RNAi mediated knock down of 3β-hydroxysteroid dehydrogenases in Digitalis lanata shoot cultures

Cardenolides are well-established substances for the treatment of atrial fibrillation or cardiac insufficiency. Latest research suggests that cardenolides could also be used in the treatment of various types of cancer [1].

Cardiac glycosides are still isolated after extraction from plants. For achieving higher amounts, e.g., in planta it is fundamental to understand cardenolide biosynthesis. We here focused on the investigation of 3β-hydroxysteroid dehydrogenase (3β-HSD) that is supposed to be involved in cardenolide biosynthesis. We screened D. lanata for the occurrence of 3β-HSD genes, determined their expression and established RNAi-mediated knock-down mutants. 3β-HSD knock-down mutants will be further investigated for a direct connection between 3β-HSD expression and cardenolide content.

Up to now, two 3β-HSDs (DlHSD1 [2] and DlHSD2), that share an amino acid sequence identity of 68.9% and a nucleotide sequence identity of 71.5% were identified. DlHSD2 was recently found by a function-based search in the transcriptome database of D. purpurea [3]. DlHSD2 is isolated, cloned and expressed in E. coli and 3βHSD (oxidation and reduction) activity with pregnenolone and 5β-pregane-3,20-dion is demonstrated. RNAi mediated knock down of the respective two D. lanata 3β-HSDs is verified by qPCR and stable transformed shoot cultures are now about to be characterized with regard to their cardenolide contents.

• References

• 1 Newman RA, Yang P, Pawlus AD, Block KI. Cardiac glycosides as novel cancer therapeutic agents. Mol Interventions 2008; 8: 36-49
• 2 Finsterbusch A, Lindemann P, Grimm R, Eckerskorn C, Luckner M. Delta(5)-3beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. - a multifunctional enzyme in steroid metabolism? Planta 1999; 209: 478-486
• 3 http://medicinalplantgenomics.msu.edu