Gene Expression Profiling in Taxus baccata L. Seedlings and Cell Cultures

Abstract

Limited native resources of paclitaxel from Taxus trees initiated the research to produce this compound by biotechnology. In vitro plant cell culture systems have been used for large-scale production of paclitaxel and related taxanes. In the past decade, several genes involved in the taxane biosynthetic pathway have already been sequenced and cloned. This protocol details how to derive cell cultures of Taxus baccata L. from young stems of mature trees and from all parts of in vitro- grown seedlings such as root segments, hypocotyls, and cotyledons. The time-course of expression of two genes - dbat and dbtnbt - coding for two enzymes of the later steps of paclitaxel biosynthesis and the intracellular taxane accumulation has been investigated through a 64-day subculture interval of T. baccata cell cultures, during germination, and in early stages of seedling development. The expression level is measured by using quantitative real-time reverse transcriptase polymerase chain reaction. The intracellular content of baccatin III and paclitaxel is quantified by high-performance liquid chromatography HPLC.We have shown that although the increase in transcriptional activity of dbat and dbtnbt positively correlate with callus growth, the intracellular accumulation of paclitaxel varies during subculture with the maximum between the late linear and stationary phase. The expression of both genes peaks on day 8 of germination, followed by a decrease in the post-germination phase and during seedling growth. The increase of the steady-state mRNA level of both genes is followed by corresponding metabolite accumulation with a delay of approximately 14-28 d.