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Ichihara Kensuke

Field Science Center for Northern Biosphere　Aquatic Research Station　Muroran Marine StationAssociate Professor

Researcher basic information

■ Degree
  • 博士（理学）, 北海道大学
■ URL
researchmap URLホームページURL■ Various IDs
Researcher number
  • 60610095
J-Global ID■ Research Keywords and Fields
Research Keyword
  • 海藻類
  • ゲノム編集
  • Sexual reproduction
  • Asexual reproduction
  • Evolution
  • Environmental adaptation
Research Field
  • Life Science, Aquatic life science, 海藻類、有性生殖、無性生殖、ゲノム編集、環境適応
■ Educational Organization

Career

■ Career
Career
  • Nov. 2025 - Present
    Hokkaido University, Field Science Center for Northern Biosphere Aquatic Research Station, 准教授
  • Apr. 2021 - Oct. 2025
    Hokkaido University, Field Science Center for Northern Biosphere Muroran Marine Station, 助教
  • Apr. 2017 - Mar. 2021
    Hokkaido University, Field Science Center for Northern Biosphere Muroran Marine Station, 特任助教
  • 2016 - 2017
    Senshu University, School of Commerce, 非常勤講師
  • 2014 - 2017
    The University of Tokyo, Graduate School of Frontier Sciences, 学振特別研究員（PD）
  • Jun. 2015 - Jun. 2016
    Toho University, Faculty of Science, 非常勤講師
  • 2013 - 2014
    Japan Women's University, Faculty of Science, 学術研究員
  • 2011 - 2013
    Toho University, Faculty of Science, 博士研究員
  • 2008 - 2011
    Hokkaido University, Graduate School of Science, 学振特別研究員（DC1）
Committee Memberships
  • Jan. 2023 - Present
    日本藻類学会, 和文誌編集委員会 副委員長, Society
  • Jan. 2023 - Dec. 2024
    日本藻類学会, 評議員, Society
  • Apr. 2018 - Dec. 2022
    日本藻類学会, 和文誌編集委員会, Society

Research activity information

■ Awards
  • Mar. 2014, 日本藻類学会, 第10回日本藻類学会研究奨励賞
    市原健介
■ Papers
  • Efficient targeted gene knock-in in Ulva using Cas9 RNPs and long single-stranded DNA donors
    Kensuke Ichihara; Chikako Nagasato; Tomokazu Yamazaki; Shigeyuki Kawano
    Phycology Journal, 2, 1, Springer Science and Business Media LLC, 03 Aug. 2026, [Peer-reviewed], [Invited], [Lead author, Corresponding author]
    Scientific journal, Abstract

    Background

    CRISPR-Cas9 ribonucleoprotein (RNP)-mediated genome editing has recently been established in the green seaweed Ulva . However, achieving precise and efficient targeted gene insertion remains challenging due to the low frequency of homology-directed repair (HDR) and suboptimal donor DNA design. In this study, we attempted to optimize a knock-in strategy by co-delivering Cas9 RNPs and donor DNA templates to target the highly expressed RbcS gene for EGFP insertion, while simultaneously disrupting the adenine phosphoribosyltransferase ( APT ) gene for robust selection. We compared the efficacy of single-stranded (ssDNA) versus double-stranded (dsDNA) donors with varying homology arm (HA) lengths.

    Results

    We found that ssDNA donors significantly outperformed dsDNA templates. Furthermore, 50-nt HAs were ineffective, while ssDNA donors with 300-nt HAs achieved the highest insertion efficiency. Sequence analysis revealed the loss of a donor-specific deletion, suggesting that Ulva utilizes synthesis-dependent strand annealing (SDSA) or mismatch repair pathways, rather than the microhomology-mediated mechanisms prevalent in Chlamydomonas . The APT -based co-targeting strategy effectively enriched the candidate population, enabling a discovery rate of approximately 3% for EGFP-positive strains among resistant individuals, achieving the first successful generation of a targeted double mutant in this species. Additionally, using tandem 2 A peptides (P2A-T2A) significantly improved ribosomal skipping efficiency compared to single 2 A systems, facilitating effective polycistronic expression.

    Conclusions

    Collectively, this study establishes a streamlined and highly efficient framework for precise insertional mutagenesis and double-mutant generation in Ulva , thereby expanding the genetic engineering toolkit for this macroalga.
  • Plasmodesmata formation in the early development of Saccharina japonica sporophytes
    Kengo Sawa; Taizo Motomura; Kensuke Ichihara; Karen Miya; Ko Yoshimura; Chika Kosugi; Chikako Nagasato
    PHYCOLOGICAL RESEARCH, 19 Jun. 2026, [Peer-reviewed]
    English, Scientific journal
  • In a grain of sand: An overlooked over-summering habitat of macroalgae.
    Yusuke Horinouchi; Kosei Mochizuki; Kensuke Ichihara; Tatsuya Togashi
    Ecology, e4447, 07 Oct. 2024, [Peer-reviewed], [International Magazine]
    English, Scientific journal
  • Cas9 ribonucleoproteins and single-strand oligodeoxynucleotides enable targeted insertional mutagenesis in Ulva
    Kensuke Ichihara; Tomokazu Yamazaki; Shigeyuki Kawano
    Algal Research, 81, Jul. 2024, [Peer-reviewed], [Lead author, Corresponding author]
    English, Scientific journal
  • Application of CRISPR-Cas9 genome editing by microinjection of gametophytes of Saccharina japonica (Laminariales, Phaeophyceae)
    Yuan Shen; Taizo Motomura; Kensuke Ichihara; Yusuke Matsuda; Ko Yoshimura; Chika Kosugi; Chikako Nagasato
    Journal of Applied Phycology, Springer Science and Business Media LLC, 27 Mar. 2023, [Peer-reviewed]
    English, Scientific journal
  • Gamete dimorphism of the isogamous green alga (Chlamydomonas reinhardtii), is regulated by the mating type-determining gene, MID
    Ryoya Innami; Shinichi Miyamura; Masako Okoshi; Tamotsu Nagumo; Kensuke Ichihara; Tomokazu Yamazaki; Shigeyuki Kawano
    Communications Biology, 5, 1, Springer Science and Business Media LLC, 06 Dec. 2022, [Peer-reviewed]
    English, Scientific journal, Abstract

    The gametes of chlorophytes differ morphologically even in isogamy and are divided into two types (α and β) based on the mating type- or sex-specific asymmetric positioning of the mating structure (cell fusion apparatus) with respect to the flagellar beat plane and eyespot, irrespective of the difference in gamete size. However, the relationship between this morphological trait and the mating type or sex determination system is unclear. Using mating type-reversed strains of the isogamous alga Chlamydomonas reinhardtii, produced by deletion or introduction of the mating type-determining gene MID, we revealed that the positioning of the mating structure is associated with conversion of mating types (mt– and mt+), implying that this trait is regulated by MID. Moreover, the dominant mating type is associated with the type β phenotype, as in the chlorophyte species Ulva prolifera. Our findings may provide a genetic basis for mating type- or sex-specific asymmetric positioning of the chlorophyte mating structure.
  • Genome editing using a DNA-free clustered regularly interspaced short palindromic repeats-Cas9 system in green seaweed Ulva prolifera
    Kensuke Ichihara; Tomokazu Yamazaki; Shigeyuki Kawano
    Phycological Research, 70, 1, 50, 56, Jan. 2022, [Peer-reviewed], [Lead author, Corresponding author]
    Scientific journal
  • Changes in Cell Wall Structure During Rhizoid Formation of Silvetia babingtonii (Fucales, Phaeophyceae) Zygotes.
    Rina Yonamine; Kensuke Ichihara; Shiro Tsuyuzaki; Cécile Hervé; Taizo Motomura; Chikako Nagasato
    Journal of phycology, 57, 4, 1356, 1367, 01 May 2021, [Peer-reviewed], [International Magazine]
    English, Scientific journal, We examined the ultrastructure of the cell wall and immunolocalization of alginates using specific antibodies against M-rich alginates and MG blocks during rhizoid formation in fucoid zygotes, Silvetia babingtonii. The thallus region of 24-h-old zygotes had a cell wall made of three layers with different fiber distribution. In the 12-h-old zygotes, three layers in the thallus were observed before rhizoid formation, namely the inner, middle, and outer layers. During rhizoid elongation, only the inner layer was apparent close to the rhizoid tip area. Immunoelectron microscopy detected M-rich blocks of alginate on the inner half of the cell wall, irrespective of the number of layers in the thallus and rhizoid regions. The MG blocks were seen to cover a slightly wider area than M-rich alginate blocks. It was suggested that parts of M in mannuronan would be rapidly converted to G, and MG-blocks are generated. Transcriptome analysis was performed using 3 -, 10 -, and 24-h-old zygotes after fertilization to examine the relationship between gene expression and alginate synthesis over time. The expression of two mannuronan C5-epimerase homologs that convert mannuronic acid into guluronic acid in alginates was upregulated or downregulated over the course of the examination.
  • Asexual thalli originated from sporophytic thalli via apomeiosis in the green seaweed Ulva
    Kensuke Ichihara; Tomokazu Yamazaki; Shinichi Miyamura; Masanori Hiraoka; Shigeyuki Kawano
    Scientific Reports, 9, 1, 13523, 18 Sep. 2019, [Peer-reviewed], [Lead author, Corresponding author]
    English, Scientific journal, AbstractApomixis is an asexual reproduction system without fertilization, which is an important proliferation strategy for plants and algae. Here, we report on the apomeiosis in the green seaweed Ulva prolifera, which has sexual and obligate asexual populations. Genomic PCR of mating type (MT)-locus genes revealed asexual thalli carrying both MT genomes. Observation of the chromosomes during the formation of each type of reproductive cell revealed that cells in asexual thalli performed apomeiosis without chromosome reduction. Moreover, genotyping revealed that laboratory-cultured sporophytic thalli produced not only each type of gametophyte but also diploid thalli carrying the mt− and mt+ genome (mt± thallus strains). The mt± thallus strain released diploid biflagellate zoids, with ultrastructure and behavior similar to mt+ gametes. Additionally, a transcriptomic analysis revealed that some meiosis-related genes (Mei2L and RAD1) were highly expressed in the quadriflagellate zoosporoids. Our results strongly suggest that asexual thalli originally evolved via apomeiosis in sporophytic thalli.
  • Visualization of Gamete Mating Structure of Marine Green Alga by FE-SEM.
    Shinichi Miyamura, Kensuke Ichihara, Tomokazu Yamazaki, Kazuyoshi Kuwano & Shigeyuki Kawano.
    Cytologia, 84, 3, 191, 192, Sep. 2019, [Peer-reviewed]
  • Metabolome profiling of various seaweed species discriminates between brown, red, and green algae
    Shahlizah Sahul Hamid; Masataka Wakayama; Kensuke Ichihara; Katsutoshi Sakurai; Yujin Ashino; Rie Kadowaki; Tomoyoshi Soga; Masaru Tomita
    Planta, 01 Jan. 2019, [Peer-reviewed]
  • Taxonomic reassessment of Ulva prolifera (Ulvophyceae, Chlorophyta) based on specimens from the type locality and Yellow Sea green tides
    Jianjun Cui; Alvin P. Monotilla; Wenrong Zhu; Yoshihito Takano; Satoshi Shimada; Kensuke Ichihara; Tohru Matsui; Peimin He; Masanori Hiraoka
    Phycologia, 57, 692, 704, 21 Sep. 2018, [Peer-reviewed]
  • Genomic structure and evolution of the mating type locus in the green seaweed Ulva partita
    Tomokazu Yamazaki; Kensuke Ichihara; Ryogo Suzuki; Kenshiro Oshima; Shinichi Miyamura; Kazuyoshi Kuwano; Atsushi Toyoda; Yutaka Suzuki; Sumio Sugano; Masahira Hattori; Shigeyuki Kawano
    SCIENTIFIC REPORTS, 7, Sep. 2017, [Peer-reviewed]
    English, Scientific journal
  • Examination of species delimitation of ambiguous DNA-based Ulva (Ulvophyceae, Chlorophyta) clades by culturing and hybridisation
    Masanori Hiraoka; Kensuke Ichihara; Wenrong Zhu; Satoshi Shimada; Naohiro Oka; Jianjun Cui; Shuntaro Tsubaki; Peimin He
    PHYCOLOGIA, 56, 5, 517, 532, 2017, [Peer-reviewed]
    English, Scientific journal
  • Molecular phylogeny and detailed morphological analysis of two freshwater Rhizoclonium strains from contrasting spring types in Egypt and Italy
    A. A. Saber; K. Ichihara; M. Cantonati
    PLANT BIOSYSTEMS, 151, 5, 800, 812, 2017, [Peer-reviewed]
    English, Scientific journal
  • Two new green algae, Rhizoclonium fractum sp nov and R-umbraticum sp nov., from tropical and subtropical brackish waters of Japan
    Kensuke Ichihara; Satoshi Shimada; Kazuyuki Miyaji
    PHYTOTAXA, 266, 4, 231, 249, Jun. 2016, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Candidate key genes for low-salinity adaptation identified by RNA-seq comparison between closely related Ulva species in marine and brackish waters.
    Yuka Masakiyo; Atsushi Ogura; Kensuke Ichihara; Kei Yura; Satoshi Shimada
    Algal Resources 9: 61-76., 2016, [Peer-reviewed]
  • Phylogeography of macroalgal species distributed in brackish water: Ulva Prolifera (ulvophyceae) and Pyropia tenera (bangiophyceae)
    Satoshi Shimada; Kensuke Ichihara; Yuka Masakiyo; Riko Kawaguchi; Norio Kikuchi
    Seaweed Phylogeography: Adaptation and Evolution of Seaweeds under Environmental Change, 345, 360, Springer Netherlands, 01 Jan. 2016, [Peer-reviewed]
    English, In book
  • Ulva partita sp nov., a Novel Enteromorpha-Like Ulva Species from Japanese Coastal Areas
    Kensuke Ichihara; Ryogo Suzuki; Tomokazu Yamazaki; Shuhei Ota; Yuko Mogi; Yayoi Kagami; Kazuyoshi Kuwano; Shigeyuki Kawano
    CYTOLOGIA, 80, 3, 261, 270, Sep. 2015, [Lead author, Corresponding author]
    English, Scientific journal
  • Taxonomic reinvestigation of Petalonia (Phaeophyceae, Ectocarpales) in southeast of Honshu, Japan, with a description of Petalonia tenuis sp. nov.
    Kaoru Matsumoto; Kensuke Ichihara; Satoshi Shimada
    PHYCOLOGIA, 53, 2, 127, 136, 2014, [Peer-reviewed]
    English, Scientific journal
  • Systematics of Rhizoclonium-like algae (Cladophorales, Chlorophyta) from Japanese brackish waters, based on molecular phylogenetic and morphological analyses
    Kensuke Ichihara; Satoshi Shimada; Kazuyuki Miyaji
    Phycologia, 52, 5, 398, 410, Sep. 2013, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Comparing the low-salinity tolerance of Ulva species distributed in different environments
    Kensuke Ichihara; Kazuyuki Miyaji; Satoshi Shimada
    PHYCOLOGICAL RESEARCH, 61, 1, 52, 57, Jan. 2013, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Morphological variations of the seagrass species, Halophila nipponica (Hydrocharitaceae, Alismatales)
    Satoshi Shimada; Maki Watanabe; Kensuke Ichihara; Masayuki Uchimura
    Coastal Marine Science, 35, 1, 85, 90, International Coastal Research Center, Atmosphere and Ocean Research Institute, the University of Tokyo, 2012, [Peer-reviewed]
    English, During recent field surveys in the Okinawa Islands, southern Japan, we found a peculiar seagrass growing at sandy ground 10 m in depth at one of the Islands, Ie Island (27 June 2010). The overall gross morphology of this plant was very similar to that of Halophila species, while the leaves were much narrower, up to 1 mm in width (up to 3 cm in length). The cross veins were not recognized with naked eyes and under dissecting microscope, but under optical microscope 2-4 cross veins and intramarginal vein were observed in the leaves. In cross section, the leaves were undulated each side of the midrib. To clarify the exact taxonomic status of this plant, the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA was analyzed. As the result, this plant was included in the H. nipponica clade and possessed the identical ITS sequence with the plants from Nakagusuku Bay, Okinawa Main Island having broad linear leaves, and from Ooura Bay, Ishigaki Island having elliptical leaves, typical forms in the H. nipponica clade. Our present study therefore suggests that H. nipponica has a marvelously wide range of morphological plasticity and that the gross morphology of leaves might be of little significance in specific descrimination.Proceedings of the Horiba International Conference "New Direction of Ocean Research in the Western Pacific" : Past, Present and Future of UNESCO/IOC/WESTPAC Activity for 50 years and the JSPS Project "Coastal Marine Science"Section I: Research Articles/Biodiversity
  • Threatened species Nemalionopsis tortuosa (Thoreales, Rhodophyta) in Japan, new locality and current condition of its all reported habitats.
    Satoshi Shimada; Kensuke Ichihara; Yuka Masakiyo; Masafumi Iima; Tadao Yoshida; Shigeru Kumano
    Algal resource, 5, 9, 15, 2012, [Peer-reviewed]
    English, Scientific journal
  • ISOLATION AND TEMPORAL EXPRESSION ANALYSIS OF FRESHWATER-INDUCED GENES IN ULVA LIMNETICA (ULVALES, CHLOROPHYTA)
    Kensuke Ichihara; Frederic Mineur; Satoshi Shimada
    JOURNAL OF PHYCOLOGY, 47, 3, 584, 590, Jun. 2011, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Culture and Hybridization Experiments on an Ulva Clade Including the Qingdao Strain Blooming in the Yellow Sea
    Masanori Hiraoka; Kensuke Ichihara; Wenrong Zhu; Jiahai Ma; Satoshi Shimada
    PLOS ONE, 6, 5, May 2011
    English, Scientific journal
  • Enteromorpha-like Ulva (Ulvophyceae, Chlorophyta) Growing in the Todoroki River, Ishigaki Island, Japan, with Special Reference to Ulva meridionalis Hoiromoto et Shimada, sp. nov.
    Rika Horimoto; Yuka Masakiyo; Kensuke Ichihara; Satoshi Shimada
    Bulletin of the National Museum of Nature and Science. Series B, Botany, 37, 4, 155, 167, National Museum of Nature and Science, 2011, [Peer-reviewed]
    English, Scientific journal
  • Phylogeographic analysis of the genus Ulva (Ulvales, Chlorophyta), including bloom sample in Qingdao, China
    Satoshi Shimada; Moe Nagano; Masanori Hiraoka; Kensuke Ichihara; Frederic Mineur; Wenrong Zhu
    Coastal Marine Science, 34, 1, 117, 122, 2010, [Peer-reviewed]
    English
  • New species of freshwater Ulva, Ulva limnetica (Ulvales, Ulvophyceae) from the Ryukyu Islands, Japan
    Kensuke Ichihara; Shogo Arai; Masayuki Uchimura; Etienne J. Fay; Hiroki Ebata; Masanori Hiraoka; Satoshi Shimada
    PHYCOLOGICAL RESEARCH, 57, 2, 94, 103, Jun. 2009, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • cDNA cloning of a lectin-like gene preferentially expressed in freshwater from the macroalga Ulva limnetica (Ulvales, Chlorophyta)
    Kensuke Ichihara; Shogo Arai; Satoshi Shimada
    PHYCOLOGICAL RESEARCH, 57, 2, 104, 110, Jun. 2009, [Peer-reviewed], [Lead author]
    English, Scientific journal
■ Other Activities and Achievements
■ Books and other publications
■ Lectures, oral presentations, etc.
■ Syllabus
  • 水圏科学特論Ⅰ, 2024年, 修士課程, 環境科学院
  • 大学院共通授業科目（一般科目）：自然科学・応用科学, 2024年, 修士課程, 大学院共通科目
  • 大学院共通授業科目（一般科目）：自然科学・応用科学, 2024年, 修士課程, 大学院共通科目
  • 科学・技術の世界（1単位）, 2024年, 学士課程, 全学教育
  • 一般教育演習(ﾌﾚｯｼｭﾏﾝｾﾐﾅｰ), 2024年, 学士課程, 全学教育
  • 海藻学実習, 2024年, 学士課程, 理学部
■ Research Themes
  • 緑色海藻の生殖細胞に性差を生み出す分子機構の解明
    科学研究費助成事業
    01 Apr. 2024 - 31 Mar. 2028
    市原 健介
    日本学術振興会, 基盤研究(C), 北海道大学, 24K09512
  • 褐藻における鉄摂取機構の分子基盤解明
    科学研究費助成事業
    Jun. 2025 - Mar. 2028
    長里 千香子; 市原 健介; 米山 明男
    日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 25K22376
  • 緑藻スジアオノリ配偶子の微細な性差から解く、細胞間の相互認識と融合の仕組み
    科学研究費助成事業 基盤研究(C)
    01 Apr. 2021 - 31 Mar. 2024
    市原 健介
    本研究の目的は、アオノリ配偶子間の「認識」と「融合」に重要な役割をもつタンパク質の実体と機能を明らかにすることを通して、雌雄性というものがどのような構造の違いや性質の違いから始まったものであるかを明らかにすることである。当該年度は以前行ったスジアオノリ雌雄配偶子の細胞膜および鞭毛のプロテオーム解析の結果から、配偶子間の「認識」と「融合」に関与すると思われるタンパク質を探索した。アノテーション用のデータベースには雌雄配偶子の形成過程で経時的にサンプリングしたRNAseqの結果をde novo assemblyしたものを利用した。解析の結果、鞭毛からは緑藻クラミドモナスで発見された鞭毛に存在し、雌雄や種の認識に機能すると考えられているアグルチニン様タンパク質が発見された。また、雄の細胞膜に特異的なタンパク質として、植物の受精因子として知られるGEX2遺伝子のホモログも発見することができた。
    このUpGEX2に対して、gRNAをデザインしCas9 RNPをPEG法により雌雄配偶子へ導入することで、複数のgex2欠損変異配偶体を得ることが出来た。雄のgex2欠損変異配偶体由来の配偶子は雌配偶子との接合子形成が全く見られないのに対し、雌のgex2欠損変異配偶体由来の配偶子は雄配偶子との間で正常に接合子形成が進んだ。このことからアオサ藻綱においても、GEX2は雄側の受精因子として機能していることが明らかになった。また現在、細胞融合因子として知られるGCS1についても複数の欠損変異体を得ることが出来ており今後表現型解析を行う予定である。さらに雌の配偶子膜から発見された膜貫通ドメインを持つEGF-domainを含むタンパク質やLLR(Leucine-Rich Repeats)を持つキナーゼ等についても欠損変異体を作成する予定である。
    日本学術振興会, 基盤研究(C), 北海道大学, 21K06242
  • The mechanism of recognition and fusion in Ulva gametes
    Grants-in-Aid for Scientific Research Grant-in-Aid for Early-Career Scientists
    Apr. 2018 - Mar. 2021
    ICHIHARA Kensuke
    In this study, the proteins involved in the recognition and the fertilization between male and female gametes in Ulvophyceae, whose molecular mechanisms of reproductive processes have been poorly understood, were searched. At first, transcriptomic analysis during gametogenesis and proteomic analysis in flagellar and plasma membrane of male and female gamete was carried out. Through these analyses, genes involved in fertilization in land plants, such as GCS1 and GEX2 were detected. Furthermore, the genome-editing experiment for gene function analysis in Ulva was tried to establish. Polyethylene glycol (PEG)-mediated transfection method made it possible to efficiently deliver the preassembled CRISPR/Cas9 RNP to Ulva gametes. By this genome-editing method, gex2 deficient male mutants were obtained and male gametes released from these mutants can't fertilize with female gametes. The results suggest that GEX2 is a conserved gene that controls fertilization in the green plant lineage.
    Japan Society for the Promotion of Science, Grant-in-Aid for Early-Career Scientists, Hokkaido University, 18K14690
  • Molecular Basis of Adaptive Evolution from Brackish to Freshwater in the Green Seaweed Ulva
    Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows
    Apr. 2014 - Mar. 2017
    市原 健介
    1) 四万十川採集個体でのRAD-seq解析
    昨年度に四万十川で採集し、SNPやマイクロサテライト部位などのゲノム中の多型情報を得るために、RAD-seq解析をおこなった。
    2) スジアオノリの配偶子および2本鞭毛型、4本鞭毛型無性胞子のRNA-seq解析
    スジアオノリの両接合型の配偶子、および2本鞭毛型無性胞子（無性配偶子）、4本鞭毛型無性胞子（無性胞子）を生殖細胞誘導によって、回収し、これらの細胞で発現している遺伝子の比較をおこなうためにRNA-seq解析をおこなった。真核生物における減数分裂コア遺伝子が保存されていることがわかり、このうち姉妹染色分体の結合に機能しているコヒーシン関連遺伝子(SYN3)が無性胞子や無性配偶子で発現量が低くなっており、前年度に観察された無性個体の生殖細胞形成時の第一減数分裂時に姉妹染色分体が乖離する現象と一致しているように思われた。
    3) スジアオノリとヒラアオノリの全ゲノム解析
    スジアオノリのゲノム解析を進め、現在までにドラフトゲノムの状態ではあるが、オスのゲノムサイズが約70 Mbpで遺伝子数は8,244個であることがわかっている。近縁種ヒラアオノリとスジアオノリのゲノムを比較したところ、ゲノム全体ではシンテニーが高度に保存されていることが確認できたが、性染色体領域ではシンテニーが失われており、この領域では遺伝子の転座等が起こりやすく、進化速度が速い領域であることが示唆された。またヒラアオノリで単離された性特異的な領域の遺伝子の多くは，スジアオノリにおいても，特定のスカッホールドにクラスター化して存在していることが明らかとなり，この性特異的な染色体領域はアオノリの性決定に保存的に機能していると考えられた。
    Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, The University of Tokyo, 14J08330
  • 海藻の『淡水』・『汽水』への適応に関する分子進化学的研究
    Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows
    2008 - 2010
    市原 健介
    本研究はアオサ属における海水域から汽水・淡水域への適応進化の分子機構を総合的に理解することを最終的な目標としている。実験は沖縄県で発見したアオサ属に含まれる唯一の淡水産種であるUlva limneticaを用いている。これまでの実験で本種は淡水・海水両条件で良好な生育を示すことが確認できている。
    今年度は前年度に単離した淡水誘導性遺伝子群についてRT-PCRを用い、淡水環境での発現上昇の確認と淡水移行後の継時的な発現量の推移(1、4、24時間、3日、7日後)について解析を行った。cDNAサブトラクション法によって単離した217個の異なる淡水誘導性遺伝子のうち39個の遺伝子に関してRT-PCRを用いて淡水条件下で発現が上昇していることを確認した。またこれらの遺伝子についてその発現量の推移を解析した。その結果、39個の遺伝子群には発現上昇のパターンが異なる3つのグループが存在することが明らかになった。一つ目のグループは淡水移行直後の短い期間(1～12時間後)にのみ発現が上昇し、その後海水条件と同じ程度の発現量まで減少した。2つ目のグループでは淡水移行後に見られた発現の上昇が7日目まで維持された。また、3つ目のグループでは1つ目のグループと似た発現パターンを示したが、発現量のピークはより遅い3、7日目に見られた。2つ目のグループは急激なイオン・浸透圧の変化に対するストレス応答など、1,3つ目のグループは淡水環境に藻体を順応させ、維持していくために必要な遺伝子群がそれぞれ含まれているのではないかと考えている。ここまでの部分は論文としてまとめJournal of Phycologyに受理されている。また、これらの遺伝子のうちアスコルビン酸ペルオキシダーゼ、トリオースリン酸イソメラーゼ、リボソームタンパク質などは汽水産種スジアオノリ、海産種ウスバアオノリ、アナアオサでオーソログの単離を行い、現在、それぞれの低塩濃度条件での発現量の推移をrealtime-PCRによって調査している。
    Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, Hokkaido University, 08J04039