Suppression of Taxoid 14?-hydroxylase Gene Expression in Taxus?media via RNA Interference / 中国生物工程杂志

ABSTRACT

Taxoid 14?-hydroxylase directs a side-route of taxol pathway that leading to a large number of 14?-hydroxy taxoid side products. A RNAi vector was used to suppress the expression of taxoid 14?-hydroxylase (14OH) gene in Taxus?media suspension cells. The dsRNA expression unit was introduced into Taxus?media cells via Agrobacterium tumefaciens-mediated transformation approach. PCR-Southern blot analysis of transgenic cells showed that the dsRNA unit was integrated into the genome of Taxus?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?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.