Molecular Mechanism of the Biosynthesis of Flavonoids Involved in Leguminous Plant-Microbe Interactions

豆科植物黄酮类化合物在病原和共生植物与微生物的相互作用中起重要作用。1.6′-脱氧查尔酮合成酶（DOCS）：主要的生物活性类黄酮为5-脱氧（iso）类黄酮。它们是由查尔酮合成酶（CHS）和聚酮还原酶（PKR）共同作用合成的6′-脱氧查尔酮衍生物。从甘草诱导细胞cDNA文库中克隆了两个组成酶的cDNA。将重组酶与蒲公英花青素产生细胞的无细胞提取物结合，证实了PKR的催化活性。CHS和PKR在大肠杆菌中的共表达将有助于阐明DOCS反应的分子机制。o -甲基转移酶（OMT）：苜蓿根系中的甲氧基查尔酮是一种强烈的化学信号，向土壤中输送更多的细菌以形成共生固氮根瘤。紫花苜蓿甲氧基查尔酮生物合成中的查尔酮OMT （CHMT）表现出额外的甘草二酮甲基转移酶（LMT）活性，该活性作为激发剂处理引起的防御反应的一部分在甘草细胞中表达。从甘草cDNA文库中克隆出一个具有LMT和CHMT活性的OMT cDNA。为了了解omt在甘草中的作用，目前正在对不含CHMT活性的LMT进行纯化。细胞色素p450 (p450)：不同的p450作用于一个共同的底物（利尿素），负责生物合成分支到具有不同生理功能的最终产物。从甘草cDNA文库中分离到P450 cDNA的PCR片段。进一步筛选得到全长P450 cDNA，其中包括肉桂酸4-羟化酶cDNA和功能未知的克隆。它们在诱导剂处理下的表达表明其中一些克隆参与了植物与微生物的相互作用。少

Flavonoids of leguminous plants play important roles in pathogenic and symbiotic plant-microbe interactions. The reaction mechanisms and physiological functions of the key enzymes in the biosynthesis of these biologically-active flavonoids were explored.1.6'-Deoxychalcone synthase (DOCS) : Major biologically-active flavonoids are 5-deoxy (iso) flavonoids. They are derived from 6'-deoxychalcone, which is synthesized by the co-action of chalcone synthase (CHS) and a polyketide reductase (PKR). From the cDNA library of elicitor-induced licorice (Glycyrrhiza echinata) cells, cDNAs for both component enzymes were cloned. Catalytic activity of PKR was demonstrated by the combination of the recombinant enzyme and cell-free extracts of anthocyanin-producing dandelion cells. Co-expression of CHS and PKR in E.coli will be beneficial to elucidate the molecular mechanism of DOCS reaction.2. O-Methyltransferases (OMT) : A methoxychalcone of alfalfa roots is a strong chemical signal toward a soil be … More cterium to form symbiotic nitrogen-fixing root nodules. A chalcone OMT (CHMT) of alfalfa in methoxychalcone biosynthesis displayd additional licodione methyltransferase (LMT) activity, which is expressed in licorice cells as a part of defense reactions caused by elicitor treatment. From the licorice cDNA library an OMT cDNA was cloned, the translated protein of which had both LMT and CHMT activities. To understand the roles of OMTs in licorice, purification of LMT devoid of CHMT activity is underway.3.Cytochrome P450s (P450s) : Distinct P450s acting on a common substrate (liquiritigenin) are responsible for the biosynthetic branching to end products with different physiological functions. PCR fragments of P450 cDNAs were isolated from the licorice cDNA library. Further screening yielded full-length P450 cDNAs, amongwhich were cinnamate 4-hydroxylase cDNA and functionally-unknown clones. Their expression in response to elicitor-treatment indicated that some of the clones are involved in plant-microbe interactions. Less