Abstract

The enzyme taxoid 14β-hydroxylase (14OH) directs a side-route of taxol pathway to 14β-hydroxy taxoids. Suppression of this side-route could increase the production of taxol. To suppress taxoid 14β-hydroxylase gene (14OH) expression in the Taxus× media TM3 cell line, antisense RNA inhibition approach was used in this study. Following the construction of an antisense RNA expression vector of 14OH fromTaxus 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 × media cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the 14OHmRNA 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.

Key words: Taxus × media, taxoid 14β-hydroxylase, antisense, gene suppression.

Abbreviation

RT-PCR, Reverse transcription-polymerase chain reaction; HYG, hygromycin phosphotransferase gene; BA, 6-benzyladenine, 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; 14OH, 14β-hydroxylase.