Alkaloids form an important class of plant compounds with many pharmaceutically active compounds. The plant species Catharanthus roseus produces among other pharmaceuticals the widely used anti-cancer compounds vincristine and vinblastine. Alkaloid biosynthesis in Catharanthus is stimulated by the plant stress hormone jasmonic acid and related signalling compounds, collectively known as JAs. JAs activate the expression of alkaloid biosynthesis genes via a jasmonate- and elicitor-responsive element (JE RE) in their promoters.
This JERE interacts with two members of the AP2-domain transcription factor family called ORCA2 and ORCA3. ORCA3 has been shown to regulate multiple genes involved in alkaloid biosynthesis, and its over-expression in Catharanthus cells leads under certain feeding conditions to a significant increase in alkaloid levels. The ORCA genes are themselves induced by JAs. Analysis of the ORCA3 promoter has revealed a novel JA-responsive element, indicating that JA-responsive ORCA gene expression is mediated by an unidentified upstream transcription factor. The latter protein can be considered a true master regulator of alkaloid metabolism, since it probably regulates both ORCA genes and thereby multiple alkaloid biosynthesis genes.
This proposal aims at the isolation of this upstream transcription factor. The proposal is to run protein extracts from C. roseus cell suspensions over DNA affinity purification columns containing the JERE from the ORCA3 promoter. Isolated proteins will be sequenced, and the corresponding DNA sequences will be isolated via RT-PCR. This upstream transcription factor has enormous potential as a tool to engineer alkaloid production in plant cells, and thereby increase the production of important pharmaceuticals. Technically it uses DNA affinity chromatography, highly uncommon in the plant field, with new improvements, combined with state of the art protein sequencing techniques.
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