Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells

Abstract

Background: Taxol(®) (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control.

Results: Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day) to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO) analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line.

Conclusions: This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

Figure 1

Expression pattern of clones from up-regulated libraries. Macroarrays spotted with randomly selected clones (probes) from all six SSH libraries were hybridized with labelled RNA targets prepared from mock elicited and elicited cells at three time points: 6h, 18h, and 5 day. Expression profile plots for the set of probes corresponding to each up-regulated library are shown: a, 6h library probes b, 18h library probes c, 5 day library probes. Expression is given as the fold-change in gene expression in the elicited culture over the un-elicited culture. Only results from probes showing a statistically significant change in expression (P < 0.05) are shown.

Figure 2

Heat map showing overall expression patterns of differentially regulated genes from up-regulated libraries. Hierarchical clustering of significantly MJ up-regulated genes was performed using average linkage and Euclidean distance as a measurement of similarity. Only results from probes showing a statistically significant change in expression for at least one time point (P < 0.05) are shown.

Figure 3

Semi-quantitative RT-PCR analysis of 12 selected up-regulated unigenes. Semi-quantitative RT-PCR was carried out using RNA from the Taxus cuspidata P93AF cell line. RNA samples were taken at 6h, 18h and 5 day after MJ elicitation, and from mock elicited cells at the same time points. Lane 1: Mock elicited for 18h, Lane 2-4: elicited with 100 μM MJ for 6h, 18h or 5 day, respectively. Gapdh and Actin1 were used as normalization controls.