| Scope and method of study. Taxol, originally isolated form the bark of Taxus brevifolia, is regarded as one of the best anti-cancer medicines developed from plant sources. However, its supply has been problematic since its discovery and has not been completely solved yet. The purpose of this study was to improve the production of Taxol by biotechnological approaches. As the first step, high performance liquid chromatography (HPLC) was employed to screen high-taxoid-yielding Taxus x media plants from 17 cultivars with different growth characteristics. Agrobacterium tumefaciens-mediated genetic transformation was then performed to transfer the high-efficiency-promoter-driven gene of taxadiene synthase, which is assumed to catalyze the first committed and rate-limiting step in the Taxol biosynthesis pathway, into tobacco leaf disks, in vitro Taxus suspension cells and in planta Taxus x media somatic cells. Lastly, gene expression profiling by cDNA-AFLP was conducted to detect alterations of gene expression in yew suspension cells upon methyl jasmonate (MeJA) and salicylic acid (SA) elicitation.; Findings and conclusions. For the T. x media cultivars screening, three cultivars with fast growth rate and upright growing form were found to contain the highest Taxol content ('Coleana', 378 mug/g; 'Hicksii', 322 mug/g; and 'Stovekenii', 309 mug/g). The in planta genetic transformation of 'Hicksii' plant with taxadiene synthase over-expression significantly increased Taxol production in the A. tumefaciens-induced gall tissues. The expression of the integrated green fluorescent protein (GFP) gene was visualized in the transformed 'Hicksii' suspension culture cells; and the integration and transcription of the taxadiene synthase gene was also detected in the tissues developed from the tobacco leaf disk transformation. For the cDNA-AFLP gene expression profiling, a total of 157 differentially expressed gene fragments were isolated, some of which were found to represent pathogenesis or stress-related genes based on BLAST search. |
