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

(1)从培养的葛根细胞中克隆了黄酮类合成关键酶查尔酮合成酶的基因组基因。在脱氧查尔酮生成过程中，还分离到了3个与查尔酮合成酶共同作用的还原酶基因组克隆。由于查尔酮合成酶和还原酶共同作用，查尔酮合成酶和还原酶基因的启动子区域应该是相似的。然而，它们没有整体的相似性，但有几个长度在20 bp左右的相似序列。2-羟基异黄酮脱水酶的纯化已在这一时期完成，其cDNA的克隆正在进行中。(2)从化合物水平、酶水平和RNA水平研究了培养的P.lobata细胞对酵母提取物、茉莉酸和CuCl_2等激发剂的反应。结果表明，酵母浸膏和茉莉酸在任何水平下都能快速响应，而CuCl_2响应较慢。(3)利用农杆菌和ti质粒系统将马萝卜过氧化物酶基因启动子区与肉毒素cDNA嵌合基因导入烟草植株，获得具有抗菌活性的植株。(4)从地曲霉中克隆到了催化苯酚分子内氧化偶联反应的二氢土豆素加氧酶基因。将该锥形基因转化到细粒曲霉中，并以酶活性形式大规模表达。

(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.