文献类型：期刊文献

英文题名：Antisense-induced suppression of taxoid 14 beta-hydroxylase gene expression in transgenic Taxus x media cells

作者：Li, Feng-Lan[1,2] Ma, Xiao-Jun[1,2] Hu, Xin-Ling[3] Hoffman, Angela[4] Dai, Jun-Gui[5,6] Qiu, De-You[3]

第一作者：Li, Feng-Lan

通信作者：Ma, XJ[1]

机构：[1]Chinese Acad Med Sci, Inst Med Plant Dev, Beijing 100094, Peoples R China;[2]Peking Union Med Univ, Beijing 100094, Peoples R China;[3]Chinese Acad Forestry, Res Inst Forestry, State Key Lab Tree Genet & Breeding, Beijing 100091, Peoples R China;[4]Univ Portland, Dept Chem, Portland, OR 97203 USA;[5]Chinese Acad Med Sci, Inst Mat Med, Beijing 100050, Peoples R China;[6]Peking Union Med Coll, Beijing 100050, Peoples R China

年份：2011

卷号：10

期号：44

起止页码：8720-8728

外文期刊名：AFRICAN JOURNAL OF BIOTECHNOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000299733300007)】；

基金：We thank Dr. Iain W. Wilson (CSIRO Plant Industry, Australia) for his critical reading the manuscript. This work is supported by National High-Tech Program (2007AA10Z182) (D.Q.), the Natural Science Foundation of China (30671698 and 30440033) (D.Q.) and The Special Research Fund for Non-profit Research Institute (2007-01) (D.Q.).

语种：英文

外文关键词：Taxus x media; taxoid 14 beta-hydroxylase; antisense; gene suppression

摘要：The enzyme taxoid 14 beta-hydroxylase (14OH) directs a side-route of taxol pathway to 14 beta-hydroxy taxoids. Suppression of this side-route could increase the production of taxol. To suppress taxoid 14 beta-hydroxylase gene (14OH) expression in the Taxus x media TM3 cell line, antisense RNA inhibition approach was used in this study. Following the construction of an antisense RNA expression vector of 14OH from Taxus chinensis, the antisense 14OH cDNA (as14OH) was introduced into TM3 cells by Agrobacterium tumefaciens-mediated transformation. Southern blot analysis of hygromycin phosphotransferase gene (HYG) revealed that this selection gene was integrated successfully into the genome of Taxus x media cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the 14OH mRNA level in transgenic cells dropped dramatically, suggesting that the expression of endogenous14OH gene was significantly suppressed by the exogenous as14OH gene. Correspondingly, the total yield of three major C-14 oxygenated taxoids (yunnanxane, taxuyunnanine C, sinenxan C) was markedly reduced in the silenced cell lines when compared with those of the non-transgenic controls. These results indicated that the antisense RNA strategy is a useful tool in suppressing the expression of genes in Taxus and this method could be used to silence other important genes that divert Taxol pathway to side-route metabolites.