Nucleic acids that interact with endocrine disruptor receptors and their applications

Abstract

The present invention evaluate various endocrine disrupting activities with high sensitivity even at low doses. Provided is a nucleic acid which comprises the nucleotide sequence represented by SEQ ID NO: 1, has a full length of 20 to 60 base pairs, and can exhibit excellent responsiveness to various endocrine disruptors.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid that interacts with an endocrine disruptor receptor, that is, exhibits responsiveness to an endocrine disruptor via the endocrine disruptor receptor, a vector and a transformant comprising the nucleic acid, and a method for evaluating a test substance using the nucleic acid. [Background Art]

[0002] Endocrine disruptors are substances that disrupt the endocrine function of organisms and may cause reproductive function inhibition, malignant tumors and the like. Endocrine disruptors are also called Hormonally Active Agents, and even when incorporated into a living body in an extremely small amount, they affect normal hormonal action. For example, endocrine disruptors exert their effects on living bodies by binding to receptors to which hormones should originally bind. Effects of endocrine disruptors on living bodies include cases where an effect similar to that of a hormone is produced, and cases where an effect opposite to that of the hormone is produced.

[0003] For example, components such as agricultural chemicals, insecticides, paints, and rust preventives include compounds known as endocrine disruptors that exhibit estrogen-like action or androgen-like action, and there are concerns about their effects on the human body.

[0004] On the other hand, chemicals that control the function of juvenile hormone (JH), which is unique to arthropods, are being developed as insecticides. Juvenile hormone is a characteristic hormone that controls metamorphosis and reproduction in various arthropods. In insects, when juvenile hormone is received by the receptor methoprene-tolerant (MET) (Gce (Germ-cell-expressed) is also present in Drosophila), it leads to the expression of final physiological functions via transcriptional activation of downstream Kruppel homolog 1 (Kr-h1). Therefore, if juvenile hormone does not function properly, arthropods that possess juvenile hormone cannot survive. Mammals do not possess juvenile hormone, so it has been thought that using chemicals that control the function of juvenile hormone as insecticides is highly safe for humans. There are several types of juvenile hormone; JHIII is known in many insects such as Coleoptera, Hymenoptera, and Diptera, JHI and JHII in Lepidoptera, and methyl farnesenoate in crustaceans.

[0005] For example, Patent Document 1 discloses the discovery of a response sequence (juvenile hormone-responsive sequence (JH response sequence)) in silkworms that activates the transcription of downstream genes in response to juvenile hormone (JH), and the construction of a reporter assay to evaluate JH responsiveness by using the JH response sequence in combination with a reporter gene. The above JH response sequence has a CACGTG base called the E-box. In addition, it has been revealed that CACGCG, which is located upstream of Kr-h1 and is called the C-box, also functions as a JH response sequence (Non-Patent Document 1).

[0006] Incidentally, while Daphnia generally reproduce asexually, producing only female individuals, it is known that when the environment deteriorates, they produce male individuals, fertilize them, and produce resting eggs, which are resistant to environmental changes. In crustaceans such as Daphnia, methyl farnesenoate, a precursor of JHIII, is used as a juvenile hormone. In Daphnia, it has been reported that exposure to juvenile hormone or juvenile hormone-like substances, or an increase in the amount of juvenile hormone in the Daphnia body, leads to the production of male individuals (Non-Patent Literature 2 and Non-Patent Literature 3). Substances that act as ligands for the juvenile hormone receptor or inhibit the function of the juvenile hormone receptor can also be considered as new endocrine disruptors.

[0007] One method for evaluating juvenile hormone activity is reproductive toxicity testing using Daphnia magna individuals, but this method has problems such as requiring a long testing period and demanding technical skills for rearing and evaluation (Non-Patent Literature 4). A system for evaluating juvenile hormone activity using cultured cells has also been established, but this also has problems such as requiring technical skills for rearing and subculturing. Furthermore, candidate nucleotide sequences for the juvenile hormone receptor gene (MET gene) and JH response element in Daphnia magna have been disclosed in Non-Patent Literature 5 and Non-Patent Literature 6, but the construction of a practical evaluation system has not yet been achieved. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Patent No. 5754681 [Non-patent literature]

[0009] [Non-Patent Document 1] Qianyu He, et al., Heat Shock Protein 83 (Hsp83) Facilitates Methoprene-Tolerant (Met) Nuclear Import to Modulate Juvenile Hormone Signaling Journal of Biological Chemistry. 289 , ( 2014 ) p. 27874-2

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[0010] As mentioned above, endocrine disruptors exist for each of the various hormones, including juvenile hormones. Therefore, determining the endocrine-disrupting effects of a particular compound required preparing separate evaluation systems for each hormone. Thus, evaluating the endocrine-disrupting effects of a particular compound required a great deal of effort and resulted in high evaluation costs. Furthermore, as mentioned above, even minute amounts of endocrine disruptors can affect living organisms, so there is a need for an evaluation system that can detect endocrine-disrupting effects with high sensitivity even at low doses.

[0011] Therefore, in view of the above-mentioned circumstances, the present invention aims to provide a nucleic acid that can be used in an evaluation system to evaluate various endocrine-disrupting effects with high sensitivity even at low doses, a vector having said nucleic acid, a transformant, and a method for evaluating a test substance using said nucleic acid. [Means for solving the problem]

[0012] In order to achieve the above-mentioned objectives, the inventors diligently conducted research and succeeded in identifying nucleic acids that exhibit excellent responsiveness to various endocrine disruptors, thus completing the present invention.

[0013] This invention encompasses the following:

[0014] (1) A nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 1, having a full length of 20 to 60 bases.

[0015] (2) The nucleic acid according to (1), wherein any 4 bases (nnnn) contained in the nucleotide sequence set forth in SEQ ID NO: 1 are kmkk (provided that k represents g or t, and m represents a or c).

[0016] (3) The nucleic acid according to (1), wherein any 4 bases (nnnn) contained in the nucleotide sequence set forth in SEQ ID NO: 1 are GCGG or TATT.

[0017] (4) The nucleic acid according to any one of (1) to (3), which has nucleotide sequences of a predetermined base length on the 3'-terminal side and 5'-terminal side of the nucleotide sequence set forth in SEQ ID NO: 1.

[0018] (5) The nucleic acid according to (1), consisting of one nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 34, 37, 40, 43, 46, 49 and 52.

[0019] (6) A vector comprising the nucleic acid according to any one of (1) to (5) above.

[0020] (7) The vector according to (6), wherein the nucleic acid is taken as one unit, and a plurality of units of the nucleic acid are linked together.

[0021] (8) The vector according to (6), which comprises a reporter gene on the 3'-terminal side of the sense strand of the nucleic acid.

[0022] (9) A transformant obtained by introducing the nucleic acid according to any one of (1) to (5) into a host.

[0023] (10) The transformant according to (9), wherein the nucleic acid is taken as one unit, and a plurality of units of the nucleic acid are linked together.

[0024] (11) The transformant according to (9), which comprises a reporter gene on the 3'-terminal side of the sense strand of the nucleic acid.

[0025] (12) The transformant according to (9), characterized in that a nucleic acid encoding an endocrine disruptor receptor that interacts with the above nucleic acid is introduced.

[0026] (13) The transformant according to (12), characterized in that the endocrine disruptor receptor is a juvenile hormone receptor in arthropods.

[0027] (14) The transformant according to (13), characterized in that the juvenile hormone receptor in the arthropod is a juvenile hormone receptor of a crustacean or insect.

[0028] (15) The transformant according to (14), characterized in that the juvenile hormone receptor of the crustacean is the juvenile hormone receptor of Daphnia.

[0029] (16) The transformant according to (15), characterized in that the juvenile hormone receptor of the Daphnia is the protein of (a) or (b) below. (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 12 (b) A protein having juvenile hormone receptor transcription factor activity, consisting of an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 12.

[0030] (17) The transformant according to (9), characterized by further introduction of nucleic acids encoding transcriptional co-factors.

[0031] (18) The transformant according to (9), characterized in that the host is yeast.

[0032] (19) A method for evaluating a test substance, comprising the steps of: introducing a nucleic acid described in any of (1) to (5) above and a reporter gene to the 3' end of the sense strand of the nucleic acid into a host, and contacting a transformant expressing an endocrine disruptor receptor that interacts with the nucleic acid; and measuring the expression of the reporter gene, wherein the interaction between the test substance and the endocrine disruptor receptor is evaluated based on the expression level of the reporter gene.

[0033] (20) The evaluation method according to (19), characterized in that if the expression level of the reporter gene increases after contact with the test substance, it is determined to be an agonist of the endocrine disruptor receptor.

[0034] (21) The evaluation method according to (19), characterized in that the above-mentioned 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 disruptor receptors that interact with the above-mentioned endocrine disruptor receptor, and if the expression level of the reporter gene is lower compared to when the substance is brought into contact with the transformant alone, the above-mentioned test substance is determined to be an antagonist of the above-mentioned endocrine disruptor receptor.

[0035] (22) The evaluation method according to (19), characterized in that the nucleic acid described above is a single unit, and multiple units of the nucleic acid described above are linked together.

[0036] (23) The evaluation method according to (19), characterized in that the transformant has nucleic acid encoding the endocrine disruptor receptor introduced into it.

[0037] (24) The evaluation method according to (19), characterized in that the endocrine disruptor receptor is a juvenile hormone receptor in arthropods.

[0038] (25) The evaluation method according to (24), characterized in that the juvenile hormone receptor in the arthropod is the juvenile hormone receptor of a crustacean or insect.

[0039] (26) The evaluation method according to (25), characterized in that the juvenile hormone receptor of the crustacean is the juvenile hormone receptor of Daphnia.

[0040] (27) The evaluation method according to (26), characterized in that the juvenile hormone receptor of the Daphnia described above is the protein of (a) or (b) below. (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 12 (b) A protein having juvenile hormone receptor transcription factor activity, consisting of an amino acid sequence that is 70% or more identical to the amino acid sequence shown in Sequence ID No. 12.

[0041] (28) The evaluation method according to (19), characterized in that the transformant further has nucleic acids encoding transcriptional co-factors introduced into it.

[0042] (29) The evaluation method according to (19), characterized in that the host is yeast.

[0043] (30) A kit for measuring endocrine disruptors, comprising the vector described in (8) above or the transformant described in (11) above. [Effects of the Invention]

[0044] According to the present invention, it is possible to provide nucleic acids that respond to low doses of endocrine disruptors, vectors having said nucleic acids, and transformants.

[0045] Furthermore, by using the nucleic acids according to the present invention, the endocrine-disrupting effect of the test substance can be evaluated by analyzing the expression of a reporter gene. [Modes for carrying out the invention]

[0046] [Response array] The nucleic acid according to the present invention (hereinafter sometimes referred to as the response sequence) contains the base sequence shown in Sequence ID No. 1 and is a nucleic acid with a total length of 20 to 60 base pairs. Hereafter, when referring to the response sequence, it does not mean the sequence itself as information, but rather the nucleic acid formed by the binding of four types of nucleotides, adenine (A), guanine (G), thymine (T), and cytosine (C), in a predetermined order. This response sequence interacts with the juvenile hormone receptor (MET protein) of Daphnia magna, which is bound to juvenile hormone, and has the function of positively regulating the expression of downstream genes at the transcriptional level. The base sequence shown in Sequence ID No. 1 has any four bases (i.e., NNNN) between CACGCG (C-box) and CACGTG (E-box). These four bases are not particularly limited, but are preferably kmkk (where k means G or T, and m means A or C). The sequences represented by kmkk include 16 types, such as GAGG, GCGG, TATT, TCTT, TAGG, TCGG, GATT, and GCTT. That is, the four bases between CACGCG (C-box) and CACGTG (E-box) are preferably one sequence selected from these 16 types. Among these, the four bases between CACGCG (C-box) and CACGTG (E-box) are preferably GCGG or TATT. A base sequence in which any four bases of the base sequence shown in Sequence ID No. 1 are GCGG (i.e., CACGCGGCGGCACGTG) is included, for example, in the promoter sequence located upstream of the Vrille gene in Daphnia magna. The bases in the region of the response sequence excluding Sequence ID No. 1 can be arbitrarily selected. The total length of the response sequence is 20 to 60 bases, but is preferably 30 to 60 bases, more preferably 40 to 60 bases, and even more preferably 40 to 50 bases.

[0047] Here, the nucleotide sequence (16 nucleotides) shown in Sequence ID No. 1 in the response sequence is preferably located at positions excluding both ends of the total length of 20 to 60 nucleotides. That is, the response sequence preferably has nucleotide sequences of predetermined lengths at the 3' and 5' ends of the nucleotide sequence shown in Sequence ID No. 1. The predetermined nucleotide length can be, for example, 1 to 43 nucleotides, preferably 5 to 30 nucleotides, more preferably 5 to 20 nucleotides, and even more preferably 7 to 20 nucleotides.

[0048] In the response sequence, the nucleotide sequence of the region excluding sequence number 1 is not particularly limited and can be any nucleotide sequence. For example, it is preferable to select from a nucleotide sequence adjacent to the nucleotide sequence present in the upstream region of the Vrille gene in Daphnia magna (i.e., CACGCGGCGGCACGTG).

[0049] As an example, the response sequence containing the nucleotide sequence shown in SEQ ID NO: 1 can be a nucleic acid consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 34, and 37. Among these, the response sequence consisting of the nucleotide sequence shown in SEQ ID NO: 2 is preferred.

[0050] The response sequence according to the present invention can also be a nucleotide sequence having 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity with a nucleotide sequence that includes a region adjacent to the nucleotide sequence present in the upstream region of the Vrille gene in Daphnia magna (i.e., CACGCGGCGGCACGTG). In particular, the nucleotide sequences shown in SEQ ID NO: 40 and SEQ ID NO: 43, which differ by 3 nucleotides from SEQ ID NO: 2, SEQ ID NO: 49, which differs by 4 nucleotides, SEQ ID NO: 46, which differs by 6 nucleotides, and SEQ ID NO: 52, which differs by 10 nucleotides are preferred.

[0051] The response sequence according to the present invention positively regulates the expression of a gene located downstream of it (towards the 3' end of the sense strand) at the transcriptional level, but a configuration with multiple response sequences is also possible. Specifically, multiple response sequences can be placed upstream of a gene whose expression is regulated at the transcriptional level by arranging multiple units directly adjacent to each other, or by arranging multiple units with spacers in between, with the above-mentioned base sequence as a single unit.

[0052] By arranging multiple response sequences, the expression of downstream genes can be more strongly positively controlled. The number of response sequences to be placed upstream of a given gene is not particularly limited, but can be, for example, 2 to 8, preferably 2 to 5, and more preferably 3 to 4.

[0053] Furthermore, when arranging multiple response sequences, each response sequence may consist of the same base sequence, each response sequence may consist of different base sequences, or each response sequence may consist of the same base sequence and the remaining sequences may consist of different base sequences.

[0054] Furthermore, the spacers placed between the response sequences are not particularly limited, but can consist of one or more arbitrary base sequences. The base length of the spacers is not particularly limited, but can be 1 to 100 bases long, preferably 1 to 90 bases long, preferably 1 to 80 bases long, preferably 1 to 70 bases long, preferably 1 to 60 bases long, preferably 1 to 50 bases long, preferably 1 to 40 bases long, preferably 1 to 30 bases long, preferably 1 to 20 bases long, and preferably 1 to 10 bases long. [vector] On the other hand, the vector according to the present invention includes the response element described above. This vector can incorporate a target gene whose expression is positively regulated at the transcriptional level by the response element. Specifically, the vector has a cloning site downstream of the response element for incorporating a target gene whose expression is positively regulated at the transcriptional level. The vector according to the present invention is not particularly limited as long as it can control the transcription of a downstream gene by the response element in a suitable host cell. Examples of such vectors include plasmid vectors, phage vectors, cosmid vectors, and donor vectors used in genome editing. Furthermore, shuttle vectors that allow gene exchange with other host strains can also be used.

[0055] Plasmids include those derived from E. coli, Bacillus subtilis, and yeast, while phages include lambda phages. Furthermore, retroviruses, animal viruses such as vaccinia viruses or adenoviruses, and insect virus vectors such as baculoviruses can also be used.

[0056] To insert a response sequence into a vector, methods such as first cleaving the purified response sequence with an appropriate restriction enzyme and inserting it into a suitable restriction enzyme site or multi-cloning site in the vector, and then ligating it into the vector, are employed. As a method that does not use restriction enzymes, homologous recombination methods such as CRISPR / Cas9, Red / ET, or Gateway are also employed.

[0057] In the vector according to the present invention, the response sequence described above is located in an expression regulatory region that controls the transcription of a downstream gene. An expression regulatory region is a region that controls the transcription of a downstream gene, and usually refers to the upstream region of the gene (the 5' region of the sense strand). More specifically, the expression regulatory region can usually be in the range of several thousand bases upstream from the transcription start site of the downstream gene, for example, within 5000 bases (b) upstream from the transcription start site, preferably within 4000b, 3000b, 2000b, 1000b, 500b, or 300b.

[0058] Furthermore, the expression regulatory region may include, in addition to the response sequence described above, regions to which transcription factors bind, enhancer regions, and so on. For example, the expression regulatory region containing the response sequence described above can be a region of a predetermined length containing the response sequence, such as a 50b region, a 60b region, a 70b region, an 80b region, a 90b region, a 100b region, a 120b region, a 150b region, or a 200b region containing the response sequence.

[0059] The vector according to the present invention can include a gene to be regulated downstream of the expression regulatory region having a response element, as configured as described above. Examples of genes to be regulated include reporter genes. Reporter genes are not particularly limited and include, for example, the chloramphenicol acetyltransferase (CAT) gene, the lacZ gene, the luciferase gene, the β-glucuronidase (GUS) gene, the green fluorescent protein (GFP) gene, drug resistance genes, and nutritional requirement genes. Note that the reporter gene is not limited to the conventionally known genes described above, and the Vrille gene described above may also be used as a reporter gene. In other words, any gene can be used as a reporter gene as long as its expression level at the transcriptional level can be monitored. [Transformed body] The transformant according to the present invention is obtained by introducing the above-described response sequence into a host. In the transformant, for example, the juvenile hormone receptor of Daphnia magna bound to juvenile hormone interacts with the above-described response sequence, thereby increasing the expression of a gene located downstream of the response sequence at the transcriptional level. The above-described response sequence can be introduced into a host, for example, using the above-described vector. In particular, it is preferable that the transformant according to the present invention is introduced with the above-described response sequence, a gene whose expression is upregulated at the transcriptional level by the response sequence (for example, a reporter gene), and a nucleic acid encoding an endocrine disruptor receptor such as the juvenile hormone receptor that interacts with the response sequence.

[0060] Here, an endocrine disruptor receptor refers to a receptor on which a specific hormone acts as a ligand, and which is also acted upon by an endocrine disruptor having hormone-like activity. The term endocrine disruptor receptor includes both cell membrane receptors and nuclear receptors. Preferably, the endocrine disruptor receptor is a receptor that interacts with the response element described above in the presence of a specific hormone or endocrine disruptor and exhibits transcription factor activity.

[0061] The term "endocrine disruptor" is not particularly limited and includes both substances that are publicly known to have endocrine-disrupting effects and substances that are suspected to have endocrine-disrupting effects. For example, endocrine disruptors can include substances that have agonist or antagonist effects on female hormones (estrogen and progesterone), and substances that have agonist or antagonist effects on male hormones (androgens). Furthermore, the term "endocrine disruptor" is not limited to substances that disrupt these female or male hormones, but also includes substances that disrupt hormones such as thyroid hormones, growth hormones, adrenocortical hormones, or insulin, as well as neurotransmitters such as acetylcholine, norepinephrine, adrenaline, or dopamine.

[0062] Therefore, endocrine disruptor receptors can be defined as the receptors for the various hormones mentioned above, such as estrogen receptors, androgen receptors, progesterone receptors, thyroid hormone receptors, growth hormone receptors, adrenocortical hormone receptors, or insulin receptors.

[0063] Furthermore, endocrine disruptors include substances that have agonist or antagonist activity against juvenile hormones, which are specifically present in arthropods. Therefore, it is particularly preferable to use the juvenile hormone receptor (MET) as the endocrine disruptor receptor.

[0064] The juvenile hormone receptor is not particularly limited, but can be a juvenile hormone receptor found in arthropods, including insects, crustaceans, spiders, and centipedes. In particular, the juvenile hormone receptor in arthropods is preferably a juvenile hormone receptor found in crustaceans or insects.

[0065] Examples of insects include the Coleoptera (beetles and ground beetles), Lepidoptera (butterflies and moths), Diptera (flies, mosquitoes and horseflies), Hymenoptera (bees and ants), Hemiptera (true bugs), Orthoptera (grasshoppers and crickets), and Odonata (dragonflies). The transformants according to the present invention can use nucleic acids that encode juvenile hormone receptors derived from these insects.

[0066] More specifically, nucleic acids encoding the juvenile hormone receptor of the fruit fly Drosophila melanogaster, which belongs to the order Diptera, can be used (see He Q et al.; J Biol Chem. 289 (40), p. 27874-27885 (2014)).

[0067] Furthermore, examples of crustaceans include animals belonging to the subphylum Crustacea, such as shrimp, crabs, krill, barnacles, and water fleas. The transformants according to the present invention can use nucleic acids that encode juvenile hormone receptors derived from these crustaceans.

[0068] In particular, among nucleic acids encoding juvenile hormone receptors derived from crustaceans, it is especially preferable to use nucleic acids encoding juvenile hormone receptors derived from animals belonging to the Daphnia family.Animals belonging to the Daphnia family include animals belonging to the genus Ceriodaphnia, such as Ceriodaphnia cornuta, Ceriodaphnia reticulata, Ceriodaphnia dubia, Ceriodaphnia megalops, Ceriodaphnia pulchella, and Ceriodaphnia quadrangular; Daphnia similis, Daphnia magna, Daphnia pulex, Daphnia pulicaria, Daphnia ambigua, Daphnia obtuse, and Daphnia Animals belonging to the genus Daphnia, such as Daphnia biwaensis, Daphnia longispina, Daphnia rosea, Daphnia hyaline, Daphnia galeata, Daphnia ezoensis, Daphnia cuculata, Daphnia cristata and Daphnia longiremis; animals belonging to the genus Scapholeberis, such as Scapholeberis mucronata and Scapholeberis kingi; and Simocephalus Examples of animals belonging to the genus Simocephalus include Simocephalus serrulatus, Simocephalus exspinosus, Simocephalus vetulus, Simocephalus vetuloides, and Simocephalus japonica.

[0069] Among these, it is particularly preferable to use nucleic acids encoding the juvenile hormone receptor of animals belonging to the genus Daphnia, and even more preferable to use nucleic acids encoding the juvenile hormone receptor of Daphnia magna. The amino acid sequence of the juvenile hormone receptor of Daphnia magna 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.

[0070] However, the transformant according to the present invention is not limited to having a nucleic acid encoding a juvenile hormone receptor consisting of the amino acid sequence shown in SEQ ID NO: 12, but may also have a nucleic acid encoding a protein having juvenile hormone receptor transcription factor activity, consisting of an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 12, preferably 80% or more identity, more preferably 90% or more identity, even more preferably 95% or more identity, and most preferably 98% or more identity.

[0071] The identity value between amino acid sequences can be calculated using programs such as BLASTN or BLASTX, which implement the Basic Local Alignment Search Tool (BLAST) algorithm (default settings). The identity value is calculated by determining the percentage of perfectly matching amino acid residues when a pair of amino acid sequences are analyzed using pairwise alignment, and then comparing these residues to the total number of amino acid residues.

[0072] Furthermore, the transformant according to the present invention is not limited to having a nucleic acid encoding a juvenile hormone receptor consisting of the amino acid sequence shown in SEQ ID NO: 12, but may also have a nucleic acid encoding a protein having juvenile hormone receptor transcription factor activity, which is encoded by a nucleic acid that hybridizes under stringent conditions to all or part of the complementary strand of the nucleic acid consisting of the base sequence of SEQ ID NO: 11. "Stringent conditions" means conditions under which a specific hybrid is formed and a nonspecific hybrid is not formed, and can be appropriately determined by referring to, for example, Molecular Cloning: A Laboratory Manual (Third Edition). Specifically, stringency can be set by the temperature and salt concentration in the solution during Southern hybridization, and the temperature and salt concentration in the solution during the washing step of Southern hybridization. More specifically, 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, the solution is 5×SSC (83mM NaCl, 83mM sodium citrate) at a temperature of 42°C.

[0073] Here, whether a protein encoded by a nucleic acid consisting of a predetermined base sequence different from SEQ ID NO: 11, or a protein containing amino acids different from SEQ ID NO: 12, possesses juvenile hormone receptor transcription factor activity can be evaluated as follows. First, a transformant is prepared by introducing the nucleic acid to be evaluated or the nucleic acid encoding the protein to be evaluated, along with the response sequence described above and a reporter gene placed downstream thereof. Then, the transformant is cultured in the presence or absence of juvenile hormone, and the expression of the reporter gene is measured. If the expression level of the reporter gene in the presence of juvenile hormone is significantly higher than the expression level of the reporter gene in the absence of juvenile hormone, then the nucleic acid to be evaluated encodes a protein with juvenile hormone receptor transcription factor activity, and the protein to be evaluated possesses juvenile hormone receptor transcription factor activity.

[0074] Therefore, the juvenile hormone receptor transcription factor activity described above can be rephrased as activity that interacts with the response element according to the present invention described above. Alternatively, the juvenile hormone receptor transcription factor activity described above can also be rephrased as activity that binds to juvenile hormone and interacts with the response element according to the present invention described above.

[0075] Incidentally, the transformant according to the present invention may further have nucleic acids encoding transcription co-factors in addition to the nucleic acids, response sequences, and reporter genes described above. Examples of transcription co-factors, though not particularly limited, include Taiman (Tai), Steroid receptor coactivator (SRC), β-FTZ-F1 (fushi tarazu binding factor 1), interacting steroid receptor coactivator (FISC), CREB binding protein (CBP), P300, transcriptional mediators / intermediary factor 2 (TIF2), amplified in breast cancer (AIB), 70 kDa androgen receptor coactivator (ARA70), activating signal co-integrator 2 (ASC2), and 140 kDa estrogen receptor-associated protein (ERAP140). By introducing nucleic acids encoding these transcription co-factors, the transcriptional activity of the reporter gene by endocrine disruptor receptors can be further activated.

[0076] Among these, it is particularly preferable to introduce nucleic acids encoding SRC or Tai, and more preferably to introduce nucleic acids 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 said SRC is shown in SEQ ID NO: 13.

[0077] However, the transcriptional co-factors that can be used in the transformants according to the present invention are not limited to proteins consisting of the amino acid sequence shown in SEQ ID NO: 14, but may also be proteins having transcriptional co-factor activity, consisting of an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 14, preferably 80% or more identity, more preferably 90% or more identity, even more preferably 95% or more identity, and most preferably 98% or more identity.

[0078] On the other hand, the host for the transformant according to the present invention may be Escherichia coli, Bacillus subtilis, budding yeast, cultured cells derived from arthropods or mammals, undifferentiated plant cells (callus), nematodes (C. elegans), arthropods such as Daphnia and Drosophila, fish such as zebrafish and medaka, amphibians such as African clawed frogs and newts, mammals such as mice and rats, or higher plants such as Arabidopsis thaliana and rice, but budding yeast is preferred. The method for introducing the above-mentioned nucleic acids, response sequences, reporter genes, vectors, etc. into the host is not particularly limited, and conventionally known methods can be applied. Examples of methods for introducing nucleic acids include the lithium acetate method, the calcium phosphate method, the liposome method, electroporation, the viral vector method, and the micropipette injection method. In the transformant according to the present invention, the introduction of the nucleic acids, response sequences, and reporter genes described above may be transient, introduced by integration into the host chromosome, or introduced as an autonomously replicating and distributable artificial chromosome or plasmid, provided that it can be used for analysis.

[0079] In the transformant according to the present invention configured as described above, the endocrine disruptor receptor to which a predetermined hormone or endocrine disruptor is bound interacts with the response element, thereby increasing the expression of a gene located downstream of the response element (e.g., a reporter gene) at the transcriptional level. Therefore, by measuring the expression of a gene located downstream of the response element, the interaction between the endocrine disruptor receptor and the response element can be evaluated. 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 element. Specifically, for example, the binding ability of the test substance to the endocrine disruptor receptor and the effect of the test substance on the binding of the endocrine disruptor receptor to its agonist can be evaluated based on the expression of a gene located downstream of the response element. In particular, when using the transformant according to the present invention, since the above-described response element is used, it is possible to evaluate the presence or absence of endocrine disruptive activity for the test substance without being limited to a specific endocrine disrupting effect.

[0080] Here, the test substance is not particularly limited and may be any substance. Examples of test substances include low molecular weight compounds, high molecular weight compounds, peptides, single compounds, compositions containing multiple compounds, cultures, extracts, natural products, and synthetic compounds.

[0081] Specifically, the transformants are cultured in the presence or absence of the test substance, and the expression of the reporter gene located downstream of the response element is measured in each case. 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, it can be concluded that the test substance interacts with endocrine disruptor receptors and has an agonist effect that positively regulates gene expression via the response element described above. In other words, in this case, it can be concluded that there is a high probability that the test substance is an endocrine disruptor with agonist activity.

[0082] Alternatively, the transformants are cultured in the presence or absence of the test substance and a substance having agonist activity against endocrine disruptor receptors, and the expression of the reporter gene located downstream of the response element is measured in each case. 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, it can be determined that the test substance inhibits agonist activity against endocrine disruptor receptors and has antagonist activity that negatively regulates gene expression via the response element described above. In other words, in this case, it can be determined that the test substance is highly likely to be an endocrine disruptor with antagonist activity. Note that a substance having agonist activity against endocrine disruptor receptors refers to a substance selected from hormones that act on endocrine disruptor receptors, endocrine disruptors corresponding to those hormones, and agonist compounds that act on endocrine disruptor receptors.

[0083] As described above, by using the transformant according to the present invention, the endocrine-disrupting effect of the test substance can be evaluated with very simple operations. In other words, the transformant according to the present invention can be used for the measurement and evaluation of endocrine-disrupting substances, and can be used as an endocrine-disrupting substance measurement kit for evaluating the endocrine-disrupting effect of the test substance.

[0084] In particular, when transformants are used that have been introduced with nucleic acids encoding the juvenile hormone receptor in Daphnia magna as an endocrine disruptor receptor, along with the aforementioned response sequence and reporter gene, the endocrine disrupting effect of the test substance on animals belonging to the Daphniidae family, especially Daphnia magna, can be properly evaluated.

[0085] Furthermore, when yeast is used as the host in the transformant according to the present invention, the effects of degradation of the test substance by drug metabolism systems such as p450 present in animal cells are eliminated, and the endocrine-disrupting effects of the test substance can be accurately evaluated. [Examples]

[0086] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples. [Preparation of transformed yeast] In this example, each reporter plasmid, each transcription factor (endocrine disruptor receptor) plasmid, and each transcription co-factor plasmid were prepared and introduced into budding yeast strains to produce transformed yeast. [Reporter plasmid creation] OligoDNA (SEQ ID NO: 3) containing a response sequence (43 bases) consisting of the base sequence shown in SEQ ID NO: 2, and oligoDNA (SEQ ID NO: 4) partially complementary to SEQ ID NO: 3 were prepared, and these oligoDNAs were annealed. During annealing, the cells were incubated at 95°C for 2 minutes, and then the temperature was gradually lowered from 90°C to 37°C over 30 minutes. In this example, "partially complementary" means that the portion of the base sequence identified by SEQ ID NO: excluding the few bases at the 5' end is complementary.

[0087] After annealing, the response sequences annealed in sequences of one, two, three, four, or five consecutive copies were purified. Then, the response sequences annealed in sequences of one, two, three, four, or five consecutive copies shown in Sequence ID No. 2 were inserted into the SpeI site of plasmid pRW95-3, which contains the β-galactosidase gene and was prepared according to the method described in Wolf SS et al., Biotechniques. 20 (4), p. 568-573 (1996).

[0088] Plasmids were also prepared using a similar method, inserting response sequences consisting of the nucleotide sequence shown in SEQ ID NO: 5 (54 nucleotides), SEQ ID NO: 8 (30 nucleotides), SEQ ID NO: 34 (22 nucleotides), SEQ ID NO: 37 (36 nucleotides), SEQ ID NO: 40 (43 nucleotides), SEQ ID NO: 43 (43 nucleotides), SEQ ID NO: 46 (43 nucleotides), SEQ ID NO: 49 (43 nucleotides), or SEQ ID NO: 52 (43 nucleotides).The manufacturing method is as follows, except that the oligoDNA of SEQ ID NO: 3 is replaced with the oligoDNA 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, and the oligoDNA of SEQ ID NO: 4 is replaced with oligoDNA partially complementary to SEQ ID NO: 6 (SEQ ID NO: 7), partially complementary to SEQ ID NO: 9 (SEQ ID NO: 10), partially complementary to SEQ ID NO: 35 (SEQ ID NO: 36), partially complementary to SEQ ID NO: 38 (SEQ ID NO: 39), partially complementary to SEQ ID NO: 41 (SEQ ID NO: 42), partially complementary to SEQ ID NO: 44 (SEQ ID NO: 45), partially complementary to SEQ ID NO: 47 (SEQ ID NO: 48), partially complementary to SEQ ID NO: 50 (SEQ ID NO: 51), or partially complementary to SEQ ID NO: 53 (SEQ ID NO: 54). Similarly, plasmids were created with three consecutive response sequences shown in SEQ ID NO: 5, one, two, three, or four consecutive response sequences shown in SEQ ID NO: 8, one, two, three, four, or five consecutive response sequences shown in SEQ ID NO: 34, one, two, three, or four consecutive response sequences shown in SEQ ID NO: 37, one, two, three, or four consecutive response sequences shown in SEQ ID NO: 40, one, two, three, or four consecutive response sequences shown in SEQ ID NO: 43, one, two, or three consecutive response sequences shown in SEQ ID NO: 46, one, two, three, or four consecutive response sequences shown in SEQ ID NO: 49, and one, two, or three consecutive response sequences shown in SEQ ID NO: 52.The nucleotide sequences shown in SEQ ID NOs. 2, 5, 8, 34, and 37 are located in the upstream region of the Vrille gene in Daphnia magna. The nucleotide sequence shown in SEQ ID NOs. 40 is the same as the nucleotide sequence in SEQ ID NOs. 2, but with the three bases outside the 5' end of the C-box (CACGCG) altered. The nucleotide sequence shown in SEQ ID NOs. 43 is the same as the nucleotide sequence in SEQ ID NOs. 2, but with the three bases outside the 3' end of the E-box (CACGTG) altered. The nucleotide sequence shown in SEQ ID NOs. 46 is the same as the nucleotide sequence in SEQ ID NOs. 2, but with the 5' end of the C-box (CACGCG) altered. The sequence shown in Sequence ID No. 49 is a sequence in which the three bases outside the end and the three bases outside the 3' end of the E-box (CACGTG) are all changed. The sequence shown in Sequence ID No. 2 is a sequence in which the four bases between the C-box (CACGCG) and E-box (CACGTG) (linker region) are changed. The sequence shown in Sequence ID No. 52 is a sequence in which the three bases outside the 5' end of the C-box (CACGCG), the three bases outside the 3' end of the E-box (CACGTG), and the four bases between the C-box and E-box (linker region) are all changed.

[0089] For comparison with the reporter plasmid containing this response sequence, a plasmid was constructed in which only the 16-base nucleic acid (i.e., CACGCGGCGGCACGTG) present in the upstream region of the Vrille gene in Daphnia magna was inserted. These 16 bases include the E-box (CACGTG) and C-box (CACGCG), which are considered juvenile hormone response sequences in many insect species. Plasmids were constructed in which one, two, three, or four consecutive copies of this 16-base nucleic acid were inserted, following the method described above.

[0090] For further comparison, a plasmid containing the juvenile hormone response region (DmJHRR) of the Kr-h1 gene in Drosophila melanogaster was constructed. DmJHRR is described in He Q et al.; J Biol Chem. 289 (40), p. 27874-27885 (2014). The construction method was the same as described above, except that the oligo DNA of SEQ ID NO: 3 was replaced with oligo DNA containing the DmJHRR sequence (SEQ ID NO: 15), and the oligo DNA of SEQ ID NO: 4 was replaced with oligo DNA partially complementary to SEQ ID NO: 15 (SEQ ID NO: 16). The plasmid was constructed with three consecutive DmJHRR sequences inserted.

[0091] For further comparison, in order to compare reporter activity with and without the C-box (CACGCG) and E-box (CACGTG) contained in the base sequence of SEQ ID NO: 2, base sequences with different C-box portions (SEQ ID NO: 25), different E-box portions (SEQ ID NO: 28), and base sequences with both different C-box and E-box portions (SEQ ID NO: 31) were prepared. Specifically, oligo DNA containing the base sequence shown in SEQ ID NO: 25 (SEQ ID NO: 26) and DNA partially complementary to SEQ ID NO: 26 (SEQ ID NO: 27), oligo DNA containing the base sequence shown in 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 base sequence shown in SEQ ID NO: 31 (SEQ ID NO: 32) and DNA partially complementary to SEQ ID NO: 32 (SEQ ID NO: 33) were prepared, and plasmids were prepared by inserting four consecutive base sequences of the base sequence shown in SEQ ID NO: 25, four consecutive base sequences of the base sequence shown in SEQ ID NO: 28, and four consecutive base sequences of the base sequence shown in SEQ ID NO: 31, respectively. [Production of transcription factor expression plasmids] To prepare the Daphnia magna juvenile hormone receptor expression plasmid (hereinafter referred to as the DampaMET plasmid), a CEN6 / ARS4 fragment was first prepared. A DNA fragment containing the yeast autonomous origin of replication (ARS) and centromere sequence (CEN) was amplified by PCR using the reporter plasmid pYTβ as a template, with the CEN / ARS amplification primers pUdp6 I-SceI CEN6 FW (SEQ ID NO: 17) and pUdp6 I-SceI ARS4 RE (SEQ ID NO: 18). PCR was performed for 35 cycles, with cycles of 20 seconds at 94°C, 20 seconds at 58°C, and 1 minute 30 seconds at 72°C.

[0092] PCR fragments amplified by PCR were cleaved with the restriction enzyme AatII to form a linear plasmid, pUdp6, which was then introduced into wild-type yeast strain (Saccharomyces cerevisiae) W303a (MATa, ade2, his3, leu2, trp1, ura3) using the lithium acetate method. pUdp6 is a plasmid containing bidirectional promoter regions gal1 and gal10, a CYC terminator downstream of gal10, an ADH terminator downstream of gal1, and the uracil selection marker URA3 gene. A plasmid in which the CEN6 / ARS4 fragment was inserted into pUdp6 by homologous recombination was extracted from yeast and further introduced into E. coli DH5α strain to obtain a low-copy episomal vector, which was designated as pUdp13. Furthermore, the CEN6 / ARS4 sequence has an 18bp restriction enzyme I-SceI recognition sequence introduced outside it. After cloning the target gene to a multi-cloning site, the CEN6 / ARS4 fragment can be easily converted to a genome insertion type by cleaving it with I-SceI.

[0093] Next, the open reading frame (ORF) of the DampaMET gene cDNA was amplified by PCR using the cDNA of an egg-bearing adult Daphnia magna as a template, with MET FW2 (SEQ ID NO: 19) and MET REV-3 (SEQ ID NO: 20) as DampaMET primers. PCR was performed for 35 cycles, consisting of 20 seconds at 94°C, 20 seconds at 58°C, and 2 minutes 30 seconds at 72°C.

[0094] The amplified cDNA of the DampaMET gene was introduced into wild yeast strain W303a using the lithium acetate method, along with the plasmid pUdp13, which had been cleaved with restriction enzymes BamHI and HindIII. Using this method, the cDNA of the DampaMET gene was inserted downstream of the GAL10 promoter in plasmid pUdp13 to form pUdp13-DapmaMet. The pUdp13-DapmaMet constructed in yeast cells was recovered and amplified in E. coli strain DH5α.

[0095] Next, pUdp13-DapmaMet was cleaved with restriction enzyme I-SceI to extract the CEN6 / ARS4 sequence, thereby converting it to a genome insertion type. This yielded a DampaMET plasmid containing the DampaMET gene and a promoter upstream of it.

[0096] Similarly, a Drosophila melanogaster juvenile hormone receptor expression plasmid (hereinafter referred to as the DmMET plasmid) was constructed. First, the ORF of the DmMET gene cDNA was amplified by PCR using DmMET primers DmMET Fwd (SEQ ID NO: 21) and DmMET Rev (SEQ ID NO: 22) with Drosophila melanogaster larval cDNA (Clontech) as a template. PCR was performed for 25 cycles, consisting of 10 seconds at 98°C, 30 seconds at 55°C, and 1 minute 30 seconds at 68°C.

[0097] The amplified cDNA of the DmMET gene was cleaved with the restriction enzymes SmaI and EcoRI. The cleaved cDNA of the DmMET gene was inserted downstream of the GAL10 promoter of the plasmid pUdp6, which had been cleaved with the restriction enzymes SmaI and EcoRI. This yielded a DmMET plasmid containing the DmMET gene and having a promoter upstream of it. [Production of transcriptional co-factor expression plasmids] In this example, we prepared and used a transcription factor expression plasmid containing the Drosophila melanogaster Tai (DmTai) gene (hereinafter referred to as the DmTai plasmid) and a Daphnia magna transcriptional co-factor expression (SRC) plasmid (hereinafter referred to as the DampaSRC plasmid). The DmTai plasmid was prepared according to Ito-Harashima S et al. FEBS Open Bio. 7(7): p. 995-1008 (2017).

[0098] Furthermore, the DampaSRC plasmid was prepared using the following method. First, the ORF of the cDNA of the Daphnia magna SRC (DampaSRC) gene was amplified by PCR using the cDNA of an egg-bearing adult Daphnia magna as a template, with the SRC primers SRC-F1F-pESC (SEQ ID NO: 23) and SRC-F4short-pESC (SEQ ID NO: 24). PCR was performed for 35 cycles, consisting of 20 seconds at 94°C, 10 seconds at 58°C, and 7 minutes at 72°C.

[0099] Next, the plasmid pESC-Leu (Agilent Technology) was cleaved with restriction enzymes SpeI and PacI, and the amplified cDNA of the DampaSRC gene was introduced into wild yeast strain W303a using the lithium acetate method to obtain a DampaSRC plasmid in which the cDNA of the DampaSRC gene was inserted downstream of the gal1 promoter region of plasmid pESC-Leu. The DampaSRC plasmid constructed in yeast cells was recovered and introduced into E. coli strain DH5α for amplification. [Preparation of transformed yeast] First, the cells of budding yeast strain W303a were cultured in YPD medium (1% yeast extract, 2% peptone, 2% D(+)-glucose) at 30°C until the turbidity (OD595) was 0.7-0.8. After washing the cultured cells twice with sterile water, they were resuspended using a pipette in 0.1 mol / L lithium acetate solution, which was one-tenth the volume of the pre-culture solution. The cell suspension was dispensed in 100 μL portions into 1.5 mL microcentrifuge tubes, centrifuged, and the supernatant was removed. 75 μL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) containing 1 μg of linearized DampaMET plasmid treated with restriction enzyme EcoRV and 50 μg of carrier DNA (product name SALMON TESTES DNA for hybridization, SIGMA) was added. Then, 240 μL of 50% polyethylene glycol 3350 solution and 36 μL of 0.1 mol / L lithium acetate solution were added and thoroughly mixed.

[0100] The mixture was incubated at 30°C for 30 minutes, then heat-treated at 42°C for 22 minutes, followed by centrifugation at 9,000 rpm for 1 minute to collect yeast cells from the mixture. The collected yeast cells were suspended in 300 μL of sterile water, and 100 μL of the suspension was spread onto selective media shown in Tables 1 and 2, to which 42 mg of tryptophan, 62 mg of leucine, and 2% agar had been added, and the mixture was cultured. Yeast strains that showed uracil non-requiring were selected as transformed yeasts in which the cDNA regions of the gal1 promoter and MET gene were integrated onto the chromosome.

[0101] Next, the DampaSRC plasmid was introduced into the transformed yeast into which the DampaMET plasmid had been introduced. For this process, the selective culture medium for the transformed yeast was the pre-culture medium shown in Tables 1 and 2, to which 100 mg of tryptophan had been added.

[0102] [Table 1]

[0103] [Table 2]

[0104] Next, a reporter plasmid containing one nucleotide sequence from Sequence ID No. 2 was introduced into yeast cells into which the DampaMET plasmid and DampaSRC plasmid had been introduced, using the lithium acetate method. The pre-culture media shown in Tables 1 and 2 were used as the selection medium. This yielded yeast cell 1 of the present invention. In the same manner, yeast cells 2-38 and comparative yeast cells 1-9, each possessing the response sequence according to the present invention as shown in Table 3, were prepared.

[0105] [Table 3]

[0106] The following experimental examples were carried out using these transformed yeasts. [Test Example 1] Measurement of reporter activity of transformed yeast against juvenile hormone The reporter activity for methyl farnesenoate, a juvenile hormone substance, was measured using yeast 1 of the present invention. First, yeast 1 of the present invention was pre-cultured at 30°C for 18 hours using the pre-culture medium shown in Table 1, until the turbidity (OD595) of the pre-culture medium was approximately 1.0. 10 μL of the pre-cultured cell suspension and 1 μL of methyl farnesenoate solution diluted to various concentrations with dimethyl sulfoxide (DMSO) were mixed with 90 μL of the main culture medium shown in Table 4 in a 96-well plate, and the mixture was incubated at 30°C for 18 hours to prepare the reaction solution. This was used as the treatment group.

[0107] [Table 4]

[0108] The reaction solution to which 1 μL of DMSO was added instead of a diluted methyl farnesenoate solution was used as the untreated control. 10 μL of each prepared reaction solution was taken and dispensed into each well of a new 96-well plate. Then, 100 μL of a measurement reagent, consisting of a mixture of lysate (Z buffer: 60 mM Na2HPO4, 40 mM NaH2PO4, 1 mM MgCl2, 10 mM KCl, 2 mM dithiothreitol, 0.20% N-Lauroylsarcosine sodium salt) and 1 mg / mL ONPG (orthonitrophenylgalactopyranoside), was dispensed into each well, and the mixture was reacted at 37°C for 30 minutes. Subsequently, the absorbance (OD405) and turbidity (OD595) of each reaction solution at a wavelength of 405 nm were measured using a microplate reader (trade name iMark microplate reader, Bio-Rad Laboratories). The increase of induction was then calculated using the formula shown below.

[0109]

number

[0110] The induced increase is described in Ito-Harashima S et al., FEBS Open Bio. 7(7): p. 995-1008 (2017). A positive induced increase indicates that the yeast has reporter activity for that substance. The experiment was conducted using 1-, 3-, 5-, or 15-segment systems, and the average induced increase was calculated. Comparison yeast 1 was treated similarly, and reporter activity was compared. The results are shown in Table 5.

[0111] [Table 5]

[0112] As shown in Table 5, it can be seen that in yeast 1 of the present invention, which has the response sequence of the present invention, the expression of the reporter gene is induced in the presence of methyl farnesenoate, a juvenile hormone substance. On the other hand, reporter activity could hardly be measured in comparative yeast 1. [Test Example 2] Measurement of reporter activity of transformed yeast against juvenile hormone Similar to Test Example 1, the reporter activity for methyl farnesenoate was measured for yeast strains 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 of the present invention. The test method was the same as in Test Example 1, and the reporter activity was compared with comparative yeast strains 2, 3, and 4. The results are shown in Tables 6 and 7.

[0113] [Table 6]

[0114] [Table 7]

[0115] As shown in Tables 6 and 7, it can be seen that the expression of the reporter gene was clearly strongly induced in the yeast strains 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 of the present invention in the presence of juvenile hormone (methyl farnesenoate). On the other hand, comparative yeast strains 2, 3, and 4 showed negative values ​​for the induced increase at all treatment doses and did not exhibit reporter activity. From these results, it has become clear that reporter activity to methyl farnesenoate, one of the juvenile hormones, can be evaluated with high sensitivity by using a response sequence that includes the base sequence shown in Sequence ID No. 1 and has a total length of 20 to 60 bases. [Test Example 3] Effect of core sequence on reporter activity of transformed yeast The effect of the core sequence on reporter activity for methyl farnesenoate was evaluated using comparative yeast 7, comparative yeast 8, comparative yeast 8, comparative yeast 9, comparative yeast 9, comparative yeast 8, comparative yeast 8, comparative yeast 8, comparative yeast 9, comparative yeast 8, comparative yeast 8, comparative yeast 9, comparative yeast 8, comparative yeast 9, comparative yeast 8, comparative yeast 9, comparative yeast 8, comparative yeast 9, comparative yeast 8, comparative yeast 9, comparative yeast 9, comparative yeast 8, comparative yeast 9, comparative yeast 1, comparative yeast 2, comparative yeast 3, comparative yeast 4, comparative yeast 4, comparative yeast 7, comparative yeast 8, comparative yeast 9, comparative yeast 1, comparative yeast 2, comparative yeast 8, comparative yeast 9, comparative yeast 1, comparative yeast 2, comparative yeast 8, comparative yeast 9, comparative yeast 1, comparative yeast 2, comparative yeast 9, comparative yeast 1, comparative yeast 2, comparative yeast 3, comparative yeast 9, comparative yeast 1, comparative yeast 2, comparative yeast 8, comparative yeast 1, comparative yeast 9, comparative yeast 1, comparative yeast 2, comparative yeast 9, comparative yeast 1, comparative yeast 1, comparative yeast 2, comparative yeast 8, comparative yeast 9, comparative yeast 1, comparative yeast 1, comparative yeast 21, comparative yeast

[0116] [Table 8]

[0117] As shown in Table 8, yeast 4 of the present invention induced high reporter activity in response to methyl farnesenoate. On the other hand, comparative yeasts 7, 8, and 9, which had different base sequences in the C-box and / or E-box regions, were found to have significantly lower reporter activity compared to yeast 4 of the present invention. [Test Example 4] Measurement of reporter activity of transformed yeast to chemicals suspected of having sex hormone and endocrine-disrupting effects. Reporter activity was measured for the sex hormones 17β-estradiol and testosterone, the insect juvenile hormone III, and chemical substances suspected of having endocrine-disrupting effects: 4-nonylphenol, bisphenol A, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (DDE), and dieldrin, using yeasts 4 and 8 of the present invention. The test method followed that of Test Example 1. All test substances were treated with 10 μM. For comparison, reporter activity for each compound was measured using comparative yeasts 5 and 6, which incorporated the JH response element of Drosophila melanogaster, a known juvenile hormone receptor response element. The results are shown in Table 9.

[0118] [Table 9]

[0119] As shown in Table 9, in yeast strains 4 and 8 of the present invention, reporter gene expression was induced in all systems regardless of the type of endocrine disruptor, confirming that the response sequence of the present invention is responsive to all endocrine disruptors tested. Yeast strain 8 of the present invention is a transformant possessing the Drosophila melanogaster transcription factor (DmMET), and the response sequence of the present invention showed responsiveness not only to crustaceans but also to the Drosophila melanogaster transcription factor, a species of insect. In contrast, comparative yeast strains 5 and 6, which possess the Drosophila melanogaster juvenile hormone response sequence (DmJHRR), only induced reporter gene expression in systems using juvenile hormone III and 4-nonylphenol, and DmJHRR did not show responsiveness to other endocrine disruptors. From these results, it is possible to detect endocrine disruptors exhibiting diverse effects by reporter assays using the response sequence of the present invention. [Test Example 5] Measurement of reporter activity for juvenile hormone receptor antagonists The juvenile hormone receptor antagonist activity was evaluated using yeast strain 4 of the present invention at 10 μM concentrations of endrin, aldrin, and dieldrin. The test was performed in the presence of 10 nM methyl farnesenoate, and other procedures were the same as in Test Example 1. The results are shown in Table 10.

[0120] [Table 10]

[0121] As shown in Table 10, it was found that the increase in reporter activity by methyl farnesenoate was reduced for all substances tested in yeast 4 of the present invention. From this result, it is possible to detect endocrine disruptors exhibiting antagonist activity by using a transformant incorporating the response sequence of the present invention. [Test Example 6] Measurement of reporter activity of transformed yeast against existing pesticide active ingredients Following the procedure in Test Example 1, the endocrine-disrupting effects of the existing pesticide active ingredients mepanipyrim, bentiavalicarb isopropyl, pyribencarb, pyroxasulfone, and fenquinotrione were measured using yeast 4 of the present invention. The treatment concentration of the existing pesticide active ingredients was set to 100 μM for all tests. All of the existing pesticide active ingredients tested are substances that have been shown not to exhibit endocrine-disrupting effects. The results are shown in Table 11.

[0122] [Table 11]

[0123] As shown in Table 11, in transformants having the response sequence of the present invention, no induction of reporter gene expression was observed at a concentration of 100 μM for all existing pesticide active ingredients tested. This result demonstrates that transformants into which nucleic acids encoding endocrine disruptor receptors with transcription factor activity, along with the response sequence and reporter gene according to the present invention, can selectively detect endocrine disruptors. [Test Example 7] Effect of the peripheral and linker regions of the core sequence on the reporter activity of transformed yeast Yeast strains 21, 22, 23, and 24 of the present invention have a different nucleotide sequence from the nucleotide sequence of Sequence ID No. 2, with the three nucleotides outside the 5' end of the C-box (CACGCG) in the response sequence being different, and yeast strains 25, 26, 27, and 28 of the present invention have a different nucleotide sequence from the three nucleotides outside the 3' end of the E-box (CACGTG) portion being different, and yeast strains 25, 26, 27, and 28 of the present invention have a different nucleotide sequence from the nucleotide sequence of Sequence ID No. 2, with the three nucleotides outside the 5' end of the C-box (CACGCG) and the three nucleotides outside the 3' end of the E-box (CACGTG) portion being different. The effect of the core sequence on reporter activity for methyl farnesenoate was evaluated using yeast strains 29, 30, 31, 32, 33, 34, 35 of the present invention, in which the linker region between the C-box and E-box had different nucleotide sequences, and yeast strains 36, 37, 38 of the present invention, in which the three nucleotides outside the 5' end of the C-box (CACGCG), the three nucleotides outside the 3' end of the E-box (CACGTG), and the linker region between the C-box and E-box had all different nucleotide sequences. The treatment concentrations of methyl farnesenoate were 100 nM and 10 μM. The test method was in accordance with Test Example 1. The results are shown in Table 12.

[0124] [Table 12]

[0125] As shown in Table 12, it can be seen that the expression of the reporter gene was clearly strongly induced in the yeast strains 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 of the present invention in the presence of juvenile hormone (methyl farnesenoate). From these results, it became clear that even if the four bases between the C-box (CACGCG) and E-box (CACGCG) (linker region) are kmkk (where k means G or T, and m means A or C), for example, TATT in addition to GCGG, reporter activity for methyl farnesenoate, one of the juvenile hormones, can be evaluated with high sensitivity as long as the total length of the base sequence is 20 to 60 bases. Furthermore, even if the base sequence surrounding the base sequence shown in Sequence ID No. 1 is different from the base sequence shown in Sequence ID No. 2, reporter activity for juvenile hormone-like substances can be evaluated with similar high sensitivity.

Claims

1. A nucleic acid comprising a nucleotide sequence with a total length of 22 to 60 nucleotides, including the nucleotide sequence shown in Sequence ID No. 1, which interacts with the juvenile hormone receptor and has the function of positively regulating the expression of genes located in the 3' end direction of its sense strand at the transcriptional level.

2. The nucleic acid according to claim 1, characterized in that any four bases (nnnn) included in the base sequence shown in Sequence ID No. 1 are kmkk (where k means g or t, and m means a or c).

3. The nucleic acid according to claim 1, characterized in that any four bases (nnnn) included in the base sequence shown in Sequence ID No. 1 are GCGG or TATT.

4. The nucleic acid according to any one of claims 1 to 3, characterized in that it has a base sequence of a predetermined base length at the 3' end and 5' end of the base sequence shown in Sequence ID No. 1 above.

5. The nucleic acid according to claim 1, characterized in that it consists of one base sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 34, 37, 40, 43, 46, 49, and 52.

6. A vector comprising the nucleic acid described in claim 5.

7. A vector comprising the nucleic acid according to any one of claims 1 to 5, characterized in that the nucleic acid is a single unit and a plurality of units of the nucleic acid are linked together.

8. A vector comprising the nucleic acid according to any one of claims 1 to 5, characterized in that a reporter gene is included at the 3' end of the sense strand of the nucleic acid.

9. A transformant obtained by introducing the vector according to any one of claims 6 to 8 into a host.

10. The transformant according to claim 9, characterized in that a nucleic acid encoding a juvenile hormone receptor that interacts with the above vector is introduced.

11. The transformant according to claim 10, characterized in that the above-mentioned juvenile hormone receptor is a juvenile hormone receptor in arthropods.

12. The transformant according to claim 11, characterized in that the juvenile hormone receptor in the arthropod is a juvenile hormone receptor of a crustacean or insect.

13. The transformant according to claim 12, characterized in that the juvenile hormone receptor of the crustacean is the juvenile hormone receptor of Daphnia.

14. The transformant according to claim 13, characterized in that the juvenile hormone receptor of the above-mentioned Daphnia is the protein (a) or (b) below. (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 12 (b) A protein having juvenile hormone receptor transcription factor activity, comprising an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No.

12.

15. The transformant according to claim 9, further characterized by the introduction of a nucleic acid encoding a transcription co-factor.

16. The transformed organism according to claim 9, characterized in that the host is yeast.

17. A method for evaluating a test substance, comprising the steps of: contacting a transformant with a nucleic acid according to any one of claims 1 to 5, which is obtained by introducing a nucleic acid and a reporter gene to the 3' end of the sense strand of the nucleic acid into a host, and which expresses a juvenile hormone receptor and its transcription cofactor that interact with the nucleic acid; and measuring the expression of the reporter gene, wherein the interaction between the test substance and the juvenile hormone receptor is evaluated based on the expression level of the reporter gene.

18. The evaluation method according to claim 17, characterized in that if the expression level of the reporter gene increases after contact with the test substance, it is determined to be an agonist of the immature hormone receptor.

19. The evaluation method according to claim 17, characterized in that the above-mentioned 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 the juvenile hormone receptor that interact with the juvenile hormone receptor, and if the expression level of the reporter gene is lower compared to when the substance is brought into contact with the transformant alone, the test substance is determined to be an antagonist of the juvenile hormone receptor.

20. The evaluation method according to claim 17, characterized in that the above nucleic acid is considered as one unit, and multiple units of the above nucleic acid are linked together.

21. The evaluation method according to claim 17, characterized in that the transformant has been introduced with nucleic acid encoding the juvenile hormone receptor.

22. The evaluation method according to claim 17, characterized in that the above-mentioned juvenile hormone receptor is a juvenile hormone receptor in arthropods.

23. The evaluation method according to claim 22, characterized in that the juvenile hormone receptor in the arthropod is the juvenile hormone receptor of a crustacean or insect.

24. The evaluation method according to claim 23, characterized in that the juvenile hormone receptor of the crustacean is the juvenile hormone receptor of Daphnia.

25. The evaluation method according to claim 24, characterized in that the juvenile hormone receptor of the above-mentioned Daphnia is the protein of (a) or (b) below. (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 12 (b) A protein having juvenile hormone receptor transcription factor activity, comprising an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No.

12.

26. The evaluation method according to claim 17, characterized in that the above-mentioned transformant is further introduced with a nucleic acid encoding a transcription co-factor.

27. The evaluation method according to claim 17, characterized in that the host is yeast.

28. A kit for measuring endocrine disruptors, comprising the vector according to claims 6 to 8 or the transformant according to claim 9.

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