Generation of Transgenic Medicinal Plants with Altered Saponin Productivity

(1)OSC cDNA的分子克隆及其酶功能鉴定利用同源性PCR技术，从多种高等植物中克隆了20多个具有不同产物特异性的氧化角鲨烯环化酶（OSC）cDNA。它们显示出超过60%的序列同一性，即使它们来源于属于不同植物科的植物并产生不同的环化产物。通过对酿酒酵母（Saccharomycescerevisiae）羊毛甾醇合成酶缺失突变株转化子产物分析，确定了它们分别为环阿屯醇、β-香树脂醇、羽扇豆醇、葫芦二烯醇的脱氢酶。β-香树脂素合成酶克隆的分离，产生最流行的齐墩果烷型三萜，将有效地应用于转化药用重要的皂苷生产植物。(2)羽扇豆醇合成酶反义基因转化拟南芥及其化学型分析将羽扇豆醇合酶基因导入A.通过35 S启动子驱动的pBIX载体系统，利用植物体内渗透法，在平板上选择转基因植物并进一步在人工土壤中生长。T1代表现出明显的矮化现象。通过LC-MS方法分析T2后代中的三萜含量，揭示转基因植物中完全不存在羽扇豆醇。这些结果，结合（1）中所述的那些，用这些三萜合酶cDNA的转基因技术将导致开发出过量生产具有生物活性的皂苷。

(1) Molecular cloning of OSC cDNAs and identification of their enzyme functions. By employing homology based PCRs, more than twenty oxidosqualene cyclase (OSC) cDNAs with different product specificities have been cloned from various higher plants. They show more than 60% sequence identity even though they are derived from Plants belonging to different Plant families and giving rise to different cyclization products. Identification of their enzyme functions as synthases of cycloartenol, β-amyrin lupeol, cucurbitadienol were established by product analysis of transformants of Saccharomyces cerevisiae mutant, which lacks lanosterol synthase and accumulates oxidosqualene in the cells carrying expression plasmid with respective cDNA. Isolation of β-amyrin synthase clone, giving rise to most popular oleanane type triterpenes, would be efficiently applied for transformation of medicinally important saponin producing plants. (2) Transgenic Arabidopsis thaliana with antisense LUP1 lupeol synthase DNA and its chemotype analysis. Antisense LUP1 lupeol synthase DNA was introduced into A. thaliana via pBIX vector system driven by 35S promoter by means of in planta infiltration method. Transgenic plants were selected on plates and further grown in artificial soil. T1 progeny showed marked dwarfism. Triterpene contents in T2 progeny were analyzed by means of LC-MS method revealing the complete absence of lupeol in the transgenic plants. These results, combined with those described in (1), transgenic technology with these triterpene synthase cDNAs would lead to the development of overproducers of pharmacologically active saponins.