{"created":"2023-06-20T16:35:28.009951+00:00","id":4801,"links":{},"metadata":{"_buckets":{"deposit":"7d009f5c-b7e3-407d-b87f-bd44921034f2"},"_deposit":{"created_by":3,"id":"4801","owners":[3],"pid":{"revision_id":0,"type":"depid","value":"4801"},"status":"published"},"_oai":{"id":"oai:kindai.repo.nii.ac.jp:00004801","sets":["14:923:1379:1401","21:3039:3040:3043"]},"author_link":["6438","6439","6437","6441","6440","6436"],"item_2_alternative_title_3":{"attribute_name":"その他（別言語等）のタイトル","attribute_value_mlt":[{"subitem_alternative_title":"Direct RT-PCR amplification of mature mRNAs in cytoplasm micropipetted from barley coleoptile epidermal cell-A model system for analyzing gene expression in host cells attacked by powdery mildew"}]},"item_2_biblio_info_21":{"attribute_name":"書誌情報","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"2005-01-01","bibliographicIssueDateType":"Issued"},"bibliographicIssueNumber":"38","bibliographicPageEnd":"18","bibliographicPageStart":"9","bibliographic_titles":[{"bibliographic_title":"近畿大学農学部紀要"},{"bibliographic_title":"Memoirs of the Faculty of Agriculture of Kinki University","bibliographic_titleLang":"en"}]}]},"item_2_description_33":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"[Author abstract]Single-cell RT-PCR was used to detect gene expression in situ in single selected cells of detached barley coleoptile epidermis inoculated with Blumeria graminis f. sp. hordei. The cytoplasm was removed with a micropipette using a light microscope and directly used for RT-PCR, followed by nested PCR. Three intron-containing genes, vacuolar ATPase B-subunit gene, cytosolic triosephosphate isomerase gene, and glycolitic glyceraldehyde-3-phosphate dehydrogenase gene were constantly expressed in this tissue and were therefore used as indicators, because shorter-size PCR-products produced by splicing are easy to detect. By simultaneously amplifying both target and indicator genes, the transcription of some stimuli-induced genes such as chitinase 2 and acidic β-1,3-glucanase genes could be precisely detected in powdery-mildew-invaded and noninvaded, neightboring cells of the coleoptile epidermis. In addition, micropipetting only the cytoplasm without the nucleus prevented contamination with genomic DNA, which leads to miss-amplification of corresponding genomic DNA sequences of the intron-less genes during RT-PCR and subsequent nested PCR. Thus, this technique can be successfully used with coleoptile epidermal cells on which the powdery mildew fungus has attempted to invade and/or form a haustorium or with epidermal cells in the vicinity of infected cells.[著者抄録]オオムギうどんこ病菌(Blumeria graminis f.sp.hordai)は,宿主植物であるオオムギに接触すると,速やかに宿主認識を行い,数分後には第一次発芽管を伸長する。その後,植物組織へ付着する器官として新たに付着器を分化し,表皮細胞への侵入を試みるとともにその細胞内に栄養吸収器官である吸器を形成する。一方,本病原菌の感染を受けた表皮細胞は,その感染過程の進行に対応し,異なる反応を示すと考えられている。しかしながら,これらの宿主細胞の反応が感染を受けている細胞に限定されること,また,その反応が数分単位で進行することから,病原菌の感染過程で誘導される遺伝子の発現を解析するためには,顕微鏡下で感染過程を連続的に観察し,感染を受けている特定細胞において遺伝子発現を検出する細胞レべルの解析系が必須となる。そこで本実験では,顕微鏡下で標的とした単一細胞からマイクロピペットを用いて細胞内容物を吸引し,そこに存在するmRNAを鋳型とした単一細胞RT-PCR/nested PCR法を適用することとした。実験材料としては,うどんこ病菌の感染過程が顕徴鏡下で観察可能なオオムギ子葉鞘の内表皮細胞を供試した。まず,既に報告されているオオムギの構成発現遺伝子を検索し,Vacuolar ATPase B subunit(ATP-B),Cytosolic triosephosphate isomerase(cTPI),Glycolytic glyceraldehyde-3-phosphate dehydrogenase(gGAPDH)について,その塩基配列をもとにプライマーを構築した。また,核由来の増幅産物とmRNA由来の産物を識別するため,増幅産物にイントロン配列が含まれる位置にプライマーを設計し,スプライスの有無からその由来を判別することとした。顕微鏡下で標的細胞から細胞内容物を吸引する際には,細胞内に核を残存させ,核を含まない内容物を鋳型としてRT-PCR/nested PCRを行った。その結果,イントロン配列がスプライスされた増幅産物のみが検出され,核を含まない細胞質を吸引することにより実際に発現している遺伝子のPCR検出が可能となった。次に,病原菌の感染によって発現が誘導される遺伝子を検索し,Chitinase 2(CHI2),β-1,3-glucanase(GLU),について,前述と同様にイントロン配列を含む位置にプライマーを設計した。誘導型遺伝子の発現を厳密に評価するため,PCRを行う際に先の構成的発現遺伝子を増幅するプライマーを混合し,指標遺伝子として同時に増幅することにより,誘導型遺伝子の発現検出を行った。その結果,病原菌の感染を受けていない細胞およびその感染を受けている両者の細胞において指標とした構成的発現遺伝子は検出された。誘導型遺伝子として使用したCHI2遺伝子およびGLU遺伝子は病原菌の感染を受けていない細胞においてその発現が検出され、本菌の感染過程において制御される傾向にあった。","subitem_description_type":"Abstract"}]},"item_2_description_36":{"attribute_name":"内容記述","attribute_value_mlt":[{"subitem_description":"記事区分：原著","subitem_description_type":"Other"}]},"item_2_description_41":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_2_publisher_14":{"attribute_name":"出版者 名前","attribute_value_mlt":[{"subitem_publisher":"近畿大学農学部"}]},"item_2_source_id_22":{"attribute_name":"ISSN","attribute_value_mlt":[{"subitem_source_identifier":"04538889","subitem_source_identifier_type":"ISSN"}]},"item_2_text_7":{"attribute_name":"著者(英)","attribute_value_mlt":[{"subitem_text_language":"en","subitem_text_value":"Moriura, Nobuyuki"},{"subitem_text_language":"en","subitem_text_value":"Matsuda, Yoshinori"},{"subitem_text_language":"en","subitem_text_value":"Fujita, Kazuhisa"},{"subitem_text_language":"en","subitem_text_value":"Wada, Mizuki"},{"subitem_text_language":"en","subitem_text_value":"Kakutani, Koji"},{"subitem_text_language":"en","subitem_text_value":"Toyoda, Hideyoshi"}]},"item_2_text_8":{"attribute_name":"著者 所属","attribute_value_mlt":[{"subitem_text_value":"近畿大学農学部"},{"subitem_text_value":"近畿大学農学部"},{"subitem_text_value":"近畿大学農学部"},{"subitem_text_value":"近畿大学農学部"},{"subitem_text_value":"近畿大学薬学総合研究所"},{"subitem_text_value":"近畿大学農学部"}]},"item_2_text_9":{"attribute_name":"著者所属(翻訳)","attribute_value_mlt":[{"subitem_text_value":"Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kinki University"},{"subitem_text_value":"Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kinki University"},{"subitem_text_value":"Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kinki University"},{"subitem_text_value":"Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kinki University"},{"subitem_text_value":"Pharmaceutical Research and Technology Institute, Kinki University"},{"subitem_text_value":"Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kinki University"}]},"item_2_version_type_12":{"attribute_name":"版","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_be7fb7dd8ff6fe43","subitem_version_type":"NA"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"森浦,  展行"},{"creatorName":"モリウラ, ノブユキ","creatorNameLang":"ja-Kana"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"松田,  克礼"},{"creatorName":"マツダ, ヨシノリ","creatorNameLang":"ja-Kana"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"藤田,  和久"},{"creatorName":"フジタ, カズヒサ","creatorNameLang":"ja-Kana"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"和多,  泉季"},{"creatorName":"ワダ, ミズキ","creatorNameLang":"ja-Kana"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"角谷,  晃司"},{"creatorName":"カクタニ, コウジ","creatorNameLang":"ja-Kana"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"豊田,  秀吉"},{"creatorName":"トヨダ, ヒデヨシ","creatorNameLang":"ja-Kana"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-18"}],"displaytype":"detail","filename":"AN00064044-20050331-0009.pdf","filesize":[{"value":"1.1 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"AN00064044-20050331-0009.pdf","url":"https://kindai.repo.nii.ac.jp/record/4801/files/AN00064044-20050331-0009.pdf"},"version_id":"1c7aed91-5150-40a8-9171-cfe505f4d385"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"departmental bulletin 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植物病原菌感染宿主細胞における遺伝子発現解析のモデルシステム"],"weko_creator_id":"3","weko_shared_id":-1},"updated":"2023-06-21T00:58:51.776495+00:00"}