Browsing by Author "Rinas, Anastasia"

Gerbera hybrida, a common ornamental plant, has natural resistance to fungal diseases. While there may be several bioactive compounds behind this trait, this master’s thesis focuses on two of them: gerberin and parasorboside. The gerberin/parasorboside biosynthesis has been profoundly investigated by Gerbera Laboratory at University of Helsinki. Gerberin and parasorboside are polyketide derivatives, a vast group of bioactive metabolites. The pathway that produces these compounds involves several enzymes: a polyketide synthase (PKS), two polyketide reductases (PKRs) and a glycosyl transferase. For gerberin to be synthesized, it requires presence of three enzymes: (1) G2PS1 (Gerbera 2-pyrone synthase 1) which initiates the chain by synthesizing the carbon backbone, and (2) an unknown first acting reductase, that continues the chain by reduction, lactonization, and (3) addition of a sugar molecule. Parasorboside production requires also a fourth enzyme, a reductase described by Zhu et al. (2022). This master’s thesis delves into post-modification of gerberin and parasorboside intermediates by the PKRs, which have not yet been verified. Until now, there has only been PKR-candidates for this position, derived from gerbera transcriptome library. The genes behind the proposed reductases were known from previous research. The objective of this master’s thesis is to demonstrate which of the proposed reductases coexpressed along with the G2PS1-reductase will produce the desired compounds (gerberin and parasorboside) in a model plant tobacco (Nicotiana tabacum in stable transformation and Nicotiana benthamiana in transient expression). The gene combinations were constructed into plasmids via PCR and Golden Gate cloning. Enzyme production was analysed by western blot and the secondary metabolites by HPLC. The hypothesis for this work was to produce the aglycones of gerbera’s natural products gerberin and parasorboside in the model plant tobacco via constructing the plasmids carrying our genes of interest. This master's thesis documents the successful construction of double and triple fusion plasmids, their integration into agrobacteria, and subsequent transformation into stably transgenic tobacco. Gerberin production in tobacco was observed with certain gene combinations, parasorboside production was not analysed yet in this work. Through meticulous work, the desired combination of genes for gerberin synthesis was identified in both agroinfiltrated plants with co-expressed genes and stable transgenic tobacco lines expressing genes from a single transcript cleaved by 2A-peptides.