Adaptation of Leguminous Plants to Environmental Microbes through Expression of Genes Encoding Enzymes of Flavonoid Biosynthetic Pathway

豆科植物黄酮类化合物具有独特的结构(5-脱氧、异黄酮类等)，在与根瘤菌共生固氮过程中起到防御植物病原菌和化学信号的作用。本研究通过cDNA的分子克隆，研究了基因表达与植物对环境微生物的反应之间的关系，并对构建这些结构的酶进行了研究。从一个豆科植物棘果甘草中克隆了编码O-甲基转移酶的cDNA和类黄酮途径的细胞色素P450。通过在异源真核细胞中的表达，确定了P450的功能：作为共生信号参与黄酮类化合物骨架构建的黄烷酮2-羟基酶；2-羟基异黄酮合酶和异黄酮2‘-羟基酶，这两种酶都是植物保卫素生物合成所必需的。此外，从甘草中克隆了一个编码与产生紫花苜蓿共生信号类似的OMT的基因，并被证明参与了后查尔酮的生物合成。查尔酮产物在甘草细胞中是不稳定的。此外，对查尔酮异构酶同工酶进行了研究，提出了植物保保素前体芒柄花素生物合成的新方案。对豆科植物莲子保鲜蛋白进行了鉴定，并克隆了其生物合成途径中部分酶的基因。发现了一株能产生异黄酮类植物保卫素的甘草新菌株。在这些植物材料中，类黄酮生物合成基因的协调表达证实了类黄酮在豆科植物中的重要防御作用。最后，建立了冬虫夏草等非豆科植物的转化方法，并构建了利用转基因对环境生物进行类黄酮反应的实验系统。

Legume flavonoids possessing characteristic structures (5-deoxy, isoflavonoid, etc) function as defense compounds against phytopathogens and chemical signals during the symbiotic nitrogen fixation with Rhizobia. In this study, the enzymes responsible for the construction of theses structures were characterized through molecular cloning of cDNAs, and the relationship between the gene expression and the response of the plant against the environmental microorganisms was studied.From a leguminous plant licorics (Glycyrrhiza echinata), cDNAs encoding O-methyltransferases and cytochrome P450s of flavonoid pathway were cloned. By expressing in heterologous eucaryotic cells, the functions of P450s were determined : i.e., flavanone 2-hydroxylase involved in the construction of the skeleton of flavones acting as symbiotic signals ; 2-hydroxyisoflavanone synthase and isoflavone 2'-hydroxylase both of which are essential in phytoalexin biosynthesis. Also, a cDNA encoding an OMT similar to that producing the alfalfa symbiotic signal was cloned from licorice, and was shown to be involved in retrochalcone biosynthesis. The instability of the chalcone product in licorice cells was demonstrated. Furthermore, chalcone isomerase isozymes were characterized, and a new scheme of the biosynthesis of formononetin, a precursor of phytoalexins, was proposed.A phytoalexin of the model legume Lotus japanicus was identified, and cDNAs of some of the enzymes in its biosynthetic pathway were cloned. A new culture line of licorice was found to produce isoflavonoid phytoalexins. In these plant materials, coordinated expression of flavonoid biosynthetic genes were demonstrated, confirming the essential defensive roles of flavonoids in legumes. Finally, transformation methods of L. japonicus and other non-legume plants were established, and experimental systems for the flavonoid response to environmental organisms using transgenes are being constructed.