Method for improving fleshiness of fish

Abstract

The present invention addresses the problem of providing a method for improving the fleshiness of fish. The problem is solved by a method for improving the fleshiness of fish, the method including suppressing the expression of the function of myostatin in fish.

Description

Methods for improving fish meat quality

[0001] The present disclosure relates to a method for improving fish meat quality, etc. More specifically, the present disclosure relates to a method for improving fish meat quality, etc., which includes suppressing the functional expression of myostatin in fish.

[0002] Currently, efforts are being actively made to develop fish with desired characteristics through genome editing and other methods (Patent Document 1, etc.).

[0003] Patent No. 7350385

[0004] Kishimoto et al., 2018, Aquaculture, Volume 495, 2018, 415-427 Nakada Hisashi et al., Nippon Suisan Gakkaishi 63(5), 728-733(1997)

[0005] An object of the present disclosure is to provide a method for improving the meat quality of fish.

[0006] The inventors have discovered that suppressing the functional expression of myostatin improves the quality of fish meat (more specifically, the amount of lactic acid in the fish meat is reduced, the decrease in pH in the fish meat is inhibited, the amount of dripping from the fish meat is reduced, the amount of collagen in the fish meat is reduced, and / or the hardness of the fish meat is reduced), and have made further improvements.

[0007] The present disclosure includes, for example, the subject matter described in the following sections: Item 1. A method for improving fish meat quality, comprising suppressing the functional expression of myostatin in fish. Item 2. The method described in Item 1, in which the suppression of functional expression is achieved by introducing a loss-of-function mutation in the myostatin gene. Item 3. The method described in Item 2, in which the introduction of a loss-of-function mutation is achieved by at least one genome editing system selected from the Clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9 (CRISPR / Cas9) system, the CompoZr Zinc Finger Nuclease (ZFN) system, and the TAL effector nuclease (TALEN) system. Item 4. Item 1. The method of Item 1, wherein functional expression is suppressed by suppressing expression of the myostatin gene. Item 5. The method of Item 4, wherein gene expression is suppressed by at least one RNA molecule selected from the group consisting of siRNA, shRNA, and miRNA that target myostatin mRNA, or at least one selected from the group consisting of vectors capable of expressing said RNA molecule. Item 6. The method of Item 1, wherein functional expression is suppressed by suppressing the function of myostatin protein. Item 7. The method of Item 6, wherein protein function is suppressed by at least one antibody selected from the group consisting of antibodies that specifically bind to myostatin and suppress the functional expression of myostatin through said binding. Item 8. The method of any of Items 1 to 7, wherein the improvement in fish meat quality is at least one selected from the group consisting of a reduction in the amount of lactic acid in the fish meat, suppression of a decrease in pH in the fish meat, a reduction in the amount of drip from the fish meat, a reduction in the amount of collagen in the fish meat, and a reduction in fish meat hardness. Item 9. Item 8. The method according to any one of Items 1 to 7, wherein the improvement in fish meat quality is at least one selected from the group consisting of suppressing putrefaction of fish meat, improving the texture of heat-processed fish meat products, and improving the texture of fish meat.

[0008] The quality of fish meat can be improved (more specifically, the amount of lactic acid in the fish meat can be reduced, the decrease in pH in the fish meat can be suppressed, the amount of dripping from the fish meat can be reduced, the amount of collagen in the fish meat can be reduced, and / or the hardness of the fish meat can be reduced).

[0009] 1 shows the results of measuring the moisture content in fish meat after 5 days of storage. WT represents fish meat collected from an individual that does not lack the myostatin gene, and KO represents fish meat collected from an individual that lacks the myostatin gene. 1 shows the results of measuring the amount of lactic acid in fish meat after 0, 1, and 4 days of storage. WT represents fish meat collected from an individual that does not lack the myostatin gene, and KO represents fish meat collected from an individual that lacks the myostatin gene. 1 shows the results of measuring the pH in fish meat after 0, 1, and 7 days of storage. WT represents fish meat collected from an individual that does not lack the myostatin gene, and KO represents fish meat collected from an individual that lacks the myostatin gene. 1 shows the results of measuring the amount of drip in fish meat after 1, 3, and 7 days of storage. WT represents fish meat collected from an individual that does not lack the myostatin gene, and KO represents fish meat collected from an individual that lacks the myostatin gene. 1 shows an electron microscope photograph of fish meat. WT represents fish meat collected from an individual without a myostatin gene deficiency, and KO represents fish meat collected from an individual lacking the myostatin gene. Measurement results of the collagen content in fish meat after 1 and 3 days of storage are shown. WT represents fish meat collected from an individual without a myostatin gene deficiency, and KO represents fish meat collected from an individual lacking the myostatin gene. Measurement results of the breaking strength of fish meat after 0, 1, and 3 days of storage are shown. WT represents fish meat collected from an individual without a myostatin gene deficiency, and KO represents fish meat collected from an individual lacking the myostatin gene. Measurement results of the moisture content in fish meat after 5 days of storage are shown. WT represents fish meat collected from an individual without a myostatin gene deficiency, and KO represents fish meat collected from an individual lacking the myostatin gene. Measurement results of the pH in fish meat after 1 and 7 days of storage are shown. WT represents fish meat collected from an individual without a myostatin gene deficiency, and KO represents fish meat collected from an individual without a myostatin gene deficiency. The results of measuring the amount of drip from fish meat after 1 and 7 days of storage are shown. WT represents fish meat collected from an individual without a myostatin gene deficiency, and KO represents fish meat collected from an individual without a myostatin gene deficiency.

[0010] Each embodiment of the present disclosure is described in more detail below. The present disclosure includes a method for improving the meat quality of fish, which comprises suppressing the functional expression of myostatin in fish. In this specification, this method may be referred to as the "method of the present disclosure."

[0011] In the present specification, the term "fish" is not particularly limited. In the present specification, the term "fish" may include saltwater fish, freshwater fish, brackish water fish, diadromous fish, etc.

[0012] Examples of the fish include Sparidae (sea bream and porgies), Tetraodontidae (puffers), Ostracidae (boxfish), Paralichthys, Cichlidae, Salmonidae, Cypriniidae, Ictaluroidea, Siluroidea, Bagroidea, Serranidae (sea bass), and the like. basses), Oryziidae (medakas), Filefish (Monacanthiidae), Smeridae, Mackerel (Scombridae), Flatfish (Pleuronectidae), Carangidae, Perch (Lateolabrax), Moronidae, Latidae, Coridae (Rachycentridae), Cynoglossidae, Eels (Anguilliidae), Conger eels (Congridae), and the like. In one embodiment, the fish is preferably a fish of the Sparidae, Tetraodontidae, Paralichthyidae, Cichlidae, Salmonidae, Grouper, Filefish, or Scombridae families.

[0013] The fish of the Sparidae family may include, for example, fish of the Pagrus genus, such as red sea bream (Pagrus major) and gilthead sea bream (Pagrus auratus); fish of the Acanthopagrus genus, such as black porgy (Acanthopagrus schlegelii) and yellow porgy (Acanthopagrus latus); fish of the Dentex genus, such as yellow sea bream (Dentex tumifrons); and fish of the Sparus genus, such as gilthead sea bream (Sparus aurata). In one embodiment, the fish of the Sparidae family is preferably a fish of the Pagrus genus, and more preferably a red sea bream.

[0014] Examples of the fish of the Tetraodontidae family include fish of the genus Takifugu, such as the tiger pufferfish (Takifugu rubripes), the red pufferfish (Takifugu porphyreus), and the grass pufferfish (Takifugu niphobles); and fish of the genus Lagocephalus, such as the white mackerel pufferfish (Lagocephalus wheeleri). In one embodiment, the fish of the Tetraodontidae family is preferably a fish of the genus Takifugu, and more preferably a tiger pufferfish.

[0015] The fish of the family Oligodontidae may include, for example, fish of the genus Oligodontidae, such as the boxfish (Ostracion immaculatus).

[0016] The fish of the family Paralichthys may include, for example, Paralichthys olivaceus (TEMMINCK et SCHLEGEL), flounders belonging to the genus Paralichthys (such as Paralichthys sole, Paralichthys parasitoid ... olivaceus is preferred.

[0017] The cichlid fish may include, for example, fish of the genus Oreochromis, such as Nile tilapia (Oreochromis niloticus), cichlid fish (Oreochromis mossambicus), blue tilapia (Oreochromis aureus), etc. In one embodiment, the cichlid fish is preferably Nile tilapia.

[0018] Examples of the fish of the Salmonidae family include fish of the genus Salmon, such as rainbow trout (Oncorhynchus mykiss), king salmon (Oncorhynchus tshawytscha), cherry salmon (Oncorhynchus masou), satsukimasu (Oncorhynchus masou), kunimasu (Oncorhynchus kawamurae), pink salmon (Oncorhynchus gorbuscha), and salmon (Oncorhynchus keta); brown trout (Salmo trutta), sockeye salmon (Oncorhynchus nerka), and coho salmon (Oncorhynchus fish of the genus Salmonidae, such as Salvelinus kisutch (Salmo salar) and Atlantic salmon (Salmo salar); fish of the genus Salvelinus, such as Dolly Varden trout (Salvelinus malamus), Arctic char (Salvelinus alpinus), Salvelinus leucomaenis (Salvelinus leucomaenis), brook trout (Salvelinus fontinalis), and lake trout (Salvelinus namaycush); fish of the genus Sakhalin, such as Sakhalin taimen (Human huchen) (Parahucho perryi). In one embodiment, the fish of the Salmonidae family is preferably a fish of the genus Salmonidae or Sakhalin, and more preferably a rainbow trout, masu salmon, satsuki salmon, coho salmon, or Atlantic salmon.

[0019] Examples of the fish of the Cyprinidae family include the Japanese carp (Gnathopogon caerulescens), the silver carp (Hypophthalmichthys molitrix), the common carp (Cyprinus carpio), the grass carp (Ctenopharyngdon idellus), the bighead carp (Hypophthalmichthys nobilis), the European crucian carp (Carassius carassius), the black porgy (Cyprinus catla), the Japanese bluefin tuna (Mylopharyngdon pieseus), the Japanese amberjack (Cirrhinus molitorella), and the Mrigal carp (Cirrhinus cirrhosus), Catla catla, Labeo rohita, Megalobrama amblycephala, and the like.

[0020] The fish of the superfamily Ictaluroidea may include, for example, channel catfish (Ictalurus punctatus), blue catfish (Ictalurus furcatus), and the like.

[0021] Examples of fish of the superfamily Siluroidea include 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.

[0022] The fish of the superfamily Bagroidea may include, for example, the Korean catfish (Pseudobagrus fulvidraco), the Mekong giant catfish (Pangasianodon gigas), the pangasius (Pangasius bocourti), the sea bass (Pangasianodon hypophthalmus), and the like.

[0023] Examples of the fish of the family Grouper include the red spotted grouper (Epinephelus septemfasciatus), the grouper (Epinephelus bruneus), the red spotted grouper (Epinephelus akaara), the yellow spotted grouper (Epinephelus malabaricus), the white grouper (Epinephelus aeneus), the black spotted grouper (Epinephelus samblicephalus), the red spotted grouper (Epinephelus areolatus), the yellow spotted grouper (Epinephelus bleekeri), the white spotted grouper (Epinephelus bontoides), and the white spotted grouper (Epinephelus chlorostigma), orange-spotted grouper (Epinephelus coiodes), red grouper (Epinephelus fasciatus), red-spotted grouper (Epinephelus fuscoguttatus), starry grouper (Epinephelus labriformis), grouper (Epinephelus lanceolatus), white-spotted grouper (Epinephelus maculatus), yellow spotted grouper (Epinephelus malabricus), dusky grouper (Epinephelus marginatus), and spotted grouper (Epinephelus Epinephelus species such as spotted grouper (Epinephelus ongus), spotted grouper (Epinephelus polyphekadion), yellow grouper (Epinephelus quoyanus), black-finned grouper (Epinephelus sexfasciatus), Nassau grouper (Epinephelus striatus), single-spotted grouper (Epinephelus tauvina), potato grouper (Epinephelus tukula), etc.; Epinephelus species such as Cromileptes altivelis, etc.; Plectropomus species In one embodiment, the fish of the grouper family is preferably a fish of the genus Epinephelus or Epinephelus, and more preferably a yellow-spotted grouper, a grouper, a red spotted grouper, a yellow spotted grouper, a yellow spotted grouper, or a grouper.

[0024] The fish of the medaka family may include, for example, fish of the genus Oryzias, such as medaka (Oryzias latipes, Oryzias sakaizumii) and Javan medaka (Oryzias javanicus).

[0025] The fish of the filefish family may include, for example, fish of the genus Stephanolepis such as the filefish (Stephanolepis cirrhifer), fish of the genus Thamnaconus such as the black filefish (Thamnaconus modestus), and fish of the genus Alterus such as the white filefish (Aluterus monoceros). In one embodiment, the fish of the filefish family is preferably a fish of the genus Malvaceae or Alterus, and more preferably a filefish or a black filefish.

[0026] The fish of the family Ayuidae may include, for example, fish of the Plecoglossinae subfamily (Plecoglossinae) such as sweetfish (Plecoglossus altivelis), the smelt subfamily (Hypomesus nipponensis) and the Japanese whiting (Hypomesus japonicus), and the smelt subfamily (Osmerinae) such as smelt (Osmerus mordax dentex), shishamo (Spirinchus lanceolatus), and icefish (Spirinchus lanceolatus).

[0027] Examples of the fish of the Scombridae family include the genus Scombrini, such as chub mackerel (Scomber japonicus), Atlantic mackerel (Scomber scombrus), and yellow mackerel (Scomber australasicus), 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 scombrini). Fish of the genus Thunnini such as Euthynnus tonggol, fish of the genus Euthynnus such as Euthynnus affinis and Euthynnus alletteratus, fish of the genus Katsuwonus such as Katsuwonus pelamis, fish of the genus Scomberomorini, fish of the genus Auxis, fish of the genus Sardini, fish of the genus Gymnosarda, and hybrids between fish of the Scombridae family. In one embodiment, the fish of the Scombridae family is preferably a fish of the genus Scombridae, the genus Thunnus, or the genus Scombridae, and more preferably a chub mackerel, Pacific bluefin tuna, or Scombridae.

[0028] Examples of the Pleuronectidae fish include Pseudopleuronectes herzensteini, Pleuronectes yokohamae, Kareius bicoloratus, Hippoglossus stenolepis, Verasper moseri, and other fish.

[0029] The fish of the Carangidae family may include, for example, fish of the Seriola genus such as amberjack (Seriola dumerili), yellowtail (Seriola lalandi), long-finned amberjack (Seriola rivoliana), and Japanese amberjack (Seriola quinqueradiata), fish of the Trachurus genus such as horse mackerel (Trachurus japonicus) and striped jack (Pseudocaranx dentex), fish of the Trachinotus genus such as Japanese amberjack, and fish of the Kaiwari genus such as Japanese amberjack (Kaiwarinus equula), as well as hybrids between fish of the Carangidae family. In one embodiment, the fish of the Carangidae family is preferably a fish of the genus Seriola, Carassius, or Scombrida, and more preferably amberjack, yellowtail, Japanese amberjack, horse mackerel, striped jack, or Japanese amberjack.

[0030] The fish of the family Lateolabrax may include, for example, blackfin bass (Lateolabrax latus) and maculatus bass (Lateolabrax maculatus), and the fish of the family Moronidae may include, for example, European sea bass (Dicentrarchus labrax).

[0031] The fish of the family Latidae may include fish of the genus Lates, such as barramundi (Lates calcarifer) and Nile perch (Lates niloticus).

[0032] The fish of the family Rachycentridae can include, for example, fish such as the cobia (Rachycentron canadum).

[0033] The fish of the family Cynoglossidae may include, for example, fish such as red tongue sole (Cynoglossus joyneri) and red tongue sole (Cynoglossus semilaevis).

[0034] The fish of the family Anguillididae may include, for example, fish such as the Japanese eel (Anguilla japonica) and the European eel (Anguilla anguilla).

[0035] The fish of the Conger eel family may include, for example, fish such as the Japanese conger eel (Conger myriaster) and the Japanese black conger eel (Conger japonicus).

[0036] As used herein, the fish may be a fixed species or a hybrid, which may include, for example, a hybrid resulting from intergeneric crossing.

[0037] The "fish" is preferably a farmed fish. Furthermore, the "farmed fish" may include fish farmed for purposes such as food, breeding, and ornamental purposes.

[0038] The myostatin mRNA of each fish is not limited as long as it is transcribed from the myostatin gene present in the genome of each fish, i.e., the myostatin mRNA of each fish may include variants transcribed from the myostatin gene.

[0039] Specific examples of myostatin genes in various fish species include those shown in Table 1 below.

[0040]

[0041] The method for inhibiting the functional expression of myostatin is not limited, and can be achieved by, for example, introducing a loss-of-function mutation in myostatin, inhibiting the expression of the myostatin gene, or inhibiting the function of the myostatin protein.

[0042] The method for introducing a loss-of-function mutation into myostatin is not limited as long as it can introduce a loss-of-function mutation into the myostatin 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; and the like.

[0043] 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, 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 nucleic acids 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 the myostatin gene. 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 racrRNA.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.

[0044] The sequence (hereinafter also referred to as "target sequence") that targets the myostatin 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 a sequence that can be introduced into a cell as a guide RNA containing a sequence complementary to the target sequence to recombine the myostatin gene. Generally, it is said that the target sequence can be selected from 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 myostatin 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 publicly known design tool.

[0045] In the case of red sea bream, an example of a sequence that targets the myostatin gene is 5'-GGACGATGAGCACGCCATCACGG-3' (SEQ ID NO: 1). In the case of tiger pufferfish, an example of a sequence that targets the myostatin gene is 5'-GGACGACGAGCACGCCATCACGG-3' (SEQ ID NO: 2). In the case of flounder, an example of a sequence that targets the myostatin gene is 5'-TCTGAGCAAACTGCGAATGAAGG-3' (SEQ ID NO: 3). In the case of Nile tilapia, an example of a sequence that targets the myostatin gene is 5'-TCTACTGGGGTGCTGACGGAAGG-3' (SEQ ID NO: 4). Preferably, if single nucleotide polymorphisms (SNPs) are present in the PAM sequence, it is preferable to avoid such sequences. If the SNPs of an individual are known, it is preferable to optimize the target sequence for each SNP. If a decrease in genome editing efficiency is confirmed for the target sequence, another sequence within the gene can be selected.

[0046] The 5' terminal region of the target sequence may be shorter by 1, 2, 3, or 4 bases, preferably 1, 2, or 3 bases.

[0047] 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.

[0048] 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.

[0049] 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 methods of introducing mutations using genome editing technology, see, for example, the Examples described below.

[0050] Examples of random mutagenesis methods include irradiation treatment with radiation such as α-rays, β-rays, γ-rays, and X-rays; chemical treatment with mutagens such as ethyl methanesulfonate (EMS) and ethynylnitrosourea (ENU); and heavy ion beam treatment.

[0051] Methods for suppressing myostatin gene expression include introducing at least one selected from the group consisting of an RNA molecule targeting myostatin mRNA or a vector capable of expressing said RNA molecule. The term "RNA molecule targeting myostatin mRNA" is not limited, as long as it targets myostatin mRNA and can suppress the expression of the myostatin protein. Examples include siRNA, shRNA, dsRNA, miRNA, and other molecules that have the effect of degrading the target mRNA and / or those that suppress the translation of the target mRNA. The sequences of these RNA molecules can be appropriately designed by those skilled in the art using known techniques based on the base sequence information of the target gene. Furthermore, the RNA molecule may be prepared using known techniques, or commercially available molecules may be obtained and 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 myostatin mRNA is not particularly limited as long as it can express an RNA molecule that suppresses the expression of the myostatin 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 base sequence downstream of a promoter base 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 base sequence that forms a loop structure after transcription; an antisense strand DNA base sequence that can bind in whole or in part to the sense strand DNA base sequence in a complementary manner; and a terminator sequence. Examples of vectors include plasmid vectors, adenovirus vectors, retrovirus vectors, and lentivirus vectors.

[0052] 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 about 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 about 10 pg to 80 pg, and more preferably about 10 pg to 50 pg per fertilized egg. The vector can be linearized as necessary.

[0053] The function of myostatin protein can be inhibited using at least one antibody selected from the group consisting of antibodies that specifically bind to myostatin and inhibit the functional expression of myostatin through this binding. The antibody may be either a polyclonal antibody or a monoclonal antibody. Both polyclonal and monoclonal antibodies can be prepared appropriately by methods known in the art. The antibody may also be an antibody fragment such as Fab, F(ab)2, diabody, scFv, minibody, peptibody, or mimetibody. The antibody can be injected in the range of 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably 10 pg to 50 pg per fertilized egg.

[0054] Larvae and fry hatched from fertilized eggs are reared for 1 to 5 months, and individuals with induced mutations in the myostatin gene can be selected using mutation analysis methods, such as heteroduplex mobility analysis, Cel1 assay, T7 endonuclease assay, and sequence analysis. The water temperature during rearing can be appropriately selected depending on the fish species. For example, red sea bream or tiger pufferfish can be reared at 10 to 32°C, preferably 16 to 28°C, and more preferably 20 to 24°C. For example, flounder can be reared at 8 to 30°C, preferably 14 to 26°C, and more preferably 18 to 22°C. For example, Nile tilapia can be reared at 15 to 37°C, preferably 20 to 32°C, and more preferably 26 to 30°C. Daylight hours can be maintained under natural conditions. Other rearing conditions can be the same as those for general fish farming. Food generally provided in aquaculture can also be used.

[0055] In this specification, "improvement of meat quality" includes, for example, a reduction in the amount of lactic acid in fish meat, suppression of a decrease in pH in fish meat, a reduction in the amount of drip from fish meat, a reduction in the amount of collagen in fish meat, and / or a reduction in fish meat hardness, etc. These may be used alone or in combination of two or more.

[0056] For example, the method of the present disclosure can reduce the amount of lactic acid in fish meat by suppressing the functional expression of myostatin in fish. In other words, the method of the present disclosure also encompasses a method for reducing the amount of lactic acid in fish meat. The amount of lactic acid in fish meat is measured by the method shown in the Examples below. Specifically, the amount of lactic acid in fish meat is evaluated by homogenizing fish meat (e.g., fillets) with 10% perchloric acid, neutralizing the resulting supernatant with 1N KOH, and measuring the amount of lactic acid in the resulting supernatant.

[0057] For example, according to the method of the present disclosure, a decrease in pH in fish meat can be suppressed by suppressing the functional expression of myostatin in fish. In other words, the method of the present disclosure also encompasses a method for suppressing a decrease in pH in fish meat. The pH in fish meat (more specifically, intracellular muscle pH) is measured by the method shown in the Examples below. Specifically, the pH in fish meat (e.g., fillets, etc.) (solid) is evaluated by measuring it with a pH meter (e.g., a handheld pH meter Testo 206-pH2 (Testo, Lenzkirch, Germany)).

[0058] For example, according to the method of the present disclosure, the amount of dripping from fish meat can be reduced by suppressing the functional expression of myostatin in fish. In other words, the method of the present disclosure also encompasses a method for reducing the amount of dripping from fish meat. Fish meat dripping is measured by the method shown in the Examples below. Specifically, the amount of dripping from fish meat (e.g., fillets, etc.) during storage is measured, and is evaluated, for example, by calculating [(fish meat weight on day 0 after death (at the start of storage) - fish meat weight after storage (e.g., day 3 or 7 after storage)) / (fish meat weight on day 0 after death (at the start of storage)] x 100 (%).

[0059] For example, the method of the present disclosure can reduce the amount of collagen in fish meat by suppressing the functional expression of myostatin in fish. In other words, the method of the present disclosure also encompasses a method for reducing the amount of collagen in fish meat. The amount of collagen in fish meat is measured by the method shown in the Examples below. Specifically, fish meat (e.g., fillets) is suspended in 6 M HCl, and the suspension is heated under vacuum at 150°C for 1 hour to hydrolyze it, and the hydroxyproline (Hyp) content of the resulting hydrolysate is measured by liquid chromatography. The collagen content is evaluated by multiplying the Hyp content by a coefficient (collagen weight / Hyp weight) obtained from the amino acid composition of the fish collagen to convert it into collagen content.

[0060] For example, according to the method of the present disclosure, the hardness of fish meat can be reduced by suppressing the functional expression of myostatin in fish. In other words, the method of the present disclosure also encompasses a method for reducing fish meat hardness. Fish meat hardness is measured by the method shown in the Examples below. Specifically, using a creep meter equipped with a 20 N load cell (e.g., RHEONER II Creep Meter RE2-33005B (Yamaden, Tokyo, Japan)), a cylindrical plunger (8 mm in diameter) is inserted into fish meat (e.g., fillets) parallel to the direction of the muscle fibers at a speed of 1 mm / s, and the maximum load value (N) at which the plunger breaks the fish meat is evaluated as the breaking strength (N) of the fish meat.

[0061] Without wishing to be bound by theory, according to the method of the present disclosure, as will be shown in the examples described below, the amount of lactic acid in the fish meat is reduced, and thereby the decrease in pH is suppressed, thereby reducing drips (free water). As a result, it is predicted that the reduction in free water that causes spoilage will have an effect of improving meat quality, such as preserving the quality of the fish meat (for example, inhibiting spoilage of the fish meat).

[0062] Without wishing to be bound by theory, according to the method of the present disclosure, as will be shown in the examples described below, the amount of lactic acid in the fish meat is reduced, and the decrease in pH is inhibited, thereby reducing dripping, including free water that has a weak bond to proteins such as muscle fibers, and maintaining moisture in the fish meat, including bound water that has a strong bond to proteins such as muscle fibers. This is expected to have an effect of improving meat quality, such as improving the texture of heated fish meat products (for example, moisture remains in the fish meat even when heated, resulting in a plump texture without dryness).

[0063] Without wishing to be bound by theory, according to the method of the present disclosure, as shown in the examples described below, the amount of lactic acid in the fish meat is reduced, and the decrease in pH is inhibited, thereby reducing dripping, including free water that has a weak bond to proteins such as muscle fibers, and maintaining moisture in the fish meat, including bound water that has a strong bond to proteins such as muscle fibers, and / or increasing muscle fibers and reducing the amount of connective tissue such as collagen, thereby reducing the amount of connective tissue such as collagen per unit meat weight that determines the firmness of the fish meat. Therefore, it is predicted that the method will have an effect of improving meat quality, such as improving the texture of the fish meat (for example, obtaining a chewy, soft texture).

[0064] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure encompasses any and all combinations of the constituent elements described in this specification.

[0065] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0066] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass."

[0067] 1. Evaluation of meat quality in red sea bream lacking the myostatin gene Using genome editing technology, we created red sea bream lacking the myostatin gene and compared them with normal red sea bream to examine changes in meat quality.

[0068] (1) Creation of red sea bream with a mutation in the myostatin gene The myostatin gene was deleted using the method described in Non-Patent Document 1. Specifically, sexually mature male and female red sea bream were subjected to abdominal compression to collect eggs (unfertilized eggs) and sperm. The resulting eggs and sperm were artificially inseminated to obtain fertilized eggs. Cas9 mRNA and guide RNA were used to introduce the mutation by genome editing. The target gene sequence was 5'-GGACGATGAGCACGCCATCACGG-3' (SEQ ID NO: 1) contained in the red sea bream myostatin gene. A solution containing 2-10 pg Cas9 mRNA and 1-5 pg guide RNA was introduced into the cytoplasm of one-cell stage fertilized eggs by microinjection to introduce a mutation into the myostatin gene. The fertilized eggs into which the Cas9 mRNA and guide RNA had been introduced were reared under normal rearing conditions and hatched to obtain the F0 generation. The F0 generation individuals were allowed to sexually mature, and the F1 offspring were obtained with homozygous deletion of the myostatin gene. The modified sequence of the target gene was a 14-base deletion (5'-ACGCCATCACGGAG-3') in the red sea bream myostatin gene, resulting in a functional loss of the myostatin gene.

[0069] The F1 generation of red sea bream was subcultured to produce an F3 generation, which was confirmed to have the 14-base deletion and to have a functional defect in the myostatin gene, accompanied by skeletal muscle hypertrophy and increased obesity.

[0070] (2) Evaluation of meat quality in myostatin gene-deficient fish (KO). The F3 generation of myostatin gene-deficient fish (KO) was compared with myostatin-deficient individuals (WT). (2-1) Measurement of moisture content in fish meat. To confirm moisture content after storage, analysis was outsourced to Bureau Veritas FEAC Co., Ltd. Muscle fillets were collected from 3-year-old red sea bream cultured in the same tank. The moisture generated during storage at 4°C was removed with a wipe. On the fifth day of storage, fillets collected from five KO and five WT individuals were mixed and analyzed. Moisture content was quantified using the loss-on-drying method after drying at 105°C for 5 hours. Results were expressed in grams per 100 g of wet weight of raw fillets.

[0071] The results are shown in Figure 1. As shown in Figure 1, it was revealed that the moisture content in KO was significantly higher than that in WT after 5 days of storage.

[0072] (2-2) Measurement of Lactic Acid Content in Fish Meat Three-year-old red sea bream cultured in the same tank were fasted for one day, then brain-deprived and bled. Fillets were prepared and stored refrigerated (4°C). A portion of meat (1 g) was collected from each dorsal muscle of five fish from each group and homogenized with 5 mL of 10% perchloric acid. The homogenate was centrifuged at 15,000 g for 10 minutes at 4°C. The supernatant was neutralized with 1N KOH on ice and centrifuged at 15,000 g for 10 minutes at 4°C. The lactic acid concentration in the supernatant was measured using a commercially available kit, ENZYTEC Liquid L-lactic acid (J.K. International, Tokyo, Japan).

[0073] The results are shown in Figure 2. As shown in Figure 2, it was revealed that the amount of lactate in KO tended to decrease at day 0 compared to WT, and decreased significantly after storage for 1 day or more.

[0074] (2-3) Measurement of pH in fish meat Three-year-old red sea bream cultured in the same tank were fasted for one day, then brain-deprived and bled, then fillets were prepared and stored refrigerated (4°C). Measurements were taken using the same six fish on days 0 and 1 of storage, and five fish different from those used on days 0 and 1 on day 7. Intracellular muscle pH was measured at two different sites for each sample using a handheld pH meter Testo 206-pH2 (Testo, Lenzkirch, Germany), and the average value was calculated.

[0075] The results are shown in Figure 3. As shown in Figure 3, it was revealed that the pH in KO tended to be higher than that in WT on day 0, and was significantly higher after storage for 1 day or more.

[0076] (2-4) Measurement of fish meat drip volume Three-year-old red sea bream cultured in the same tank were fasted for one day, then brain-deprived and bled. 40 g fillets were prepared and stored refrigerated (4°C). Excess water was removed from the fillets with a wipe, and the initial weight (A) was recorded. Subsequently, body weight was measured every 24 hours until the seventh day after death (B). The amount of drip at each time point was calculated as [(A-B) / A] x 100%.

[0077] The results are shown in Figure 4. As shown in Figure 4, the amount of drip in the KO fish tended to be lower than that in the WT fish on day 0, and was significantly lower after one day of storage. This suggests that the moisture content in the fish meat can be maintained.

[0078] (2-5) Observation of Fish Meat Cross Sections On day 0 of storage, a portion (1 g) was excised from the dorsal muscle and fixed in 4% formalin at room temperature for 24 hours, then stored at 4°C until analysis. The tissue was dehydrated with increasing concentrations of ethanol, placed in a gelatin capsule, crushed in liquid nitrogen, and freeze-dried in tert-butyl alcohol. The specimens were gold-coated using a JFC-1100E sputter ionizer (JEOL), and the muscle fiber structure was observed using an MT-3030 scanning electron microscope (SEM) at an accelerating voltage of 15 kV (Hitachi, Ltd.).

[0079] The results are shown in Figure 5. As shown in Figure 5, it was revealed that the muscle fibers in KO were larger and the spaces between the muscle fibers were narrower.

[0080] (2-6) Measurement of Fish Meat Collagen Content. A portion (1 g) was collected from each dorsal muscle on days 1 and 3 of storage. The muscle samples were freeze-dried and suspended in 2 mL of 6 M HCl. The suspension was hydrolyzed by heating at 150°C under vacuum for 1 hour, and the hydrolysate was diluted to 50 mL with water. The hydroxyproline (Hyp) content of the hydrolysate was measured using an LC-20 liquid chromatography system (Shimadzu Corporation, Kyoto, Japan). The Hyp content was converted to collagen content by multiplying it by a coefficient (collagen weight / Hyp weight) derived from the amino acid composition of red sea bream collagen. Because there was significant variation between individuals depending on the muscle fiber arrangement during slice preparation, five fish were measured in each group, and the individuals with the highest and lowest values ​​were excluded, and three fish from each group were analyzed. Results were expressed in grams per 100 g of dorsal muscle.

[0081] The results are shown in Figure 6. As shown in Figure 6, it was revealed that the amount of collagen contained in the fish meat (connective tissue) of KO was significantly low, and that this was maintained even after storage.

[0082] (2-7) Measurement of Fish Meat Breaking Strength. Three-year-old red sea bream cultured in the same tank were fasted for one day, then brain-deprived and bled. Fillets were prepared and stored refrigerated (4°C). On days 0, 1, and 4, 5-mm-thick fish meat slices were cut from the dorsal muscle perpendicular to the muscle fiber direction. The breaking strength of the slices was evaluated using a RHEONER II creep meter RE2-33005B (Yamaden, Tokyo, Japan) equipped with a 20 N load cell. A cylindrical plunger (8 mm diameter) simulating a molar was inserted into the slice parallel to the muscle fiber direction at a speed of 1 mm / s. The breaking strength of the slice (N) was expressed as the maximum load (N) at which the plunger broke the slice. The breaking strain (mm) of each slice was defined as the penetration distance (mm) from the contact point of the slice to the breaking point. Because there was a large variation between and within individuals depending on how the muscle fibers were arranged when the slices were prepared, three slices per fish were used to calculate the average value for each individual, and five fish in each group were measured. The individuals with the highest and lowest values ​​were excluded, and three fish from each group were analyzed.

[0083] The results are shown in Figure 7. As shown in Figure 7, the breaking load of KO tended to be lower than that of WT even without storage, and was significantly lower after storage. This suggests that the amount of collagen (connective tissue amount) per unit meat weight, which determines the firmness of fish meat, is reduced.

[0084] 2. Evaluation of meat quality in tiger pufferfish lacking the myostatin gene Using genome editing technology, we created tiger pufferfish lacking the myostatin gene and examined changes in meat quality.

[0085] (1) Creation of F0 Generation with a Mutation in the Myostatin Gene Sexually mature male and female pufferfish were administered hormones, followed by abdominal compression to collect eggs (unfertilized eggs) and sperm. The resulting eggs and sperm were artificially inseminated to obtain fertilized eggs. Note that artificially fertilized pufferfish eggs can be produced using hormones (Non-Patent Document 2). Cas9 mRNA and guide RNA were used to introduce mutations via genome editing. The target gene sequence was 5'-GGACGACGAGCACGCCATCACGG-3' (SEQ ID NO: 2) contained in the pufferfish myostatin gene. A solution containing 2-10 pg Cas9 mRNA and 1-5 pg guide RNA was introduced into the cytoplasm of one-cell stage fertilized eggs via microinjection to introduce a mutation into the myostatin gene. The fertilized eggs into which the Cas9 mRNA and guide RNA were introduced were reared under normal rearing conditions and hatched to obtain F0 generation male individuals.

[0086] (2) Creation of F1 generation with a mutation in the myostatin gene Female pufferfish were mated with F0 generation male pufferfish with a mutation in the myostatin gene obtained in (1) above, to obtain an F1 generation carrying a heterozygous deletion of 8 bases (5'-CACGCCAT-3') in the pufferfish myostatin gene.

[0087] (3) Creation of F2 Generation with Mutations in the Myostatin Gene The F1 generation obtained in (2) above was mated with males and females to obtain an F2 generation homozygous for the 8-base deletion in the tiger pufferfish myostatin gene described in (2). It was confirmed that the F2 generation tiger pufferfish had a functional defect in the myostatin gene, accompanied by increased skeletal muscle hypertrophy and obesity compared to normal tiger pufferfish.

[0088] (4) Evaluation of Meat Quality of Myostatin Gene-Deficient Tiger Pufferfish The meat quality of the myostatin gene-deficient tiger pufferfish obtained in (3) above was evaluated using the same method as described above. (4-1) Measurement of Moisture Content in Fish Meat To confirm the moisture content after storage, analysis was outsourced to Bureau Veritas FEAC Co., Ltd. Moisture generated during storage at 4°C was removed with a wipe from muscle fillets collected from 2-year-old tiger pufferfish cultivated in the same tank. On the 5th day of storage, fillets collected from KO2 and WT2 individuals were mixed and analyzed. Moisture content was quantified using the loss-on-drying method after drying at 105°C for 5 hours. Results were expressed in grams per 100g of wet weight of raw fillets.

[0089] The results are shown in Figure 8. As shown in Figure 8, it was revealed that the water content in KO was significantly higher than that in WT after 5 days of storage.

[0090] (4-2) Measurement of pH in fish meat Two-year-old tiger pufferfish cultured in the same tank were fasted for one day, then brain-deprived and bled, then fillets were prepared and stored refrigerated (4°C). Measurements were performed using the same four wild-type fish and three koi fish on days 1 and 7 of storage. Intracellular muscle pH was measured at two different sites for each sample using a handheld pH meter, Testo 206-pH2 (Testo, Lenzkirch, Germany), and the average value was calculated.

[0091] The results are shown in Figure 9. As shown in Figure 9, it was revealed that the pH of the KO was comparable to that of the WT at day 1, but was significantly higher after 7 days of storage.

[0092] (4-3) Measurement of fish meat drip amount Two-year-old tiger pufferfish cultured in the same tank were fasted for one day, then brain-deprived and bled, after which 40 g fillets were prepared and stored refrigerated (4°C). Excess water was removed from the fillets with a wipe, and the initial weight (A) was recorded. Then, the weight was measured on the first and seventh days of storage (B). The amount of drip at each time point was calculated as [(A-B) / A] x 100%.

[0093] The results are shown in Figure 10. As shown in Figure 10, it was clear that the amount of dripping in the KO fish was significantly lower than that in the WT fish after 1 and 7 days of storage. This suggests that the moisture content in the fish meat can be maintained.

[0094] 8 to 10, the myostatin gene-deficient pufferfish obtained in (3) above also had improved meat quality compared to pufferfish that did not lack the myostatin gene. Furthermore, when the meat quality of the myostatin gene-deficient pufferfish obtained in (3) above and that of the fish meat that had been stored at 4°C for about three days and aged were evaluated, it was confirmed that spoilage of the fish meat was inhibited, and the texture of the heated fish meat was improved.

[0095] 3. Evaluation of meat quality in flounder lacking the myostatin gene Using genome editing technology, flounder lacking the myostatin gene were produced and changes in meat quality were examined.

[0096] (1) Creation of F0 Generation with a Mutation in the Myostatin Gene Eggs (unfertilized eggs) and sperm were collected from sexually mature male and female flounders by compressing their abdomens. The resulting eggs and sperm were artificially inseminated to obtain fertilized eggs. Cas9 protein and guide RNA were used to introduce mutations by genome editing. The target gene sequence was 5'-TCTGAGCAAACTGCGAATGAAGG-3' (SEQ ID NO: 3) contained in the flounder myostatin gene. A solution containing 10-50 pg of Cas9 protein and 1-5 pg of guide RNA was introduced into the cytoplasm of one-cell stage fertilized eggs by microinjection to introduce a mutation into the myostatin gene. The fertilized eggs into which the Cas9 protein and guide RNA had been introduced were reared and hatched under normal rearing conditions to obtain the F0 generation.

[0097] (2) Creation of F1 generation with mutations in the myostatin gene Sperm was collected from the F0 generation males and artificially inseminated with eggs collected from sexually mature female flounder to obtain fertilized eggs. The fertilized eggs were reared and hatched under normal rearing conditions to obtain the F1 generation. F1 generation males were obtained from the F1 generation carrying a heterozygous deletion of four bases (5'-AATG-3') in the flounder myostatin gene.

[0098] (3) Generation of F2 generation with mutations in the myostatin gene. The F1 generation males were artificially inseminated with unfertilized eggs from sexually mature female flounder to generate F2 generation females. The myostatin gene mutation in the F2 generation was confirmed to be heterozygous for the four-base deletion, the same as in the F1 generation.

[0099] (4) Creation of F3 Generation with a Mutation in the Myostatin Gene F2 female individuals heterozygous for the four-base deletion in the myostatin gene described in (3) above and F0 male individuals with a mutation in the myostatin gene described in (1) above were sexually matured and artificially inseminated to obtain an F3 generation. From the F3 generation, F3 individuals homozygous for the four-base deletion in the myostatin gene described in (2) above were obtained. It was confirmed that the F3 generation flounder exhibited a functional defect in the myostatin gene accompanied by increased skeletal muscle hypertrophy and obesity compared to normal flounder.

[0100] (5) Evaluation of the meat quality of myostatin gene-deficient flounder The meat quality of the myostatin gene-deficient flounder obtained in (4) above, and the fish meat after storing and aging at 4°C for approximately 3 days, was evaluated, and it was confirmed that the fish meat was prevented from spoiling, the texture of heated fish meat products was improved, and the texture of the fish meat was also improved.

[0101] 4. Evaluation of meat quality in Nile tilapia lacking the myostatin gene We used genome editing technology to create Nile tilapia lacking the myostatin gene and examined changes in meat quality.

[0102] (1) Creation of F0 Generation with a Mutation in the Myostatin Gene After hormone administration, sexually mature Nile tilapia were harvested by compressing their abdomens to collect eggs (unfertilized eggs) and sperm. The resulting eggs and sperm were artificially inseminated to obtain fertilized eggs. The Cas9 protein and guide RNA were used to introduce mutations via genome editing. The target gene sequence was 5'-TCTACTGGGGTGCTGACGGAAGG-3' (SEQ ID NO: 4) contained in the Nile tilapia myostatin gene. A solution containing 10-50 pg of Cas9 protein and 1-5 pg of guide RNA was introduced into the cytoplasm of one-cell stage fertilized eggs via microinjection to introduce a mutation into the myostatin gene. The fertilized eggs introduced with the Cas9 protein and guide RNA were reared and hatched under normal rearing conditions to obtain the F0 generation.

[0103] (2) Creation of F1 generation with a mutation in the myostatin gene Sperm was collected from the F0 generation male individuals and artificially inseminated with eggs collected from sexually mature female Nile tilapia to obtain fertilized eggs. The fertilized eggs were reared and hatched under normal rearing conditions to obtain the F1 generation (heterozygous deficiency). Similarly, sperm was collected from the F0 generation male individuals and artificially inseminated with eggs collected from sexually mature female Nile tilapia of the F0 generation to obtain fertilized eggs. The fertilized eggs were reared and hatched under normal rearing conditions to obtain the F1 generation (homozygous deficiency). F1 generation individuals carrying a 14-base (5'-GCCTTCCGTCAGCA-3') or 13-base (5'-CCTTCCGTCAGCA-3') deletion in the Nile tilapia myostatin gene were obtained from the F1 generation individuals.

[0104] (3) Creation of F2 Generation with Mutations in the Myostatin Gene The F1 generation individuals were allowed to reach sexual maturity to produce F2 and subsequent generations. F2 generation individuals were obtained that homozygously carried either the 14-base or 13-base deletion described in (2) above.

[0105] (4) Evaluation of the meat quality of myostatin gene-deficient Nile tilapia When the texture of the meat quality of the myostatin gene-deficient Nile tilapia obtained in (3) above was evaluated, an improvement in texture was observed.

Claims

1. A method for improving the meat quality of fish, comprising inhibiting the functional expression of myostatin in fish.

2. The method of claim 1, wherein the suppression of functional expression is achieved by introducing a loss-of-function mutation in the myostatin gene.

3. The method of claim 2, wherein the loss-of-function mutation is introduced using at least one genome editing system selected from the group consisting of a clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9 (CRISPR / Cas9) system, a CompoZr Zinc Finger Nuclease (ZFN) system, and a TAL effector nuclease (TALEN) system.

4. The method according to claim 1, wherein the inhibition of functional expression is achieved by inhibiting the expression of the myostatin gene.

5. The method described in claim 4, wherein the gene expression is suppressed using at least one RNA molecule selected from the group consisting of siRNA, shRNA, and miRNA that target myostatin mRNA, or at least one vector selected from the group consisting of a vector capable of expressing said RNA molecule.

6. The method according to claim 1, wherein the inhibition of functional expression is achieved by inhibiting the function of myostatin protein.

7. The method according to claim 6, wherein the inhibition of the protein function is carried out by at least one antibody selected from the group consisting of antibodies that specifically bind to myostatin and inhibit the functional expression of myostatin through this binding.

8. The method according to claim 1, wherein the improvement in fish meat quality is at least one selected from the group consisting of a reduction in the amount of lactic acid in the fish meat, suppression of a decrease in pH in the fish meat, a reduction in the amount of dripping from the fish meat, a reduction in the amount of collagen in the fish meat, and a reduction in the hardness of the fish meat.

9. The method according to claim 1, wherein the improvement in fish meat quality is at least one selected from the group consisting of suppressing putrefaction of fish meat, improving the texture of heat-processed fish meat products, and improving the texture of fish meat itself.

Images