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

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

Technical Field

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

Background Art

[0002] Non-Patent Document 1 describes that in XY Nile tilapia, although an increase in gonadal somatic index is observed 240 days after hatching, a decrease in duct area index occurs. - / - XY Nile tilapiaにおいて、孵化から240日後に、gonadal somatic indexの上昇が認められるものの、duct area indexの減少が起こることが記載されている。

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Conventional cultured fish use individuals equivalent to wild fish with no advanced breeding, or varieties produced by selective breeding that are subcultured using growth rate etc. as an index from within a population. The former has no genetic traits for improving production efficiency or commercial value in aquaculture, so its market value is extremely low. The latter also requires subculturing for several generations for variety production, which takes a long time and depends on accidental mutations, so the accuracy of variety establishment is low and very few varieties have been put into practical use. Therefore, conventional cultured fish have problems such as low survival rate due to competition between individuals, slow growth, and only a limited amount of high-value white fish (testes) can be obtained. In particular, male individuals tend to be more aggressive than female individuals due to the influence of male hormones etc., and their aggressiveness tends to increase during the maturation period. One objective of this invention is to provide male fish with low aggression. Another objective of this invention is to provide male fish with better growth efficiency. [Means for solving the problem]

[0005] The present invention may include the following embodiments. Item 1. Male fish individuals in which the functional expression of CYP19a1b (cytochrome P450 family 19 subfamily A polypeptide 1b) is suppressed. Item 2. Male individuals as described in Item 1, in which the suppression of functional expression is achieved by introducing a loss-of-function mutation in the CYP19a1b gene. Item 3. Male individuals as described in Item 2, wherein the introduction of a loss-of-function mutation is performed by 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. Item 4. Male individuals as described in Item 2, in which a loss-of-function mutation is introduced by radiation, heavy ion beam, or chemical mutagens. Item 5. A male individual as described in Item 1, wherein the suppression of functional expression is carried out by suppressing the expression of the CYP19a1b gene, and the suppression of the CYP19a1b gene expression is carried out 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 a vector capable of expressing said RNA molecule. Item 6. Male individuals as described in Item 1 having at least one of the following phenotypes (a) to (c): (a) reduced aggression compared to individuals with wild-type CYP19a1b function, (b) continuing to grow beyond sexual maturity. (c) Compared to individuals with wild-type CYP19a1b function, testicular weight per unit body weight is increased. Item 7. A male individual as described in Item 1, wherein the suppression of functional expression is carried out by suppressing the function of the CYP19a1b protein, and the suppression of the function of the CYP19a1b protein is carried out by an antibody that binds to the CYP19a1b protein and suppresses the functional expression of the target by binding. Item 8. A method for producing a male fish individual 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 individual. Item 9. The method according to item 7, wherein a male individual has at least one of the following phenotypes (a) to (d): (a) Compared to control males with CYP19a1b function, aggression is reduced; (b) Compared to control males with CYP19a1b function, the rate of weight gain after sexual maturity is higher. (c) Compared to control male individuals with CYP19a1b function, testicular weight per unit body weight is increased. (d) Compared to control male individuals with CYP19a1b function, the number of robule structures in the testes is increased. [Effects of the Invention]

[0006] According to the present invention, it is possible to provide male fish with low aggression. Furthermore, according to the present invention, it is possible to provide male fish with better growth efficiency. [Brief explanation of the drawing]

[0007] [Figure 1A] The nucleotide sequences of the mutated site and the nucleotide sequences of the mutated site (SEQ ID NO: 1 is the wild type, SEQ ID NO: 2 is the mutant type) are shown. [Figure 1B] The amino acid sequence of CYP19a1b in wild-type medaka (SEQ ID NO: 3) and the amino acid sequence of the detected mutant CYP19a1b (SEQ ID NO: 4) are shown below. [Figure 2A] This shows the changes in body weight for male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -). [Figure 2B]This shows the changes in body length between wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -) males. [Figure 3] The results of the aggression evaluation for male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -) individuals are shown. [Figure 4A] The testicular weights of male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -) individuals are shown. [Figure 4B] The graph shows the testicular weight per unit body weight for male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -). [Figure 4C] The figure shows longitudinal sections of the testes of male wild-type (+ / +) and homozygous mutant (- / -) individuals. [Figure 4D] The number of roburetic structures in the testes of male wild-type (+ / +), heterozygous mutant (+ / -), and homozygous mutant (- / -) individuals is shown. [Modes for carrying out the invention]

[0008] 1. Method for producing male fish individuals in which the functional expression of CYP19a1b is suppressed. One embodiment of the present invention relates to a method for producing male fish in which the functional expression of CYP19a1b is suppressed. In this specification, the term "fish" is not particularly limited. In this specification, "fish" may include freshwater fish, saltwater fish, brackish water fish, and marine fish such as circadian fish.

[0009] The aforementioned freshwater fish may include fish of the families Oryziidae (medakas), Cyprinidae (cyprinidae), Ictaluroidea (Ictaluroidea), and Siluroidea (Siluroidea). The aforementioned fish of the family Oryziidae may include, for example, fish of the genus Oryzias, such as the Japanese rice fish (Oryzias latipes, Oryzias sakaizumii) and the Javan rice fish (Oryzias javanicus).

[0010] The Cyprinidae fish may include, for example, Gnathopogon caerulescens, Hypophthalmichthys molitrix, Cyprinus carpio, Ctenopharyngodon idellus, Hypophthalmichthys nobilis, Carassius carassius, Cyprinus catla, Mylopharyngodon piceus, Cirrhinus molitorella, Cirrhinus cirrhosus, Catla catla, Labeo rohita, Megalobrama amblycephala, etc.

[0011] The Ictaluroidea fish may include, for example, Ictalurus punctatus, Ictalurus furcatus, etc.

[0012] The Siluroidea fish may include, for example, Silurus asotus, Silurus biwaensis, Silurus lithophilus, Silurus glanis, Clarias fuscus, Clarias batrachus, etc.

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

[0014] The fish of the family Paralichthys may include, for example, Paralichthys olivaceus (TEMMINCK et SCHLEGEL), fish belonging to the genus Megalobrama (Megalobrama, Megalobrama nantoensis, Megalobrama skolops, Megalobrama terminalis, Megalobrama tamensis, Megalobrama amblycephala, Megalobrama formosana, etc.), the alaskan halibut belonging to the genus Anoplopoma fimbria, the silver-sided flounder belonging to the genus Atheresthes stomias, the sand sole belonging to the genus Psettichthys melanostictus, etc. Preferably, the fish of the family Paralichthys is Paralichthys olivaceus (TEMMINCK et SCHLEGEL).

[0015] The aforementioned pufferfish may include, for example, fish belonging to the genus Takifugu (Takifugu rubripes, Takifugu porphyreus, Takifugu niphobles, etc.) and fish belonging to the genus Lagocephalus (Lagocephalus wheeleri, etc.). Preferably, the pufferfish of the family Takifugu is Takifugu rubripes. Fish of the family Ostracidae may include, for example, the boxfish (Ostracion immaculatus), which belongs to the genus Ostracion.

[0016] The aforementioned fish of the Sparidae family may include, for example, fish belonging to the genus Pagrus (such as red sea bream (Pagrus major) and Australian red sea bream (Pagrus auratus)), fish belonging to the genus Acanthopagrus (such as black sea bream (Acanthopagrus schlegelii) and yellowfin sea bream (Acanthopagrus latus)), yellowback sea bream (such as Dentex tumifrons) belonging to the genus Dentex, and European sea bream (Sparus aurata) belonging to the genus Sparus.

[0017] The aforementioned grouper species include, for example, fish 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 septemfasciatus) (e.g., fuscoguttatus), starry grouper (Epinephelus labriformis), giant grouper (Epinephelus lanceolatus), white-spotted grouper (Epinephelus maculatus), yellowfin grouper (Epinephelus malabricus), dusky grouper (Epinephelus marginatus), spotted grouper (Epinephelus ongus), spotted grouper (Epinephelus polyphekadion), patterned grouper (Epinephelus quoyanus), black-spotted grouper (Epinephelus sexfasciatus), Nassau grouper (Epinephelus striatus), eye-spotted grouper (Epinephelus tauvina), potato grouper (Epinephelus tukula), etc.), and the spotted grouper (Cromileptes) belonging to the genus Cromileptes This may include species such as *Plectropomus altivelis*, *Plectropomus leopardus* belonging to the genus *Plectropomus*, and hybrids of fish from the family Serranidae.

[0018] The aforementioned fish of the family Monacanthidae may include, for example, the triggerfish (Stephanolepis cirrhifer) belonging to the genus Stephanolepis, and the filefish (Thamnaconus modestus) belonging to the genus Thamnaconus.

[0019] The aforementioned fish of the Scombridae family include, for example, fish belonging to the genus Scombrini (such as Pacific mackerel (Scomber japonicus), Atlantic mackerel (Scomber scombrus), spotted mackerel (Scomber australasicus), etc.), fish belonging to the genus Thunnini (such as Pacific bluefin tuna (Thunnus orientalis), Atlantic bluefin tuna (Atlantic bluefin tuna), Southern bluefin tuna (Thunnus maccoyii), Bigeye tuna (Thunnus obesus), Yellowfin tuna (Thunnus albacares), Albacore tuna (Thunnus alalunga), Longtail tuna (Thunnus tonggol), etc.), and fish belonging to the genus Euthynnus (such as Japanese bonito (Euthynnus affinis), Atlantic yellowtail (Euthynnus This may include fish such as *Alletteratus*, skipjack tuna (*Katsuwonus pelamis*) belonging to the genus *Katsuwonus*, fish belonging to the genus *Scomberomorini*, fish belonging to the genus *Auxis*, fish belonging to the genus *Sardini*, and fish belonging to the genus *Gymnosarda*.

[0020] The aforementioned flatfish may include, for example, the flathead flounder (Pseudopleuronectes herzensteini), the marbled flounder (Pleuronectes yokohamae), the stone flounder (Kareius bicoloratus), the Pacific halibut (Hippoglossus stenolepis), and the Japanese flounder (Verasper moseri).

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

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

[0023] The aforementioned fish of the family Soleidae may include, for example, the red sole (Cynoglossus joyneri) and the black sole (Cynoglossus semilaevis). The aforementioned conger eel species may include, for example, the Japanese conger eel (Conger myriaster) and the black conger eel (Conger japonicus).

[0024] Early codfish may include, for example, Pacific cod (Gadus macrocephalus), Atlantic cod (Gadus morhua), and walleye pollock (Gadus chalcogrammus).

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

[0026] The aforementioned salmonid fish include, for example, fish belonging to the genus Oncorhynchus (rainbow trout (Oncorhynchus mykiss), king salmon (Oncorhynchus tshawytscha), cherry salmon (Oncorhynchus masou), Satsuki salmon (Oncorhynchus masou), Kunimasu (Oncorhynchus kawamurae), pink salmon (Oncorhynchus gorbuscha), salmon (Oncorhynchus keta), etc.), fish belonging to the genus Salmo (brown trout (Salmo trutta), sockeye salmon (Oncorhynchus nerka), coho salmon (Oncorhynchus kisutch), Atlantic salmon (Salmo salar), etc.), and fish belonging to the genus Salvelinus (Dolly Varden trout (Salvelinus malma), Arctic char (Salvelinus This may include species such as alpinus, char (Salvelinus leucomaenis), brook trout (Salvelinus fontinalis), lake trout (Salvelinus namaycush), and the huchen (Parahucho perryi) belonging to the genus Hucho.

[0027] The aforementioned Osmeridae fish may include, for example, the ayu (Plecoglossus altivelis) belonging to the Plecoglossinae subfamily, fish belonging to the Hypomesinae subfamily (such as the smelt (Hypomesus nipponensis) and the pond smelt (Hypomesus japonicus), and fish belonging to the Osmerinae subfamily (such as the smelt (Osmerus mordax dentex), the capelin (Spirinchus lanceolatus), and the icefish (Spirinchus lanceolatus)).

[0028] The aforementioned eel species may include, for example, the Japanese eel (Anguilla japonica) and the European eel (Anguilla anguilla). CYP19a1b is an abbreviation for cytochrome P450 family 19 subfamily A polypeptide 1b.

[0029] For example, the mRNA sequence of CYP19a1b in the Japanese rice fish (Oryzias latipes) is registered as NCBI Reference Sequence: AB591736. For example, the mRNA sequence of CYP19a1b in the Japanese pufferfish (Takifugu rubripes) is registered as NCBI Reference Sequence: NM_001173496. Specific examples of CYP19a1b in fish include the CYP19a1b gene shown in Table 1 below.

[0030] [Table 1]

[0031] There are no restrictions on the methods used to suppress the functional expression of CYP19a1b. For example, suppression of functional expression can be achieved by suppressing the expression of the CYP19a1b gene or by suppressing 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 introduces a loss-of-function mutation into the target gene in the genome of the target fish. Such methods are well known. For example, methods for introducing loss-of-function mutations include genome editing, site-directed mutagenesis such as homologous recombination, and random mutagenesis.

[0033] Genome editing methods include methods for introducing proteins and nucleic acids that constitute genome editing technology, or vectors that encode them. An example of such a protein is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzyme. 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, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, etc. The nucleic acids mentioned above include, for example, crRNA and tracrRNA, or single-stranded nucleic acids in which these are linked via a linker. In this case, the nucleic acids are designed such that, for example, the base sequence that anneals with the target sequence in the crRNA is a base sequence complementary to the base sequence encoding CYP19a1b. The nucleic acids may be used individually or in combination of two or more types. An example of a genome editing system using the CRISPR enzyme is 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 introduces gRNA in the form of 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 on the same vector. The promoter for activating CRISPR is not particularly limited, but the U6 promoter is preferred. The promoter for activating Cas9 is not particularly limited, but a promoter expressed in mammalian cells, such as the cytomegalovirus promoter, is preferred. A commercially available vector such as the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector can be used as the CRISPR / Cas9 system.

[0034] The sequence that targets the CYP19a1b gene and is incorporated into the CRISPR sequence (hereinafter also referred to as the "target sequence") is not limited as long as it is a sequence that can be incorporated into guide RNA (also called gRNA, sgRNA, or crRNA) by the CRISPR / Cas9 system and transcribed, or introduced into cells as a guide RNA containing a sequence complementary to the target sequence, thereby recombining the CYP19a1b gene. Generally, it is said that the target sequence can be selected from a sequence of approximately 20 bases in the upstream 5' region of the base sequence "NGG" (PAM sequence: N is one of the partial clayotides A, G, T, or C) present in the CYP19a1b gene. Target sequences can be designed using publicly available design tools such as 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)), 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 / (China Central Agricultural University)), and CRISPR RGEN Tools (http: / / www.rgenome.net / (Seoul National University)).

[0035] Furthermore, preferably, if single nucleotide polymorphisms (SNPs) are present in the PAM sequence, it is preferable to avoid such sequences. With respect to the target sequence, if the SNPs of an individual are known, it is preferable to optimize the sequence to match each SNP. The 5' terminal region of the target sequence may be 1, 2, 3, or 4 bases shorter, preferably 1, 2, or 3 bases shorter.

[0036] Furthermore, while the CRISPR / Cas9 system may be introduced into cells as a vector, it may also be introduced into cells by combining artificially synthesized or in vitro synthesized gRNA, crRNA, trans-activating crRNA (tracrRNA), and RNA encoding Cas9. Alternatively, the Cas9 protein may be introduced into cells by combining it with guide RNA.

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

[0038] The genome editing system can be injected into the cytoplasm of a fertilized egg, preferably a one-cell stage fertilized egg, by microinjection. For example, when introducing the Cas9 protein, the amount injected per fertilized egg can range from 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably 10 pg to 50 pg. At this time, the guide RNA can be injected in the range of 0.1 pg to 50 pg, preferably 0.5 pg to 20 pg, and more preferably 1 pg to 5 pg. For an example of how to introduce mutations using genome editing technology, see Example 1 described below.

[0039] Examples of random mutagenesis methods include radiation treatment with alpha rays, beta rays, gamma rays, and X-rays; chemical treatment with chemical mutagens such as ethyl methanesulfonate (EMS) and ethynylnitrosourea (ENU); and heavy ion beam treatment. By employing methods such as the tilling method as a random mutagenesis method, mutation induction by chemical mutagens and mutation screening can be performed efficiently.

[0040] One method for suppressing the repression of CYP19a1b gene expression is to introduce at least one selected from the group consisting of an RNA molecule that targets CYP19a1b mRNA or a vector capable of expressing said RNA molecule. The term "RNA molecule that targets CYP19a1b mRNA" is not limited as long as it targets CYP19a1b mRNA and can suppress the expression of the CYP19a1b protein. Examples include those that degrade target mRNA, such as siRNA, shRNA, dsRNA, and miRNA, and / or those that suppress the translation of target mRNA. The sequences of these RNA molecules can be appropriately designed by a person skilled in the art using known methods based on the nucleotide sequence information of the target gene. Alternatively, the RNA molecule may be prepared using known methods, or it may be obtained from the market and used. Preferred RNA molecules are siRNA, shRNA, and miRNA, with siRNA and shRNA being particularly preferred. The vectors capable of expressing RNA molecules targeting the CYP19a1b mRNA described above are not particularly limited as long as they can express RNA molecules that suppress the expression of the CYP19a1b protein within an individual's body or cell. For example, hairpin RNA expression vectors can be cited. A hairpin RNA expression vector includes, for example, a promoter sequence suitable for the expression of short-chain RNA, such as a U6 promoter, and further downstream, a sense strand DNA sequence having the same sequence as the sense strand of the target mRNA (however, 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 complementarily to the sense strand DNA sequence in whole or in part; and a terminator sequence. Examples of vectors include plasmid vectors, adenovirus vectors, retrovirus vectors, lentivirus vectors, and the like.

[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 approximately 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably 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 approximately 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably 10 pg to 50 pg per fertilized egg. The vector can be linearized as needed.

[0042] The suppression of protein function can be achieved using at least one antibody selected from the group consisting of antibodies that bind to CYP19a1b and suppress the functional expression of the target by 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 methods known to those skilled in the art. Furthermore, the antibody may be an antibody fragment such as Fab, F(ab)2, diabody, scFv, minibody, peptibody, or mimetibody. Antibodies can be injected in a range of approximately 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably 10 pg to 50 pg per fertilized egg.

[0043] Larvae and juvenile fish hatched from fertilized eggs are reared from larvae or juveniles to adults and then to broodstock. Individuals with induced mutations in the CYP19a1b gene are selected using mutation analysis methods, such as heterozygous double-stranded mobility analysis, Cel 1 assay, T7 endonuclease assay, and sequencing analysis. Rearing conditions can be the same as those for general fish farming. In addition, feed commonly given in aquaculture can be used. 2. Male fish individuals in which the functional expression of CYP19a1b is suppressed. Male fish individuals in which the functional expression of CYP19a1b is suppressed have, for example, at least one of the following phenotypes: (a) Compared to control males with CYP19a1b function, aggression is reduced; (b) Compared to control males with CYP19a1b function, the rate of weight gain after sexual maturity is higher. (c) Compared to control male individuals with CYP19a1b function, testicular weight per unit body weight is increased. (d) Compared to control male individuals with CYP19a1b function, the number of robule structures in the testes is increased.

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

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

[0046] Aggression can be assessed by at least one aggressive behavior selected from, for example, chasing, intimidation, parallel positioning, striking, and biting. Aggressive behavior can be assessed by placing at least two male individuals in a single tank and counting the number of times they perform the above aggressive behaviors within a predetermined time period (e.g., 20 to 60 minutes). Reduced aggression is defined as the number of aggressive behaviors in CYP19a1b-suppressed male individuals being 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 30, 1 / 50, or 1 / 100 or less compared to control male individuals.

[0047] The evaluation of the number of attacks is preferably performed statistically by counting the number of attacks in a control male population containing two or more individuals and a CYP19a1b function-suppressed male population containing two or more individuals.

[0048] The rate of weight gain after sexual maturity is calculated by using the average weight at the average age of sexual maturity in weeks or months for each fish species as a baseline (hereinafter also referred to as "baseline weight") to determine how much weight has increased.

[0049] A high rate of weight gain in male individuals with suppressed CYP19a1b function 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 that of control male individuals.

[0050] The rate of weight gain is preferably evaluated statistically by counting data for a control male population containing two or more individuals and a CYP19a1b-suppressed male population containing two or more individuals. The testicular weight per unit body weight can be calculated from the body weight of the individual before orchiectomy and the weight of the testes removed from the individual.

[0051] An increase in testicular weight per unit body weight in male individuals with suppressed CYP19a1b function means that the testicular weight per unit body weight is 1.1 times, 1.2 times, 1.3 times, or 1.4 times or more compared to control male individuals. The number of roburetic structures in the testis can be counted by analyzing tissue sections of the excised testis.

[0052] An increase in the number of roburetic structures in the testes of male individuals with suppressed CYP19a1b function means that the testicular weight per unit body weight is 1.1 times, 1.2 times, 1.3 times, or 1.4 times greater than that of control male individuals. [Examples]

[0053] The embodiments of the present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

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

[0055] (2) Introduction of mutations CYP19a1b-deficient medaka were prepared using the tilling method. A library of 5760 medaka individuals chemically mutagenicated with N-ethyl-N-nitrosourea (ENU) (Taniguchi et al. https: / / genomebiology.biomedcentral.com / articles / 10.1186 / gb-2006-7-12-r116) was screened for mutations in exons 3, 4, and 5 of CYP19a1b using direct sequencing. Figure 1A shows the nucleotide sequences of the mutant sites and the nucleotide sequences of the mutant sites (SEQ ID NO: 1 is wild type, SEQ ID NO: 2 is mutant). Figure 1B shows the amino acid sequence of CYP19a1b in wild-type medaka (SEQ ID NO: 3) and the amino acid sequence of the detected mutant CYP19a1b (SEQ ID NO: 4). F0 generations with a nonsense mutation (K105*) in exon 4 (* represents a stop codon) were identified and backcrossed with the wild-type medaka d-rR strain for more than six generations to remove background mutations. Heterozygous males and females were crossed to create wild-type, heterozygous, and homozygous siblings, which were used in the experiment. DNA extracted from the caudal fins of the aforementioned medaka was analyzed, and the nucleotide sequences of individuals with loss-of-function mutations in the CYP19a1b gene were analyzed by PCR and nucleotide sequence analysis. The loss-of-function mutation in the CYP19a1b gene was a single nucleotide substitution (A>T) line. Sequence ID 5 of the sequence list described later shows the nucleotide sequence of the wild-type CYP19a1b gene, and Sequence ID 6 shows the nucleotide sequence of the loss-of-function mutation in the CYP19a1b gene.

[0056] In the following, individuals with a loss-of-function mutation in one allele of the CYP19a1b gene may be simply referred to as heterozygous mutants. Conversely, individuals with loss-of-function mutations in both alleles of the CYP19a1b gene may be simply referred to as homozygous mutants. Furthermore, wild-type mutants are sometimes denoted as "+ / +", heterozygous mutants as "+ / -", and homozygous mutants as "- / -".

[0057] 2. Phenotypes of wild-type, heterozygous, and homozygous mutants (1)Growth The growth curves for wild-type, heterozygous, and homozygous medaka 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 wild-type medaka, gray circles represent heterozygous medaka, and black circles represent homozygous medaka. The black inverted triangles in Figure 2 indicate the onset of sexual maturation.

[0058] As shown in Figure 2A, male wild-type medaka and heterozygous mutants showed a slowdown in weight gain after 9 weeks of age, but homozygous mutants continued to increase in weight even after 9 weeks of age. Furthermore, as shown in Figure 2B, male homozygous mutants also showed an increasing trend in body length compared to wild-type and heterozygous mutants. Generally, in medaka, male growth slows down after the onset of sexual maturity, while females continue to grow at the same pace. As a result, females eventually become significantly larger. This study demonstrated that male homozygous mutants do not experience a slowdown in growth even after sexual maturity. This revealed that loss-of-function mutations in both alleles of the CYP19a1b gene promote male growth.

[0059] (2) Aggression Male wild-type, heterozygous, and homozygous mutants were grouped into sets of four, each placed in a separate tank, and their aggression was observed for 30 minutes. Aggression was assessed using the following criteria: chasing, intimidation, 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 represent wild-type, gray bars represent heterozygous mutants, and black bars represent homozygous mutants. The dot plots in each graph represent the raw data. In Figure 3, * indicates a statistically significant difference of p<0.05, and ** indicates a statistically significant difference of p<0.01.

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

[0062] This suggests that loss-of-function mutations in both alleles of the CYP19a1b gene may reduce or eliminate aggressiveness in males. (3) Phenotype of the male gonads

[0063] We observed the phenotypic characteristics of male gonads. The evaluation items were male testicular weight, testicular weight per unit body weight, and histological evaluation (morphological observation of the tissue, number of roburetic structures in the testis).

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

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

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

[0067] Based on these results, it was concluded that having loss-of-function mutations in both alleles of the CYP19a1b gene can lead to increased growth and testicular enlargement in male fish. Furthermore, the reduced aggression is expected to improve farming efficiency, as multiple fish can be raised in a single tank without injuring each other.

[0068] II. Creation of CYP19a1b-deficient tiger pufferfish and observation of male growth, aggression, and maturation 1. Creation of tiger pufferfish with a functional loss 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 collected eggs and sperm were then artificially inseminated to obtain fertilized eggs.

[0069] (2) Introduction of mutations Mutations were introduced using Cas9 protein and guide RNA. The target gene sequence is 5'-GCTGGAGTGTATCGGGATGGAGG-3' (SEQ ID NO: 7). Mutations were introduced into the Cyp19a1b gene by microinjecting a solution prepared with 2-10 pg Cas9 protein and 1-5 pg guide RNA into the cytoplasm of one-cell stage fertilized eggs. 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 growth rate of male cyp19a1b-deficient troughs We confirmed that the introduction of the above mutation improved the growth potential of males.

[0071] (4) Observation of aggressiveness in male pufferfish with Cyp19a1b dysfunction We confirmed that the introduction of the above mutation suppressed aggressiveness in males.

[0072] (5) Confirmation of maturation of male pufferfish with Cyp19a1b dysfunction We confirmed that the introduction of the above mutation accelerates the maturation of males.

[0073] Based on these results, it is expected that introducing a loss-of-function mutation into the Cyp19a1b gene of the tiger pufferfish will result in larger male tiger pufferfish, particularly those with larger milt. Furthermore, the lack of aggression will eliminate the need for tooth trimming, improving aquaculture efficiency and commercial value.

[0074] <array list> Sequence ID 1: CCATGTTTTGAAGAGCGCCCA Sequence ID 2: CCATGTTTTGATGAGCGCCCA Sequence ID 3: MIQQEVQTLDELFHSVSQVTLFLLLLMLILLFINTWRQSHLSHIPGPSFLAGLGPVLSYARFIWMGIGTACNYYNKKYGSIVRVWINGEETLILSRSSEVYHVLKSAHYTSRFGSQKGLQCIGMDGRGIIFNCDVPLWKKTRMYFSKALSGPSLQRTVGICVSSTAKHLARLQDVTDSSGHVDALNLLRAIVVDISNRLFLRVPLNEKELLQKIHNYFETWQAVLIKPDVFFKMGWLYNKHKRAAQELQDS MESLLEIKRKMINEAEKLDDDLDFATELIFAQNHGELSADNVRQCVLEMVIAAPDTLSISLFFMLMLLKQNPDVELQVVEEMNSVLDEKATESIDYESLRVLESFINESMRFHPVVDFTMRKALEDDEIEGTKIRRGTNIILNIGLMHKTEFFPKPTEFSLANFEKTVPSRFFQPFGCGPRSCVGKHIAMVMMKVILITLLSRYTVCPRQGCTLNSIRQTNNLSQQPVEDEHSLTMRFIPRTSQPHLA* (Registered as GenBank: AB591736.1) Sequence ID 4: MIQQEVQTLDELFHSVSQVTLFLLLLMLILLFINTWRQSHLSHIPGPSFLAGLGPVLSYARFIWMGIGTACNYYNKKYGSIVRVWINGEETLILSRSSEVYHVL* Sequence number 6: ggcagaggaagcaggttaccttaaaccagggaagggaagtctgaatcccctcaaacccaacttgcatttcaggggtaggagaagatctgtgagacccatccacctgcagacaggataaagtgagtttagtgtgagtttctgcagagacaagaagATGATCCAGCAAGAGGTGCAAACCCTCGACGAGTTGTTTCACAGTGTGTCTCAAGTCACACTTTTCTTACTTCTTCTGATGCTGATACTCTGTTTTATTAACACGTGGAGACAAAGTCACCTTTTCACACATACCAGGTCCTTTCCTTTGGCAGGACTTGGTCCTGTCCTGTCCTATGCAAGATTTATCTGGATGGGATTGGGAACAGCATGTAACTACTACAAATAAAAGTAGGCAGCATTGTGAGGGTATGGATTATTGAATGGGGGAGGACTCTCATATTGAGCAGATCTTCTGAGGTTGACCATGTTTTG T Sequence ID 7: GCTGGAGTGTATCGGGATGGAGG

Claims

1. Male individuals of fish selected from marine fish, brackish water fish, anadromous fish, and fish of the family Adrianichthyidae, in which the functional expression of CYP19a1b (cytochrome P450 family 19 subfamily A polypeptide 1b) is suppressed.

2. The male individual according to 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 according to claim 2, wherein the introduction of a loss-of-function mutation is performed by 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 introduction of a loss-of-function mutation is carried out by radiation, a heavy ion beam, or a chemical mutagens.

5. The male individual according to claim 1, wherein the suppression of functional expression is performed by suppressing the expression of the CYP19a1b gene, and the suppression of the expression of the CYP19a1b gene is performed by at least one selected from the group consisting of a vector capable of expressing at least one RNA molecule selected from the group consisting of siRNA, shRNA, and miRNA that targets the mRNA of CYP19a1b.

6. A male individual according to claim 1, having at least one of the following phenotypes (a) to (c): (a) Compared to individuals with wild-type CYP19a1b function, aggression is reduced. (b) Continues to grow even after sexual maturity, (c) Compared to individuals with wild-type CYP19a1b function, the number of roburetic structures in the testes is increased and testicular weight per unit body weight is increased.

7. A method for producing male fish individuals in which the functional expression of CYP19a1b (cytochrome P450 family 19 subfamily A polypeptide 1b) is suppressed, wherein the fish is selected from marine fish, brackish water fish, anacromigratory fish, and fish of the family Adrianichthyidae.

8. The method according to claim 7, wherein a male individual has at least one of the following phenotypes (a) to (c): (a) Compared to control male individuals with CYP19a1b function, aggression is reduced. (b) Compared to control male individuals with CYP19a1b function, the rate of weight gain after sexual maturity is higher. (c) Compared to control male individuals with CYP19a1b function, the number of robule structures in the testes is increased and the testicular weight per unit body weight is increased.

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