Abstract
Cephalotaxus hainanensis, an endangered plant, is known to contain several metabolites with anti-cancer activity. Despite its clinical impact, the alkaloid metabolism of this species has remained largely uncharacterized. The potential of Cephalotaxus for metabolic engineering of medically interesting compounds has, so far, not been exploited, due to the almost complete lack of molecular information. We have therefore performed a high throughput RNA-seq analysis and assembled the transcriptome de novo. Raw reads comprising 4.3 Gbp were assembled de novo into 39,416 unique sequences (unigenes) with a mean length of 1,089.8 bp and a total assembly size of 45.8 Mbp, which equals to more than 50 times the number of Cephalotaxaceae sequences currently deposited in the GenBank (as of August 2013). As proof of principle for medically interesting pathways, gene fragments related to paclitaxel biosynthesis were searched and detected. To verify their functionality, the metabolic product paclitaxel, and its precursor baccatin III, were identified in the leaves of C. hainanensis by HPLC, and shown to be induced by MeJA. This finding demonstrates exemplarily the potential of the annotated transcriptome as information resource for the biotechnological exploitation of plant secondary metabolism.
Highlights
IntroductionMost of these metabolites modulate the interaction of plants with other organisms and many of them are pharmaceutically active
Plants generate around 106 specific secondary metabolites [1]
Preparation of plant samples and RNA isolation Seedlings of C. hainanensis were collected in the greenhouse of the Institute of Tropical Crop Genetic Resources, Chinese Academy of Tropical Agricultural Sciences (CATAS) originating from Jianfengling in Ledong county, Hainan province, China
Summary
Most of these metabolites modulate the interaction of plants with other organisms and many of them are pharmaceutically active Often, those compounds are medically relevant, but cannot be synthetized technically and have to be extracted and purified from their natural source. The underlying metabolic pathways are complex and often require the interaction of different cell types until the active compound is stored in specialized tissues [2], often even in specialized secretory cells. This renders extraction cumbersome, inefficient and costly. The technological breakthroughs in high-throughput technologies, especially next-generation sequencing, have allowed extending molecular analysis to more ‘‘exotic’’ models that so far had remained out of scope
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