Male fish individual having suppressed functional expression of CYP19a1b, and method for producing male fish individual

Abstract

The present invention addresses the problem of providing a male fish individual having low aggressiveness. The present invention also address the problem of providing a male fish individual having higher growth efficiency. The present invention provides a male fish individual having suppressed functional expression of CYP19a1b (cytochrome P450, family 19, subfamily A, polypeptide 1b).

Description

Male fish in which functional expression of CYP19a1b is suppressed, and method for producing said male fish

[0001] The present specification discloses male fish in which the functional expression of CYP19a1b is suppressed, and a method for producing such male fish.

[0002] Non-patent document 1 describes cyp19a1b －／－ It has been reported that in XY Nile tilapia, 240 days after hatching, an increase in the gonadal somatic index is observed, but a decrease in the duct area index occurs.

[0003] Mol Reprod Dev. 2019;86:1224-1235.; DOI: 10.1002 / mrd.23237

[0004] Traditionally, farmed fish are either individuals equivalent to wild fish, for which breeding has not progressed, or varieties developed through selective breeding, where growth rate and other indicators are used. The former have extremely low market value because they lack any genetic traits that would improve production efficiency or commercial value through farming. The latter also require multiple generations of breeding, which requires a long period of time and relies on random mutations, resulting in low succession rates and very few commercially viable varieties. Therefore, traditional farmed fish have problems such as low survival rates due to fighting between individuals, slow growth, and limited yields of commercially valuable milt (testes). Male individuals, in particular, tend to be more aggressive than females due to the influence of androgen and other factors, and their aggressiveness increases during maturity. One objective of the present invention is to provide male fish with low aggressiveness. Another objective of the present invention is to provide male fish with better growth efficiency.

[0005] The present invention may include the following embodiments: Item 1. A male fish in which the functional expression of CYP19a1b (cytochrome P450 family 19 subfamily A polypeptide 1b) is suppressed. Item 2. The male fish according to Item 1, in which the functional expression is suppressed by introducing a loss-of-function mutation into the CYP19a1b gene. Item 3. Item 3. The male individual according to Item 2, wherein the loss-of-function mutation is introduced using at least one genome editing system selected from the group consisting of Clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9 (CRISPR / Cas9) system, Zinc Finger Nuclease (ZFN) system, and TAL effector nuclease (TALEN) system. Item 4. The male individual according to Item 2, wherein the loss-of-function mutation is introduced using radiation, a heavy ion beam, or a chemical mutagen. Item 5. Item 6. The male individual according to Item 1, wherein the suppression of functional expression is achieved by suppressing expression of the CYP19a1b gene, and the suppression of CYP19a1b gene expression is achieved by at least one RNA molecule selected from the group consisting of siRNA, shRNA, and miRNA that target CYP19a1b mRNA, or at least one selected from the group consisting of vectors capable of expressing the RNA molecule. Item 7. The male individual according to Item 1, wherein the male individual has at least one of the following phenotypes (a) to (c): (a) reduced aggressiveness compared to individuals with wild-type CYP19a1b function, (b) continued growth even after sexual maturity, and (c) increased testis weight per unit body weight compared to individuals with wild-type CYP19a1b function. Item 8. A male fish according to Item 1, wherein the suppression of functional expression is achieved by suppressing the function of the CYP19a1b protein, and the suppression of the function of the CYP19a1b protein is achieved by an antibody that binds to the CYP19a1b protein and suppresses the functional expression of the target protein through this binding. Item 9. A method for producing a male fish in which the functional expression of CYP19a1b is suppressed, comprising suppressing the functional expression of CYP19a1b (cytochrome P450 family 19 subfamily A polypeptide 1b) in a male fish.Item 9. The method according to Item 7, wherein the male individual has at least one of the following phenotypes (a) to (d): (a) decreased aggression compared to a control male individual having CYP19a1b function, (b) a higher rate of body weight gain after sexual maturity compared to a control male individual having CYP19a1b function, (c) an increased testis weight per unit body weight compared to a control male individual having CYP19a1b function, and (d) an increased number of robule structures in the testis compared to a control male individual having CYP19a1b function.

[0006] According to the present invention, it is possible to provide male fish individuals that are less aggressive and have higher growth efficiency.

[0007] The nucleotide sequence of the mutation site and the nucleotide sequence of the mutation site (SEQ ID NO: 1 is for the wild type, and SEQ ID NO: 2 is for the mutant type) are shown in Figure 1. The amino acid sequence of wild-type medaka CYP19a1b (SEQ ID NO: 3) and the amino acid sequence of the detected mutant CYP19a1b (SEQ ID NO: 4) are shown in Figure 1. The changes in body weight of male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -) are shown. The changes in body length of male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -) are shown. The results of evaluating aggressiveness of male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -) are shown. The testis weight of male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -) are shown. The testis weight per unit body weight of male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -) are shown. Longitudinal sections of male wild-type (+ / +) and homozygous mutant (- / -) testes are shown. The number of robule structures in the testes of male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -) testes is shown.

[0008] 1. Method for Producing Male Fish in Which Functional Expression of CYP19a1b is Suppressed

[0014] One embodiment of the present invention relates to a method for producing male fish in Which functional expression of CYP19a1b is suppressed. As used herein, the term "fish" is not particularly limited. As used herein, the term "fish" may include freshwater fish, saltwater fish, brackish water fish, diadromous fish, and other marine fish.

[0009] The freshwater fish may include fish of the family Oryziidae (medakas), family Cyprinidae, superfamily Ictaluroidea, and superfamily Siluroidea. The fish of the family Oryziidae may include fish of the genus Oryzias, such as killifish (Oryzias latipes, Oryzias sakaizumii), and Java killifish (Oryzias javanicus).

[0010] Examples of the fish of the Cyprinidae family include Honmoroko (Gnathopogon caerulescens), Silver carp (Hypophthalmichthys molitrix), Common carp (Cyprinus carpio), Grass carp (Ctenopharyngodon idellus), Bighead carp (Hypophthalmichthys nobilis), European crucian carp (Carassius carassius), Catla (Cyprinus catla), Japanese bluefin tuna (Mylopharyngodon piceus), Kenhi (Cirrhinus molitorella), Mrigal carp (Cirrhinus cirrhosus), Catla (Catla catla), Rohita (Labeo rohita), and Slenderhead bream (Megalobrama amblycephala).

[0011] The fish of the superfamily Ictaluroidea may include, for example, channel catfish (Ictalurus punctatus), blue catfish (Ictalurus furcatus), and the like.

[0012] The fish of the superfamily Siluroidea may include, for example, catfish (Silurus asotus), Biwa catfish (Silurus biwaensis), rock catfish (Silurus lithophilus), European catfish (Silurus glanis), longfin catfish (Clarias fuscus), walking catfish (Clarias batrachus), and the like.

[0013] Examples of the marine fish include fishes from the family Paralichthys, Tetraodontidae (puffers), Ostraciidae (boxfishes), Sparidae (sea breams and porgies), Serranidae (sea basses), Monacanthidae, Scombridae, Pleuronectidae, Carangidae, Lateolabrax, Moronidae, Rachycentridae, Cynoglossidae, Conger eels (Congridae), and Gadidae. Examples of the brackish water fish include fishes from the family Latidae. The diadromous fish may include fish from the Salmonidae, Osmeridae, and Anguillidae families.

[0014] Examples of fish of the family Paralichthys include Paralichthys olivaceus (TEMMINCK et SCHLEGEL), fish of the genus Paralichthys (such as sole, Pacific flounder, Korean flounder, Japanese flounder, Pacific flounder, Taiwan flounder, etc.), sole of the genus Paralichthys, etc., sole of the genus Paralichthys, flathead flounder of the genus Paralichthys, small flounder of the genus Paralichthys, and small flounder of the genus Paralichthys. Paralichthys olivaceus (TEMMINCK et SCHLEGEL) is preferred as a fish of the family Paralichthys.

[0015] The fish of the Tetraodontidae family may include, for example, fish belonging to the genus Takifugu (such as Takifugu rubripes, Takifugu porphyreus, and Takifugu niphobles), and the white-spotted pufferfish (Lagocephalus wheeleri) belonging to the genus Lagocephalus. A preferred fish of the Tetraodontidae family is Takifugu rubripes. The fish of the Picodontidae family may include, for example, the boxfish (Ostracion immaculatus) belonging to the Picodontidae genus.

[0016] Examples of fish of the Sparidae family include fish belonging to the Pagrus genus (Pagrus major, Pagrus auratus, etc.), fish belonging to the Acanthopagrus genus (Acanthopagrus schlegelii, Acanthopagrus latus, etc.), Dentex genus (Dentex tumifrons, etc.), and Sparus genus (Sparus, Sparus aurata, etc.).

[0017] Examples of the fish of the family Epinephelus include fishes belonging to the genus Epinephelus (Epinephelus septemfasciatus, Epinephelus bruneus, Epinephelus akaara, Epinephelus malabaricus, Epinephelus aeneus, Epinephelus amblycephalus, Epinephelus areolatus, Epinephelus bleekeri, Epinephelus bontoides, Epinephelus chlorostigma, Epinephelus coiodes, Epinephelus fasciatus, Epinephelus fuscoguttatus ... labriformis), grouper (Epinephelus lanceolatus), white-spotted grouper (Epinephelus maculatus), yellowtail grouper (Epinephelus malabricus), dusky grouper (Epinephelus marginatus), spotted grouper (Epinephelus ongus), spotted grouper (Epinephelus polyphekadion), brown grouper (Epinephelus quoyanus), black-finned grouper (Epinephelus sexfasciatus), Nassau grouper (Epinephelus striatus), single-spotted grouper (Epinephelus tauvina), potato grouper (Epinephelus tukula), etc.), grouper (Cromileptes altivelis), etc. belonging to the grouper genus), leopard grouper (Plectropomus leopardus), etc. belonging to the grouper genus, and hybrids between fishes of the family Serranidae.

[0018] Examples of fish of the Filefish family include Stephanolepis cirrhifer, which belongs to the Stephanolepis genus, and Thamnaconus modestus, which belongs to the Thamnaconus genus.

[0019] Examples of the fish of the Scombrini family include fishes belonging to the genus Scombrini (such as chub mackerel (Scomber japonicus), Atlantic mackerel (Scomber scombrus), and bluefin tuna (Scomber australasicus)), fishes belonging to the genus Thunnini (such as Pacific bluefin tuna (Thunnus orientalis), Atlantic bluefin tuna (Thunnus orientalis), southern bluefin tuna (Thunnus maccoyii), bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares), albacore tuna (Thunnus alalunga), and longfin tuna (Thunnus tonggol)), and fishes belonging to the genus Euthynnus (such as Euthynnus affinis and Euthynnus tonggol). alletteratus, etc.), skipjack tuna (Katsuwonus pelamis, etc.) belonging to the genus Katsuwonus, fish belonging to the genus Scomberomarini, fish belonging to the genus Auxis, fish belonging to the genus Sardini, fish belonging to the genus Gymnosarda, etc.

[0020] The fish of the family Pleuronectidae may include, for example, flathead flounder (Pseudopleuronectes herzensteini), marbled flounder (Pleuronectes yokohamae), stone flounder (Kareius bicoloratus), Pacific halibut (Hippoglossus stenolepis), and barfin flounder (Verasper moseri).

[0021] The fish of the Carangidae family may include, for example, fish belonging to the Seriola genus (such as Seriola dumerili, Seriola lalandi, Seriola rivoliana, and Seriola quinqueradiata), fish belonging to the Pseudocaranx genus (such as Japanese horse mackerel (Trachurus japonicus) and Japanese striped jack (Pseudocaranx dentex)), and fish belonging to the Trachinotus genus (such as Japanese jack mackerel).

[0022] The fish of the family Perciformes (Lateolabrax) may include, for example, blackfin sea bass (Lateolabrax latus) and Japanese sea bass (Lateolabrax maculatus). The fish of the family Moronidae may include, for example, European sea bass (Dicentrarchus labrax). The fish of the family Rachycentridae may include, for example, Japanese cedar (Rachycentron canadum).

[0023] The fish of the family Cynodontidae may include, for example, the red tongue sole (Cynoglossus joyneri), the orange tongue sole (Cynoglossus semilaevis), etc. The fish of the family Conger eel may include, for example, the Japanese conger eel (Conger myriaster), the Japanese black conger eel (Conger japonicus), etc.

[0024] Protogadiidae fish may include, for example, Pacific cod (Gadus macrocephalus), Atlantic cod (Gadus morhua), Alaska pollock (Gadus chalcogrammus), and the like.

[0025] The fish of the family Latidae may include, for example, fish of the genus Lates, such as barramundi (Lates calcarifer) and Nile perch (Lates niloticus).

[0026] Examples of the fish of the Salmonidae family include fish belonging to the genus Salmon (rainbow trout (Oncorhynchus mykiss), king salmon (Oncorhynchus tshawytscha), cherry salmon (Oncorhynchus masou), Japanese salmon (Oncorhynchus masou), Japanese jack masu (Oncorhynchus kawamurae), pink salmon (Oncorhynchus gorbuscha), and salmon (Oncorhynchus keta)), fish belonging to the genus Salmon (brown trout (Salmo trutta), sockeye salmon (Oncorhynchus nerka), coho salmon (Oncorhynchus kisutch), and Atlantic salmon (Salmo salar)), and fish belonging to the genus Salvelinus (Doll vardendorff trout (Salvelinus malma), Arctic char (Salvelinus alpinus), and Japanese char (Salvelinus leucomaenis), brook trout (Salvelinus fontinalis), lake trout (Salvelinus namaycush), etc.), and the huchen (Parahucho perryi) belonging to the genus Huchen.

[0027] The fish of the Smeltidae family may include, for example, the sweetfish (Plecoglossus altivelis) belonging to the Plecoglossinae subfamily, fish of the Hypomesinae subfamily (Hypomesus nipponensis, Hypomesus japonicus, etc.), and fish of the Osmerinae subfamily (Osmerinae) (Osmerus mordax dentex, Spirinchus lanceolatus, Spirinchus lanceolatus, etc.).

[0028] The fish of the family Anguillididae can include, for example, the Japanese eel (Anguilla japonica), the European eel (Anguilla anguilla), etc. CYP19a1b is an abbreviation for cytochrome P450 family 19 subfamily A polypeptide 1b.

[0029] For example, the mRNA sequence of CYP19a1b from medaka (Oryzias latipes) has been registered as NCBI Reference Sequence: AB591736. For example, the mRNA sequence of CYP19a1b from tiger pufferfish (Takifugu rubripes) has been registered as NCBI Reference Sequence: NM_001173496. Specific examples of CYP19a1b from fish include the CYP19a1b genes shown in Table 1 below.

[0030]

[0031] The method for inhibiting the functional expression of CYP19a1b is not limited, and can be achieved by, for example, inhibiting the expression of the CYP19a1b gene or inhibiting the function of the CYP19a1b protein.

[0032] The method for introducing a loss-of-function mutation into CYP19a1b is not limited as long as it can introduce a loss-of-function mutation into the target gene in the genome of the target fish. Such methods are known. For example, methods for introducing a loss-of-function mutation include site-specific mutagenesis methods such as genome editing and homologous recombination; random mutagenesis, etc.

[0033] Genome editing methods include methods of introducing proteins and nucleic acids constituting genome editing technology, or vectors encoding them. Examples of the proteins include CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzymes. Specifically, examples of the CRISPR enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, and Cs Examples of the nucleic acid include Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4. Examples of the nucleic acid include crRNA and tracrRNA, or single-stranded nucleic acids in which these are linked via a linker. In this case, the nucleic acid is designed, for example, so that the base sequence that anneals to the target sequence in crRNA is complementary to the base sequence encoding CYP19a1b. One type of nucleic acid may be used alone, or two or more types may be used in combination. Genome editing systems using CRISPR enzymes include the Clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9 (CRISPR / Cas9) system. Other genome editing systems include at least one method selected from the CompoZr Zinc Finger Nuclease (ZFN) system and the TAL effector nuclease (TALEN) system. Preferably, the CRISPR / Cas9 system introduces Cas9 as mRNA or protein, and gRNA as sgRNA or crRNA and tracrRNA.Furthermore, in a CRISPR / Cas9 system using a vector, the nucleic acid encoding CRISPR and the nucleic acid encoding Cas9 may be on different vectors or may be on a single vector. The promoter for driving CRISPR to function is not particularly limited, but the U6 promoter is preferred. The promoter for driving Cas9 to function is not particularly limited, but a promoter expressed in mammalian cells, such as a cytomegalovirus promoter, is preferred. Commercially available vectors such as the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector can be used as the CRISPR / Cas9 system.

[0034] The sequence (hereinafter also referred to as "target sequence") that targets the CYP19a1b gene and is incorporated into the CRISPR sequence is not limited as long as it is incorporated into a guide RNA (also referred to as gRNA, sgRNA, or crRNA) by the CRISPR / Cas9 system and transcribed, or is introduced into a cell as a guide RNA containing a sequence complementary to the target sequence to recombine the CYP19a1b gene. Generally, it is said that the target sequence can be a sequence of about 20 bases upstream of the 5' side of the base sequence "NGG" (PAM sequence: N is any of the bases A, G, T, or C) present in the CYP19a1b gene. The target sequences were identified using the Optimized CRISPR design tool (Massachusetts Institute of Technology, ZhangLab webpage (http: / / crispr.mit.edu / )), E-CRISP (http: / / www.e-crisp.org / E-CRISP / (German Cancer Research Center)), ZiFiT Targeter (http: / / zifit.partners.org / ZiFit / (Zing Finger Consortium)), and Cas9 design (http: / / cas9.cbi.pku.edu.cn (Peking University)), CRISPRdirect (http: / / crispr.dbcls.jp (University of Tokyo)), CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / (Hua Zhong Agricultural University)), CRISPR RGEN Tools (http: / / www.rgenome.net / (Seoul National University)) and the like. It can be designed using a known design tool published in.

[0035] In addition, if single-nucleotide polymorphisms (SNPs) exist in the PAM sequence, it is preferable to avoid such sequences. When the SNPs of an individual are known for the target sequence, it is preferable to optimize the sequence for each SNP. The 5'-terminal region of the target sequence may be shortened by 1, 2, 3, or 4 bases, preferably 1, 2, or 3 bases.

[0036] The CRISPR / Cas9 system may be introduced into cells as a vector, or may be introduced into cells in combination with artificially synthesized or in vitro transcribed gRNA, crRNA, trans-activating crRNA (tracrRNA), and RNA encoding Cas9. Alternatively, the Cas9 protein and guide RNA may be combined and introduced into cells.

[0037] Furthermore, in the genome editing system, donor oligo DNA such as single-stranded oligonucleotides (ssODNs) may be co-introduced. ssODNs can be designed according to known methods.

[0038] The genome editing system can be injected by microinjection into the cytoplasm of a fertilized egg, preferably a one-cell stage fertilized egg. For example, when introducing Cas9 protein, it can be injected in a range of about 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably about 10 pg to 50 pg per fertilized egg. In this case, the guide RNA can be injected in a range of about 0.1 pg to 50 pg, preferably 0.5 pg to 20 pg, and more preferably about 1 pg to 5 pg. For a method of introducing a mutation using genome editing technology, see, for example, Example 1 described below.

[0039] Examples of random mutagenesis methods include irradiation treatment with α-rays, β-rays, γ-rays, X-rays, etc.; chemical treatment with chemical mutagens such as ethyl methanesulfonate (EMS) and ethynylnitrosourea (ENU); heavy ion beam treatment, etc. By employing, for example, the tiling method as a random mutagenesis method, it is possible to efficiently induce mutations using chemical mutagens and screen for mutations.

[0040] Methods for suppressing the expression of the CYP19a1b gene include introducing at least one selected from the group consisting of an RNA molecule targeting CYP19a1b mRNA or a vector capable of expressing the RNA molecule. The term "RNA molecule targeting CYP19a1b mRNA" is not limited as long as it targets CYP19a1b mRNA and can suppress the expression of the CYP19a1b protein. Examples include siRNA, shRNA, dsRNA, miRNA, and other molecules that degrade target mRNA and / or those that suppress the translation of target mRNA. The sequences of these RNA molecules can be appropriately designed by those skilled in the art using known methods based on the base sequence information of the target gene. Furthermore, the RNA molecule may be prepared using known methods, or commercially available molecules may be used. Preferred RNA molecules include siRNA, shRNA, and miRNA, with siRNA and shRNA being particularly preferred. The vector capable of expressing the RNA molecule targeting the CYP19a1b mRNA is not particularly limited as long as it can express an RNA molecule that inhibits the expression of the CYP19a1b protein in an individual's body or cells. Examples include hairpin RNA expression vectors. A hairpin RNA expression vector includes at least a sense strand DNA sequence downstream of a promoter sequence suitable for expressing short-stranded RNA, such as a U6 promoter, that has the same sequence as the sense strand of the target mRNA (except that uracil in the mRNA is replaced with thymine); a loop sequence that forms a loop structure after transcription; an antisense strand DNA sequence that can bind in whole or in part to the sense strand DNA sequence in a complementary manner; and a terminator sequence. Examples of vectors include plasmid vectors, adenoviral vectors, retroviral vectors, and lentiviral vectors.

[0041] At least one RNA molecule selected from the group consisting of siRNA, shRNA, and miRNA can be injected by microinjection in a range of about 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably about 10 pg to 50 pg per fertilized egg. At least one RNA molecule selected from the group consisting of siRNA, shRNA, and miRNA, or a vector capable of expressing said RNA molecule, can be injected by microinjection in a range of about 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably about 10 pg to 50 pg per fertilized egg. The vector can be linearized as necessary.

[0042] The inhibition of protein function can be carried out using at least one antibody selected from the group consisting of antibodies that bind to CYP19a1b and inhibit the functional expression of the target protein through this binding. The binding is preferably specific. The antibody may be either a polyclonal antibody or a monoclonal antibody. Both polyclonal and monoclonal antibodies can be prepared by those skilled in the art using known methods. The antibody may also be a Fab, F(ab) or other antibody. ２ The antibody may be an antibody fragment such as a diabody, scFv, minibody, peptibody, mimetibody, etc. The antibody can be injected in the range of about 5 pg to 100 pg, preferably 10 pg to 80 pg, more preferably 10 pg to 50 pg per fertilized egg.

[0043] Larvae and fry hatched from fertilized eggs are raised to larvae or fry, adult fish, and parent fish, and individuals in which mutations have been induced in the CYP19a1b gene are selected by mutation analysis methods, such as heteroduplex mobility analysis, Cel 1 assay, T7 endonuclease assay, sequence analysis, etc. The rearing conditions can be the same as those for general fish farming. Furthermore, food generally given in aquaculture can be used. 2. Male fish in which the functional expression of CYP19a1b is suppressed Male fish in which the functional expression of CYP19a1b is suppressed have, for example, at least one of the following phenotypes (a) to (c): (a) reduced aggression compared to control male individuals with CYP19a1b function, (b) a higher rate of weight gain after sexual maturity compared to control male individuals with CYP19a1b function, (c) an increased testis weight per unit body weight compared to control male individuals with CYP19a1b function, and (d) an increased number of robule structures in the testes compared to control male individuals with CYP19a1b function.

[0044] Here, "CYP19a1b functional expression is suppressed" means that the function of CYP19a1b derived from both alleles of the CYP19a1b gene on the chromosome is suppressed. Such a male individual is also referred to as a "CYP19a1b function-suppressed male individual." Furthermore, a male individual in which the function of CYP19a1b derived from one allele of the CYP19a1b gene on the chromosome is suppressed is also referred to as a "CYP19a1b function-suppressed male individual."

[0045] Control male individuals having CYP19a1b function (also simply referred to as "control male individuals") may include wild-type male individuals and / or CYP19a1b semi-function-suppressed male individuals.

[0046] Aggression can be evaluated, for example, by at least one aggressive behavior selected from chasing, threatening, parallel positioning, striking, and biting. Aggressive behavior can be evaluated by placing at least two male individuals in an aquarium and counting the number of times the above-mentioned aggressive behaviors are performed within a predetermined period of time (e.g., about 20 to 60 minutes). "Decreased aggression" means that the number of aggressive behaviors in CYP19a1b function-suppressed male individuals is 1 / 2 or less, 1 / 5 or less, 1 / 10 or less, 1 / 20 or less, 1 / 30 or less, 1 / 50 or less, or 1 / 100 or less compared to control male individuals.

[0047] The number of attacks is preferably evaluated statistically by counting the number of attacks for a control male population including two or more individuals and a CYP19a1b function-suppressed male population including two or more individuals.

[0048] The rate of weight gain after sexual maturity is calculated by using the weight at the average age at sexual maturity in weeks or months for each fish species as the standard (hereinafter also referred to as "standard weight") and calculating the amount of weight gain.

[0049] The phrase "the weight gain rate of a male individual in which CYP19a1b function is suppressed is high" means that the weight gain rate is 1.1 times or more, 1.2 times or more, 1.3 times or more, or 1.4 times or more compared to a control male individual.

[0050] The body weight gain rate is preferably evaluated statistically by counting a control male population including 2 or more individuals and a CYP19a1b function-suppressed male population including 2 or more individuals. The testis weight per unit body weight can be calculated from the body weight of the individual before castration and the weight of the testis excised from the individual.

[0051] "The testis weight per unit body weight of a CYP19a1b function-suppressed male individual is increased" means that the testis weight per unit body weight is 1.1 times or more, 1.2 times or more, 1.3 times or more, or 1.4 times or more compared to that of a control male individual. The number of robule structures in the testis can be counted by analyzing tissue sections of the excised testis.

[0052] The phrase "the number of robule structures in the testes of a male individual with suppressed CYP19a1b function is increased" means that the testis weight per unit body weight is 1.1 times or more, 1.2 times or more, 1.3 times or more, or 1.4 times or more compared to a control male individual.

[0053] The following examples will be used to explain the present invention in more detail, but the present invention should not be construed as being limited to these examples.

[0054] I. Creation of Cyp19a1b-deficient medaka and observation of male growth, aggressiveness, and maturation 1. Creation of medaka with a loss-of-function mutation in the CYP19a1b gene (1) Creation of fertilized eggs Fertilized eggs were obtained by placing sexually mature medaka in the same tank and allowing them to mate naturally. The collected eggs were used to remove any threads adhering to the egg membrane using tweezers or the like, and then used for mutagenesis.

[0055] (2) Mutation Introduction: CYP19a1b loss-of-function medaka were generated by the tiling method. A library of 5,760 medaka (Taniguchi et al. https: / / genomebiology.biomedcentral.com / articles / 10.1186 / gb-2006-7-12-r116) chemically mutagenized with N-ethyl-N-nitrosourea (ENU) was screened for mutations in exons 3, 4, and 5 of CYP19a1b by direct sequencing. Figure 1A shows the nucleotide sequence of the mutation site and the nucleotide sequence of the mutation site (SEQ ID NO: 1 for wild-type and SEQ ID NO: 2 for mutant). Figure 1B shows the amino acid sequence of wild-type medaka CYP19a1b (SEQ ID NO: 3) and the amino acid sequence of the detected mutant CYP19a1b (SEQ ID NO: 4). An F0 clone carrying a nonsense mutation (K105*) (* denotes a stop codon) in exon 4 was identified and backcrossed with the wild-type medaka d-rR strain for six or more generations to eliminate background mutations. Male and female heterozygotes were mated to produce wild-type, heterozygote, and homozygote siblings, which were used in the experiments. DNA extracted from the caudal fins of the medaka was analyzed, and the nucleotide sequences of individuals with loss-of-function mutations introduced into the CYP19a1b gene were analyzed by PCR and nucleotide sequence analysis. The loss-of-function mutation in the CYP19a1b gene was a single-base substitution (A>T) lineage. The nucleotide sequence of the wild-type CYP19a1b gene is shown in SEQ ID NO:5, and the nucleotide sequence of the loss-of-function mutation in the CYP19a1b gene is shown in SEQ ID NO:6 in the sequence list described below.

[0056] Hereinafter, an individual having a loss-of-function mutation in one allele of the CYP19a1b gene may be simply referred to as a heterozygous mutant. Furthermore, an individual having loss-of-function mutations in both alleles of the CYP19a1b gene may be simply referred to as a homozygous mutant. Furthermore, a wild type may be referred to as "+ / +", a heterozygous mutant as "+ / -", and a homozygous mutant as "- / -".

[0057] 2. Phenotypes of wild-type, heterozygous mutant, and homozygous mutant (1) Growth Growth curves of wild-type, heterozygous mutant, and homozygous mutant are shown in Figure 2. Figure 2A shows the change in body weight. Figure 2B shows the change in body length. In Figure 2, white circles represent the results for wild-type medaka, gray circles represent the results for heterozygous mutant medaka, and black circles represent the results for homozygous mutant medaka. In addition, the black inverted triangle in Figure 2 indicates the onset of sexual maturity.

[0058] As shown in Figure 2A, male wild-type medaka and heterozygous mutants showed a slowdown in body weight gain after about 9 weeks of age, while the homozygous mutants continued to increase their body weight even after 9 weeks of age. Furthermore, as shown in Figure 2B, male homozygous mutants also showed a tendency to increase in body length compared to wild-type and heterozygous mutants. Generally, in medaka, male growth slows after the onset of sexual maturity, while females continue to grow at the same pace. As a result, females eventually become significantly larger. It was shown that male homozygous mutants did not show a slowdown in growth even after sexual maturity. This demonstrates that loss-of-function mutations in both alleles of the CYP19a1b gene promote male growth.

[0059] (2) Aggression Male wild-type, heterozygous, and homozygous mutant mice were grouped into groups of four, and each group was placed in an independent tank and observed for aggression for 30 minutes. The aggression was evaluated by chasing, threatening, parallel positioning, striking, and biting, and the number of occurrences of each behavior was recorded.

[0060] The results are shown in Figure 3. In Figure 3, white bars indicate wild-type, gray bars indicate heterozygous mutants, and black bars indicate homozygous mutants. The dot plots in each graph show raw data. In Figure 3, * indicates a significant difference of p<0.05, and ** indicates a significant difference of p<0.01.

[0061] As is clear from Figure 3, the homozygous mutant showed almost no aggressive behavior in any of the items examined. This phenomenon was significant compared to wild-type and heterozygous mutants, especially wild-type medaka. However, no such difference in aggressiveness was observed in females.

[0062] These results suggest that loss-of-function mutations in both alleles of the CYP19a1b gene may reduce or eliminate male aggression.

[0063] The phenotype of male gonadal tissue was observed. Evaluation items were testis weight, testis weight per unit body weight, and tissue evaluation (morphological observation of tissue, number of lobule structures in the testis).

[0064] The results are shown in Figure 4. In Figure 4, white bars indicate wild-type mice, gray bars indicate heterozygous mutants, and black bars indicate homozygous mutants. The dot plots in each graph show raw data. ** indicates a significant difference of p<0.01, and *** indicates a significant difference of p<0.001. Figure 4A shows testis weight, and Figure 4B shows testis weight per unit body weight. For both evaluation items, the homozygous mutants showed an increase compared to the wild-type and heterozygous mutants. These results indicated that loss-of-function mutations in both alleles of the CYP19a1b gene not only increased male growth but also led to enlargement of the testes themselves.

[0065] Figure 4C shows longitudinal cross sections of wild-type and homozygous mutant testes. The homozygous mutant testes were significantly larger than the wild-type testes.

[0066] Figure 4D shows the number of robule structures in the testes of wild-type, heterozygous, and homozygous mutants. The number of robule structures in the testes of homozygous mutants was significantly increased compared to the wild-type and heterozygous mutants.

[0067] These results suggest that the loss-of-function mutation in both alleles of the CYP19a1b gene is expected to increase growth and testicular enlargement in male fish. Furthermore, the reduced aggressiveness may prevent multiple fish from harming each other, even when reared in a single tank, improving farming efficiency.

[0068] II. Creation of CYP19a1b-deficient pufferfish and observation of male growth, aggressiveness, and maturation 1. Creation of pufferfish with a deletion mutation in the CYP19a1b gene (1) Creation of fertilized eggs Eggs (unfertilized eggs) and sperm were collected from sexually mature male and female pufferfish by compressing their abdomens. The eggs and sperm obtained were artificially inseminated to obtain fertilized eggs.

[0069] (2) Introduction of Mutations Cas9 protein and guide RNA were used to introduce mutations. The sequence of the target gene is 5'-GCTGGAGTGTATCGGGGATGGAGG-3' (SEQ ID NO: 7). A solution prepared by microinjecting 2-10 pg Cas9 protein and 1-5 pg guide RNA into the cytoplasm of a one-cell stage fertilized egg was used to introduce a mutation into the Cyp19a1b gene. The fertilized eggs introduced with the Cas9 protein and guide RNA were reared under normal rearing conditions and hatched to obtain the F0 generation.

[0070] (3) Observation of male growth performance in Cyp19a1b-deficient trough mice It was confirmed that the introduction of the above mutation improved male growth performance.

[0071] (4) Observation of male aggressiveness in Cyp19a1b-deficient tiger pufferfish It was confirmed that the introduction of the above mutation suppressed male aggressiveness.

[0072] (5) Confirmation of male maturation in Cyp19a1b-deficient tiger pufferfish It was confirmed that the introduction of the above mutation promoted male maturation.

[0073] These results suggest that introducing a deletion mutation into the Cyp19a1b gene of tiger pufferfish will enable the production of male tiger pufferfish with larger bodies, particularly larger milt. Furthermore, because they are not aggressive, there is no need for tooth clipping, which will improve aquaculture efficiency and commercial value.

[0074]

Claims

1. A male fish in which the functional expression of CYP19a1b (cytochrome P450 family 19 subfamily A polypeptide 1b) is suppressed.

2. The male individual described in claim 1, wherein the suppression of functional expression is achieved by introducing a loss-of-function mutation in the CYP19a1b gene.

3. The male individual described in claim 2, wherein the loss-of-function mutation is introduced using at least one genome editing system selected from the clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9 (CRISPR / Cas9) system, the zinc finger nuclease (ZFN) system, and the TAL effector nuclease (TALEN) system.

4. The male individual according to claim 2, wherein the loss-of-function mutation is introduced by radiation, heavy ion beam, or chemical mutagen.

5. The male individual described in claim 1, wherein the inhibition of functional expression is achieved by suppressing expression of the CYP19a1b gene, and the suppression of expression of the CYP19a1b gene is achieved by at least one RNA molecule selected from the group consisting of siRNA, shRNA and miRNA targeting CYP19a1b mRNA, or at least one vector selected from the group consisting of vectors capable of expressing the RNA molecule.

6. A male individual as described in claim 1, having at least one of the following phenotypes (a) to (c): (a) reduced aggression compared to individuals with wild-type CYP19a1b function; (b) continued growth after sexual maturity; and (c) increased testis weight per unit body weight compared to individuals with wild-type CYP19a1b function.

7. The male individual described in claim 1, wherein the inhibition of functional expression is achieved by inhibiting the function of CYP19a1b protein, and the inhibition of the function of CYP19a1b protein is achieved by an antibody that binds to CYP19a1b protein and inhibits the functional expression of its target by said binding.

8. A method for producing a male fish in which the functional expression of CYP19a1b (cytochrome P450 family 19 subfamily A polypeptide 1b) is suppressed, the method comprising suppressing the functional expression of CYP19a1b in a male fish.

9. The method of claim 7, wherein the male individual has at least one of the following phenotypes (a) to (d): (a) decreased aggression compared to control male individuals having CYP19a1b function; (b) a higher rate of weight gain from sexual maturity compared to control male individuals having CYP19a1b function; (c) increased testis weight per unit body weight compared to control male individuals having CYP19a1b function; and (d) an increased number of robure structures in the testes compared to control male individuals having CYP19a1b function.