ANALYSIS OF STRESS RESPONCES IN SOME MEDICINAL PLANT CELL CULTURES

1.在野葛（Pueraria lobata）中参与β-胡萝卜素生物合成的酶中，已克隆并测序了编码查尔酮合酶（CHS）和与CHS共同作用的还原酶（CHR）的互补基因以及基因组DNA。在这两个基因组克隆的启动子区，激发子响应元件中报道的菜豆进行了鉴定。构建了含有该500 bp区域的质粒，该区域连接至作为报告基因的β-葡萄糖醛酸酶（GUS）基因。用该质粒转化烟草的毛状根表达GUS蛋白，以响应诱导胁迫。这类启动子可用于外源基因在植物中的时间和组织特异性表达.诱导子诱导的野葛代谢包括两个阶段。在第一阶段（诱导后0-4小时），组成型β-丙二酰葡糖苷（IMG）通过木质化与细胞壁结合。IMG在第二阶段（诱导后6-24小时）的再积累是通过从头生物合成引起的。在第一阶段中发现的快速木质化是IMG和水解酶亚细胞区室化破坏的结果。这种类型的反应在豆科植物中从未有过报道，然而，它可能是一种先于其他需要从头合成酶的快速防御机制.从二穗麻黄（Ephedra distachya）培养物中分离纯化了诱导对香豆酰氨基酸产生的酵母提取物活性成分，并鉴定为甘露糖蛋白。这是首次证明以甘露聚糖结构为主要糖成分的活性激发子，还发现D-丙氨酸是酵母提取物的激发子活性成分。在二穗桉培养物中检测到一种新的D-氨基酸特异性对香豆酰转移酶，它是对香豆酰氨基酸生物合成的关键酶。

1. Among the enzymes involved in isoflavonoid biosynthesis in Pueraria lobata, complementary as well as genomic DNAs encoding chalcone synthase (CHS) and reductase (CHR) co-acting with CHS have been cloned and sequenced. In the promoter regions of both genomic clones, elicitor responding elements reported in Phaseolus vulgaris were identified. Plasmid containing this 500 bp region connected to beta-glucuronidase (GUS) gene as a reporter was constructed. Hairy roots of tobacco plant transformed with this plasmid expressed GUS protein responding to the elicitation stress. This type of promoter is useful for the time and tissue specific expressions of foreign genes in plants.2. Elicitor inducible isoflavonoid metabolism in Pueraria lobata consisted of two distinct phases. In the first phase (0-4 hr after elicitation), constitutive isoflavone malonylglucosides (IMG) were bound to cell walls by lignification. Reaccumulation of IMG in the second phase (6-24 hr after elicitation) was brought about by de novo biosynthesis. The rapid lignification of isoflavonoid found in the first phase as a consequence of disruption of subcellular compartmentation of IMG and hydrolytic enzymes. This type of responce has never been reported in Legume plants, however, it can possibly function as a rapid defense mechanism prior to others which require de novo biosynthesis of enzymes.3. Elicitor active components of yeast extract which induce p-coumarolyamino acids in Ephedra distachya cultures were separated, purified and identified as mannanglycoprotein. This is the first demonstration of active elicitor having mannan structure as the main sugar component D-Alanine was also found to be an elicitor active component of yeast extract. A new D-aminoacid specific p-coumaroyltransferase was detected in E.distachya cultures which serves as a key enzyme in the biosynthesis of p-coumarolyamino acids.