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

细胞色素p450和三萜合成酶（氧化角鲨烯环化酶，OSCs）对植物成分的显著多样性负责。本项目对其蛋白/基因结构、催化模式、特异性表达、生理生态功能及其分子进化机制进行了研究。以模式豆科植物荷花为主要实验材料。通过对粳稻P450基因的分类，分析了构建豆科植物异黄酮骨架的关键酶P450 IFS基因的结构，明确了IFS基因在粳稻基因组中与异黄酮通路OMT的特异性串联定位。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，通过蛋白质工程方法鉴定了P450 IFS反应中独特芳基迁移的关键氨基酸残基。对日本l.a japonicus的OSC基因进行了全面分析，鉴定出β-amyrin、lupeol和cycloartenol合成酶8个基因和1个假基因。与日本l.a japonicus和其他植物OSC基因的分子系统发育分析表明，三萜骨架的显著多样性主要是通过β-amyrin合成酶基因的突变引起的。为了研究OSCs基因的生理功能，目前正在研究OSCs基因过表达或被抑制的转基因日本l.a japonicus植株，并设想对OSCs进行蛋白质工程以阐明其产生骨骼多样性的催化机制。为了对代谢酶的转录和产物的积累进行全面分析，还对枇杷器官和培养细胞的成分进行了分析。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

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