SWAP Mutagenesis in GDFR1-2 vs. GDFR1-3 to Explore Substrate Specificity of DFR in the Anthocyanin Pathway of Gerbera

Anthocyanins are an important class of flavonoids under the class of phenolic compounds and contribute to flower color variation. Gerbera hybrida is a flowering plant of Asteraceae family having mainly two colors of flowers – orange and red. Dihydroflavonol 4-reductase (DFR) is a key enzyme catalyzing a reaction in anthocyanin biosynthesis, the reduction of dihydroflavonols to leucoanthocyanidins. GDFR1-2 and GDFR1-3 are two allelic forms of gerbera DFR differing in substrate specificity for the dihydroflavonols - dihydrokaempferol, dihydroquercetin and dihydromyricetin and also differ in 13 amino acids where eight are considered to be important for substrate specificity. GDFR1-2 has strong preference for dihydrokaempferol and GDFR1-3 doesn’t have any preference for the three substrates. In order to find out the amino acids responsible for substrate specificity, swap mutations were generated between GDFR1-2 and GDFR1-3 by two PCR methods– first, running separate PCR from the templates of GDFR1-2 and GDFR1-3, making a heteroduplex by mixing separate PCR where non-matching nucleotides are expected to be corrected by E. coli and, second, by running PCR from mixed templates with short extension time of PCR to make swaps by template switching. The second method was found more effective than the first method. 81 lines (named GDAT1-81) were sequenced and 35 unique swap mutants were found. In this work the DFR assay was done from six randomly picked GDAT lines where GDAT5 had a swap in one amino acid showing still a similar pattern of substrate specificity as the reference (GDFR1-3) indicating that the mutated amino acid doesn’t have any role in substrate specificity. GDAT14 had an extra mutation (S167P) along with 2 swaps showing incapability of reducing dihydrokaempferol, demonstrating that the mutated amino acids are important and other 4 lines were identical to either GDFR1-2 or to GDFR1-3. This was a preliminary test with 6 lines. In order to get more explanations about the roles of amino acids in substrate specificity, DFR assay was done for all the 81 lines in experiments outside of this thesis and five patterns of substrate specificity were identified indicating that substrate specificity of DFR can be altered by changing only three important amino acids. The amino acids at the position 85,135 and 181 in DFR coding sequence have been identified having important roles in substrate specificity. In addition, the amino acid at position 167 may have a function in making the gerbera DFR able to reduce dihydrokaempferol