Transcriptional regulation of terpenoid genes in transformed roots of Artemisia annua L.

摘要

Two distinct biosynthetic pathways have been characterized in higher plants leading to the biosynthesis of isopentenyl diphosphate, the universal precursor of terpenoids: the cytosolic mevalonate pathway and the mevalonate-independent pathway, postulated to be located in plastids. The overall goal of this study was to further our fundamental understanding of the regulation of this branched pathway by using hairy root cultures of Artemisia annua L. and the production of artemisinin as a model. Using a cDNA library made from these roots, we isolated two cDNAs: the 2.6 kb deoxy-D-xylulose-5-phosphate synthase (DXPS) gene and the 2.2 kb deoxy-D-xylulose-5-phosphate reductoisomerase (DXPR) gene.; These cDNAs were used to analyze tissue-specific gene expression in greenhouse-grown A. annua (strain YU) plants. HMGR transcripts accumulated preferentially in roots and stems while DXPS expression was localized mainly in stems and to a certain extend in leaves.; Since artemisinin accumulation varies during growth and aging of the in vitro cultures of A. annua transformed roots, we also investigated the temporal regulation of these five terpenoid enzymes. While relative levels of HMGR mRNAs remained quite constant throughout the entire culture cycle, steady-state levels of DXPS transcripts were strongly affected by the culture age of the transformed roots.; Light-mediated changes in gene expression of these terpenoid enzymes were also investigated. Only the DXPS transcripts showed a marked difference with nearly three times greater levels in light grown versus dark grown cultures while roots grown continuously in light had similar mRNA levels of HMGR, DXPR, FPS, and SQS as roots grown in continuous dark.; The expressions of all the genes tested were also affected by culture methods. We compared shake flasks to bubble column, and nutrient mist reactors. Furthermore, we characterized the spatial heterogeneity in gene expression throughout the two types of bioreactors. Overall, higher HMGR, DXPS, DXPR, and SQS mRNA accumulation was found in the middle and top zones from both the wall and core regions of the bubble column reactor. In contrast, mRNA accumulation in transformed roots of A. annua grown in the nutrient mist reactor was generally higher in the wall region compared to the core. (Abstract shortened by UMI.)