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フロンティア科学総合研究センター 実験支援部門遺伝資源分野 |
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Ng H.M., Gondo T., Tanaka H., Akashi R.
Plant Cell Reports 43 ( 2 ) 50 2024年2月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Plant Cell Reports
Key Message: Genome editing by CRISPR/Cas9 can be applied to Z. matrella ‘Wakaba’, and knockout mutants of ZmNYC1 gene exhibited stay-green phenotype and reduced tillering. Abstract: Zoysia matrella is a widely used C4 warm-season turfgrass for landscaping, golf courses, and sports fields. Here, we used the CRISPR/Cas9 system to target the Non-Yellow Coloring1 (ZmNYC1) gene in the highly heterozygous allotetraploid Z. matrella ‘Wakaba’, aiming to generate a novel stay-green variety. Of 441 Agrobacterium-infected calli, 22 (5.0%) were transformed, and 14 of these (63.6%) showed targeted mutations through cleaved amplified polymorphic sequences analysis. Sequencing analysis revealed mutations mostly consisting of 1 or 2 bp indels, occurring 2 to 4 bp upstream of the PAM sequence. Regenerated plants exhibited five ZmNYC1 target locus genotypes, including homozygous mutants with a complete knockout of all four alleles in the T0 generation. Under dark treatment, ZmNYC1-mutated plants displayed suppressed chlorophyll b (Chl b) degradation, leading to higher chlorophyll content and Chl b, with a lower chlorophyll a/chlorophyll b ratio compared to the wild type (WT). However, the ZmNYC1 mutation also inhibited plant growth in homozygous mutant genotypes, exhibiting reduced tillering compared to WT. Additionally, during winter simulation, mutant with a complete knockout retained greenness longer than the WT. This is the first successful use of CRISPR/Cas9 genome editing in zoysiagrass. The mutants of the ZmNYC1 gene would serve as valuable breeding material for developing improved zoysiagrass varieties that can maintain their green color for longer periods, even during winter dormancy.
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Ng H.M., Gondo T., Yamada T., Tanaka H., Akashi R.
Grassland Science 70 ( 2 ) 67 - 76 2024年
掲載種別：研究論文（学術雑誌） 出版者・発行元：Grassland Science
Bahiagrass (Paspalum notatum Flügge) is a warm-season grass where high lignin content limits its forage quality, negatively affecting animal performance. To create a new breeding method by genome editing, isolation and characterization of lignin biosynthesis genes and identification of their molecular relationships are essential. The caffeic acid O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD) cDNA clones were isolated from bahiagrass, and their protein sequences showed high similarity to other C4 monocot species through phylogenetic analysis. Gene expression analysis of phenylalanine ammonia-lyase (PAL), COMT and CAD, involved in crucial stages of lignin biosynthesis and lignin content, was conducted at different plant developmental stages. The highest gene expression levels of these genes were observed in the vegetative (V) and early reproductive stages, while the lignin content increased until the middle reproductive stage and remained constant thereafter. Additionally, to further understand the molecular relationships in lignin biosynthesis, the effect of CAD downregulation was analyzed in transgenic bahiagrass lines introduced with sorghum CAD antisense and RNAi vector obtained from a previous report. This led to a reduced lignin content and affected the expression of PAL and COMT working upstream of CAD. In the V stage, PAL expression was lower in transgenic lines compared to wild type (WT), while COMT expression showed no significant differences. However, PAL and COMT expression of transgenic lines in the middle reproductive stage (R2) was significantly higher than in the WT. These findings suggest that the downregulation of CAD gene expression affects PAL and COMT expression and induces a feedback system in the R2 stage. Lignin content influenced the phenotype of the transgenic plants with significantly reduced lignin, exhibiting a dwarf phenotype with shorter plant heights. The findings of this study can be applied to genome editing for the development of practical new breeding materials with improved digestibility in bahiagrass.
DOI： 10.1111/grs.12419
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Narushima J., Kimata S., Shiwa Y., Gondo T., Akimoto S., Soga K., Yoshiba S., Nakamura K., Shibata N., Kondo K.
Genes to Cells 27 ( 12 ) 706 - 718 2022年12月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Genes to Cells
Genome-editing using the CRISPR-Cas9 system has the potential to substantially accelerate crop breeding. Since off-target editing is one of problems, a reliable method for comprehensively detecting off-target sites is needed. A number of in silico methods based on homology to on-target sequence have been developed, however the prediction without false negative is still under discussion. In this study, we performed a SITE-Seq analysis to predict potential off-target sites. SITE-Seq analysis is a comprehensive method that can detect double-strand breaks in vitro. Furthermore, we developed a systematic method using SITE-Seq in combination with web-based Galaxy system (Galaxy for Cut Site Detection), which can perform reproducible analyses without command line operations. We conducted a SITE-Seq analysis of a rice genome targeted by OsFH15 gRNA-Cas9 as a model, and found 41 candidate off-target sites in the annotated regions. Detailed amplicon-sequencing revealed mutations at one off-target site in actual genome-edited rice. Since this off-target site has an uncommon protospacer adjacent motif, it is difficult to predict using in silico methods alone. Therefore, we propose a novel off-target assessment scheme for genome-edited crops that combines the prediction of off-target candidates by SITE-Seq and in silico programs and the validation of off-target sites by amplicon-sequencing.
DOI： 10.1111/gtc.12985
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Jiamtae P., Hashiguchi M., Lelapiyamit I., Harada K., Gondo T., Tanaka H., Akashi R.
Euphytica 218 ( 12 ) 2022年12月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Euphytica
Soybean (Glycine max L. Merr.) seeds are highly sensitive to the environmental conditions experienced in STORAGE. The objectives of this study were to identify QTLs for soybean seed storability by evaluating seed viability and seed vigor and investigating candidate genes for these traits in detected QTL regions. These objectives will aid in developing soybean cultivars with a high capacity for storability. The seeds of 109 and 90 recombinant inbred lines (RILs) derived from a cross between Misuzudaizu and Moshidou Gong 503 in 2010 and 2019, respectively, were used to evaluate seed viability by a germination test and seed vigor by an accelerated aging test, after storage under two temperature conditions (25 °C and 35 °C) for six months. Seed viability and seed vigor of Moshidou Gong 503 were higher than those of Misuzudaizu in both temperature conditions. The average seed viability and seed vigor of RILs decreased when stored at the higher temperature. A total of five QTLs were found for the two traits, seed viability and seed vigor, located in chromosomes 2, 6 and 8. The Misuzudaizu alleles decreased seed viability and seed vigor at all detected QTLs. Most QTLs in this study were found near loci controlling seed viability, maturity, germination, seed hardness, and seed surface micromorphology, indicating that seed storability is related to these traits. In addition, two new QTLs were found that are associated with seed storability.
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Muguerza M.B., Gondo T., Ishigaki G., Shimamoto Y., Umami N., Nitthaisong P., Rahman M.M., Akashi R.
Plants 11 ( 9 ) 2022年5月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Plants
Warm-season grasses are C4 plants and have a high capacity for biomass productivity. These grasses are utilized in many agricultural production systems with their greatest value as feeds for livestock, bioethanol, and turf. However, many important warm-season perennial grasses mul-tiply either by vegetative propagation or form their seeds by an asexual mode of reproduction called apomixis. Therefore, the improvement of these grasses by conventional breeding is difficult and is dependent on the availability of natural genetic variation and its manipulation through breeding and selection. Recent studies have indicated that plant tissue culture system through somatic em-bryogenesis complements and could further develop conventional breeding programs by micro-propagation, somaclonal variation, somatic hybridization, genetic transformation, and genome ed-iting. This review summarizes the tissue culture and somatic embryogenesis in warm-season grasses and focus on current status and above applications including the author’s progress.
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形質転換プロトコール（植物編）「2章2-3バヒアグラス」
権藤 崇裕、明石 良（ 担当： 分担執筆）
化学同人 2012年9月
総ページ数：412 担当ページ：97-105 記述言語：日本語 著書種別：学術書
MISC 【 表示 ／ 非表示 】
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暖地型イネ科牧草類のバイオ技術 ―組織培養・遺伝子組換え技術の確立からゲノム編集技術の開発まで― 招待あり
権藤 崇裕
アグリバイオ 3 ( 10 ) 975 - 980 2019年9月
記述言語：日本語 掲載種別：記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）
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バイオリソース＆データベース活用術〜ミヤコグサ・ダイズ モデル植物から作物への展開 招待あり
橋口正嗣、権藤崇裕、明石 良
細胞工学 別冊 212 - 215 2009年7月
記述言語：日本語 掲載種別：記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）
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バイオテクノロジーを利用した暖地型イネ科牧草の育種(<特集>イネ科牧草の遺伝育種の展開)
明石 良, 権藤 崇裕, 川村 修
日本草地学会誌 49 ( 1 ) 79 - 87 2003年
記述言語：日本語 掲載種別：記事・総説・解説・論説等（学術雑誌） 出版者・発行元：日本草地学会
DOI： 10.14941/grass.49.79
講演・口頭発表等 【 表示 ／ 非表示 】
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2017年中に発表されたゲノム編集イネのオフターゲット効果に関する評価
成島純平、中村公亮 他
日本農芸化学会大会
開催年月日： 2020年3月25日 - 2020年3月28日
記述言語：日本語 会議種別：口頭発表（一般）
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バヒアグラスにおけるリグニン生合成に関わるCAD遺伝子のゲノム編集
権藤崇裕、湯浅玲奈、明石良
第76回日本草地学会
開催年月日： 2020年
記述言語：日本語 会議種別：口頭発表（一般）
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Asparaginyl-tRNA synthetase gene (SYNC1) characterized by lotus corniculatus FOX-superroot lines has effects on plant morphology and amino acid contents of seed in soybean
Azeri Gautama Arifin, Takahiro Gondo, Ryo Akashi
第37回日本植物細胞分子生物学会
開催年月日： 2019年9月7日 - 2019年9月8日
記述言語：英語 会議種別：口頭発表（一般）
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バヒアグラス遺伝子組換え体におけるCAD遺伝子の発現抑制がPAL、COMT遺伝子の発現とリグニン含量に及ぼす影響
権藤崇裕、山田智仁、湯浅玲奈、明石良
日本草地学会大会
開催年月日： 2019年
記述言語：日本語 会議種別：口頭発表（一般）
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コウシュンシバにおけるアグロバクテリウム法による遺伝子組換え系の確立
牛山真里、張震、明石良、権藤崇裕
第36回日本植物細胞分子生物学会
開催年月日： 2019年
記述言語：日本語 会議種別：ポスター発表
科研費（文科省・学振・厚労省）獲得実績 【 表示 ／ 非表示 】
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ゲノム編集による高消化性暖地型イネ科牧草の作出とその遺伝子拡散リスク評価
研究課題/領域番号：23K05524 2023年04月 - 2027年03月
独立行政法人日本学術振興会 科学研究費補助金 基盤研究(C)
担当区分：研究代表者
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タイヌビエ多剤抵抗性発現の鍵となる除草剤代謝遺伝子群の一斉発現システムの解明
研究課題/領域番号：22H02347 2022年04月 - 2026年03月
独立行政法人日本学術振興会 科学研究費補助金 基盤研究(B)
担当区分：研究分担者
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植物の遺伝子組換え技術について学ぼう！
研究課題/領域番号：99999999 2019年
科学研究費補助金 研究成果公開促進費・研究成果公開発表(B)
担当区分：研究代表者
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植物の遺伝子組換え技術について学ぼう！
研究課題/領域番号：99999999 2018年
科学研究費補助金 研究成果公開促進費・研究成果公開発表(B)
担当区分：研究代表者
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牧草・芝草におけるゲノム編集による新たな育種技術の基盤構築
研究課題/領域番号：17K07608 2017年 - 2020年03月
科学研究費補助金 基盤研究(C)
担当区分：研究代表者
研究・技術シーズ 【 表示 ／ 非表示 】
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暖地型イネ科牧草の品質向上に関する研究
ダイズの収量性向上に関する研究
遺伝子組換え作物における生物多様性影響評価ホームページ： 草類遺伝資源・育種学研究室
技術相談に応じられる関連分野：・植物の組織培養および遺伝子組換え
・遺伝子組換え作物の生物多様性影響評価メッセージ：・共同研究の希望テーマ：植物の遺伝子組換えと生物多様性影響評価
・「植物の大量増殖および遺伝子組み換え作物の評価」というニーズがあれば、ぜひ教えてください。