Functional characterization of alternative oxidase genes from Digitalis ferruginea and investigation of their association with secondary metabolite production

Alternative oxidases (AOX)s serve as one of the terminal oxidases in plant mitochondrial electron transport chain. They have a very important role in mitochondrial retrograde signaling and are very well known for their function on the helping in stress signaling in plants. As a sesille organism, plants undergo several environmental stresses and to fight with these stresses they adapt themselves by producing diverse metabolites because of their secondary metabolism. Digitalis ferruginea is a well-known species for the production of secondary metabolites; especially, cardenolides. In this thesis, the relationship between alternative oxidase activity on the secondary metabolite production was evaluated in the D. ferruginea undifferentiated callus suspension culture. Tree AOX gene family members belonging to D. ferruginea were determined using transcriptome data. Their functions were characterized as terminal oxidase and structural properties of the putative protein sequences were compared with each other using bioinformatic methods in silico. Interested genes were further amplified using RT-PCR method. Determined and verified dfsAOX genes were later cloned into S. cerevisiae using Gateway cloning method. Recombinant S. cerevisiae cells were investigated for their protein expression capacity using Western-blot, RT-PCR, Spot Assay and Confocal microscopy methods. Later, prepared callus suspension cultures were treated with SHAM, SA, and AA and than examined for their secondary metabolite accumulation and AOX gene expression studies. Combines results commened as AOX can play a regulatory role in sekonder metabolite accumulation in plan but further analyses needed substantiate this conclusion.