权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Generation of Transgenic Medicinal Plants with Altered Saponin Productivity

具有改变皂苷生产力的转基因药用植物的产生

基本信息

批准号：
10470464
负责人：
EBIZUKA Yutaka
金额：
$ 6.14万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470464/
关键词：
Triterpene Saponin Triterpene Synthase Oxidosqualene cDNA PCR Transgenic Plant Medicinal Plant オタネニンジンカンゾウ

项目摘要

(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.

(1) OSC cdna的分子克隆及其酶功能鉴定。利用同源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。尽管它们来自不同的植物科，产生不同的环化产物，但它们仍具有60%以上的序列同一性。通过对缺失羊毛甾醇合成酶和积累氧化角鲨烯表达质粒的酿酒酵母突变体转化体的产物分析，确定了它们的酶功能分别为环青蒿醇合成酶、β-amyrin lupeol和葫芦二烯醇合成酶。β-amyrin合成酶克隆的分离可产生最常见的齐墩烷型三萜，可有效地应用于药用皂素生产植物的转化。(2)带反义LUP1 luppeol合成酶DNA的转基因拟南芥及其化学型分析。利用35S启动子驱动的pBIX载体体系，采用植物内浸润法将反义LUP1 luppeol合成酶DNA导入拟南芥。在平板上选择转基因植物，并在人工土壤中进一步种植。T1子代有明显的侏儒症。用LC-MS法分析了T2子代的三萜含量，结果表明转基因植株中完全不含lupeol。这些结果与(1)中描述的结果相结合，使用这些三萜合成酶cdna的转基因技术将导致过量生产具有药理活性的皂苷。