The Research on the Production of Usefull Compounds by the Control of Regulations in the Biosynthesis of Secondary Metabolism

(1)从葛根细胞中克隆了黄酮生物合成关键酶查尔酮合成酶的基因组基因。此外，还分离到了在脱氧查尔酮形成过程中与查尔酮合酶协同作用的3个还原酶基因组克隆。由于查尔酮合酶和还原酶共同作用，查尔酮合酶和还原酶基因的启动子区域被认为是相似的。然而，它们没有总体上的相似性，而是有几个长度约为20bp的相似序列。在此期间，2-羟基异黄酮脱水酶的纯化已经完成，其cDNA的克隆也在进行中。(2)从化合物水平、酶水平和RNA水平研究了培养的对虾细胞对酵母提取物、茉莉酸和CuCl2等激发子的响应。结果表明，酵母膏和茉莉酸在任何水平上都有很快的反应速度，而CuCl2的反应较慢。(3)将萝卜过氧化物酶基因启动子区域与肉毒素基因的嵌合基因导入烟草植株，获得了具有抗真菌活性的植物。(4)从土曲霉中克隆了催化分子内苯酚氧化偶联反应的二氢大豆黄素加氧酶基因。将该基因转化到尼杜拉曲霉中，并以具有酶活性的形式大规模表达。

(1) The genomic gene encoding chalcone synthase which was the key enzyme of flavonoid biosynthesis was cloned from cultured Pueraria lobata cells. And also the three genomic clones of reductase which was co-acting with chalcone synthase during the formation of deoxychalcone formation were isolated from P.lobata. Since chalcone synthase and reductase are acting together, the promoter regions of chalcone synthase and reductase genes are supposed to be similar. However they have no over-all similarity, but have several similar sequences in the length of about 20 bp. The purification of 2-hydroxyisoflavone dehydratase was achieved in this period and the cloning of its cDNA is proceeding.(2) The response of cultured P.lobata cells to elicitors such as yeast extracts, jasmonic acid and CuCl_2 was investigated at compound level, enzyme level and RNA level. The results clearly showed that yeast extracts and jasmonic acid responsed very fast at any level, on the other hand CuCl_2 responsed slowly.(3) The chimeric gene of the promoter region of horse radish peroxidase gene and Sarcotoxin cDNA was introduced to Tobacco plant using the Agrobacterium and Ti-Plasmid system to produce the plant having the anti-batcerial activity.(4) The gene encoding dihydrogeodin oxygenase which catalyzed intramolecular phenol oxidative coupling reaction was cloned from Aspergillus terreus. The coned gene was transformed to Aspergillus nidulans and expressed in the enzymatically active form in large scale.