Plant Metabolomics towards understanding of the molecular diversity and functional specificity of the P450 containing electron trnasfer chain

植物代谢组学旨在了解含有电子转移链的 P450 的分子多样性和功能特异性

• 批准号: 14560288
• 负责人: OHTA Daisaku
• 金额: $ 2.3万
• 依托单位: Osaka Prefecture University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560288/
• 关键词: cytochrome P450; metabolomics; mass spectrometry; plat; Arabidopsis; FT-MS; ミトクロムP450; フェレドキシン; フェレドキシン還元酵素; ミトコンドリア; シロイヌナズナ

Among a number of potential ferredoxin reductase genes in Arabidopsis genome, we have identified AtMFDR to correspond to a homologue of adrenodoxin reductase (mitochondrial ferredoxin reductase), and AtMFDX1 and AtMFDX2 as the genes for ferredoxins highly similar to adrenodoxin (mitochondrial ferredoxin). The deduced primary structures of AtMFDR and AtMFDXs contained the signature sequences involved in the electron transfer, and the recombinant AtMFDX1 and AtMFDR proteins were fully active to reconstitute the electron-transfer activity with NAD(P) H as the electron donor. Protein-gel blot analysis demonstrated constant accumulation of AtMFDR protein in leaf, stem, and flower. Clarification of their subcellular localization is prerequisite to identify physiological partner proteins in planta. A subcellular fractionation analysis demonstrated membrane association of AtMFDR protein, and specific localization could not be elucidated through a series of immunohistochemical analyses. Detailed analysis is in progress to clarify the membrane structure where the proteins exert their physiological functions.During the course of molecular characterization of the electron transfer chain, we have also established a metabolomics platform on a basis of combination of GC/MS, LC/MS, and FT/MS. The metabolome analysis includes the non-biased high throughput analysis of plant crude extracts and data analysis program. The established method will open the possibilities to explore "silent phenotypic alterations" caused by disruption of genes of unknown functions.

在拟南芥基因组中的许多潜在的铁氧还蛋白还原酶基因中，我们已经确定AtMFDR对应于肾上腺素还蛋白还原酶（线粒体铁氧还蛋白还原酶）的同源物，并且AtMFDX 1和AtMFDX 2作为与肾上腺素还蛋白（线粒体铁氧还蛋白）高度相似的铁氧还蛋白的基因。AtMFDR和AtMFDXs的一级结构中含有参与电子传递的特征序列，重组蛋白AtMFDX1和AtMFDR能够以NAD（P）H为电子供体重建电子传递活性。蛋白质凝胶印迹分析表明AtMFDR蛋白在叶、茎和花中不断积累。阐明它们的亚细胞定位是鉴定植物生理伴侣蛋白的前提。亚细胞分级分析表明，AtMFDR蛋白的膜协会，并通过一系列的免疫组化分析无法阐明具体的定位。在电子传递链的分子表征过程中，我们还建立了基于GC/MS、LC/MS和FT/MS相结合的代谢组学平台，包括植物粗提物的无偏高通量分析和数据分析程序。建立的方法将打开的可能性，探索“沉默的表型改变”所造成的破坏基因的未知功能。

项目成果

Takuno, K., Morikawa, T., Nonaka, Y., et al.: "Identification and molecular characterization of mitochondrial ferredoxins and ferredoxin reductase from Arabidopsis"Plant Molecular Biology. 52. 817-830 (2003)

Takuno, K.、Morikawa, T.、Nonaka, Y. 等人：“来自拟南芥的线粒体铁氧还蛋白和铁氧还蛋白还原酶的鉴定和分子表征”植物分子生物学。

Takubo K, Morikawa T, Nonaka Y, Mizutani M, Takenaka S, Takabe K, Takahashi MA, Ohta D.: "Identification and molecular characterization of mitochondrial ferredoxins and ferredoxin reductase from Arabidopsis"Plant Molecular Biology. 52. 817-830 (2003)

Takubo K、Morikawa T、Nonaka Y、Mizutani M、Takenaka S、Takabe K、Takahashi MA、Ohta D.：“拟南芥线粒体铁氧还蛋白和铁氧还蛋白还原酶的鉴定和分子特征”植物分子生物学。

Takubo K, Morikawa T, Nonaka Y et al.: "Identification and molecular characterization of mitochondrial ferredoxins and ferredoxin reductase from Arabidopsis"Plant Molecular Biology. 52. 817-830 (2003)

Takubo K、Morikawa T、Nonaka Y 等人：“来自拟南芥的线粒体铁氧还蛋白和铁氧还蛋白还原酶的鉴定和分子特征”植物分子生物学。