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

豆科黄酮类化合物具有特殊的结构（5-脱氧、异黄酮等），在与根瘤菌共生固氮过程中具有防御植物病原体和化学信号的作用。本研究通过cdna的分子克隆对这些结构的构建酶进行了表征，并研究了基因的表达与植物对环境微生物的反应之间的关系。从豆科植物甘草（glycyrhiza echinata）中克隆了编码类黄酮途径o -甲基转移酶和细胞色素p450的cdna。通过在异种真核细胞中表达，确定了P450s的功能：即参与黄酮骨架构建的黄酮2-羟化酶，作为共生信号；2-羟基异黄酮合成酶和异黄酮2'-羟化酶都是植物抗菌素生物合成中必需的酶。此外，从甘草中克隆了一个编码OMT的cDNA，该cDNA与产生苜蓿共生信号的OMT相似，并被证明参与后查尔酮的生物合成。证实了查尔酮产物在甘草细胞中的不稳定性。此外，对查尔酮异构酶同工酶进行了表征，并提出了植物抗毒素前体刺芒柄花素的生物合成新方案。鉴定了一种模式豆科植物日本莲的植物抗毒素，并克隆了其生物合成途径中部分酶的cdna。发现了一种新的甘草培养系，可产生异黄酮类植物抗毒素。在这些植物材料中，类黄酮生物合成基因的协同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L. japonicus和其他非豆科植物的转化方法，并构建了转基因黄酮类化合物对环境生物响应的实验体系。

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.