{"created":"2023-06-19T07:19:00.476746+00:00","id":4671,"links":{},"metadata":{"_buckets":{"deposit":"d744fd43-b76e-47ad-8a4c-3c319ccb97e7"},"_deposit":{"created_by":4,"id":"4671","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"4671"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00004671","sets":["17:232:208","17:253:315","17:253:325","31:181:490"]},"author_link":["20302","20301","20306","20303","20305","20304"],"item_12_biblio_info_16":{"attribute_name":"書誌情報","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"2005-03-31","bibliographicIssueDateType":"Issued"},"bibliographicPageEnd":"166","bibliographicPageStart":"161","bibliographicVolumeNumber":"9/10","bibliographic_titles":[{"bibliographic_title":"麻布大学雑誌"},{"bibliographic_title":"Journal of Azabu University","bibliographic_titleLang":"en"}]}]},"item_12_description_13":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"近年,内分泌撹乱物質としてのダイオキシン類の環境汚染が社会問題となってきており,汚染の有効的な除去法として従来の方法より低コストのバイオレメディエーションが注目を集めている。そこで,本研究では,従来からダイオキシン類の生分解についての報告の多い白色腐朽菌の産生するリグニン分解系酵素に着目し,これらの酵素群をコードする遺伝子を導入したトランスジェニックシロイヌナズナを作成するためのクローニングに取り組んだ。リグニン分解酵素群の中でも有害汚染物質の分解に関する報告の多い,リグニンペルオキシダーゼ(LiP),マンガンペルオキシダーゼ(MnP),ラッカーゼ(Lac)の3種類の酵素をコードする遺伝子に着目し,そのクローニングを行った。まず,これまでに報告のあったリグニン分解酵素群をコードするcDNAの塩基配列を参考に,数種類のプライマーを作製し,白色腐朽菌,P. chrysosporium(UAMH3641)およびT. versicolor(UAMH 8272)のcDNAをテンプレートとしてRT-PCR法を行い,数種類の増幅産物を得た。これらの増幅産物のcDNA断片の塩基配列をDNAシークエンサーで解析し,得られた配列についてNCBI-BLASTのDNAライブラリで相同性の検索を行った。その結果,P. chrysosporiumのLiP及びMnP, T. versicolorのLacのそれぞれcDNA中に,ライブラリ中の配列と高い相同性を有するものが見い出された。そこで,バクテリオファージラムダと大腸菌ゲノム間の類似組み換え反応を応用した遺伝子クローニングシステム(Gateway Technology/Invitrogen)を用いて,LiP, MnPおよびLacをコードする完全長cDNAのクローニングを試みた結果,それぞれの完全長cDNAのクローンを得ることができた。これらのcDNAの塩基配列は,ライブラリ上の配列と95%以上の高い相同性を有していた。また,3種の完全長cDNAの塩基配列をアミノ酸に変換し,上述のライブラリの塩基配列を同じくアミノ酸に変換したものと比較したところ,97%以上の高い相同性を示した。しかし,今回同定したアミノ酸配列は,どのライブラリ配列とも数%の差が見られ,これらの結果からは,今回塩基配列を決定した3種類の酵素をコードするcDNAは,これまでに報告されている対象遺伝子の変異体である可能性が考えられた。さらに,LiPとMnPのアミノ酸配列中に約10アミノ酸残基からなる共通の配列が見られ,この配列部位がペルオキシダーゼとしての酵素活性に必要な触媒ドメイン,あるいは基質結合ドメインである可能性が示唆された。また,今回同定したLacは,他菌種由来のLacのアミノ酸配列との相同性も高かったことから,異菌種由来のLacタンパク質間で構造が類似していると考えられた。これらの遺伝子を,タンパク質発現用ベクターpDEST15に組換え反応し,大腸菌細胞中での酵素遺伝子の発現を試みた。その結果,LiP,MnPおよびLacについて,GST融合タンパク質として予想されるサイズのバンドをSDS-PAGEで確認することができた。さらに,植物形質転換用Tiプラスミドベクターであり,なおかつGatewayに対応したベクターpPDB-GBを作成した。","subitem_description_type":"Abstract"}]},"item_12_description_14":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"It has been of great concern that halogenated aromatic hydrocarbons cause the disruption in the endocrine system of animals. Among them, the serious toxicity of dioxin-related compounds is becoming more severe especially in Japan. To clean up the polluted environment, biological remediation system using plants, so-called phytoremediation, is expected to solve the environmental pollution problem. The degradation of dioxin-related compounds by white-rot fungi has been extensively studied in the process of lignin degradation. As a result unique extracellular oxidative enzymes, lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and laccase (Lac) were found to be responsible for degrading a wide variety of organic recalcitrant. Thus, these enzymes are thought to be useful in bioremediation of dioxin-related compounds. In this study, the cloning of the genes of lignin-degrading enzymes was tried to construct the transgenic Arabidopsis thaliana involving these genes in the genome. According to the sequence data base, specific primers for cloning of genes for lignin-degrading enzymes were constructed, and then full-length cDNAs were prepared using the RT-PCR system with full-length mRNAs from white-rot fungi as templates. The nucleotide sequence of each mRNA of lignin-degrading enzymes was determined using the DNA sequencer and the homology searching was carried out in the DNA library of NCBL-BLAST. As a result, mRNA for each LiP and MnP, and Lac was captured from Phanerochaete chrysosporium UAMH 3641, and Trametes versicolor UAMH 8272, respectively with about 95% homology. Therefore, cloning for each gene was tried using a universal cloning method based on the sitespecific recombination system of bacteriophage lambda, Gateway technology (Invitrogen). As a result, the PCR band equivalent to the full base length for each enzyme was cloned with the homology of 95% or 97% for base sequence or amino acid alignment, respectively. The expression of cloned gene for each lignindegrading enzyme was investigated using protein-expression vector system in E. coli, and then the protein band equal to the size for each LiP, MnP and Lac was detected with SDS-PAGE. The vector for transformation of plant, pPDB-GB that involves each lignin-degrading enzyme gene was constructed to apply this kind of vector for creating the transgenic plant.","subitem_description_type":"Other"}]},"item_12_description_2":{"attribute_name":"ページ属性","attribute_value_mlt":[{"subitem_description":"P(論文)","subitem_description_type":"Other"}]},"item_12_description_3":{"attribute_name":"記事種別","attribute_value_mlt":[{"subitem_description":"特集","subitem_description_type":"Other"}]},"item_12_description_34":{"attribute_name":"format","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_12_description_4":{"attribute_name":"Type","attribute_value_mlt":[{"subitem_description":"FEATURE ARTICLES","subitem_description_type":"Other"}]},"item_12_publisher_19":{"attribute_name":"出版者","attribute_value_mlt":[{"subitem_publisher":"麻布大学"}]},"item_12_publisher_20":{"attribute_name":"Publisher","attribute_value_mlt":[{"subitem_publisher":"Azabu University"}]},"item_12_source_id_21":{"attribute_name":"ISSN","attribute_value_mlt":[{"subitem_source_identifier":"1346-5880","subitem_source_identifier_type":"ISSN"}]},"item_12_source_id_23":{"attribute_name":"書誌レコードID","attribute_value_mlt":[{"subitem_source_identifier":"AA11561468","subitem_source_identifier_type":"NCID"}]},"item_12_text_10":{"attribute_name":"Author ID","attribute_value_mlt":[{"subitem_text_value":"000120"},{"subitem_text_value":"000235"},{"subitem_text_value":"000121"}]},"item_12_text_11":{"attribute_name":"所属機関","attribute_value_mlt":[{"subitem_text_value":"環境保健学研究科"},{"subitem_text_value":"環境保健学研究科"},{"subitem_text_value":"環境保健学研究科"}]},"item_12_text_12":{"attribute_name":"Institution or Company","attribute_value_mlt":[{"subitem_text_value":"Graduate School of Environmental Health Science"},{"subitem_text_value":"Graduate School of Environmental Health Science"},{"subitem_text_value":"Graduate School of Environmental Health Science"}]},"item_12_text_9":{"attribute_name":"著者ID","attribute_value_mlt":[{"subitem_text_value":"000120"},{"subitem_text_value":"000235"},{"subitem_text_value":"000121"}]},"item_12_version_type_29":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_12_version_type_30":{"attribute_name":"text version","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"其木, 茂則"},{"creatorName":"ソノキ, シゲノリ","creatorNameLang":"ja-Kana"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"佐俣, 哲郎"},{"creatorName":"サマタ, テツロウ","creatorNameLang":"ja-Kana"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"堂ヶ崎, 知格"},{"creatorName":"ドウガサキ, チカク","creatorNameLang":"ja-Kana"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"Sonoki, Shigenori","creatorNameLang":"en"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"Samata, tetsurou","creatorNameLang":"en"}],"nameIdentifiers":[{}]},{"creatorNames":[{"creatorName":"Dougasaki, chikaku","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2004-01-01"}],"displaytype":"detail","filename":"bull_jau_vol9-10-161.pdf","filesize":[{"value":"666.1 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"bull_jau_vol9-10-161.pdf","url":"https://az.repo.nii.ac.jp/record/4671/files/bull_jau_vol9-10-161.pdf"},"version_id":"703afb28-c0ca-4ba0-8af9-dd4334f7683f"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"departmental bulletin paper","resourceuri":"http://purl.org/coar/resource_type/c_6501"}]},"item_title":"リグニン分解酵素遺伝子導入シロイヌナズナを用いたダイオキシン類のファイトレメディエーション","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"リグニン分解酵素遺伝子導入シロイヌナズナを用いたダイオキシン類のファイトレメディエーション"},{"subitem_title":"Phytoremediation of dioxins using the transgenic Arabidopsis thaliana genetically engineered by lignin-degrading enzyme genes","subitem_title_language":"en"}]},"item_type_id":"12","owner":"4","path":["208","315","325","490"],"pubdate":{"attribute_name":"公開日","attribute_value":"2004-01-01"},"publish_date":"2004-01-01","publish_status":"0","recid":"4671","relation_version_is_last":true,"title":["リグニン分解酵素遺伝子導入シロイヌナズナを用いたダイオキシン類のファイトレメディエーション"],"weko_creator_id":"4","weko_shared_id":4},"updated":"2023-06-19T07:48:34.235019+00:00"}