vector. It can also be inserted so that it can be operated under the control of a promoter. The shRNA expression vector is a vector that expresses the shRNA in a target organism or target cell, similar to the shRNA expression vector described above. When introduced into the host, the RNA aptamer is expressed by the activity of the promoter. It binds to the target protein in the host cell and inhibits its function. Similarly, DNA encoding RNA aptamers can be used in transposon or genome editing, as described below. Therefore, it is possible to insert (knock in) it into the genome of lepidopteran insects.

[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 can 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 a vector, insert a marker gene into the vector in the same way as for the shRNA expression vector. By preserving the marker gene, introduced individuals can be selected based on the activity of the marker gene.

[0098] (passage) As with siRNA, the suppression effect of aptamers on target genes is generally limited to one generation. However, the DNA encoding the RNA aptamer is inserted into the genome of the host lepidopteran insect. When the RNA aptamer is expressed, it can be easily induced in the progeny by subculturing the lineage. This allows the target gene to be suppressed.

[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 The expression of the nanosO / P gene is suppressed, resulting in reduced fertility in both males and females. The sterile Lepidoptera insect of the present invention is a sterile Lepidoptera insect according to the first aspect. It can be produced by insect production methods.

[0100] 2-2.Configuration Sterile lepidopteran insects exhibit suppressed expression of the nanosO / P genes in germline cells during early embryonic development. The expression of both nanosO and nanosP genes is suppressed (double knockdown). It is sufficient if the information is properly compiled.

[0101] The sterile Lepidoptera insect in which gene expression is suppressed includes, but is not limited to, the sterile Lepidoptera insect described in the first aspect. In the method for producing sterile lepidopteran insects, individuals obtained through gene knockdown methods, or is an individual in which gene expression suppression is induced in the progeny containing the gene expression suppressor in the genome. .

[0102] The type of sterile Lepidoptera insect is not limited, but preferably is an insect useful in industry, such as silkworms. Species for which silk is harvested, such as silkworms, silkworms, silkworms, silkworms, and silkworms. Examples include silkworm moth, silkworm, and silkworm moth. Preferred is silkworm.

[0103] The developmental stages of sterile lepidopteran insects are not limited to eggs, larvae, pupae, and adults. However, for the purpose of infertility, it is preferable to use a naturally reproductive It is an adult.

[0104] The sterile Lepidoptera insects of the present invention can be identified by molecular genetic techniques and / or phenotypic characteristics. They can be distinguished from fertile Lepidoptera insects.

[0105] Discrimination based on molecular genetic techniques, for example, shows that expression of the nanosO / P gene results in normal fertility. The expression level of the nanosO / P gene can be measured or detected using the same species of lepidopteran insect as a control. Methods for measuring or detecting gene expression levels include RT-PCR and Northern hybridization. In the case of the sterile Lepidoptera insect of the present invention, a method known in the art, such as the cloning method, can be used. , the expression level of the nanosO / P gene was significantly higher than that of the control individuals (e.g., p<0.05, p<0. 01, p<0.001).

[0106] Discrimination based on phenotypic characteristics is based on the observation and identification of morphological abnormalities in the reproductive organs or germ cells. In the case of the sterile Lepidoptera insect of the present invention, in the case of female adults, the number of female adults is 100 or more compared to control individuals. The number of eggs laid is significantly reduced or no eggs are laid at all. In the case of adult males, the testes become small and transparent. , resulting in decreased fertility.

[0107] 3. Lepidoptera insect sterilization composition Overview A third aspect of the present invention is a composition for sterilizing lepidopteran insects. The present invention relates to the development of a gene that specifically suppresses the expression of the nanosO gene and the nanosP gene. The sterilization composition of the present invention contains a sterilization inhibitor as an active ingredient. It can easily and efficiently sterilize Lepidoptera insects.

[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 an active ingredient, each of the nanosO / P genes. It contains at least two gene expression inhibitors that inhibit gene expression. "Gene expression inhibitors" are compounds that inhibit the expression of the target genes, nanosO gene or nanosP gene. It is an agent that specifically suppresses the expression of each of these. Gene expression inhibitors are classified into transcription product inhibitors and translation product inhibitors based on their inhibitory effect on gene expression. They can be divided into two types:

[0110] (1) Transcription inhibitors As used herein, the term "transcription product inhibitor" refers to a compound that inhibits the transcription of the target gene, the nanosO gene or the nanosP gene. It targets mRNA, the transcription product of a gene, and degrades or inactivates it, thereby inhibiting the expression of that gene. Specific examples of transcription inhibitors include RNAi agents and antisense oligonucleotides. These include antisense oligonucleotides (ASOs), and nucleic acid enzymes. The synthesis of the antibody may be carried out by the (1) RNAi method or the (2) antisense oligonucleotide method of the first aspect. (3) According to the composition of the RNAi agent, ASO, and nucleic acid enzyme detailed in the nucleic acid enzyme method, respectively Therefore, only a brief explanation will be given here.

[0111] If the transcript suppressor is an RNAi agent, it is nanosO-dsRNA or nanosO-siRNA against the nanosO gene. or nanosO-shRNA, nanosP-dsRNA, nanosP-siRNA, or nanosP-shR against the nanosP gene. For example, shRNA is an example of a transcript suppressor. When a transcript suppressor is an shRNA, it activates the nucleic acid that encodes it. It may be in the form of an expression vector containing the gene in a suitable state. In principle, the effect of RNAi agents is limited to one generation. If the expression vector can be inserted into the genome of the host lepidopteran insect, Even if the line is subcultured, the progeny carrying the expression vector do not express nanosO-shRNA or nano The effect of the RNAi agent can be achieved by inducing the expression of sP-shRNA.

[0112] If the transcription inhibitor is an ASO, it may be a nanosO-ASO for the nanosO gene or a nanosP gene. For example, nanosP-ASO is an example of an ASO. If the ASO is RNA-type, the nucleic acid that encodes it is The nanosO-asRNA expression vector and the nanosP-asRNA expression vector operably carrying the The effect of ASOs as transcription inhibitors is generally limited to one generation. However, the as expression vector can be inserted into the genome of the host lepidopteran insect. As a result, even if the line is subcultured, the progeny carrying the expression vector express nanosO-ASO. Alternatively, the effect of ASO can be achieved by inducing the expression of nanosP-ASO.

[0113] If the transcript repressor is a nucleic acid enzyme, it is the nanosO-(deoxy)ribonucleotide for the nanosO gene. Examples include the nanosP-(deoxy)ribozyme and the nanosP gene-targeted nanosP-(deoxy)ribozyme. In addition, when the nucleic acid enzyme is a ribozyme, it contains a nucleic acid encoding it in an operable state. The vectors were expressed as nanosO-ribozyme expression vectors or nanosP-ribozyme expression vectors. That's fine.

[0114] (2) Translation product inhibitors As used herein, the term "translation product inhibitor" refers to a compound that inhibits the translation of a target gene, the nanosO gene or the nanosP gene. By targeting the protein that is the translation product of the gene, it is possible to degrade or inactivate it. A specific example of a translation product inhibitor is nanosO protein. Anti-nanosO aptamers against the protein or anti-nanosP aptamers against the nanosP protein The specific structure of the aptamer is described in the first aspect of the present invention, "(4) Aptamer "The structure of the aptamer is similar to that described in detail in ", so only a brief explanation will be given here.

[0115] When the transcript inhibitor is an RNA aptamer, the nucleic acid encoding it is encapsulated in an operative state. The aptamer may be in the form of an expression vector containing the aptamer. In principle, the effect lasts for only one generation, but nanosO-RNA aptamer and nanosP-RNA aptamer If the expression vector encodes the Therefore, even if the line is subcultured, the progeny carrying the expression vector do not express the nanosO-RNA approach. The effect of the RNA aptamer can be achieved by inducing the expression of the nanosP-RNA aptamer or nanosP-RNA aptamer. can be done.

[0116] The sterilization composition of the present invention contains at least a gene expression inhibitor for the nanosO gene and a nanosP Gene expression inhibitors for genes are used in pairs.

[0117] In addition to the above reasons, the sterilization composition of the present invention is also effective in preventing the formation of steroid hormones by inhibiting either the nanosO gene or the nanosP gene. For example, the sterility agent of the present invention may contain two or more gene expression inhibitors. The compound contains two types of gene expression inhibitors, siRNA and shRNA, and It contains a total of three active ingredients, including one type of siRNA, as a gene expression inhibitor for the anosP gene. It's okay to do that.

[0118] 3-2-2.Other ingredients The sterilizing composition for Lepidoptera insects of the present invention may contain, in addition to the above-mentioned active ingredients, an entomological ingredient, if necessary. "Entomologically acceptable" may contain solvents or carriers that are acceptable in the field. is commonly used in the field of entomology and is harmless to the lepidopteran insects to which it is administered? or has little or no effect.

[0119] The solvent may be, for example, water or an aqueous solution, or an organic solvent acceptable to lepidopteran insects. Examples of aqueous solutions include buffers (phosphate buffers and sodium acetate buffers). buffer solution, saline solution, and isotonic solution.

[0120] Carriers include glucose, D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid ethylene Stells and the like.

[0121] 3-3.Administration method The method of administering the sterilizing composition of the present invention to Lepidoptera insects is the same as that of the first aspect of the gene expression suppression method. This can be done in accordance with the method for introducing each gene expression inhibitor described in the methods section. [Example]

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

[0123] (method) (1) Material The host insect, a lepidopteran insect, was the National Agriculture and Food Research Organization (NARO, Silkworms (pnd strain) that had been serially reared at the University of Tokyo (Japan) were used.

[0124] (2) Preparation of gene expression inhibitors In this example, the gene expression inhibitor used in the gene expression suppression step was a target gene inhibitor. RNAi agents were prepared against the nanosO gene and the nanosP gene, respectively.

[0125] First, the silkworm nanosO gene consisting of the base sequence shown in SEQ ID NO: 2 was used as a template, and the The primer pairs shown in SEQ ID NO: 5 were used to elucidate the base sequence shown in SEQ ID NO: 5. The PCR reaction conditions were (98°C, 10 s; 55°C, 15 s; 68°C, 60 s) × 15, followed by (98 The reaction was carried out in two steps (68°C, 10 s; 68°C, 60 s) x 25 using GXL polymerase (Takara Bio). It was.

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

[0127] Furthermore, the nanosM gene and the nanosN gene, which are paralogous nanos genes, were also identified. The nanosM gene was synthesized from the silkworm nano The sM gene was used as a template to generate the primer pair shown in SEQ ID NOs: 13 and 14 (M-Fw and M-Fw, respectively). The base sequence shown in SEQ ID NO: 12 was obtained using the nucleotide sequence of the nanosN gene shown in SEQ ID NO: 15. The primers shown in SEQ ID NOs: 17 and 18 were prepared using the silkworm nanosN gene consisting of the base sequence as a template. The base sequence shown in SEQ ID NO: 16 was amplified using the mer pairs (N-Fw and N-Rv, respectively). The PCR reaction conditions were the same as those described above.

[0128] Next, each PCR amplification product was used to prepare a Megascript RNAi kit (Ambion). Double-stranded RNA (dsRNA) derived from each nanos gene was prepared using the following method. The method was carried out according to the protocol attached to the kit.

[0129] For injection, the dsRNA of each nanos gene was evaporated to a concentration of 3 μg / μL. When simultaneously suppressing the expression of two target genes, The two types of nanos gene dsRNA were prepared in equal ratios so that each concentration was 3 μg / μL. Here, (1) nanosO-dsRNA alone, (2) nanosP-dsRNA alone, (3) 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). Five species were prepared as nanos-RNAi agents.

[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. Using a glass micropipette, add 10 nL to 20 nL of each nanos-RNAi agent to each tube. After the injection, the injection hole was sealed with instant adhesive (tree The holes were sealed with a multi-purpose, fast-hardening instant adhesive (Alpha Shoji). The eggs were laid at the same time without any transfection. Next, to create a humidified state, a kitten moistened with water was placed inside the tight box. A tin towel was laid on the surface, the petri dish was placed on top of it, and the tight box was then covered with a lid. The egg boxes were 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 and fed artificial diet (Silkmate original species 1-3 stage S, Japan) for all stages. The animals were reared in a nutrient-rich environment (Nagano Prefecture, Japan). The artificial diet was changed as needed.

[0132] After emergence, the ovaries were removed from virgin 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 by the time the females emerge.

[0133] In addition, testes were extracted from virgin male adults after emergence and the morphology of the testes was observed. The male adults obtained after the transfection 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. If the number of eggs is 100 or more, mating is considered normal. Fertilized eggs are induced by oviposition (egg development occurs several days after oviposition). The number of eggs (pigmented eggs) was counted.

[0134] Furthermore, for each individual after nanos-RNAi injection, phenotypes other than germline cells were examined. (Behavior, growth, and morphology, etc.) were also examined.

[0135] (result) Figures 1 to 5 show the number of female individuals and the number of mature eggs in their ovaries after injection, and Figure 6 The morphology of the testes excised from the mice after administration of nanosO-dsRNA alone is shown in Fig. 1. Figure 2 shows the results of nanosO-RNAi with nanosP-dsRNA alone. Figure 3 shows the results of nanosM / O-RNAi after administration of nanosM / O-dsRNA, and Figure 4 shows the results of nanosN / O-dsRNA after administration of nanosM / O-dsRNA. 5 shows the results of nanosO / P-dsRNA administered to the cells treated with nanosN / O-RNAi, and FIG. 6 shows the results of nanosO / P-dsRNA administered to the cells treated with nanosN / O-RNAi. In Figure 6, A shows the testis of a wild-type male adult, and B shows the testis of a male treated with nanosO-RNAi. A shows the testis of an adult male, and B shows the testis of an adult male treated with nanosO / P-RNAi.

[0136] (oogenesis) From the results in Figures 1 to 5, nanosO-RNAi, nanosP-RNAi, nanosM / O-RNAi, and nanosN / O-RNAi In the individuals treated with nanosO, there were no individuals with less than 150 mature eggs. The gene may be expressed alone or in combination with other nanos paralogs, the nanosM gene or the nanosN gene. This suggests that inhibition does not affect egg formation. Nakao H. and Takasu Y. (2019, cited above) ) reported that the number of mature eggs was rarely reduced in Bm-nosO gene knockout silkworms. In this example, when the expression of the nanosO gene was suppressed by nanosO-RNAi, mature eggs No abnormalities in numbers were observed, and the results of Nakao H. and Takasu Y. (2019) in which the nanosO gene was disrupted This is almost consistent with the above. In other words, suppression or inhibition of the expression of the nanosO gene alone does not affect the function of the lepidopteran This suggests that nanosO is insufficient to induce stable sterility in insects. Similar results were also confirmed when the nanosP gene expression was suppressed alone. Even if the expression of the sP gene is suppressed alone, it is not sufficient to induce stable sterility in lepidopteran insects. It was shown that

[0137] In contrast, the results of Figure 5 show that when the nanosO gene and the nanosP gene are double suppressed, It was revealed that the number of mature eggs was significantly reduced and clear abnormalities in egg formation occurred. Approximately 70% of the injected females had eggs with less than 150 eggs. The results showed that only when the expression of the nanosO / P genes was dually suppressed did the formation of mature eggs become inhibited. It has been proven that this can be done.

[0138] (Testicular morphology) As shown in Figure 6A and B, no morphological differences were observed between the testes of wild-type males and those of nanosO-RNAi-treated males. However, in the testis of the nanosO / P-RNAi-treated male shown in C, the area indicated by the arrow was In addition to the observation of partially transparent testes, several significantly dwarfed testes were observed, as indicated by the arrowheads. I was criticized.

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

[0140] [Table 1]

[0141] The rate of normal mating was significantly reduced only when nanosO / P expression was double suppressed by RNAi. On the other hand, when only the expression of nanosO or nanosP was suppressed by RNAi, the expression of nanosM / O or nanosN / O was suppressed. When the expression of nanosO / P was double-suppressed by RNAi, the normal mating rate was similar. In males where expression is double-silenced by RNAi, functional spermatogenesis is inhibited and fertility is reduced. Or it suggests that it has been lost.

[0142] These results suggest that only when the expression of the nanosO / P genes is double suppressed does the male individual also have testis formation. It was revealed that not only did this cause abnormalities in the sperm's morphology, but it also caused the sperm to lose their fertilizing ability.

[0143] (Other phenotypes) Observation of phenotypes other than germline cells (behavior, growth, morphology, etc.) throughout the breeding process However, no notable phenotypic differences were found other than infertility. They grow, molt, metamorphose, make cocoons, become adults, and do not change mating behavior. In addition, there was no difference in the size of the larvae or adults compared to the wild type. It was.

[0144] (Conclusion) These results suggest that the nanosO and nanosP genes are involved in the formation of germline cells such as primordial germ cells. It functions redundantly, and its function cannot be complemented by other nanos paralogs. and nanosO / P genes during early embryonic development stabilize germline cell formation. It was shown that the sterility of lepidopteran insects was induced by the induction of sterility. The induction requires the suppression or inhibition of nanosO / P gene expression during early embryonic development. This suggests that

[0145] <Comparative Example 1> (the purpose) The expression inhibitors of the nanos gene, both alone and in combination, which were not verified in Example 1, The fertility suppression effect of the present invention in lepidopteran insects was achieved by doubly suppressing the expression of the nanosO / P genes. Confirm that this can only be achieved when the

[0146] (method) The basic operation is the same as in Example 1, so only the differences from Example 1 will be explained here. do.

[0147] (1) Preparation of gene expression inhibitors In this comparative example, the nanosM gene and the nanosN gene of the nanos paralog genes used in Example 1 were RNs for the nanosO gene and the nanosP gene, which are the target genes of the present invention, An Ai agent was prepared according to the method described in Example 1.

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

[0149] In this comparative example, (1) nanosM-dsRNA alone (nano sM-dsRNA), (2) nanosN-dsRNA alone (nanosN-dsRNA), (3) nanosM-dsRNA and nanosP-d (4) a mixture of nanosM-dsRNA and nanosP-dsRNA (nanosN / P-dsRNA), and (5) a mixture of nanosM-dsRNA, nanosN-dsRNA, and nanosO-dsRNA (nan osM / N / O-dsRNA), (6) a mixture of nanosM-dsRNA, nanosN-dsRNA, and nanosP-dsRNA (nano sM / N / P-dsRNA) and (7) nanosM-dsRNA, nanosN-dsRNA, nanosO-dsRNA, and nanosP-dsRNA Seven types of nanos-RNAi agents were prepared: a mixture of four types (nanosM / N / O / P-dsRNA). The egg injection, rearing of silkworms, and confirmation of the phenotype of the adult gonads were performed as described in Example 1. The method described was followed.

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

[0151] From the results of Figures 7 to 13, the nano Except for the quadruple suppression of the s gene (nanosM / N / O / P-dsRNA), no RNAi was produced in individuals treated with any of the RNAi. There were no individuals with less than 150 mature eggs. These results suggest that the nanosM gene and the nanosN gene The nanosO gene was not suppressed either singly or in combination with other nanos genes. and nanosP gene double suppression, it does not affect oogenesis in lepidopteran insects. This suggests that:

[0152] Taking the results of Example 1 and this Comparative Example together, the stable sterilization effect of lepidopteran insects is It is induced only when the sO gene and the nanosP gene are double-repressed, and the other nanos genes The double suppression of the nanosO gene and the multiple suppression that does not include the nanosP gene are insufficient. It was proven that:

Claims

1. A method for producing sterile silkworms, comprising: The method includes a step of suppressing the expression of the nanosO gene and the nanosP gene using a gene knockdown method. The production method.

2. The production method according to claim 1, wherein the gene knockdown method is the RNAi method or the antisense oligonucleotide method.

3. Sterile silkworms in which the expression of the nanosO and nanosP genes was suppressed using gene knockdown techniques.

4. The sterile silkworm according to claim 3, wherein the gene knockdown method is the RNAi method or the antisense oligonucleotide method.

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