文献类型：期刊文献

中文题名：利用RNAi抑制曼地亚红豆杉细胞紫杉烷14β-羟基化酶基因的表达

英文题名：Suppression of Taxoid 14β-hydroxylase Gene Expression in Taxus×media via RNA Interference

作者：李凤岚[1,2] 邱德有[2,3] 马小军[1] 刘敏[2,3] 许德荣[2,3]

第一作者：李凤岚

机构：[1]中国医学科学院/中国协和医科大学药用植物研究所;[2]国家林业局林木培育重点实验室;[3]中国林业科学研究院林业研究所

年份：2009

卷号：29

期号：5

起止页码：55-60

中文期刊名：中国生物工程杂志

外文期刊名：China Biotechnology

收录：CSTPCD;;北大核心:【北大核心2008】；CSCD:【CSCD2011_2012】；

基金：国家“863”计划(2007AA10Z182);国家自然科学基金(30671698、30440033);中央级公益性科研院所基本科研业务费专项资金(2007-01)资助项目

语种：中文

中文关键词：曼地亚红豆杉;紫杉烷14β-羟基化酶;RNA干扰;基因表达

外文关键词：Taxus x media Taxoid 14β-hydroxylase RNA interference Gene expression

分类号：Q786

摘要：目的:利用RNA干扰技术抑制紫杉烷14β-羟基化酶(简称14OH)基因的表达。方法:以曼地亚红豆杉(Taxus×media)为材料,通过农杆菌介导的双元载体转化系统将针对14OH基因构建的RNA干扰载体导入该植物细胞中。应用PCR-Southern技术对转基因细胞进行了鉴定。另外,利用RT-PCR技术检测14OH基因mRNA表达水平,并采用HPLC技术对转基因细胞系中三种C-14氧取代的紫杉烷成分进行测定。结果:Southern检测证实转基因实验获得成功,RT-PCR结果表明转基因细胞系中14OH基因mRNA表达水平与对照组相比显著下降,HPLC分析显示C-14氧取代的紫杉烷含量也有明显降低。结论:该RNA干扰技术有效地抑制了曼地亚红豆杉细胞内14OH基因的表达,有望为提高紫杉醇的生物合成产量提供一条新途径。
Taxoid 14β-hydroxylase directs a side-route of taxol pathway that leading to a large number of 14β-hydroxy taxoid side products. A RNAi vector was used to suppress the expression of taxoid 14β-hydroxylase （14OH） gene in Taxus x media suspension cells. The dsRNA expression unit was introduced into Taxus x media cells via Agrobacterium tamefaciens-mediated transformation approach. PCR-Soutbern blot analysis of transgenic cells showed that the dsRNA unit was integrated into the genome of Taxus x media. The levels of transcripts for 14OH gene in transformed and the control cell lines were determined by using RT-PCR method. In addition, the yield of taxoids were analyzed by HPLC. The transcription of 14OH gene was suppressed and the total amount of three C-14 oxygenated taxoids （yunnanxane, sinenxan A, sinenxan C ） in Taxus x media transgenic cell lines reduced significantly as compared to those in the non-transgenic control. Inhibition of the 14OH gene via RNAi might provide a new way to enhance the production of Taxol in Taxus cells.