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  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity

Definitions

• the present invention uses nucleic acids that interact with endocrine disruptor receptors, that is, exhibit responsiveness to endocrine disruptors via endocrine disruptor receptors, vectors and transformants having the nucleic acids, and the nucleic acids. Regarding the evaluation method of the test substance.
• Endocrine disruptors are substances that disturb the endocrine action of living organisms and may cause reproductive function inhibition and malignant tumors. Endocrine disruptors are also called hormone-like agents (Hormonally Active Agents), and when taken into the body, even a very small amount affects normal hormone action. Endocrine disruptors, for example, exert their effects on living organisms by binding to receptors to which hormones should bind. As the action of the endocrine disruptor on the living body, there are cases where the action similar to that of the hormone is brought about, and there are cases where the action opposite to the hormone is brought about.
• hormone-like agents Hormonally Active Agents
• ingredients such as pesticides, pesticides, paints, and rust inhibitors contain compounds known as endocrine disruptors that exhibit estrogen-like or androgen-like effects, and there is concern about their effects on the human body. ing.
• Juvenile hormone is a characteristic hormone that controls metamorphosis and reproduction in various arthropods.
• MET Methoprene-tolerant
• Kr-h1 Kruppel homolog 1
• JHIII is known for many insects such as Hymenoptera, Hymenoptera, and Diptera
• JHI and JHII are known for Lepidoptera
• methyl farneseneate is known for Crustaceans. ing.
• Patent Document 1 a response sequence (juvenile hormone responsive sequence (JH response sequence)) that activates transcription of a downstream gene in response to juvenile hormone (JH) was found in silkworm. It is disclosed that a reporter assay for evaluating JH responsiveness was constructed by using a JH response sequence in combination with a reporter gene.
• the JH response sequence has a CACGTG base called E-box.
• CACGCG which exists upstream of Kr-h1 and is called C-box, also functions as a JH response sequence (Non-Patent Document 1).
• Daphnia pulexa usually produces only female individuals by parthenogenesis, but it is known that when the environment deteriorates, it produces male individuals and fertilizes them, and has the property of producing durable eggs, which are resistant to environmental changes. ing.
• shellfish such as mijinko, methyl farnesenoate, which is a precursor of JHIII, is used as a juvenile hormone. It has been reported that when the amount increases, male individuals are produced (Non-Patent Documents 2 and 3), and substances that act as ligands for juvenile hormone receptors or inhibit the function of juvenile hormone receptors are also newly added. It can be thought of as an endocrine disruptor.
• Non-Patent Document 4 As a method for evaluating the action of juvenile hormone, there is a reproductive toxicity test using an individual Daphnia magna, but there are problems that the test period takes a long time and that breeding and evaluation requires technology (Non-Patent Document 4). In addition, a system for evaluating the action of juvenile hormone using cultured cells has been established, but there is a problem that technology is required for breeding and passage. In addition, Non-Patent Document 5 and Non-Patent Document 6 disclose juvenile hormone receptor genes (MET genes) and base sequences that are candidates for JH response sequences in Omidinko, but construction of a practical evaluation system Has not reached.
• MET genes juvenile hormone receptor genes
• the present invention utilizes a nucleic acid that can be used in an evaluation system for evaluating various endocrine disrupting effects with high sensitivity even at a low dose, a vector having the nucleic acid, a transformant, and the nucleic acid. It is an object of the present invention to provide a method for evaluating a test substance.
• the present invention includes the following.
• nucleic acid according to (1) wherein any four bases (nnnn) contained in the base sequence shown in SEQ ID NO: 1 are GCGG or TATT.
• nucleic acid according to any one of (1) to (3) which has a base sequence having a predetermined base length on the 3'end side and the 5'end side of the base sequence shown in SEQ ID NO: 1.
• nucleic acid according to (1) which comprises one base sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 34, 37, 40, 43, 46, 49 and 52.
• test substance is brought into contact with the transformant together with at least one substance selected from the group consisting of endocrine disruptors, hormones and agonists of endocrine disruptors that interact with the endocrine disruptor receptors.
• substance selected from the group consisting of endocrine disruptors, hormones and agonists of endocrine disruptors that interact with the endocrine disruptor receptors When the expression level of the reporter gene is lower than that when the substance is contacted alone, the test substance is judged to be an antagonist of the endocrine disruptor receptor ( 19) The evaluation method described.
• a kit for measuring endocrine disruptors which comprises the vector described in (8) above or the transformant described in (11) above.
• nucleic acid that is responsive to a low dose of an endocrine disruptor, a vector having the nucleic acid, and a transformant.
• the endocrine disrupting effect of the test substance can be evaluated by analyzing the expression of the reporter gene using the nucleic acid according to the present invention.
• the nucleic acid according to the present invention (hereinafter, may be referred to as a response sequence) is a nucleic acid containing the base sequence shown in SEQ ID NO: 1 and having a total length of 20 to 60 bases.
• the term response sequence does not mean the sequence itself as information, but four types of nucleotides, adenine (A), guanine (G), thymine (T) and cytosine (C), are bound in a predetermined order.
• This response sequence has the function of interacting with the juvenile hormone receptor (MET protein) of Daphnia magna, which is bound to juvenile hormone, and positively regulates the expression of downstream genes at the transcriptional level.
• MET protein juvenile hormone receptor
• the base sequence shown in SEQ ID NO: 1 has an arbitrary 4 bases (that is, NNNN) between CACGCG (C-box) and CACGTG (E-box).
• the arbitrary four bases are not particularly limited, but are preferably kmkk (where k means G or T and m means A or C).
• the sequence represented by kmkk includes 16 types including, for example, GAGG, GCGG, TATT, TCTT, TAGG, TCGG, GATT and GCTT. That is, the 4 bases between CACGCG (C-box) and CACGTG (E-box) are preferably one sequence selected from these 16 types of sequences.
• CACGCG CACGCG
• CACGTG E-box
• a base sequence in which any four bases of the base sequence shown in SEQ ID NO: 1 are used as GCGG is included in a promoter sequence located upstream of the Vrille gene in, for example, Daphnia magna.
• the base of the region other than SEQ ID NO: 1 in the response sequence can be arbitrarily selected.
• the total length of the response sequence is 20 to 60 bases in length, preferably 30 to 60 bases in length, more preferably 40 to 60 bases in length, and even more preferably 40 to 50 bases in length.
• the base sequence (16 bases) shown in SEQ ID NO: 1 in the response sequence is preferably positioned at a position excluding both ends of the total length of 20 to 60 bases. That is, the response sequence preferably has a base sequence having a predetermined base length on the 3'end side and the 5'end side of the base sequence shown in SEQ ID NO: 1.
• the predetermined base length can be, for example, 1 to 43 base length, preferably 5 to 30 base length, more preferably 5 to 20 base length, and 7 to 20 base length. Is more preferable.
• the base sequence of the region excluding SEQ ID NO: 1 is not particularly limited and can be any base sequence. As an example, it is preferable to select from the base sequence adjacent to the base sequence (that is, CACGCGGCGGCACGTG) existing in the upstream region of the Vrille gene in Daphnia magna.
• the response sequence containing the base sequence shown in SEQ ID NO: 1 can be a nucleic acid consisting of a base sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 34 and 37. Among them, the response sequence preferably consists of the base sequence shown in SEQ ID NO: 2.
• the response sequence according to the present invention is 75% or more, 80% or more, 85% or more, 90% or more with respect to the base sequence including the region adjacent to the base sequence existing in the upstream region of the Vrille gene in Daphnia magna (that is, CACGCGGCGGCACGTG).
• a base sequence having 95% or more identity can also be used.
• the base sequences shown in SEQ ID NO: 40 and SEQ ID NO: 43 which are different from SEQ ID NO: 2, SEQ ID NO: 43, which is different from SEQ ID NO: 2, SEQ ID NO: 49, which is different from 4 bases, SEQ ID NO: 46, which is different from 6 bases, and SEQ ID NO: 52, which is different from 10 bases, are preferable.
• the response sequence according to the present invention positively controls the expression of the gene located downstream of the response sequence (in the 3'end direction of the sense strand) at the transcription level, but a configuration in which a plurality of response sequences are arranged may be used. Specifically, by arranging the above-mentioned base sequence as one unit so that a plurality of units are directly adjacent to each other, or by arranging a plurality of units via a spacer, a plurality of units are arranged upstream of a gene whose expression is controlled at the transcription level. Response sequences can be placed.
• the number of response sequences arranged upstream of a predetermined gene is not particularly limited, but can be, for example, 2 to 8, preferably 2 to 5, and more preferably 3 to 4. ..
• the response sequences consisting of the same base sequence may be arranged, the response sequences consisting of different base sequences may be arranged, or some of them may be arranged from the same base sequence.
• a response sequence consisting of base sequences having different base sequences may be arranged.
• the spacer arranged between the response sequences is not particularly limited, but may be one or a plurality of arbitrary base sequences.
• the base length of the spacer is not particularly limited, and may be 1 to 100 base length, preferably 1 to 90 base length, preferably 1 to 80 base length, and 1 to 70 base length.
• the base length is preferably 1 to 60 bases, 1 to 50 bases, 1 to 40 bases, and 1 to 30 bases. It is preferably 1 to 20 bases in length, and preferably 1 to 10 bases in length.
• the vector according to the present invention contains the above-mentioned response sequence.
• the response sequence allows the vector to incorporate a gene of interest that positively regulates expression at the transcriptional level.
• the vector has a cloning site downstream of the response sequence for incorporating the gene of interest that positively regulates expression at the transcriptional level.
• the vector according to the present invention is not particularly limited as long as it can control the transcription of the downstream gene by the response sequence in an appropriate host cell. Examples of such a vector include a plasmid vector, a phage vector, a cosmid vector, a donor vector used for genome editing, and a shuttle vector capable of exchanging genes with other host strains. Can be used.
• plasmid examples include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, and the like, and examples of the phage include ⁇ phage.
• animal viruses such as retrovirus, vaccinia virus or adenovirus, and insect virus vectors such as baculovirus can also be used.
• the purified response sequence is cleaved with an appropriate restriction enzyme, inserted into the restriction enzyme site or multicloning site of the appropriate vector, and ligated to the vector.
• an appropriate restriction enzyme As a method that does not use restriction enzymes, homologous recombination methods such as CRISPR / Cas9, Red / ET, or Gateway method are also adopted.
• the above-mentioned response sequence is arranged in an expression control region that controls transcription of a downstream gene.
• the expression control region is a region that controls transcription of the downstream gene, and usually means a region upstream of the gene (the region on the 5'side of the sense strand). More specifically, the expression control region can usually be in the range of several thousand bases upstream from the transcription initiation site of the downstream gene, for example, within 5000 bases (b) upstream from the transcription initiation site, preferably 4000b. Within, within 3000b, within 2000b, within 1000b, within 500b, within 300b.
• the expression control region may include a region to which a transcription factor binds, an enhancer region, and the like, in addition to the above-mentioned response sequence.
• the expression control region containing the response sequence described above is a region having a predetermined length including the response sequence, for example, a region of 50b, a region of 60b, a region of 70b, a region of 80b, and a region of 90b containing the response sequence. It can be a region, 100b, 120b, 150b, or 200b region.
• the vector according to the present invention can contain a gene to be controlled for expression downstream of the expression control region having a response sequence, which is configured as described above.
• Examples of the gene whose expression is controlled include a reporter gene.
• the reporter gene is not particularly limited, and is, for example, chloramphenicol acetyl transferase (CAT) gene, lacZ gene, luciferase gene, ⁇ -glucuronidase (GUS) gene, green fluorescent protein (GFP) gene, drug resistance gene, nutrition. Requirement genes and the like can be mentioned.
• the reporter gene is not limited to the conventionally known gene described above, and the Vrille gene described above may be used as the reporter gene.
• the transformant according to the present invention is obtained by introducing the above-mentioned response sequence into a host.
• the interaction between the juvenile hormone receptor of Omidinko bound to the juvenile hormone and the above response sequence enhances the expression of the gene located downstream of the response sequence at the transcription level.
• the response sequence described above can be introduced into the host using, for example, the vector described above.
• the transformant according to the present invention includes the above-mentioned response sequence, a gene whose expression is enhanced at the transcription level by the response sequence (for example, a reporter gene), and a juvenile hormone receptor that interacts with the response sequence. It is preferable that a nucleic acid encoding an endocrine disruptor receptor is introduced.
• the endocrine disruptor receptor means a receptor on which a predetermined hormone acts as a ligand, and an endocrine disruptor having a hormone-like action also acts on the receptor.
• the endocrine disruptor receptor means to include both a cell membrane receptor and a nuclear receptor.
• the endocrine disruptor receptor is preferably a receptor that interacts with the above-mentioned response sequence in the presence of a specific hormone or endocrine disruptor and exhibits transcription factor activity.
• the endocrine disrupting substance is not particularly limited, and means that it includes both a substance known to have an endocrine disrupting action and a substance suspected of having an endocrine disrupting substance.
• substances having an agonistic action or antagonistic action on female hormones follicle hormone: estrogen and luteinizing hormone: progesterone
• substances having an agonistic action on male hormone androgen
• a substance having an antagonistic action can be mentioned.
• the endocrine disruptor is not limited to substances having a disturbing effect on these female hormones or male hormones, such as hormones such as thyroid hormone, growth hormone, corticosteroid or insulin, acetylcotin, noradrenaline, adrenaline or dopamine. It also includes substances that have a disturbing effect on the neurotransmitters of.
• the endocrine disruptor receptor is an estrogen receptor, an androgen receptor, a progesterone receptor, a thyroid hormone receptor, a growth hormone receptor, an corticosteroid receptor or an insulin receptor, which are receptors for various hormones described above. I can raise my body.
• endocrine disruptors include substances that have an agonistic or antagonistic effect on juvenile hormones specifically possessed by arthropods. Therefore, it is particularly preferable to use the juvenile hormone receptor (MET) as the endocrine disruptor receptor.
• MET juvenile hormone receptor
• the juvenile hormone receptor is not particularly limited, but can be a juvenile hormone receptor in arthropods including insects, crustaceans, spiders and mucades. Among them, as the juvenile hormone receptor in arthropods, it is preferable to use the juvenile hormone receptor of crustaceans or insects.
• Insects include Coleoptera (Coleoptera) including beetles and worms, Coleoptera (Orthoptera) including butterflies and moths, Flies (Diptera) including flies, mosquitoes and abs, bees and ants. Examples include Flies (Flies), Hemiptera (Hemiptera) including semi- and Coleoptera, Orthoptera (Orthoptera) including Coleoptera and Coleoptera, and Dragonfly (Coleoptera) including Coleoptera. Nucleic acids encoding juvenile hormone receptors derived from these insects can be used in the transformants according to the present invention.
• nucleic acid encoding the juvenile hormone receptor of Drosophila melanogaster belonging to the order Flies can be used (He Q et al.; J Biol Chem. 289 (40), p. See 27874-27885 (2014)).
• crustaceans examples include animals contained in the subphylum Crustaceans, including shrimp, crabs, krill, barnacles and daphnia pulexa. Nucleic acids encoding juvenile hormone receptors derived from these crustaceans can be used in the transformants according to the present invention.
• nucleic acids encoding juvenile hormone receptors derived from crustaceans it is particularly preferable to use nucleic acids encoding juvenile hormone receptors derived from animals belonging to the Daphniidae family.
• Animals belonging to the Daphnia family include Daphnia (Ceriodaphnia cornuta), Daphnia (Ceriodaphnia reticulata), Daphnia (Ceriodaphnia dubia), Daphnia (Ceriodaphnia megalops), and Daphnia (Ceriodaphnia megalops).
• Daphnia Animals belonging to the genus Daphnia (Ceriodaphnia) such as Daphnia (Ceriodaphnia quadrangular); Daphnia similis, Daphnia magna, Daphnia pulex, Daphnia pulex, Daphnia pulicaria , Daphnia obtuse, Daphnia biwaensis, Daphnia longispina, Kawari water flea (Daphnia rosea), Usukawa water flea (Daphnia hyaline), Kabutomijinko (Daphnia hyaline) Animals belonging to the genus Daphnia (Daphnia) such as Daphnia ezoensis, Daphnia cuculata, Dap
• Animals belonging to the genus Daphnia such as (Scapholeberis kingi); as well as Daphnia (Simocephalus serrulatus), Daphnia (Simocephalus exspinosus), Daphnia (Simocephalus) Examples include animals belonging to the genus Daphnia (Simocephalus), such as Daphnia (Simocephalus japonica).
• nucleic acid encoding the juvenile hormone receptor of an animal belonging to the genus Daphnia it is particularly preferable to use a nucleic acid encoding the juvenile hormone receptor of an animal belonging to the genus Daphnia, and further to use a nucleic acid encoding the juvenile hormone receptor of Daphnia magna. Is more preferable.
• the amino acid sequence of the juvenile hormone receptor of Omidinko is shown in SEQ ID NO: 12, and the nucleotide sequence of the nucleic acid encoding the juvenile hormone receptor is shown in SEQ ID NO: 11.
• the transformant according to the present invention is not limited to those having a nucleic acid encoding a juvenile hormone receptor consisting of the amino acid sequence shown in SEQ ID NO: 12, and is 70% or more of the amino acid sequence shown in SEQ ID NO: 12. Consists of an amino acid sequence having an identity of 80% or more, more preferably 90% or more, still more preferably 95% or more, most preferably 98% or more. It may have a nucleic acid encoding a protein having juvenile hormone receptor transcription factor activity.
• the value of identity between amino acid sequences can be calculated by a BLASTN or BLASTX program that implements the Basic Local Alignment Search Tool (BLAST) algorithm (default setting).
• the value of identity is calculated as a ratio of amino acid residues that completely match when a pair of amino acid sequences are pairwise aligned and compared with each other.
• the transformant according to the present invention is not limited to one having a nucleic acid encoding a juvenile hormone receptor consisting of the amino acid sequence shown in SEQ ID NO: 12, and a complementary strand of the nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 11. All or part of it may have a nucleic acid encoded by a nucleic acid that hybridizes under stringent conditions and that encodes a protein having immature hormone receptor transcription factor activity.
• the "stringent condition” means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, it can be appropriately determined by referring to Molecular Cloning: A Laboratory Manual (Third Edition). can.
• the stringency can be set by the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution.
• the stringent conditions include, for example, a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 42 to 68 ° C, preferably 42 to 65 ° C. More specifically, it has 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate) and a temperature of 42 ° C.
• a protein encoded by a nucleic acid having a predetermined base sequence different from SEQ ID NO: 11 or a protein containing an amino acid different from SEQ ID NO: 12 has immature hormone receptor transcription factor activity is as follows. It can be evaluated as. That is, first, a transformant into which the nucleic acid to be evaluated or the nucleic acid encoding the protein to be evaluated and the above-mentioned response sequence and the reporter gene arranged downstream thereof is introduced is prepared. Then, the transformant is cultured in the presence or absence of juvenile hormone, and the expression of the reporter gene is measured.
• the nucleic acid to be evaluated exhibits the immature hormone receptor transcription factor activity. It encodes the protein to be evaluated, and the protein to be evaluated has immature hormone receptor transcription factor activity.
• the above-mentioned juvenile hormone receptor transcription factor activity can be rephrased as the above-mentioned activity that interacts with the response sequence according to the present invention.
• the above-mentioned juvenile hormone receptor transcription factor activity can be paraphrased as an activity that binds to juvenile hormone and interacts with the above-mentioned response sequence according to the present invention.
• the transformant according to the present invention may further have a nucleic acid encoding a transcriptional coupling factor in addition to the above-mentioned nucleic acid, response sequence and reporter gene.
• the transcriptional coupling factor is not particularly limited, but Taiman (Tai), Steroid receptor coactivator (SRC), ⁇ -FTZ-F1 (fushi tarazu binding factor 1), interacting steroid receptor coactivator (FISC), CREB binding protein (CBP).
• transcriptional mediators / intermediate factor 2 TNF2
• AIB amplified in breast cancer
• ARA70 70 kDa androgen receptor coactivator
• ASC2 activating signal co-integrator 2
• 140 kDa estrogen receptor-associated protein can be mentioned.
• nucleic acid encoding SRC or Tai it is particularly preferable to introduce a nucleic acid encoding SRC or Tai, and it is more preferable to introduce a nucleic acid encoding SRC of Daphnia magna.
• the amino acid sequence of Daphnia magna SRC is shown in SEQ ID NO: 14, and the nucleotide sequence of the nucleic acid encoding the SRC is shown in SEQ ID NO: 13.
• the transcriptional conjugation factor that can be used in the transformant according to the present invention is not limited to the protein consisting of the amino acid sequence shown in SEQ ID NO: 14, and is preferably 70% or more identical to the amino acid sequence shown in SEQ ID NO: 14. Consists of an amino acid sequence having 80% or more identity, more preferably 90% or more identity, even more preferably 95% or more identity, most preferably 98% or more identity, and having transcriptional conjugating factor activity. It may be a protein.
• the hosts in the transformant according to the present invention include Escherichia coli, Zebrafish, Saccharomyces cerevisiae, cultured cells derived from arthropods and mammals, undifferentiated plant cells (carus), nematodes (C. elegans), and zebrafish.
• Arthropods such as Drosophila, fish such as zebrafish and medaka, amphibians such as Xenopus laevis and newt, mammals such as mice and rats, and higher plants such as Saccharomyces cerevisiae and rice can be considered, but budding yeast is preferable. ..
• nucleic acid into the host is not particularly limited, and conventionally known methods can be applied.
• the method for introducing nucleic acid include a lithium acetate method, a calcium phosphate method, a method using liposomes, electroporation, a method using a viral vector, and a micropipette injection method.
• the above-mentioned nucleic acid, response sequence, reporter gene, etc. may be introduced into a transient type as long as it can be used for analysis, and by incorporating it into a host chromosome. It may be introduced, or an artificial chromosome or plasmid type capable of autonomous replication and distribution may be introduced.
• a gene located downstream of the response sequence is generated by the interaction of the endocrine disruptor receptor to which a predetermined hormone or endocrine disruptor is bound with the response sequence.
• Expression of eg, reporter gene
• the interaction between the endocrine disruptor receptor and the response sequence can be evaluated by measuring the expression of the gene located downstream of the response sequence. Based on this phenomenon, it is possible to evaluate how a predetermined substance (test substance) affects the interaction between the endocrine disruptor receptor and the response sequence.
• the evaluation of the binding ability of the test substance to the endocrine disruptor receptor and the evaluation of the effect of the test substance on the binding between the endocrine disruptor receptor and its agonist are located downstream of the response sequence. It can be done based on the expression of the gene.
• the transformant according to the present invention since the above-mentioned response sequence is used, the presence or absence of the endocrine disrupting effect can be evaluated for the test substance without being limited to a specific endocrine disrupting effect.
• test substance is not particularly limited and may be any substance.
• examples of the test substance include low molecular weight compounds, high molecular weight compounds, peptides, single compounds, compositions containing a plurality of compounds, cultures, extracts, natural products and synthetic compounds.
• the transformant is cultured in the presence or absence of the test substance, and the expression of the reporter gene located downstream of the response sequence in each case is measured. If the expression level of the reporter gene in the presence of the test substance is significantly higher than the expression level of the reporter gene in the absence of the test substance, the test substance interacts with the endocrine disruptor receptor. It can be determined that it acts and has an agonistic action that positively regulates gene expression via the above-mentioned response sequence. That is, in this case, it can be determined that the test substance is likely to be an endocrine disruptor having an agonistic action.
• the transformant is cultured in the presence or absence of a substance having an agonistic action on the test substance and the endocrine disruptor receptor, and the expression of the reporter gene located downstream of the response sequence in each case is measured. .. If the expression level of the reporter gene in the presence of the test substance is significantly lower than the expression level of the reporter gene in the absence of the test substance, the test substance is relative to the endocrine disruptor receptor. It can be determined that it has an antagonistic action that inhibits the agonist action and negatively controls gene expression via the above-mentioned response sequence. That is, in this case, it can be determined that the test substance is likely to be an endocrine disruptor having antagonistic activity.
• the substance having an agonistic action on the endocrine disruptor receptor means a substance selected from a hormone acting on the endocrine disruptor receptor, an endocrine disruptor corresponding to the hormone, and an agonist compound on the endocrine disruptor receptor. ..
• the transformant according to the present invention can be used for the measurement and evaluation of endocrine disruptors, and can be used as an endocrine disruptor measurement kit for evaluating the endocrine disrupting action of the test substance.
• the transformant according to the present invention eliminates the influence of the decomposition of the test substance by the drug metabolism system such as p450 possessed by the animal cell, and eliminates the influence of the test substance.
• the endocrine disrupting effect can be accurately evaluated.
• each reporter plasmid, each transcription factor (endocrine disruptor receptor) plasmid, and each transcription coupling factor plasmid shown below were prepared and introduced into a budding yeast strain to prepare a transformed yeast.
• An oligo DNA (SEQ ID NO: 3) containing a response sequence (43 bases) consisting of the nucleotide sequence shown in SEQ ID NO: 2 and an oligo DNA (SEQ ID NO: 4) partially complementary to SEQ ID NO: 3 were prepared, and these oligo DNAs were prepared. Annie ring.
• partially complementary means that the portion of the base sequence specified by the SEQ ID NO: excluding a few bases on the 5'end side is complementary.
• the response sequence consisting of the nucleotide sequence shown in SEQ ID NO: 5 54 bases
• the response sequence consisting of the nucleotide sequence shown in SEQ ID NO: 8 30 bases
• the response sequence consisting of the nucleotide sequence shown in SEQ ID NO: 34 22).
• a plasmid was also prepared.
• the method for producing the oligo DNA of SEQ ID NO: 3 is the oligo DNA of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50 or SEQ ID NO: 53.
• the oligo DNA partially complementary to SEQ ID NO: 6 SEQ ID NO: 7
• the oligo DNA partially complementary to SEQ ID NO: 9 SEQ ID NO: 10
• one to SEQ ID NO: 35 is the oligo DNA partially complementary to SEQ ID NO: 35.
• Partially complementary oligo DNA (SEQ ID NO: 36), partially complementary oligo DNA to SEQ ID NO: 38 (SEQ ID NO: 39), partially complementary oligo DNA to SEQ ID NO: 41 (SEQ ID NO: 42), to SEQ ID NO: 44 Partially complementary oligo DNA (SEQ ID NO: 45), partially complementary oligo DNA to SEQ ID NO: 47 (SEQ ID NO: 48), partially complementary oligo DNA to SEQ ID NO: 50 (SEQ ID NO: 51) or SEQ ID NO: 53
• SEQ ID NO: 54 was partially complementary to the above, and the plasmid in which three response sequences shown in SEQ ID NO: 5 were continuously inserted and one response sequence shown in SEQ ID NO: 8 were inserted.
• the plasmid, the response sequence shown in SEQ ID NO: 49 was inserted 1, 2, 3 or 4 consecutively, and the response sequence shown in SEQ ID NO: 52 was inserted 1, 2, or 3 consecutively.
• a plasmid was prepared.
• the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 34 and SEQ ID NO: 37 are the nucleotide sequences existing in the region upstream of the Vrille gene of Omijinko, and the nucleotides shown in SEQ ID NO: 40.
• the sequence is a base sequence in which 3 bases outside the 5'end of C-box (CACGCG) are changed from the base sequence of SEQ ID NO: 2, and the base sequence shown in SEQ ID NO: 43 is the base sequence of SEQ ID NO: 2.
• it is a base sequence in which 3 bases outside the 3'end of the E-box (CACGTG) part are changed, and the base sequence shown in SEQ ID NO: 46 is that of C-box (CACGCG) with respect to the base sequence of SEQ ID NO: 2.
• CACGCG 5'end of C-box
• CACGTG 3 bases outside the 3'end of the E-box
• linker part 4 between the C-box part and the E-box part (linker part) It is a base sequence in which all the bases are changed.
• a plasmid in which only the nucleic acid consisting of only 16 bases existing in the upstream region of the Vrille gene in Daphnia magna (that is, CACGCGGCGGCACGTG) was inserted was prepared.
• These 16 bases include E-box (CACGTG) and C-box (CACGCG), which are considered to be juvenile hormone response sequences in many insect species.
• the method for producing this plasmid was the same as the above method, and a plasmid in which 1, 2, 3, or 4 nucleic acids consisting of 16 bases were continuously inserted was prepared.
• a plasmid was prepared in which a region (DmJHRR) containing the juvenile hormone response sequence of the Kr-h1 gene of Drosophila melanogaster was inserted.
• DmJHRR is described in He Q et al.; J Biol Chem. 289 (40), p. 27874-27885 (2014).
• the production method is as follows: the oligo DNA of SEQ ID NO: 3 is used as an oligo DNA containing the DmJHRR sequence (SEQ ID NO: 15), and the oligo DNA of SEQ ID NO: 4 is partially complementary to SEQ ID NO: 15 (SEQ ID NO: 16). It was prepared as a plasmid in which three DmJHRR sequences were continuously inserted according to the above method except that it was changed to.
• the base sequence in which the C-box portion of SEQ ID NO: 2 is different SEQ ID NO: 25
• a base sequence having a different E-box part SEQ ID NO: 28
• a base sequence having a different C-box part and an E-box part SEQ ID NO: 31
• an oligo DNA containing the nucleotide sequence shown in SEQ ID NO: 25 (SEQ ID NO: 26), a DNA partially complementary to SEQ ID NO: 26 (SEQ ID NO: 27), and an oligo DNA containing the nucleotide sequence shown in SEQ ID NO: 28 (SEQ ID NO: 28).
• SEQ ID NO: 29) and DNA partially complementary to SEQ ID NO: 29 SEQ ID NO: 30
• oligo DNA containing the nucleotide sequence shown in SEQ ID NO: 31 SEQ ID NO: 32
• DNA partially complementary to SEQ ID NO: 32 (sequence).
• a plasmid in which four nucleotide sequences were continuously inserted was prepared.
• PCR fragment amplified by PCR was cleaved with the restriction enzyme AatII to form a linear plasmid, pUdp6, and the yeast wild strain (Saccharomyces cerevisiae) W303a (MATa, ade2, his3, leu2, trp1, ura3) was subjected to the lithium acetate method.
• pUdp6 is a plasmid that has a CYC terminator downstream of the bidirectional promoter regions gal1, gal10 and gal10, an ADH terminator downstream of gal1, and the uracil selection marker URA3 gene.
• a plasmid in which a CEN6 / ARS4 fragment was inserted into pUdp6 by homologous recombination was extracted from yeast, and further introduced into an Escherichia coli DH5 ⁇ strain to obtain a low copy episomal vector as pUdp13.
• the recognition sequence of the restriction enzyme I-SceI of the recognition sequence 18bp is introduced outside the CEN6 / ARS4 sequence, and after cloning the target gene to the multicloning site, it is cleaved with I-SceI to form a CEN6 / ARS4 fragment. By cutting out, it is possible to easily convert to the genome insertion type.
• METFW2 SEQ ID NO: 19
• METREV-3 SEQ ID NO: 20
• the amplified cDNA of the DampaMET gene was introduced into the wild yeast strain W303a by the lithium acetate method together with the plasmid pUdp13 cleaved with the restriction enzymes BamHI and HindIII.
• the cDNA of the DampaMET gene was inserted downstream of the GAL10 promoter of the plasmid pUdp13 to obtain pUdp13-DapmaMet.
• the pUdp13-DapmaMet constructed in yeast cells was recovered, introduced into the Escherichia coli DH5 ⁇ strain, and amplified.
• pUdp13-DapmaMet was cleaved with the restriction enzyme I-SceI to excise the CEN6 / ARS4 sequence, thereby converting to the genome insertion type.
• I-SceI restriction enzyme
• DmMET plasmid a Drosophila melanogaster juvenile hormone receptor expression plasmid (hereinafter referred to as DmMET plasmid) was prepared.
• DmMET plasmid a Drosophila melanogaster juvenile hormone receptor expression plasmid
• the ORF of the cDNA of the DmMET gene was amplified by PCR using Drosophila melanogaster cDNA (manufactured by Clontech) as a template and DmMET Fwd (SEQ ID NO: 21) and DmMET Rev (SEQ ID NO: 22) for DmMET.
• DmMET Fwd SEQ ID NO: 21
• DmMET Rev SEQ ID NO: 22
• the amplified DmMET gene cDNA was cleaved with restriction enzymes SmaI and EcoRI.
• the cDNA of the cleaved DmMET gene was inserted downstream of the GAL10 promoter of the plasmid pUdp6 cleaved with restriction enzymes SmaI and EcoRI.
• SmaI and EcoRI restriction enzymes SmaI and EcoRI.
• a transcription factor expression plasmid (hereinafter referred to as DmTai plasmid) containing the Drosophila melanogaster Tai (DmTai) gene and a Omizujinko transcription conjugate expression (SRC) plasmid (hereinafter referred to as DampaSRC plasmid) were prepared.
• DmTai plasmid was prepared according to Ito-Harashima S et al. FEBS Open Bio. 7 (7): p. 995-1008 (2017).
• the DampaSRC plasmid was prepared by the following method. First, the ORF of the cDNA of the Daphnia magna SRC (DampaSRC) gene is used as a template, and the SRC primers SRC-F1F-pESC (SEQ ID NO: 23) and SRC-F4short-pESC (SEQ ID NO: 24) are used using the cDNA of the adult Daphnia magna as a template. It was amplified by PCR. PCR was carried out for 35 cycles of 94 ° C. for 20 seconds, 58 ° C. for 10 seconds, and 72 ° C. for 7 minutes.
• the plasmid pESC-Leu (manufactured by Agilent technology) was cleaved with restriction enzymes SpeI and PacI, and the amplified cDNA of the DampaSRC gene was introduced into the wild yeast strain W303a by the lithium acetate method.
• a DampaSRC plasmid in which the cDNA of the DampaSRC gene was inserted downstream of the promoter region was obtained.
• the Dampa SRC plasmid constructed in yeast cells was recovered, introduced into the Escherichia coli DH5 ⁇ strain, and amplified.
• TE buffer 10 mM Tris, 1 mM EDTA, pH 8.0
• carrier DNA trade name SALMON TESTES DNA for hybridization, manufactured by SIGMA
• the mixed solution was incubated at 30 ° C. for 30 minutes, heat-treated at 42 ° C. for 22 minutes, and then centrifuged at 9,000 rpm for 1 minute to collect yeast cells from the mixed solution.
• the collected yeast cells were suspended in 300 ⁇ L of sterile water, and 100 ⁇ L of the suspension was applied to the medium shown in Tables 1 and 2 with 42 mg of tryptophan, 62 mg of leucine, and 2% agar, and cultured. bottom.
• a yeast strain showing uracil non-requirability was selected as a transformed yeast in which the cDNA region of the gal1 promoter and the MET gene was integrated on the chromosome.
• the DampaSRC plasmid was introduced into the transformed yeast into which the DampaMET plasmid was introduced.
• the selective medium for the transformed yeast a culture medium in which 100 mg of tryptophan was added to the pre-culture medium shown in Tables 1 and 2 was used.
• a reporter plasmid in which one base sequence of SEQ ID NO: 2 was inserted into yeast into which the DampaMET plasmid and DampaSRC plasmid were introduced was introduced by the lithium acetate method.
• the selective medium the preculture media shown in Tables 1 and 2 were used.
• the yeast 1 of the present invention was obtained.
• yeasts 2 to 38 of the present invention and comparative yeasts 1 to 9 having the response sequences according to the present invention shown in Table 3 were prepared.
• Test Example 1 Measurement of Reporter Activity of Transformed Yeast to Juvenile Hormone Using the yeast 1 of the present invention, the reporter activity to methyl farneseneate, which is a juvenile hormone substance, was measured. First, the yeast 1 of the present invention was pre-cultured using the pre-culture medium shown in Table 1 at 30 ° C. for 18 hours so that the turbidity (OD595) of the pre-culture medium was about 1.0.
• reaction solution to which 1 ⁇ L of DMSO was added instead of the diluted solution of methyl farneseneate was designated as an untreated group. 10 ⁇ L of each prepared reaction was collected and dispensed into each well of a new 96-well plate.
• lysate Z buffer: 60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 1 mM MgCl 2 , 10 mM KCl, 2 mM dithiothreitol, 0.20% N-Lauroylsarcosine sodium salt
• 1 mg / mL ONPG 100 ⁇ L of the measurement reagent mixed with orthonitrophenyl galactopyranoside
• the induced increase is described in Ito-Harashima S et al., FEBS Open Bio. 7 (7): p. 995-1008 (2017), and if the induced increase is a positive number, it is relative to the substance. It is analyzed that it has reporter activity. The test was carried out in a 1-series, 3-series, 5-series or 15-series system, and the average induction increase amount was calculated. Comparative yeast 1 was also treated in the same manner, and the reporter activity was compared. The results are shown in Table 5.
• the yeasts of the present invention 2 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 are It can be seen that the expression of the reporter gene is clearly strongly induced in the presence of the juvenile hormone (methyl farneseneate).
• Comparative Yeasts 2, 3 and 4 showed a negative value in the induced increase amount at any of the treated amounts and did not show reporter activity. From this result, by using the base sequence containing the base sequence shown in SEQ ID NO: 1 and having a total length of 20 to 60 bases as the response sequence, it is highly sensitive to methyl farnesenoate, which is one of the juvenile hormones. It became clear that the reporter activity could be evaluated.
• Test Example 3 Effect of core sequence on reporter activity of transformed yeast Comparative yeast 7, E-box (CACGTG), in which the C-box (CACGCG) part in the response sequence was different from the yeast 4 of the present invention.
• the effect of the core sequence on the reporter activity on methyl farnesenoate was evaluated using comparative yeast 8 having different base sequences in the) part and comparative yeast 9 having both the C-box part and the E-box part having different base sequences. bottom.
• the test method was based on Test Example 1. The results are shown in Table 8.
• Test Example 4 Measurement of reporter activity of transformed yeast against sex hormones and chemical substances suspected of having endocrine disrupting effects Using the yeasts 4 and 8 of the present invention, the sex hormones 17 ⁇ -estradiol, testosterone, and insects Immature hormone III, a class of immature hormones, 4-nonylphenol, bisphenol A, a chemical substance suspected of having endocrine disrupting effects, 1,1,1-trichloro-2,2-bis (4- Reporter activity against chlorophenyl) ethane (DDT), 2,2-bis (4-chlorophenyl) -1,1-dichloroethylene (DDE), and dildoline was measured. The test method was based on Test Example 1.
• the expression of the reporter gene is induced in all systems regardless of the type of endocrine disruptor, and the present application is applied to all the endocrine disruptors tested. It was confirmed that the response sequence was responsive.
• the yeast 8 of the present invention is a transformant having a transcription factor (DmMET) of Drosophila melanogaster, but the response sequence of the present application may be a transcription factor of Drosophila melanogaster, which is a kind of insects, as well as shellfish. It showed responsiveness.
• comparative yeasts 5 and 6 having the juvenile hormone response sequence (DmJHRR) of Drosophila melanogaster only induced the expression of the reporter gene in the system using juvenile hormones III and 4-nonylphenol.
• DmJHRR did not respond to endocrine disruptors. From this result, endocrine disruptors showing various actions can be detected by the reporter assay using the response sequence of the present application.
• Test Example 5 Measurement of reporter activity against juvenile hormone receptor antagonist substance Using the yeast 4 of the present invention, the juvenile hormone receptor antagonist activity at 10 ⁇ M of endrin, aldrin, and dieldrin was evaluated. The test method was carried out in the presence of 10 nM methyl farneseneate, and the others were in accordance with Test Example 1. The results are shown in Table 10.
• the yeasts 32, 33, 34, 35 of the present invention having different base sequences between the C-box part and the E-box part (linker part), and 3 outside the 5'end of C-box (CACGCG).
• the bases and the yeasts of the present invention 36, 37, 38 having different base sequences in the three bases outside the 3'end of the E-box (CACGTG) part and between the C-box part and the E-box part (linker part).
• the effect of the core sequence on the reporter activity on methyl farnesenoate was evaluated using.
• the treatment concentrations of methyl farneseneate were 100 nM and 10 ⁇ M.
• the test method was based on Test Example 1. The results are shown in Table 12.
• the yeasts 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 and 38 of the present invention are juvenile hormones. It can be seen that the expression of the reporter gene is clearly strongly induced in the presence of (methyl farneseneate). From this result, the 4 bases between C-box (CACGCG) and E-box (CACGCG) (linker part) are kmkk (where k means G or T and m means A or C), for example, other than GCGG. It was also clarified that TATT can evaluate the reporter activity against methyl farneseneate, which is one of the juvenile hormones, with high sensitivity if the base sequence has a total length of 20 to 60 bases. Furthermore, even when the base sequence around the base sequence shown in SEQ ID NO: 1 is different from the base sequence shown in SEQ ID NO: 2, it is possible to evaluate the reporter activity for juvenile hormone-like substances with the same high sensitivity. be.

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