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Post-genomic approaches to reaction mechanism and ecophysiological functions of P450 and terpene synthases of leguminous plants

豆科植物 P450 和萜烯合酶反应机制和生态生理功能的后基因组方法

基本信息

批准号：
14540603
负责人：
AYABE Shin-ichi
金额：
$ 1.92万
依托单位：
Nihon 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 2004
项目状态：
已结题

项目摘要

Cytochrome P450s and triterpene synthases (oxidosqualene cyclases, OSCs) are responsible for the remarkable diversity of plant constituents. In this project, their protein/gene structures, catalysis modes, specific expression and ecophysiological functions, and the mechanism of their molecular evolution were investigated. The main experimental material was the model legume Lotus japonicus.Following cataloging of P450s of L.japonicus, the gene structure of P450 IFS, the critical enzyme for the construction of legume-characteristic isoflavonoid skeleton was analyzed, and a specific tandem localization of an IFS gene with an OMT of the isoflavonoid pathway in L.japonicus genome was clarified. The gene structure and expression of a P450 which was estimated to be important in the development and functions of nitrogen fixing root nodules were also examined. Further, key amino acid residues for the unique aryl migration during the P450 IFS reaction were identified through protein engineering … More approaches.OSC genes of L.japonicus were analyzed as comprehensively as possible, and eight genes including β-amyrin, lupeol and cycloartenol synthases and a pseudogene were identified. Molecular phylogenetic analysis with L.japonicus and other plant OSC genes indicated the remarkable diversity of triterpene skeletons is mainly brought about through mutations in β-amyrin synthase genes. Transgenic L.japonicus plants in which OSCs are overexpressed or suppressed are being produced to examine the physiological functions of these genes, and protein engineering of OSCs to clarify the catalytic mechanism producing skeletal diversity is envisaged. For the overall analysis of transcription of the metabolic enzymes and accumulation of the products, profiling of the constituents of the organs and cultured cells of L.japonicus is also performed.This project yielded interesting findings especially in the protein/gene structures to produce specific natural products and also in the mechanism of molecular evolution of plant secondary metabolism. Less

细胞色素P450和三萜环化酶（oxidosqualene cyclases，OSC）负责植物成分的显著多样性。本项目主要研究了它们的蛋白质/基因结构、催化方式、特异性表达和生理生态功能，以及它们的分子进化机制。以模式豆科植物百脉根为材料，在对百脉根P450基因进行编目的基础上，分析了构建豆科植物特有的百脉根骨架的关键酶P450 IFS的基因结构，并明确了IFS基因与百脉根P450合成途径的OMT在百脉根基因组中的串联定位。此外，还研究了在固氮根瘤发育和功能中起重要作用的P450基因的结构和表达。此外，通过蛋白质工程鉴定了P450 IFS反应期间独特芳基迁移的关键氨基酸残基 ...更多信息方法。对日本落叶松OSC基因进行了全面分析，鉴定出β-香树脂醇、羽扇豆醇和环阿屯醇合酶等8个基因和一个假基因。与其他植物OSC基因的分子系统发育分析表明，β-香树脂素合成酶基因的突变是导致三萜骨架结构显著多样性的主要原因。正在生产其中OSC被过表达或抑制的转基因L. lasticum植物以检查这些基因的生理功能，并且设想了OSC的蛋白质工程以阐明产生骨骼多样性的催化机制。为了全面分析代谢酶的转录和产物的积累，还对L. lauricum的器官和培养细胞的组成进行了分析。该项目在产生特定天然产物的蛋白质/基因结构以及植物次生代谢的分子进化机制方面取得了有趣的发现。少