WO2016076240A1 - Souche de bombyx mori létale pour le ver à soie femelle - Google Patents

Souche de bombyx mori létale pour le ver à soie femelle Download PDF

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WO2016076240A1
WO2016076240A1 PCT/JP2015/081397 JP2015081397W WO2016076240A1 WO 2016076240 A1 WO2016076240 A1 WO 2016076240A1 JP 2015081397 W JP2015081397 W JP 2015081397W WO 2016076240 A1 WO2016076240 A1 WO 2016076240A1
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gene
silkworm
masc
female
promoter
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Japanese (ja)
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めぐみ 笠嶋
秀樹 瀬筒
進 勝間
隆史 木内
雅京 鈴木
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国立研究開発法人農業生物資源研究所
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a female pupal dead silkworm strain having a mutant Masc gene expression vector, and a method of eliminating female pupae using the same.
  • the silk thread spun by male silkworms (Bombyx ⁇ mori) is superior in terms of thread quality because it is slender and has less variation in fineness than silk thread spun by females. Moreover, even when the same amount of feed is ingested, it is known that the production amount of male raw silk is about 20% higher than that of females (Non-patent Document 1). Furthermore, since silkworms have lost their flying ability, they are approved for use in the first type based on the Cartagena Act, that is, use without preventing diffusion into the environment. Can not rule out the possibility of crossing with the male of the quay (Bombyx mandarina) to get. For this reason, there is a need for bred breeding for breeding only male individuals in order to maintain useful strains and prevent the spread of genetically modified organisms into the environment. Therefore, the technology for freely maleizing silkworm breeding individuals is extremely useful in industry.
  • the doublesex (dsx) gene encoding a transcription factor functions at the most downstream of the sex determination cascade, and it has been found that sex determination is made by the splicing pattern of the mRNA.
  • Attempts to overexpress silkworm dsx (Bmdsx), which shows a male splicing pattern, to make males, etc. have been made so far, but none of them have succeeded in order to establish a male breeding method. References 2 and 3).
  • Non-patent Document 4 Non-patent Document 4
  • Non-patent Document 5 since the W chromosome of silkworm has a complex structure with multiple transposons inserted (Non-patent Document 5), it is difficult to determine the base sequence over the entire region, and recombination does not occur in females. No forward genetic analysis was possible, and the identity of the female determinant remained unclear. Therefore, the technique to breed only males using the sex-determining mechanism and sex differentiation genes of silkworms has not been established so far.
  • Platinum Boy registered trademark
  • This breed is designed so that only the male individual can survive by using the two types of equilibrium lethal genes and letting the lethal gene work in the female individual, resulting in embryonic lethality (Non-patent Document 6).
  • Platinum Boy is on the market and has a reputation for the high quality of silk thread produced (Non-patent Document 7).
  • this variety has a big problem that it takes a lot of labor and time to produce. Usually, to get a Platinum Boy, you have to go back two generations before mating, and it takes a year to produce it.
  • the problem of the present invention is that it is possible to easily maintain and manage the strain without disturbing the genetic background using a sex determination mechanism or a sex differentiation gene, and it can be applied to a gene recombination technique.
  • Another object of the present invention is to establish and provide Lepidoptera insect strains, particularly silkworm strains, which can be bred male in a short and simple manner.
  • the present inventors found that when the mutant Masc gene was overexpressed in the silkworm, no abnormality was found in the male individual, but the female individual did not become male, It became clear that he died by the time of larvae. Based on this finding, the present inventors introduced a mutant Masc gene into a host cell of a lepidopteran insect, and controlled its expression to control the female lethality of the host, thereby arbitrarily controlling the domestic individual. Successfully made an individual.
  • the present invention is based on the research results and provides the following.
  • a mutant Masc gene having cleavage resistance against the Fem piRNA-Siwi complex and the activity of the wild-type Masc gene, and the Fem at positions 1391 to 1419 in the silkworm Masc gene comprising the base sequence represented by SEQ ID NO: 1.
  • the mutation is present in a region consisting of 15 consecutive bases including positions 1409 and 1410 in the Fem piRNA recognition sequence in the silkworm Masc gene, or in a corresponding region in the ortholog of the Lepidoptera insect of the silkworm Masc gene.
  • the mutant Masc gene according to (1) (3) The mutant Masc gene according to (1) or (2), wherein the base substitution is a silent mutation.
  • a mutant Masc gene expression vector comprising the mutant Masc gene according to any one of (1) to (3) operably linked downstream of a ubiquitous expression-inducible promoter.
  • the mutant Masc gene expression vector according to (4), wherein the ubiquitous expression-inducible promoter is a heat shock protein 70 promoter.
  • a first expression unit comprising a ubiquitous promoter and a gene encoding a transcriptional regulatory factor operably linked downstream of the promoter, and a functional promoter linked downstream of the target promoter of the transcriptional regulatory factor and the target promoter
  • a mutant Masc gene expression vector comprising a second expression unit comprising the mutant Masc gene according to any one of (1) to (3).
  • a female pupal dead silkworm strain comprising the mutant Masc gene expression vector according to (4) or (5).
  • a method for producing a female pupal dead silkworm comprising the step of subjecting an embryo at an early stage of development to an expression induction treatment in the female pupal dead silkworm strain according to (9).
  • mutant Masc gene and the expression vector of the present invention makes it possible to easily produce a female dead lethal silkworm strain.
  • the female dead lethal silkworm strain of the present invention a strain that can easily obtain a female dead lethal silkworm from the strain can be easily maintained or managed without disturbing the genetic background.
  • Application to genetic recombination technology is possible, and male breeding can be carried out conveniently and arbitrarily in a short period of time. Thereby, high-quality silk thread can be produced efficiently.
  • the female pupal dead male infertile silkworm strain of the present invention since it is possible to suppress crossing with natural mulberry individuals, it is possible to prevent the propagation of offspring of transgenic silkworms into the environment, Contamination can be prevented.
  • an infertile female pup lethal silkworm can be produced according to the sterilized bred breeding method of the present invention.
  • the top row shows the amino acid sequence encoded by the base in the Fem piRNA recognition sequence.
  • Masc WT and MascR represent Fem piRNA recognition sequences of the wild-type Masc gene and the mutant Masc gene, respectively.
  • the underlined base in the MascR base sequence indicates a silent mutation site and its mutant base that are preferred as a cleavage resistant mutation.
  • b represents c, g, or t.
  • d indicates a, g, or t.
  • h represents a, c, or t.
  • the arrowhead indicates the position of the Fem piRNA recognition sequence in the Masc gene. It is a figure which shows the sex ratio of each genotype in a female pupal dead silkworm.
  • a and B are genotypes of individuals living in the 5th instar larva stage among the F1 individuals obtained by crossing the sumi13 line (female lethal silkworm of the present invention) and the 193-2 line (BmA3-GAL4 line).
  • the sex ratios of C and D are the F1 individuals obtained by crossing between the sumi12 line (control silkworm line) and the 193-2 line (BmA3-GAL4 line) in the individual living in the 5th instar larva stage.
  • the genotype sex ratio, and E and F are individuals living in the 5th instar larvae stage of 193-2 line having the first expression unit BmA3-GAL4 of MascR gene expression vector used for mating with the female pupal dead silkworm line
  • the sex ratio of each genotype in is shown.
  • G + R indicates a sex ratio in a population having the second expression unit pUAS-MascR of the MascR gene expression vector of the present invention and the first expression unit BmA3-GAL4.
  • G indicates a sex ratio in a population having only the first expression unit BmA3-GAL4.
  • R represents a sex ratio in a population having only the second expression unit pUAS-MascR of the MascR gene expression vector of the present invention.
  • Negative indicates a sex ratio in a population having neither the first expression unit BmA3-GAL4 nor the MascR gene expression vector second expression unit pUAS-MascR of the present invention.
  • the black squares on each panel indicate the percentage of male individuals, and the white squares indicate the percentage of female individuals.
  • FIG. 1 shows the result of having detected the karyotype of the sex chromosome in F1 individual which has both the 2nd expression unit pUAS-MascR of the MascR gene expression vector of this invention, and 1st expression unit BmA3-GAL4 by PCR.
  • A shows the karyotype of the larva individual who died among the F1 individuals
  • B shows the karyotype of the larva individual who survived to the surviving 5th instar larva stage among the F1 individuals.
  • the form of a cocoon (A) when the silkworm of each strain including a female pupal dead silkworm is formed is shown, and the form and sex (B) of the cocoon in the cocoon.
  • Mutant Masc gene 1-1 Overview
  • a first aspect of the present invention is a mutant Masc gene.
  • the mutant Masc gene of the present invention is characterized by having cleavage resistance to the Fem piRNA-Siwi complex.
  • 1-2 Definitions The following key terms used in this specification are defined.
  • PIWI-interacting RNA is a non-coding small RNA with a length of 23-30 bases involved in germ cell formation and sex determination.
  • the piRNA is expressed in a precursor state, and after processing such as cleavage and modification, becomes a mature piRNA having a recognition region consisting of a base sequence complementary to the target RNA. After that, piRNA forms a complex with PIWI (P-element Induced Wimpy ⁇ Testis) protein, and binds to the target RNA via the recognition region, thereby converting the PIWI protein with single-stranded RNA cleavage activity into the target RNA. Induce.
  • PIWI P-element Induced Wimpy ⁇ Testis
  • the piRNA-PIWI complex is known to cleave the target RNA between the 10th and 11th bases from the 5 'end of piRNA (Brennecke, J., et al., Cell, 2007, 128, 1089). -1103; Gunawardane, L. S., et al., 2007, Science, 315, 1587-1590).
  • Fem piRNA is a small molecule RNA produced from a transcription product of the feminizer (Fem) gene, which is a female determining gene of silkworm.
  • Fem piRNA is composed of piRNA (PIWI-interacting RNA) consisting of 29 bases shown in SEQ ID NO: 3.
  • Fem piRNA forms a complex with silkworm's PIWI ortholog Siwi and cleaves the Masc gene transcript (Masc mRNA), a silkworm's male determinant gene described later, at a specific position. Suppress.
  • dsx double sex gene transcript
  • “Masc (Masculinizer) gene” is a male sex-determining gene of silkworm encoded on the Z chromosome.
  • the Masc gene encodes a zinc finger protein Masc that has two CCCH domains in tandem, and orthologs exist only in Lepidoptera insects.
  • the Masc gene is a target gene of the Fem piRNA-Siwi complex.
  • Masc mRNA is cleaved by the Fem piRNA-Siwi complex.
  • Lepidoptera refers to insects belonging to the taxonomic Lepidoptera, butterfly or moth.
  • the butterfly includes insects belonging to Nymphalidae, Papilionidae, Pieridae, Lycaenidae, and Hesperiidae.
  • the moths include Saturniidae, Bombycidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Geometridae, and idae , Insects belonging to Noctuidae, Pyralidae, Sphingidae and the like.
  • the mutant Masc gene of the present invention is a mutant Masc gene having cleavage resistance to the Fem piRNA-Siwi complex and the activity of the wild-type Masc gene.
  • the “mutant Masc gene” (often referred to herein as “MascR (Fem-piRNA-resistant Masc) gene”) is a Masc gene in which a part of the base sequence of the wild-type Masc gene has been mutated.
  • the mutant Masc protein encoded by the MascR gene (often referred to as “MascR protein” in this specification) does not necessarily have an amino acid mutation.
  • the expressed MascR protein has the same amino acid sequence as the wild-type Masc protein.
  • a “mutation having cleavage resistance to Fem piRNA-Siwi complex” is a mutation that occurs on the Masc gene and is a Fem piRNA-Siwi complex.
  • a mutation that confers cutting resistance to the Masc gene is suppressed, and as a result, cleavage of Masc mRNA by Fem piRNA-Siwi complex is inhibited. Therefore, even in the presence of Fem piRNA, a functional Masc protein can be expressed without cleavage of Masc mRNA.
  • the activity of the wild-type Masc gene means that it encodes a functional Masc protein.
  • “Functional Masc protein” refers to a Masc protein having an activity found in a wild-type Masc protein, that is, an activity that induces intracellular dsx mRNA into a male-specific splicing pattern. Whether or not the mutant Masc gene retains the activity of the wild-type Masc gene is determined, for example, by introducing an expression vector of the mutant Masc gene into a cultured cell derived from a female silkworm and expressing it in the cultured cell. It can be determined by examining whether the splicing pattern of dsxdsmRNA possessed by a cell is converted to a male-specific splicing pattern by Masc protein (Kiuchi T., et al., 2014; supra).
  • a mutant Masc gene expression vector having a drug resistance marker gene is introduced into a cultured cell derived from a female silkworm (for example, BmN4 cell derived from a silkworm ovary) by a transformation method, and then the cell is treated with a drug. Select with. Subsequently, mRNA is purified from cells that hold the above expression vector and express the mutant Masc gene according to a conventional method, and cDNA is prepared using this mRNA as a template.
  • DNA primer pair that can distinguish whether the splicing pattern of dsx mRNA is male-specific or female-specific using cDNA as a template (for example, Bmdsx-F pair shown in SEQ ID NO: 4 and Bmdsx-R pair shown in SEQ ID NO: 5) )
  • cDNA for example, Bmdsx-F pair shown in SEQ ID NO: 4 and Bmdsx-R pair shown in SEQ ID NO: 5
  • the mutant Masc gene retains the wild-type Masc gene activity. It is shown to be a gene.
  • the cleavage-resistant mutation is present in the Fem piRNA recognition sequence on the Masc gene.
  • the Fem piRNA recognition sequence corresponds to the nucleotide sequence of positions 1391 to 1419 when the a (adenine) of the start codon atg is 1 in the Masc gene consisting of the nucleotide sequence represented by SEQ ID NO: 1.
  • the base sequence corresponding to the Fem piRNA recognition sequence of the silkworm Masc gene corresponds to the silkworm Masc gene ortholog.
  • a preferred region where a cleavage resistant mutation is present is a region that forms direct base pairing by complementary binding with Fem ⁇ ⁇ ⁇ ⁇ piRNA.
  • the base sequence corresponding to the region corresponds to the silkworm Masc gene ortholog.
  • Types of cleavage resistant mutations include base substitutions other than nonsense mutations, deletions that do not cause frame shifts, or additions that do not cause frame shifts.
  • Base substitution other than nonsense mutation The “base substitution (mutation)” is a mutation in which a part of the base of the wild-type gene is replaced with another base. In the present specification, a mutation in which a part of the base of the wild type Masc gene is replaced with another base corresponds.
  • the base substitution includes a single base substitution such as a point mutation and a substitution of two or more consecutive bases. However, in the MascR gene of the present invention, any base substitution is included.
  • Base substitution includes transition mutation and transversion mutation based on the nature between bases before and after substitution.
  • Transition mutations are substitutions between purines or pyrimidines. For example, a (adenine) to g (guanine) substitution and g to a substitution.
  • Transversion mutations are substitutions between purines and pyrimidines. For example, substitution from c (cytosine) to t (thymine) and substitution from t to c can be mentioned. Any mutation may be used in the MascR gene of the present invention.
  • silent mutation is a substitution to a degenerate codon that does not result in an amino acid substitution.
  • a missense mutation is a base substitution that results in an amino acid substitution in the amino acid sequence of the Masc protein.
  • Nonsense mutations are base substitutions that result in stop codons.
  • the base substitution there is no frame shift because the number of bases does not increase or decrease before and after the base substitution occurs (for example, between the wild type Masc gene and the MascR gene).
  • a nonsense mutation it becomes a truncated protein from which a downstream amino acid has been deleted by insertion of a stop codon. Therefore, it is not preferable as the base substitution of the present invention. Therefore, the base substitution in the MascR gene of the invention may be either a silent mutation or a missense mutation as long as it is other than a nonsense mutation.
  • the MascR protein having the amino acid substitution is a functional Masc protein. Silent mutation that does not change the amino acid sequence of the Masc protein is preferable.
  • FIG. 2 shows a preferred silent mutation as a cleavage resistant mutation in the Fem piRNA recognition sequence of the silkworm Masc gene.
  • the silkworm Masc gene ortholog may be a silent mutation that does not cause an amino acid substitution in the base sequence corresponding to the Fem piRNA recognition sequence, similarly to the silkworm.
  • the amino acid substitution caused by the missense mutation may be a conservative amino acid substitution.
  • “Conservative amino acid substitution” refers to substitution between amino acids in the same amino acid group when amino acids are classified based on their properties. In the case of conservative amino acid substitution, since the properties of the amino acid before and after substitution are similar, the structure and properties substantially equivalent to the wild-type protein can be brought to the mutant protein.
  • the amino acid groups include non-polar amino acid groups (alanine (A), phenylalanine (F), leucine (L), isoleucine (I), valine (V), methionine (M), proline (P), tryptophan (W).
  • Polar amino acid groups (glycine (G), serine (S), threonine (T), cysteine (C), asparagine (N), glutamine (Q), tyrosine (Y), lysine (K), histidine (H) , Arginine (R), glutamic acid (E), aspartic acid (D)), acidic amino acid group (D, E), basic amino acid group (R, H, K), aromatic amino acid group (F, W, Y) And aliphatic amino acid groups (G, A, L, I, V) and the like.
  • Table 1 shows suitable missense mutations as cleavage-resistant mutations of the present invention within the Fem piRNA recognition sequence of the silkworm Masc gene (substantially within the range of positions 1393 to 1419 which can cause missense mutations).
  • Deletion that does not cause a frame shift is a mutation that loses the base of the wild-type gene.
  • Deletion mutations include deletions that cause frameshifts and deletions that do not. In the MascR gene of the present invention, deletion mutations that do not cause frame shift are targeted.
  • “Deletions that do not cause frameshift” are the deletion of 3n consecutive bases (n is an integer) in the wild-type gene, resulting in the deletion of one or several amino acids in the amino acid sequence encoded by the wild-type gene. It is a mutation that causes loss. This mutation does not cause a reading frame shift, that is, a frame shift downstream of the mutation site.
  • Deletion mutations suitable as cleavage-resistant mutations of the present invention result in a deletion at positions 425 to 476 (Masc-md1) when the initiation methionine is position 1 in the silkworm Masc protein shown in SEQ ID NO: 2.
  • Deletions from 1273 to 1428 in the Masc gene, deletions from 450 to 476 (Masc-md2), deletions from 1348 to 1428 in the Masc gene, and deletions from 463 to 476 (Masc-md3) Deletion of positions 1387 to 1428 in the Masc gene, and deletion of positions 1408 to 1428 in the Masc gene that results in a deletion of positions 469 to 476 (Masc-md4).
  • Addition that does not cause frame shift is a mutation in which a base is inserted into the base sequence of a wild-type gene. Addition mutations, like deletion mutations, include additions that cause frameshifts and additions that do not. In the MascR gene of the present invention, additional mutations that do not cause frame shift are targeted.
  • “Addition that does not cause frame shift” means that the insertion of one or several amino acids in the amino acid sequence encoded by the wild type gene results from the addition of 3n consecutive bases (n is an integer) in the wild type gene. It is a mutation that brings about. This mutation does not cause a frameshift downstream of the mutation site.
  • the number of cleavage resistant mutations is not particularly limited as long as it can inhibit the binding of Fem piRNA and Masc mRNA via base pairing.
  • 1 or 2 or more within the Fem piRNA recognition sequence specifically, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 3
  • the number of cleavage resistant mutations also depends on the type of mutation. In the case of single base substitution, it is preferable to have a plurality of mutations in the Fem piRNA recognition sequence in order to effectively inhibit the binding of Fem piRNA and Masc mRNA via base pairing. For example, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the number of mutations may be small if the number of bases lost by one mutation or the number of inserted bases is large.
  • each mutation when there are a plurality of cleavage resistance mutations, each mutation may be the same type of mutation or a different mutation.
  • one MascR gene may have two or more substitution mutations and one deletion mutation.
  • the MascR gene of the present invention may be inserted into a suitable cloning vector for conservation and / or cloning.
  • a method for constructing a cloning vector and a method for producing a transformant such as Escherichia coli or yeast into which the vector has been introduced may be performed using molecular biology techniques known in the art. These methods are described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc. 2.
  • a second aspect of the present invention is a mutant Masc gene expression vector (often referred to herein as “MascR gene expression vector”).
  • the MascR gene expression vector of the present invention comprises the MascR gene of the first aspect in a state capable of being expressed. According to the MascR gene expression vector of the present invention, the MascR gene can be overexpressed by introduction into the host. 2-2. Configuration
  • an “expression vector” refers to one expression system unit that contains a target gene in a state capable of being expressed and can control the expression of the gene.
  • the target gene of interest in the present invention is the MascR gene described in the first embodiment.
  • the “expressible state” means that a target gene is placed under the control of a promoter in an expression vector.
  • an autonomously replicable expression vector such as a plasmid or Bacmid, a viral vector, an expression vector capable of homologous or non-homologous recombination in a chromosome, or a part of a chromosome in which it is inserted into a host chromosome.
  • a shuttle vector that can replicate in E. coli, Bacillus subtilis, or yeast can also be used.
  • 2-2-1. Components of MascR gene expression vector The MascR gene expression vector is configured to express the MascR gene in a host cell.
  • the components of the MascR gene expression vector include the MascR gene of the first embodiment and a ubiquitous promoter as essential components.
  • a multicloning site, 5′UTR, 3′UTR, terminator, enhancer, marker gene, insulator, transposon inverted terminal repeat and the like are included as selection components.
  • the MascR gene expression vector is composed of two units, a first expression unit and a second expression unit, which will be described later, a transcription regulatory factor gene and a target promoter of the transcription regulatory factor are essential components. Include.
  • each component of the MascR gene expression vector of the present invention will be specifically described.
  • (1) MascR gene The configuration of the MascR gene is described in detail in the first embodiment, and a description thereof is omitted here.
  • Promoter The promoter included in the MascR gene expression vector of the present invention is a ubiquitous promoter.
  • the “ubiquitous promoter” is a promoter capable of expressing the target gene arranged downstream (3 ′ end side) of the promoter, that is, the MascR gene in the entire host individual into which the expression vector has been introduced. In this specification, it uses as a term corresponding to the site-specific promoter which controls the expression in a specific cell or tissue.
  • Ubiquitous promoters can be classified into constitutively active promoters, expression-inducible promoters, or time-specific active promoters based on their expression control timing.
  • a constitutively active promoter can constitutively express a target gene in a host cell.
  • constitutively active promoters in silkworms include actin 3 gene-derived actin 3 promoter (A3 promoter), silkworm heat shock protein 90 (hsp90) gene-derived heat shock protein 90 promoter (hsp90 promoter), silkworm elongation factor 1 ⁇ (Elongation Factor-1 ⁇ ) gene-derived elongation factor 1 promoter (EF-1 promoter), BmNPV (Bombyx mori nuclear polyhedrosis virus) early gene 1 (immediate-early gene 1; ie-1) promoter, etc. Can be mentioned.
  • the base sequence of A3 promoter is shown in SEQ ID NO: 6, and the base sequence of hsp90 promoter is shown in SEQ ID NO: 7.
  • An expression-inducible promoter can induce the expression of a target gene in a host cell at any time. Therefore, this type of promoter is used to maintain a host strain having a MascR gene expression vector when the MascR gene expression vector is composed of one gene expression unit in “Unit structure of MascR gene expression vector” described later. And is particularly useful.
  • a specific example of the expression-inducible promoter in the silkworm is a heat shock protein 70 promoter (hsp70 promoter) derived from the heat shock protein 70 (hsp70) gene. The base sequence of the hsp70 promoter is shown in SEQ ID NO: 8.
  • a time-specific active promoter can induce the expression of a target gene in a host cell only at a specific stage of development.
  • the MascR protein needs to function at an early stage of embryonic development. Therefore, it is desirable that a time-specific active promoter that can be included in the MascR gene expression vector of the present invention induces expression at an early stage of embryonic development.
  • any of the above ubiquitous promoters can cause overexpression of the MascR gene in the host cell. Therefore, the ubiquitous promoter in this specification may be interpreted as an overexpression promoter.
  • the donor species of the promoter is not particularly limited as long as it is operable in the host cell into which the MascR gene expression vector is introduced.
  • Preferred donor species are those that belong taxonomically with the host of the MascR gene expression vector.
  • the donor species is a species belonging to the same Lepidoptera as the silkworm, preferably a species belonging to the same family Bombycidae as the silkworm, more preferably a mulberry (Bombyx mandarina). Species belonging to the same genus Bombyx are suitable.
  • the most preferred donor species is the same silkworm.
  • Multicloning site is a cluster sequence comprising a plurality of cloning restriction enzyme sites.
  • the type and number of restriction enzyme sites to be included in the base sequence constituting the multicloning site there are no particular restrictions on the type and number of restriction enzyme sites to be included in the base sequence constituting the multicloning site.
  • the number of multi-cloning sites in the MascR gene expression vector and the position at which the multi-cloning site is arranged are not limited, but are preferably arranged within the control region of the ubiquitous promoter. This is because the MascR gene can be easily inserted into the MascR gene expression vector of the present invention by arranging at such a position.
  • 5′UTR (5'untranslated region) and 3'UTR (3'untranslated region) “5′UTR and 3′UTR” are both polynucleotides consisting of untranslated regions that do not themselves encode proteins, fragments thereof, or functional nucleic acids.
  • each UTR is preferably, but not limited to, 5′UTR and 3′UTR derived from the Masc gene. Particularly preferred are 5'UTR and 3'UTR derived nucleotide sequences of the Masc gene itself.
  • 5′UTR is located upstream (5 ′ end side) of the start codon of the MascR gene
  • 3′UTR is located downstream (3 ′ end side) of the stop codon of the MascR gene.
  • the 3 ′ UTR can contain a poly A signal.
  • Terminator is a base sequence that is located at the 3 ′ end of the MascR gene, preferably downstream of the stop codon in the MascR gene expression vector of the present invention, and is a base sequence that can terminate the transcription of the MascR gene. It is configured.
  • the hsp70 terminator consisting of the base sequence shown in SEQ ID NO: 9
  • the SV40 terminator consisting of the base sequence shown in SEQ ID NO: 10 can be mentioned.
  • Enhancer An “enhancer” consists of a base sequence that can further enhance the expression of a MascR gene by controlling a ubiquitous promoter in the MascR gene expression vector of the present invention.
  • Marker gene A “marker gene” is a gene encoding a labeled protein, also called a selection marker.
  • Labeled protein refers to a polypeptide that can determine the presence or absence of expression of a labeled gene based on its activity. Therefore, when the MascR gene expression vector contains a marker gene, the host carrying the MascR gene expression vector, that is, a transformant can be easily identified based on the activity of the marker protein.
  • “based on activity” means based on the detection result of activity. The activity may be detected directly by the activity of the labeled protein itself or indirectly by a metabolite generated by the activity of the labeled protein such as a dye. Good. Detection can be chemical detection (including enzymatic reaction detection), physical detection (including behavioral analysis detection), or sensory detection of the detector (including visual, tactile, olfactory, auditory, taste detection) Either may be sufficient.
  • the type of labeled protein encoded by the labeled gene is not particularly limited as long as its activity can be detected by a method known in the art.
  • it is a labeled protein with low invasiveness to the transformant upon detection.
  • fluorescent proteins, chromogenic proteins, photoproteins, external secreted proteins, proteins that control external morphology, and the like can be mentioned.
  • Fluorescent proteins, chromogenic proteins, photoproteins, and exocrine proteins can be visually detected under certain conditions without changing the external form of the transformant, and therefore are less invasive to the transformant.
  • it is particularly suitable because it is easy to identify and select transformants.
  • Fluorescent protein refers to a protein that emits fluorescence of a specific wavelength when irradiated with excitation light of a specific wavelength. Either a natural type or a non-natural type may be used. Further, the excitation wavelength and the fluorescence wavelength are not particularly limited. Specifically, for example, CFP, RFP, DsRed (including derivatives such as 3xP3-DsRed), YFP, PE, PerCP, APC, GFP (including derivatives such as EGFP and 3xP3-EGFP), etc. It is done.
  • Chrosome synthesis protein is a protein involved in pigment biosynthesis and is usually an enzyme.
  • the “dye” here is a low molecular compound or peptide capable of imparting a dye to a transformant, and the kind thereof is not limited.
  • a pigment that appears as an external color of an individual is preferable. Examples include melanin pigments (including dopamine melanin), omochrome pigments, and pteridine pigments.
  • photoprotein refers to a substrate protein that can emit light without the need for excitation light or an enzyme that catalyzes the light emission of the substrate protein.
  • luciferin or aequorin as a substrate protein and luciferase as an enzyme can be mentioned.
  • exocrine protein refers to a protein that is secreted extracellularly or externally, and includes exocrine enzymes, fiber proteins such as fibroin, and sericin. Exocrine enzymes include digestive enzymes in addition to enzymes that contribute to the degradation or inactivation of drugs such as blasticidin and impart drug resistance to the host.
  • the marker gene is placed in a state capable of being expressed downstream of the promoter in the MascR gene expression vector.
  • Insulator An “insulator” is a base sequence that can stably control the transcription of a gene sandwiched between sequences without being affected by chromatin of surrounding chromosomes. Examples include the cHS4 sequence of chicken and the gypsy sequence of Drosophila.
  • ITRs Inverted terminal repeat sequences of transposon “Inverted terminal repeat sequences (ITRs)” are included when the MascR gene expression vector of the present invention is used as an expression vector capable of homologous recombination. Is a selection component to obtain.
  • the inverted terminal repeats are usually used in pairs, and piggyBac, mariner, minos, etc.
  • Transcriptional regulatory factor gene is an essential component of the first expression unit described below.
  • transcriptional regulatory factor refers to a protein factor that can bind to a target promoter described later and activate the target promoter. Examples thereof include GAL4 protein, which is a galactose metabolic activation protein of yeast, tTA, which is a tetracycline-regulated transactivator, and mutants thereof.
  • Target promoter of transcription regulator is an essential element of the second expression unit described later, and the transcription regulator encoded by the first expression unit binds to it.
  • the transcriptional regulatory factor and its target promoter are in a corresponding relationship with the transcriptional regulatory factor. Normally, when a transcriptional regulatory factor is determined, the target promoter is inevitably determined. For example, when the transcriptional regulatory factor is GAL4 protein, UAS (Upstream Activating Sequence) is used.
  • GAL4 protein Upstream Activating Sequence
  • UAS Upstream Activating Sequence
  • the MascR gene expression vector When configured with a single gene expression unit, the MascR gene expression vector contains all the necessary elements for expressing the MascR gene in a host cell.
  • the components are contained within one gene expression vector. Specifically, it includes a ubiquitous promoter that is an essential component and a MascR gene operably linked downstream of the promoter.
  • the promoter is preferably an expression-inducible promoter for the maintenance and management of female lethal lines.
  • the MascR gene expression vector may contain two or more MascR genes under the control of one promoter.
  • the MascR gene expression vector is composed of one gene expression unit, the MascR gene can be overexpressed in the host cell simply by introducing the MascR gene expression vector into the host.
  • the first and second expression units function as one MascR gene expression vector only when they coexist in the host cell. That is, in the same cell, a transcriptional regulatory factor is expressed from the first expression unit by activating the promoter contained in the first expression unit, which activates the target promoter of the second expression unit, thereby obtaining the target MascR gene. Can be expressed.
  • the first and second expression units have the following configuration.
  • the “first expression unit” comprises a promoter and a gene of the above-mentioned transcription regulatory factor operably linked downstream of the promoter.
  • the aforementioned ubiquitous promoter is used as the promoter used in the first expression unit.
  • the same or different two or more transcriptional regulatory factors may be included under the control of one promoter.
  • the first expression unit may have two or more sets of promoters and transcription regulatory factor genes under the control of the promoter.
  • each set may be the same set or different sets.
  • an existing gene expression vector can also be used.
  • the “second expression unit” comprises a target promoter of a transcriptional regulatory factor encoded by the first expression unit and a MascR gene operably linked downstream of the target promoter.
  • the target promoter contained in the second expression unit selects a promoter that is activated by the transcriptional regulatory factor encoded by the first expression unit.
  • the gene of the transcriptional regulatory factor contained in the target promoter first expression unit is a GAL4 gene
  • UAS is used for the GAL4 target promoter of the second expression unit.
  • the second expression unit may include two or more identical or different MascR genes under the control of one target promoter.
  • the second expression unit may have two or more pairs of a target promoter and a MascR gene under its control.
  • each set may be the same set or different sets.
  • the second expression unit may be composed of two or more identical or different units containing the MascR gene.
  • the transcriptional regulatory factor expressed from one first expression unit can express the MascR gene contained in each second expression unit by activating the target promoters of the plurality of second expression units.
  • the MascR gene expression vector of this configuration is useful for the maintenance and management of female lethal lines. Normally, when the MascR gene is overexpressed in a host cell, it becomes female lethal and the strain cannot be maintained. However, maintenance of the first and second expression units can be facilitated by preparing a line that holds each of the first and second expression units and managing each of them individually.
  • the MascR gene expression vector of this configuration can amplify the expression of the MascR gene of the second expression unit via the transcriptional regulatory factor encoded by the first expression unit. Therefore, it is suitable for overexpression of the MascR gene in a host cell. 2-3.
  • MascR gene expression vector host introduction method A MascR gene expression vector host introduction method will be described.
  • the host into which the MascR gene expression vector is introduced is a lepidopteran insect individual, a cell derived from a lepidopteran insect (including a cell line), or a tissue derived from a lepidopterous insect. Particularly preferred are lepidopteran insects.
  • a lepidopteran insect individual a cell derived from a lepidopteran insect (including a cell line), or a tissue derived from a lepidopterous insect.
  • lepidopteran insects When introduced into a cell or tissue, the stage of development of the collected individual is not particularly limited. When introduced into an individual, there are no particular limitations on the developmental stage or sex, and any stage of an embryo, larva, pupa, or adult can be used. Preferably, it is an embryonic time when a higher effect can be expected.
  • the MascR gene expression vector can be introduced by a method known in the art depending on the MascR gene expression vector to be introduced.
  • the MascR gene expression vector is a plasmid having transposon inverted terminal repeats (ITRs) (Handler AM. Et al., 1998, Proc. Natl. Acad. Sci. USA 95: 7520-5) and introduced. If the host is a silkworm, the method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84) can be applied.
  • the MascR gene expression vector of the present invention diluted to an appropriate concentration may be injected into an early embryo of a silkworm egg together with a helper vector having a transposon transferase gene.
  • a helper vector having a transposon transferase gene For example, pHA3PIG can be used as the helper vector.
  • the MascR gene expression vector of the present invention contains a marker gene, as described above, the target transformant can be easily selected based on the expression of the gene or the like.
  • the transgenic silkworm obtained by this method the MascR gene expression vector is integrated into the chromosome via the transposon inverted terminal repeat sequence. If necessary, this transgenic silkworm may be sibling or inbred to obtain a homozygous expression vector inserted into the chromosome. 2-4.
  • the host can be made female lethal.
  • a MascR gene expression vector composed of two gene expression units it is possible to easily maintain and manage the produced female lethal line. Therefore, a MascR gene expression vector composed of two gene expression units can reduce costs and labor in producing a female lethal line.
  • summary The 3rd aspect of this invention is a puerperal lethal silkworm strain
  • the fetus lethal silkworm strain of this embodiment is a transgenic silkworm strain having the MascR gene expression vector described in the second embodiment.
  • female pupal dead silkworms can be easily obtained.
  • female-killed silkworm refers to a silkworm in which a female individual dies by the 5th instar larva stage after the start of development.
  • the “feminine lethal silkworm strain” refers to a passable genetically modified silkworm or its progeny that has the potential to kill a female individual.
  • the female mortal silkworm strain of the present invention has the MascR gene expression vector described in the second embodiment.
  • the MascR gene expression vector possessed by the female pupal dead silkworm strain may be either composed of one gene expression unit described in the second embodiment or composed of two gene expression units. In the latter case, a transgenic silkworm strain in which the transgenic silkworm has only the second expression unit is included in the female pupal dead silkworm strain of the present invention.
  • the transgenic silkworm strain having only the second expression unit is mated with the silkworm strain containing the first expression unit, and the MascR gene from the second expression unit contained in the transgenic silkworm strain having only the second expression unit. This is because a transgenic silkworm strain having only the second expression unit has the potential to kill a female individual in that it can easily produce a female dead lethal silkworm by inducing expression. .
  • the transgenic silkworm lines having only the second expression unit are exemplified by the sumi-13 lines (sumi13-1, sumi13-2) described in Example 2, but are not limited thereto.
  • a transgenic silkworm strain having only the first expression unit does not correspond to the female dead lethal silkworm strain of the present invention because it has no direct potential to kill a female individual.
  • the MascR gene expression vector may be present transiently in the silkworm cell, or may be stably and continuously present in a state of being introduced into the chromosome. Usually, it is preferable to exist stably and continuously.
  • the ubiquitous promoter that controls the expression of the MascR gene in the MascR gene expression vector is an expression-inducible promoter. It is desirable that
  • the MascR gene expression vector is composed of two gene expression units, a first expression unit and a second expression unit, and each is integrated on a silkworm chromosome
  • the first and second expression units are the same chromosome of the same silkworm individual. It may exist on the top, or may exist on different chromosomes of the same silkworm individual. Furthermore, it is desirable that the first expression unit and the second expression unit are incorporated into chromosomes of silkworm individuals or silkworm strains different from each other.
  • the first and second in F1 A female pupal dead silkworm having 2 expression units can be easily obtained.
  • any promoter can be used as long as the ubiquitous promoter contained in the first expression unit is the ubiquitous promoter described in the second embodiment.
  • the ubiquitous promoter contained in the first expression unit is preferably an expression-inducible promoter.
  • Method for producing a female pupal dead silkworm strain The method for producing a female pupal dead silkworm strain of the present invention is not particularly limited because any method capable of producing a recombinant silkworm can be used. Examples of a method for producing a recombinant silkworm include a method of introducing a MascR gene expression vector into a silkworm. The specific method is the same as the method described in the section “2-3. Method for introducing host of MascR gene expression vector” in the second embodiment.
  • the MascR gene expression vector is composed of one gene expression unit
  • the MascR gene expression vector is composed
  • the MascR gene expression vector is composed of two gene expression units, the first and second expression units.
  • Method for producing female pupal dead silkworm In order to produce female pupal dead silkworm composed only of male population using the female pupal dead silkworm strain of the present invention, for example, (1) expression induction in female pupal dead dead silkworm strain And (2) mating two transgenic silkworm strains each having the first expression unit and the second expression unit of the MascR gene expression vector, and combining both the first expression unit and the second expression unit.
  • the expression of the MascR gene is suppressed in an untreated state of expression induction, and since it does not become a female pupal dead silkworm, it can be maintained and managed in the same manner as a wild type silkworm.
  • an expression inducing treatment may be performed at the early embryo stage for this female pupal dead silkworm strain.
  • the expression induction treatment method may be appropriately selected according to the properties of the expression inducible promoter. For example, if the expression-inducible promoter is an hsp70 promoter, a female pupal dead silkworm can be produced by treating the early embryo at 42 ° C. for 30 minutes to 1 hour.
  • This method is a gene set in which the MascR gene expression vector is composed of two gene expression units, the first and second expression units, and has the first expression unit. This is carried out when there is a female silkworm dead silkworm strain having a replacement silkworm strain and a second expression unit. By mating two transgenic silkworm strains, it is possible to produce female pupal dead silkworms having first and second expression units in F1 individuals.
  • a genetically modified silkworm strain having the first expression unit and a female pupal dead silkworm strain having the second expression unit may be crossed based on a conventional method.
  • a transgenic silkworm having two expression units is preferably preliminarily sibling or inbred and made homozygous for each expression unit.
  • F1 individuals having the first and second expression units may be selected based on the activity of the labeled protein encoded by the labeled gene contained in each expression vector. 4).
  • Summary The 4th aspect of this invention is a female pupal dead dead male silkworm infertile silkworm strain
  • the female pupal dead male silkworm infertile silkworm strain of the present invention can also be understood as one form of the female pupal dead silkworm strain of the third aspect.
  • this strain it is possible to obtain a silkworm population in which not only the female individual is lethal but also the surviving male individual is infertile. 4-2. Configuration Since the basic configuration of this aspect is the same as the configuration of the third aspect, a characteristic configuration of this aspect will be described below.
  • female dead dead male sterilized silkworm refers to a silkworm in which a female individual becomes lethal at the developmental stage and a living male individual becomes infertile.
  • Male sterility or “male (individual) infertility” means that the male fertility is lost.
  • Infertile silkworms that are deadly male and female are composed only of infertile male individuals.
  • the female mortal male sterilized silkworm has first and second expression units of the MascR gene expression vector described later in the cell.
  • a female individual can be killed by the MascR protein expressed from the second expression unit by activating the promoter, and a male individual can be sterilized by the action of the transcriptional regulatory factor GAL4 protein encoded by the first expression unit.
  • female-lethal dead male sterilized silkworm strain refers to a genetically-transmissible silkworm or its progeny that can be passaged and has the potential to let female individuals become dead and males become infertile.
  • the female mortal male sterilized silkworm strain of the present invention has the MascR gene expression vector described in the second embodiment.
  • This MascR gene expression vector is composed of two gene expression units composed of first and second expression units.
  • the basic configuration of the first expression unit may be the same as the configuration of the first expression unit described in the second aspect.
  • the gene of the transcriptional regulatory factor included in the vector is limited to the GAL4 gene encoded by the GAL4 protein, which is a galactose metabolic activation protein of yeast.
  • the basic structure of the second expression unit may be the same as that of the second expression unit described in the second aspect.
  • the gene of the transcriptional regulatory factor included in the first expression unit is the GAL4 gene
  • the target promoter of the transcriptional regulatory factor included in the second expression unit must be the target promoter of GAL4.
  • An example of a target promoter for GAL4 is UAS.
  • the female pupal lethal male sterilized silkworm strain has either one of the first and second expression units. Unlike the third embodiment, in this embodiment, not only the transgenic silkworm strain having only the second expression unit but also the transgenic silkworm strain having only the first expression unit is the female pupal lethal male pupal infertile silkworm strain of the present invention. included.
  • the MascR gene expression vector may be transiently present in the silkworm cell, or may be stably and continuously present in the state introduced into the chromosome. Also good. Usually, it is preferable to exist stably and continuously. When two expression vectors of the MascR gene expression vector are present on the chromosome, each expression vector is preferably present on a different chromosome. 4-3.
  • Method for producing female slaughtered male sterilized silkworm strain The method for producing a female slaughtered male sterilized silkworm strain of the present invention is in accordance with the method for producing the female slaughtered male silkworm strain of the third aspect.
  • Example 1 Construction of MascR gene expression vector> (the purpose)
  • the MascR gene expression vector of the present invention is constructed.
  • a MascR gene expression vector composed of two gene expression units, a first expression unit and a second expression unit was constructed.
  • the first expression unit a known gene expression vector for transformation pBac [A3-GAL4: 3xP3-DsRed] having an actin 3 promoter which is a ubiquitous promoter and a GAL4 gene which is a gene of a transcriptional regulator (Uchino et al ., 2006, J. Insect Biotechnol.
  • a pUAS-MascR vector was constructed here as the second expression unit.
  • the pUAS-Masc vector containing the wild-type silkworm Musc gene was constructed as a control for the second expression unit.
  • RNA-PCR-Kit TaKaRa Bio-Inc.
  • PCR was performed using Prime STAR® GXL (TaKaRa Bio Inc.), and the composition of the reaction solution was in accordance with the standard method of the protocol attached to Prime STAR® GXL.
  • the PCR cycle conditions were 40 cycles with 98 ° C. for 10 seconds, 60 ° C. for 15 seconds and 68 ° C. for 2 minutes as one cycle.
  • the amplification product thus obtained was subjected to A addition using 10 ⁇ A-attachment Mix (TOYOBO) and then cloned into pGemTeasy (Promega) to construct a silkworm Masc gene cloning vector “Masc-pGemTeasy”.
  • PCR was performed using Masc-pGemTeasy as a template and a primer pair of Masc_hind_FlagF (SEQ ID NO: 13) and Masc_bamR (SEQ ID NO: 14). PCR conditions were in accordance with the above conditions.
  • the obtained amplification product encodes a silkworm Masc protein in which a FLAG tag is fused on the N-terminal side. This amplified fragment was inserted into the HindIII / BamHI site of pIZ / V5-His (life technologies) to construct “Masc-pIZ”.
  • the MascR gene has 5 base substitutions introduced into the FemFpiRNA recognition sequence as shown in FIG. PCR using PrimeSTAR Mutagenesis Basal Kit (TaKaRa) and Masc_ resistant1 (SEQ ID NO: 19) and Masc_ resistant2 (SEQ ID NO: 20) primer pairs using Masc-pIZ as a template, and a silkworm fused with a FLAG tag on the N-terminal side
  • a plasmid containing MascR protein was constructed and designated as “MascR-pIZ”.
  • PBacMCS [UAS-SV40, 3xP3-GFP TtoH] is a pBacMCS [UAS-SV40, 3XP3-EGFP] (Sakudoh et al., 2007, Proc Natl Acad Sci USA 104: 8941-8946.)
  • Vector 3xP3-EGFP After the fragment containing the DNA was excised from the vector by digestion with EcoRI, the direction of the 3xP3-EGFP fragment was changed and ligated again to the vector.
  • PBacMCS [UAS-MascR-SV40,3xP3-EGFP] (often referred to herein as “pUAS-MascR”) as the second expression unit of the MascR gene expression vector of the present invention and pBacMCS as the second expression unit for the control [UAS-Masc-SV40, 3xP3-EGFP] (often referred to herein as “pUAS-Masc”) was obtained.
  • the structures of pUAS-MascR and pUAS-Masc are shown in FIG.
  • the target promoter of the transcription factor in these second expression units is UAS and the terminator is SV40 terminator.
  • the marker gene is 3xP3 EGFP that expresses EGFP in embryonic, larval and adult eyes.
  • piggyBacL and piggyBacR are included as transposon inverted terminal repeats (ITRs) upstream of UAS and downstream of the marker gene, respectively.
  • Example 2 Production of a silkworm strain killed by female pupae> (the purpose) Using the MascR gene expression vector constructed in Example 1, the female pupal dead silkworm strain of the present invention is produced.
  • the MascR gene expression vector second expression unit pUAS-MascR constructed in Example 1 and its control second expression unit pUAS-Masc were purified using Qiagen Plasmid Midi Kit (Qiagen). The specific production method followed the protocol attached to the kit.
  • the purified pUAS-MascR and pUAS-Masc were microinjected together with piggyBac helper plasmids, each encoding transposon transferase, into eggs 2-8 hours after laying in silkworm w1, pnd, which is a non-dormant strain (Tamura et al.) al. 2000; supra). Eggs after injection were incubated at 25 ° C.
  • “sumi13 strain” two strains of sumi13-1 and sumi13-3) as a female dead lethal silkworm strain with pUAS-MascR integrated into the chromosome
  • “sumi12 strain” as a control strain with UAS-Masc integrated into the chromosome (2 systems of sumi12-1 and sumi12-2) were obtained.
  • the insertion of pUAS-MascR into the chromosome was confirmed by Southern blotting (not shown) for two strains of the female dead lethal silkworm strain.
  • Example 3 Verification of sex ratio in female dead lethal silkworm> (the purpose) It is verified that a female pupal dead silkworm produced using the female pupal dead silkworm strain of the present invention is only a male individual.
  • the male individuals of the sumi13 line (UAS-MascR line; female pupal dead silkworm line) and the sumi12 line (UAS-Masc; control line) established in Example 2 were respectively 193-2 lines (BmA3-GAL4 line) ( F1 individuals were obtained by mating with female individuals of Uchino et al., 2006;
  • Line 193-2 is a transgenic silkworm line in which a known gene expression vector having the structure of the first expression unit in which the silkworm actin 3 promoter and the GAL4 gene are linked is incorporated into the chromosome.
  • the eye has green fluorescence (G), in individuals with only the BmA3-GAL4 line, the eye has red fluorescence (R), and BmA3-GAL4 and UAS-MascR, Or in individuals with BmA3-GAL4 and UAS-Masc, the eyes exhibit green and red fluorescence (G + R).
  • G green fluorescence
  • R red fluorescence
  • G + R green and red fluorescence
  • no fluorescent color is seen in the eye (Negative).
  • the classified genotypes were the above four types.
  • Table 2 shows the number of embryos of each genotype, the hatching rate, and the number of male and female individuals of 5th instar (final) larvae, and FIG. 4 shows the genotype sex ratio in each line.
  • Genomic DNA from larvae dead individuals (17 individuals) in sumi13-1 ⁇ 193-2 (G + R) group and larvae dead individuals (9 individuals) in sumi13-3 ⁇ 193-2 (G + R) group Extracted.
  • the genomic DNA was extracted according to the method described in Green & Sambrook (2012; mentioned above). Gender was determined using a method of detecting the RAPD marker on the W chromosome by PCR.
  • genomic DNA was used as a template, and Musashi-A1 (SEQ ID NO: 17) and Musashi-B1 (SEQ ID NO: 18) were used as primer pairs.
  • PCR was performed using ExTaq (TaKaRa Bio Inc.), and the composition of the reaction solution was in accordance with the standard method of the protocol attached to ExTaq.
  • the PCR cycle conditions were 40 cycles, with 98 ° C for 10 seconds, 55 ° C for 30 seconds and 72 ° C for 1 minute as one cycle.
  • the amplified product was electrophoresed on a 1.5% agarose gel, and the sex was confirmed from the number of bands. (result)
  • the results are shown in FIG. 5A.
  • the majority of the dead individuals were female.
  • the growth after 2 years of age was significantly delayed compared to male individuals that grew normally (not shown). Therefore, it was suggested that MascR protein causes growth delay in the larva stage in female individuals and induces female lethality.
  • Example 5 Verification of cocoons formed by dead pupae silkworm> (the purpose) It verifies about the form etc. of the cocoon which the female pupal dead silkworm produced from the female pupal dead silkworm line of this invention forms. (Method) The surviving individuals of female pupal dead silkworms obtained in the sumi13-1 ⁇ 193-2 (G + R) group and the sumi13-3 ⁇ 193-2 (G + R) group of Example 3 were all male. . In order to confirm that these individuals were male at the sex chromosome level, the karyotype of each individual was examined.
  • the surviving individuals obtained in the sumi13-1 ⁇ 193-2 (G + R) and sumi13-3 ⁇ 193-2 (G + R) groups have wings formed, and abnormalities are observed in their morphology. I checked it. Furthermore, the cocoon was taken out of the cocoon and the morphological abnormalities and sex were confirmed. The gender of the moth was identified from the morphological difference at the tail end. (result) The results are shown in FIG. 5B and FIG.
  • FIG. 5B shows the karyotype of the sex chromosome in the 5th instar larva of the female dead lethal silkworm. It was confirmed that the surviving individuals of the female pupae dead silkworm were males with sex chromosome type ZZ.
  • FIG. 6 shows the form of a cocoon (A) and the form and sex (B) of the cocoon in the cocoon when the silkworm of each strain including a female pupal dead silkworm is formed.
  • A no morphological abnormality was observed in the pupae formed by female pupal dead silkworms (indicated by sumi13-1 (G + R) and sumi13-1 (G + R) in the figure) It was found that normal silkworms can be obtained from dead female silkworms. B also confirmed that the wings were normal and all male.
  • Example 6 Verification of male infertility in line 193-2> (the purpose)
  • the 193-2 line contains the GAL4 gene as a transcriptional regulator gene
  • the sumi13 line contains the UAS and MascR genes that are the target promoters of GAL4. It is not only a lethal silkworm strain, but also a female dead lethal male infertile silkworm strain in which a surviving male becomes infertile.
  • the 193-2 line used for the production of the female mortal male sterilized silkworm line of the present invention becomes male infertile.
  • Method It is described in Uchino et al. (Uchino et al., 2008, JIBS, Insect biochem. Mol. Biol., 38: 1165-1173) that the 193-2 line is a male infertile line. Therefore, when mating 193-2 females and white / C males, and when mating white / C females and 193-2 males, the number of eggs laid by each female was confirmed. .
  • FIG. 7 shows the egg-laying state of the female when the 193-2 line and the white / C line are crossed with each other.
  • A shows the case where 193-2 females and white / C males were crossed
  • B shows the case where white / C females and 193-2 males were crossed.
  • 193-2 females laid eggs normally and the next generation was obtained, but white / C females mated with 193-2 males could lay eggs less than 1/4 of the normal number of eggs laid. And none of them hatched.
  • FIG. 8 is a diagram showing the difference in the number of eggs laid in white / C females when white / C females and various males are crossed. 1 is unmating female, 2 is white / C female x193-2 male, 3 is white / C female xw1-pnd male, and 4 is white / C female x white / C male It is. Even when females of the same strain were used, when 193-2 males were used, the number of eggs laid by unmated females was lower, and none of the eggs hatched.

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Abstract

La présente invention concerne une souche d'un insecte appartenant à l'ordre Lepidoptera, ladite souche étant apte à être facilement maintenue et gérée sans interruption de son contexte génétique, étant applicable à une technologie de modification génétique et permettant facilement et de façon arbitraire une sélection spécifique des mâles en une courte période de temps. Une souche létale pour la femelle peut être obtenue par transfert d'un vecteur d'expression génique, ledit vecteur d'expression génique codant pour un gène Masc muté qui présente une résistance au clivage contre le complexe Fem piRNA-Siwi et maintient l'activité du gène Masc de type sauvage, chez un insecte appartenant à l'ordre Lepidoptera.
PCT/JP2015/081397 2014-11-14 2015-11-06 Souche de bombyx mori létale pour le ver à soie femelle WO2016076240A1 (fr)

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CN110117613A (zh) * 2018-02-05 2019-08-13 中国科学院上海生命科学研究院 一种制备雄性不育的鳞翅目昆虫的方法及其核酸构建物
CN110117613B (zh) * 2018-02-05 2022-10-11 中国科学院分子植物科学卓越创新中心 一种制备雄性不育的鳞翅目昆虫的方法及其核酸构建物
CN113692225A (zh) * 2019-03-05 2021-11-23 以色列农业和农村发展部农业研究组织(范卡尼中心) 经基因组编辑的鸟类
CN114317613A (zh) * 2020-09-30 2022-04-12 浙江省农业科学院 利用基因组编辑技术构建鳞翅目昆虫雌性不育品系的方法
CN114317613B (zh) * 2020-09-30 2023-12-22 浙江省农业科学院 利用基因组编辑技术构建鳞翅目昆虫雌性不育品系的方法
CN112175967A (zh) * 2020-10-10 2021-01-05 安徽农业大学 一种增强植物抵抗鳞翅目害虫的pen1基因及其应用
CN114113569A (zh) * 2021-11-25 2022-03-01 江苏科技大学 一种基于代谢组学技术建立BmNPV抗性品系家蚕筛选标准的方法
CN114113569B (zh) * 2021-11-25 2023-10-27 江苏科技大学 一种基于代谢组学技术建立BmNPV抗性品系家蚕筛选标准的方法
CN114921469A (zh) * 2022-04-12 2022-08-19 广西壮族自治区蚕业技术推广站 家蚕嗅觉受体基因BmOR56的用途
CN114921469B (zh) * 2022-04-12 2024-02-27 广西壮族自治区蚕业技术推广站 家蚕嗅觉受体基因BmOR56的用途
CN114885906A (zh) * 2022-04-28 2022-08-12 广西壮族自治区蚕业技术推广站 抗BmNPV性状与家蚕黄茧性状快速融合与纯化的方法

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