オメガ３脂肪酸代謝酵素は海産魚にとって本当に必要か？

• Project/Area Number: 16J06812
• Research Category: Grant-in-Aid for JSPS Fellows
• Allocation Type: Single-year Grants
• Section: 国内
• Research Field: Aquatic life science
• Research Institution: The University of Tokyo
• Principal Investigator: 壁谷 尚樹 東京大学, 農学生命科学研究科, 特別研究員(PD)
• Project Period (FY): 2016-04-22 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2018: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2017: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2016: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
• Keywords: 多価不飽和脂肪酸代謝 / 不飽和化酵素 / 遺伝子ノックアウト / ゲノム編集 / ノックアウト

Outline of Annual Research Achievements

海産魚への変異導入を実施するため、その準備から採卵、マイクロインジェクションによる第一世代の作出、およびその解析まで行った。実験魚には、ニベ Nibea mitsukuriiを用いた。まず、Crispr/Cas9システムを利用したゲノム編集に必要なガイドRNAのデザインを行った。不飽和化酵素遺伝子2(Fads2)をターゲットとし、3種のガイドRNAをデザインした。まず、これらガイドRNAをニベ受精卵にマイクロインジェクションし、得られた孵化仔魚に目的の変異が導入されているか確認した。その結果、75%の個体において予想した領域への変異導入が確認され、用いたガイドRNAの切断効率は良好であると判断された。その後、8月から9月にかけて親魚作出のためのインジェクションを連続して行った。この際のインジェクション翌日の浮上卵率は40～60%であり、インジェクションを行わない卵(80%以上)と比較すると低下はするものの、浮上卵の孵化率は90%以上を維持したため、十分に生残させることが可能な水準であると判断した。得られた浮上卵は100 L水槽に収容し、EPA・DHA強化したワムシおよびアルテミアを順次給餌した。なおこの間、同ロット内においても大きな体長差が現れ始めたため、状況に応じて成長の遅れが激しい個体を別水槽に移動させるなどして対応した。体長が約20 mmを超え始めた段階で配合飼料への餌付けを行い、体長が30 mm程度を超えた段階で500 L水槽への移動を行った。ここまでの生残数は41尾であった。作出した第一世代の変異導入率を解析するため、胸鰭の一部から抽出したゲノムDNAを用いて、変異導入の有無を確認した。その結果、初期ロットでは56%(9/16)の個体で目的の箇所への変異導入が確認された。今後は初期ロット以降のロットも合わせて解析を続け、次世代を作出する予定である。

Research Progress Status

平成30年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

平成30年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] University of Stirling(英国)
• [Journal Article] A complete enzymatic capacity for long-chain polyunsaturated fatty acid biosynthesis is present in the Amazonian teleost tambaqui, Colossoma macropomum (2019)
  • Author(s): Renato B Ferraz, Naoki Kabeya, Monica Lopes-Marques, Andre M Machado, Ricardo A Ribeiro, Ana L Salaro, Rodrigo Ozorio, L Filipe C Castro, Oscar Monroig
  • Journal Title: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology Volume: 227 Pages: 90-97
  • DOI: 10.1016/j.cbpb.2018.09.003
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Methyl-end desaturases with ?12 and ω3 regioselectivities enable the de novo PUFA biosynthesis in the cephalopod Octopus vulgaris (2019)
  • Author(s): Diego Garrido, Naoki Kabeya, Francisco Hontoria, Juan C Navarro, Diana B Reis, M Virginia Martin, Covadonga Rodriguez, Eduardo Almansa, Oscar Monroig
  • Journal Title: Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids Volume: 1864 Issue: 8 Pages: 1134-1144
  • DOI: 10.1016/j.bbalip.2019.04.012
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] A key metabolic gene for recurrent freshwater colonization and radiation in fishes. (2019)
  • Author(s): Ishikawa, A, N Kabeya, K Ikeya, R Kakioka, JN Cech, N Osada, MC Leal, J Inoue, M Kume, A Toyoda, A Tezuka, AJ Nagano, YY Yamasaki, Y Suzuki, T Kokita, H Takahashi, K Lucek, D Marques, Y Takehana, K Naruse, S Mori, O Monroig, N Ladd, CJ Schubert, B Matthews, CL Peichel, O Seehausen, G Yoshizaki, J Kitano
  • Journal Title: Science Volume: 印刷中
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Functional characterisation of two Δ12-desaturases demonstrates targeted production of linoleic acid as pheromone precursor in Nasonia (2019)
  • Author(s): Florian Semmelmann, Naoki Kabeya, Miriama Malcicka, Astrid Bruckmann, Bastian Broschwitz, Kristina Straub, Rainer Merkl, Oscar Monroig, Reinhard Sterner, Joachim Ruther, Jacintha Ellers
  • Journal Title: The Journal of Experimental Biology Volume: 印刷中
  • DOI: 10.1242/jeb.201038
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] “Out of the can”: A draft genome assembly, liver transcriptome and nutrigenomics of the European sardine, Sardina pilchardus. (2018)
  • Author(s): Andre M. Machado, Ole K. Torresen, Naoki Kabeya, Alvarina Couto, Bent Petersen, Monica Felicio, Paula F. Campos, Elza Fonseca, Narcisa Bandarra, Monica Lopes-Marques, Renato Ferraz, Raquel Ruivo, Miguel M. Fonseca, Sissel Jentoft, Oscar Monroig, Rute R. da Fonseca, L. Filipe C. Castro
  • Journal Title: Genes Volume: 9 Issue: 10 Pages: 485-485
  • DOI: 10.3390/genes9100485
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Desaturases and elongases involved in polyunsaturated fatty acid biosynthesis in aquatic invertebrates: a comprehensive review (2018)
  • Author(s): Oscar Monroig, Naoki Kabeya
  • Journal Title: Fisheries Science Volume: 84 Issue: 6 Pages: 911-928
  • DOI: 10.1007/s12562-018-1254-x
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Genes for de novo biosynthesis of omega-3 polyunsaturated fatty acids are widespread in animals (2018)
  • Author(s): Naoki Kabeya, Miguel M. Fonseca, David E. K. Ferrier, Juan C. Navarro, Line K. Bay, David S. Francis, Douglas R. Tocher, L. Filipe C. Castro, Oscar Monroig
  • Journal Title: Science Advances Volume: 4 Issue: 5
  • DOI: 10.1126/sciadv.aar6849
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Retention of fatty acyl desaturase 1 (fads1) in Elopomorpha and Cyclostomata provides novel insights into the evolution of long-chain polyunsaturated fatty acid biosynthesis in vertebrates (2018)
  • Author(s): Monica Lopes-Marques, Naoki Kabeya, Yu Qian, Raquel Ruivo, Miguel M Santos, Byrappa Venkatesh, Douglas R. Tocher, L Filipe C Castro, Oscar Monroig
  • Journal Title: BMC Evolutionary Biology Volume: 18 Issue: 1 Pages: 157-157
  • DOI: 10.1186/s12862-018-1271-5
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Essential fatty acid metabolism and requirements of the cleaner fish, ballan wrasse Labrus bergylta : Defining pathways of long-chain polyunsaturated fatty acid biosynthesis (2018)
  • Author(s): Kabeya Naoki、Yevzelman Simon、Oboh Angela、Tocher Douglas R.、Monroig Oscar
  • Journal Title: Aquaculture Volume: 488 Pages: 199-206
  • DOI: 10.1016/j.aquaculture.2018.01.039
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Genes for de novo biosynthesis of omega-3 polyunsaturated fatty acids are widespread in animals. (2018)
  • Author(s): Kabeya Naoki、Fonseca Miguel M.、Ferrier David E. K.、Navarro Juan C.、Bay Line K.、Francis David S.、Tocher Douglas R.、Castro L. Filipe C.、Monroig Oscar
  • Journal Title: Science Advances Volume: 4
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Two alternative pathways for docosahexaenoic acid (DHA, 22:6n-3) biosynthesis are widespread among teleost fish (2017)
  • Author(s): Oboh Angela、Kabeya Naoki、Carmona-Antonanzas Greta、Castro L. Filipe C.、Dick James R.、Tocher Douglas R.、Monroig Oscar
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 3889-3889
  • DOI: 10.1038/s41598-017-04288-2
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] 200.Cloning and functional characterization of fads2 desaturase 1 and elovl5 elongase fromJapanese flounder Paralichthys olivaceus (2017)
  • Author(s): N. Kabeya, M.Chiba, Y. Haga, S. Satoh., and G. Yoshizaki
  • Journal Title: Comparative Biochemistry and Physiology Part B Volume: 214B Pages: 36-47
  • DOI: 10.1016/j.cbpb.2017.09.002
  • Peer Reviewed
• [Journal Article] Spawning induction and seed production of Eastern little tuna, Euthynnus affinis (Cantor, 1849), in the post- and pre-spawning seasons by hormonal treatment in a semi-closed recirculation system with elevated temperature (2017)
  • Author(s): Ryosuke Yazawa, Yutaka Takeuchi, Kenta Satoh, Yuri Machida, Kotaro Amezawa, Naoki Kabeya & Goro Yoshizaki
  • Journal Title: Aquaculture Research Volume: Volume 48 Issue: 7 Pages: 3472-3481
  • DOI: 10.1111/are.13173
  • Peer Reviewed
• [Journal Article] Biosynthesis of Polyunsaturated Fatty Acids in Sea Urchins: Molecular and Functional Characterisation of Three Fatty Acyl Desaturases from Paracentrotus lividus (Lamark 1816) (2017)
  • Author(s): Kabeya Naoki、Sanz-Jorquera Alicia、Carboni Stefano、Davie Andrew、Oboh Angela、Monroig Oscar
  • Journal Title: PLOS ONE Volume: 12 Issue: 1 Pages: e0169374-e0169374
  • DOI: 10.1371/journal.pone.0169374
  • Peer Reviewed
• [Journal Article] Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a marine teleost, Nibe croaker (Nibea mitsukurii) (2016)
  • Author(s): H. Yoshikawa, Y. Takeuchi, Y. Ino, J. Wang, G. Iwata, N. Kabeya, R. Yazawa, G. Yoshizaki
  • Journal Title: Aquaculture Volume: - Pages: 35-47
  • DOI: 10.1016/j.aquaculture.2016.05.011
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Production of functionally sterile triploid Nibe croaker Nibea mitsukurii induced by cold-shock treatment with special emphasis on triploid aptitude as surrogate broodstock (2016)
  • Author(s): Y. Takeuchi, T. Yatabe, H. Yoshikawa, Y. Ino, N. Kabeya, R. Yazawa, G. Yoshizaki
  • Journal Title: Aquaculture Volume: - Pages: 0-0
  • DOI: 10.1016/j.aquaculture.2016.05.030
  • Peer Reviewed
• [Presentation] 水産生物の脂肪酸代謝酵素の機能解析とその利用 (2019)
  • Author(s): 壁谷尚樹
  • Organizer: 日本水産学会大会春季大会
  • Invited
• [Presentation] 魚類fads2遺伝子の改変による脂肪酸合成系改良の試み (2019)
  • Author(s): 柿本貴広, 潮 秀樹, 壁谷尚樹, 吉崎悟朗, 木下政人
  • Organizer: 日本水産学会大会春季大会
• [Presentation] 様々な動物種がオメガ3多価不飽和脂肪酸のde novo生合成を可能にする遺伝子を保持する (2018)
  • Author(s): 壁谷尚樹, Fonseca, M.M., Ferrier, D.E.K., Navarro, J.C., Bay, L.K., Francis, D.S., Tocher, D.R., Castro, L.F.C., Monroig O.
  • Organizer: マリンバイオテクノロジー学会
• [Presentation] マダコにおけるオメガ3多価不飽和脂肪酸のde novo生合成 (2018)
  • Author(s): 壁谷尚樹, Garrido, D., Hontoria, F., Navarro, J.C., Reis, D.B., Martin, M.V., Rodriguez, C.R., Almansa, E., Monroig, O.
  • Organizer: 日本水産学会大会秋季大会
• [Presentation] ゲノム編集技術を用いたfads2ノックインマダイの作出 (2018)
  • Author(s): 柿本貴広, 鷲尾洋平, 大濱光希, 壁谷尚樹, 吉崎悟朗, 家戸敬太郎, 木下政人
  • Organizer: 日本水産学会大会秋季大会
• [Presentation] Desaturase genes enabling de novo biosynthesis of omega-3 polyunsaturated fatty acids are widespread in animals. (2018)
  • Author(s): Kabeya, N., Fonseca, M.F., Ferrier, D.E.K., Navarro, J.C., Bay, L.K., Francis, D., Tocher, D.R., Castro, L.F.C., Monroig, O.
  • Organizer: Aoshan Forum - The 4th Workshop on Lipid Nutrition and Metabolism in Aquatic Animals
  • Int'l Joint Research / Invited
• [Presentation] Retracing the evolution of long-chain polyunsaturated fatty acids biosynthesis in vertebrates. (2018)
  • Author(s): Lopes-Marques, M., Kabeya, N., Qian, Y., Ruivo, R.F., Santos, M., Venkatesh, B., Tocher, D.R., Castro, L.F.C., Monroig, O.
  • Organizer: 31st Congress New European Society for Comparative Physiology and Biochemistry
  • Int'l Joint Research
• [Presentation] Creeping up to freshwater: complementation of the DHA biosynthetic pathway in the lineage of freshwater sole (Pleuronectiformes: Achiridae). (2018)
  • Author(s): Matsushita, Y., Sanada, S., Kabeya, N., Haga, Y., Satoh, S., Yazawa, R., Yoshizaki, G.
  • Organizer: 18th International Symposium on Fish Nutrition and Feeding
  • Int'l Joint Research
• [Presentation] Long-chain polyunsaturated fatty acid biosynthesis in Japanese eel Anguilla japonica: Cloning and functional characterization of fatty acid desaturase 1 (Fads1). (2018)
  • Author(s): Kabeya, N., Lopes-Marques, M., Santos, M., Tocher, D.R., Castro, L.F.C., Monroig, O.
  • Organizer: 18th International Symposium on Fish Nutrition and Feeding
  • Int'l Joint Research
• [Presentation] Essential fatty acid metabolism and requirements of the cleaner fish, ballan wrasse Labrus bergylta: Defining pathways of long-chain polyunsaturated fatty acid biosynthesis. (2018)
  • Author(s): Monroig, O., Yevzelman, S., Oboh, A. Tocher, D.R., Kabeya, N.
  • Organizer: 18th International Symposium on Fish Nutrition and Feeding
  • Int'l Joint Research
• [Presentation] Elucidating the biosynthesis of long-chain polyunsaturated fatty acid in a freshwater fish species, Clarias gariepinus. (2018)
  • Author(s): Oboh, A., Betancor, M.B., Kabeya, N., Navarro, J.C., Tocher, D.R., Monroig, O.
  • Organizer: 18th International Symposium on Fish Nutrition and Feeding
  • Int'l Joint Research
• [Presentation] ゲノム編集による塩基置換でゼブラフィッシュのΔ6/Δ5不飽和化酵素 をΔ4不飽和化酵素に改変する. (2018)
  • Author(s): 眞田修和, 松下芳之, 壁谷尚樹, 岸本譲太, 木下政人, 芳賀譲, 佐藤秀一, 矢澤良輔, 吉崎悟朗
  • Organizer: 日本水産学会
• [Presentation] カレイ目魚類の淡水進出とDHA合成能：アキルス科海産種のDHA合成経路 (2018)
  • Author(s): 松下芳之, 眞田修和, 壁谷尚樹, 芳賀 穣, 佐藤秀一, 矢澤良輔, 吉崎悟朗
  • Organizer: 日本水産学会
• [Presentation] Effects of dietary amino acid-imbalance on lipid metabolism in fish. (2017)
  • Author(s): Yoshinaga, H., Kabeya, N., Ushio, H., Haga, Y., Satoh, S.
  • Organizer: International Symposium "Fisheries Science for Future Generations"
  • Int'l Joint Research
• [Presentation] Diversification of Fads2 in finfish species: Implications for aquaculture. (2017)
  • Author(s): Kabeya, N.
  • Organizer: XIV International Symposium on Aquaculture Nutrition
  • Int'l Joint Research / Invited
• [Presentation] Acquiring the capacity of docosahexaenoic acid synthesis by three alternative ways in freshwater soles (Pleuronectiformes: Achiridae). (2017)
  • Author(s): Matsushita, Y., Sanada, S., Kabeya, N., Haga, Y., Satoh, S., Yazawa, R., Yoshizaki, G.
  • Organizer: International Symposium "Fisheries Science for Future Generations"
  • Int'l Joint Research
• [Presentation] Molecular and functional characterisation, and expression of desaturase and elongase genes involved in ω3 LC-PUFA biosynthesis in Sarpa salpa and Pegusa lascaris (2017)
  • Author(s): Garrido, D., Monroig, O., Galindo A., Perez J.A., Acosta N.G., Kabeya N., Betancor M.B., Rodriguez C.
  • Organizer: Aquaculture Europe 2017
  • Int'l Joint Research
• [Presentation] loning and characterization of a fatty acyl desaturase 2 (Fads2) from tambaqui Colossoma macropomum. (2017)
  • Author(s): Ferraz, R., Kabeya, N., Ribeiro, R.A., Lopez-Marques, M., Ozorio, R., Castro, L.F., Monroig, O.
  • Organizer: Aquaculture Europe 2017
  • Int'l Joint Research
• [Presentation] 魚類における 2 通りのドコサヘキサエン酸(DHA)生合成経路 (2017)
  • Author(s): 壁谷尚樹、Angela Oboh、Greta Carmona-Antonanzas、L. Filipe. C. Castro、James R. Dick、Douglas R. Tocher、Oscar Monroig
  • Organizer: 日本水産学会
• [Presentation] パプアニューギニア産ササウシノシタ科淡水カレイはα‐リノレン酸からDHAを合成できる (2017)
  • Author(s): 松下芳之、眞田修和、壁谷尚樹、芳賀穣、佐藤秀一、矢澤良輔、吉崎悟朗
  • Organizer: 日本水産学会
• [Presentation] カレイ目魚類の淡水進出とDHA合成能:不飽和化酸素遺伝子の重複と新機能獲得 (2017)
  • Author(s): 松下芳之、眞田修和、壁谷尚樹、芳賀穣、佐藤秀一、矢澤良輔、吉崎悟朗
  • Organizer: 日本水産学会
• [Presentation] 海産魚の脂肪酸不飽和化酵素の活性と進化 (2016)
  • Author(s): 吉崎悟朗、松下芳之、千葉瑞萌、矢澤良輔、壁谷尚樹
  • Organizer: マリンバイオテクノロジー学会
• [Presentation] ヨーロッパムラサキウニ Paracentrotus lividus の長鎖多価不飽和脂肪酸（LC-PUFA）不飽和化酵素の機能解析 (2016)
  • Author(s): 壁谷尚樹、Alicia Sanz-Jorquera、Stefano Carboni、Andrew Davie、Angela Oboh、Oscar Monroig
  • Organizer: 日本水産学会
• [Book] Investigacion y Desarrollo en Nutricion Acuicola (2017)
  • Author(s): Kabeya Naoki、Yoshizaki Goro、Tocher Douglas R.、Monroig Oscar
  • Total Pages: 581
  • Publisher: Universidad Autonoma de Nuevo Leon
  • ISBN: 9786072708228
• [Book] アグリバイオ 2017年4月号 (2017)
  • Author(s): 壁谷尚樹、吉崎悟朗
  • Total Pages: 102
  • Publisher: 北隆館
• [Book] 水産遺伝育種学 (2017)
  • Author(s): 中嶋 正道、荒井 克俊、岡本 信明、谷口 順彦
  • Total Pages: 256
  • Publisher: 東北大学出版会
  • ISBN: 4861632706