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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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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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Ng H.M., Gondo T., Ushiyama M., Cho S., Maemura S., Hashiguchi M., Tanaka H., Akashi R.
Grassland Science 2022年
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：Grassland Science
Zoysia matrella (L.) Merrill is a perennial C4 warm-season turfgrass grown for landscapes, golf courses, sports fields and recreation parks. To create a new breeding method by genome editing, an efficient genetic transformation system is essential. In this study, we report the efficient protocol of Agrobacterium-mediated transformation through the establishment of a stable tissue culture system for Z. matrella ‘Wakaba’. The embryogenic callus was induced from shoot apices of nodal segments incubated in Murashige and Skoog (MS) medium containing 2 mg/l 2,4-dichlorophnoxyacetic acid (2,4-D), 0.1 mg/l 6-benzylaminopurine (BAP) and 5 μM CuSO4 (MS-DBC). Repeated subculture of compact high-quality callus in MS-DBC medium produced a highly regenerative callus with dense pre-embryogenic clusters, and it was used as a transformation target. Agrobacterium strain EHA105 harboring pANIC8B vector containing the β-glucuronidase gene (GUS) and hygromycin B phosphotransferase gene was used. Agrobacterium-infected calli were cocultured for 5 days with 100 μM acetosyringone and then subjected to selection pressure of 50 mg/l hygromycin. This optimized protocol yielded transformation efficiencies of up to 6.6%. Southern blot analysis verified one to three copies of the GUS gene in different independent transgenic plants. All transgenic plants were morphologically normal, and the GUS expressions were stable. Our optimized in vitro and transgenic system will facilitate the new breeding technology of genome editing in zoysiagrass.
DOI： 10.1111/grs.12396
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Quantitative analysis of seven plant hormones in Lotus japonicus using standard addition method
Hashiguchi T., Hashiguchi M., Tanaka H., Fukushima K., Gondo T., Akashi R.
PLoS ONE 16 ( 2 February ) e0247276 2021年2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：PLoS ONE
Plant hormones have been identified to be versatile signaling molecules essential for plant growth, development, and stress response. Their content levels vary depending on the species, and they also change in response to any external stimuli. Thus, simultaneous quantification of multiple plant hormones is required to understand plant physiology. Sensitive and quantitative analysis using liquid chromatography-linked mass spectrometry (LC-MS/MS) has been used in detecting plant hormones; however, quantification without stable isotopes is yet to be established. In this study, we quantified seven representative plant hormones of Lotus japonicus, which is a model legume for standard addition method. Accurate masses for monoisotopic ions of seven phytohormones were determined for high-resolution mass spectrometry (HR-MS). Selected ion monitoring (SIM) mode based on accurate masses was used in detecting phytohormones in the roots, stems, and leaves. Evaluation of matrix effects showed ion suppression ranging from 10.2% to 87.3%. Both stable isotope dilution and standard addition methods were able to detect plant hormones in the roots, stems, and leaves, with no significant differences in using both approaches and thus a standard addition method can be used to quantify phytohormones in L. japonicus. The method will be effective, especially when stable isotopes are not available to correct for matrix effects.
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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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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：22H0234701 2022年04月 - 2026年03月
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！
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！
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
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
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植物の遺伝子組換え技術について学ぼう！
2017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