Analysis of the genes for porphyrin biosynthetic pathway in higher plants utilizing the light-sensitive mutants of Escherichia coli

利用大肠杆菌光敏突变体分析高等植物卟啉生物合成途径基因

• 批准号: 11480201
• 负责人: INOKUCHI Hachiro
• 金额: $ 2.94万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480201/
• 关键词: rice (Oryza sativa); soybean (Glycine max); hemF gene of E.coli; cucumber (Cucumis sativus); thymidylate synthase; spinach (Spinacia oleracea); ferrochelatase; protoporphyrinogen IX oxidase; ポルフィリン; PPOX; CPOX; シロイヌナズナ; キューリ; ダイズ; 大腸菌

1) By taking advantage of the ability of such a cDNA to complement the thymine auxotrophy of a mutant of Escherichia coli, a cDNA clone for thymidylate synthase (TS) of rice (Oryza sativa L.indica) was isolated. [Kanjo ＆ Inokuchi, Plant Physiol. (Gene Register), 120, 634 (1999)]2) A cDNA clone for protoporphyrinogen IX oxidase of soybean (Glycine max was isolated by functional complementation against the poor growth of BT3 delta hemG : : Km^R cells. Nucleotide sequence analysis of the cDNA insert revealed that the insert was 1745 bp in length. It included a single open reading frame that encoded a protein consisting of 502 amino acid residues. The deduced amino acid sequence of PPOX from soybean was 67 % homologous to that of mitochondrial PPOX from tobacco. It shares some amino acid residues at the N-terminus speculated to be a mitochondrial translocation signal. [Kanjo et al., Plant Physiol. (Gene Register), 121, 1384 (1999)]3) We examined poessible alternate pathways for the oxidati … More on of protoporphyrinogen IX to protoporphyrin IX, by isolating and investigating E.coli mutants that can still grow normally when the hemG gene is disrupted. It was suggested that the E.coli aerobic coproporphyrinogen oxidase has an intrinsic capacity to oxidize not only coproporphyrinogen III but also protoporhyrinogen IX.[Narita et al., Mol. Gen. (Genet., 261, 1012-1020 (1999)]4) A putative mature region of a cucumber (Cucumis sativus) ferrochelatase cDNA (hemH) was overexpressed in E.coli, purified to homogenity and examined its enzymatic properties. Immunolocalization of the enzyme examined with antibody demonstrated that the ferrochelatase was present in both hypocotyls and roots but hardly in cotyledons of cucumber. [Suzuki et al., Plant Cell Physiol., 41, 192-199 (2000)]5) We reported the first cloning of spinach (Spinacia oleracea) plastidal PPOX cDNA and analysis of the N-terminal sequence in mature plastidal PPOX purified from spinach chloroplast and discussed the mechanism of the transport of plastidal PPOX into chloroplasts. [Che et al., Plant Physiol., 124, 59-70 (2000)] Less

1)利用该基因对突变型水稻胸腺嘧啶营养缺陷症的补充作用，获得了水稻胸苷酸合成酶(TS)的基因克隆。拉丁植物志[Kanjo&Inokuchi，植物性生理学。(Gene Register)，120,634(1999)]2)针对BT3 Delta hemG：：KM^R细胞生长不良的问题，通过功能互补获得了大豆原卟啉原IX氧化酶(Glyine Max)的cDNA克隆。核苷酸序列分析表明，该插入片段全长1745个碱基对。它包括一个单一的开放阅读框架，编码一个由502个氨基酸残基组成的蛋白质。大豆花痘的氨基酸序列与烟草线粒体花痘的氨基酸序列同源性为67%。它在N端有一些氨基酸残基，推测是线粒体易位信号。[Kanjo等人，植物性生理学。(基因登记)，1211384(1999年)3)我们研究了氧化…的可能的替代途径通过分离和研究在hemG基因被破坏时仍能正常生长的大肠杆菌突变株，更多地将原卟啉原IX转化为原卟啉IX。这表明，大肠杆菌好氧共比例卟啉原氧化酶不仅具有氧化共比例卟啉原III的内在能力，而且具有氧化原甲肾上腺素IX的内在能力。(Genet，261,1012-1020(1999)]4)黄瓜(Cucumis Sativus)铁络合酶基因(HemH)在大肠杆菌中高效表达，纯化至均一，并检测其酶学性质。用抗体检测该酶的免疫定位表明，该铁络合酶在黄瓜的下胚轴和根中都存在，但在子叶中几乎不存在。5)我们首次克隆了菠菜(Spinacia Olacea)质潮PPOX基因，并对从菠菜叶绿体中提纯的成熟质潮PPOX的N末端序列进行了分析，探讨了质潮PPOX向叶绿体运输的机制。[Che等，植物生理学，124，59-70(2000)]少

项目成果

S.Narita,S.Taketani & H.Inokuchi: "Oxidation of Protoporphysinogen IX mediated by the E.Coli aerobic coprosporphysinogen Oxidase"Molec.Gen.Genet.. 261. 1012-1020 (1999)

成田 S.竹谷 S.

F.Che, N.Watanabe, M.Iwano, H.Inokuchi, S.Takayama, S.Yoshida & A.Isogai: "Molecular characterization subcellular localization of protoporphyrinogen oxidase in spinach chloroplasts."Plant Physiol.. 124. 59-70 (2000)

F.Che、N.Watanabe、M.Iwano、H.Inokuchi、S.Takayama、S.Yoshida

N.Kanjo, S.Nishio, S.Narita, K.Oeda & H.Inokuchi: "Nucleotide sequence of a cDNA clone encoding protoporphyrinogen IX oxidase from soybean."Plant Physiol. (Gene Register). 112. 1384 (1999)

N.Kanjo、S.Nishio、S.Narita、K.Oeda

F.Che et.al.,: "Molecular characterization subcellular localization PPOX in spinach chlosoplasts"Plant Physiol.,. 124. 59-70 (2000)

F.Che 等人：“菠菜叶绿体中 PPOX 的分子表征亚细胞定位”植物生理学。

S.Nasita et al.,: "Oxidation of Protoporphysinogen IX mediated by the E.coli aerobic coproposphysinogen oxidase"Molec.Gen.Genet.. 261. 1012-1020 (1999)

S.Nasita 等人：“大肠杆菌需氧粪卟啉原氧化酶介导的原卟啉原 IX 的氧化”Molec.Gen.Genet.. 261. 1012-1020 (1999)