Donma-eri̇me stresi̇ne di̇rençli̇ Saccharomyces cerevisiae laboratuvar ve endüstri̇yel suşlarinin gen anlatim düzeyleri̇ndeki̇ farkliliklarinin qRT-PCR i̇le anali̇zi̇

The aim of this thesis study was to analyze gene expression level differences between freeze-tolerant laboratory and industrial strains of Saccharomyces cerevisiae, to gain insight into the molecular mechanisms of freeze tolerance in this yeast. For this purpose, S. cerevisiae laboratory strain '905', industrial strain 'R625' and their freeze-thaw resistant mutant individuals 'F1' and 'P8' were used. Freeze-thaw stress-related genes were chosen according to whole-genome transcriptomic analysis results of 905 and F1, which have been obtained previously. S.cerevisiae is a small, unicellular fungus that has been used as a model eukaryotic organism in biological research for a long time. Also, it has been commonly used in industrial processes since ancient times. The ability to ferment sugars makes the budding yeast is commercially important in bread-making and wine industries. The organism is also being used in bioethanol and fuel ethanol production, recently. S. cerevisiae is also named as baker's yeast because of its ability to leaven dough. It is also known that the yeast cells are exposed to freeze-thaw stress during long term storage conditions. Thus, cryopreservation process is a stressful condition for yeast cells. The freeze-thaw resistant mutants F1 and P8 were obtained in a previous study by an inverse metabolic engineering approach, based on random mutation and selection under freeze-thaw stress conditions. Whole-genome transcriptomic analyses of 905 and F1 were also done previously. To verify the transcriptomic analysis results obtained by DNA microarray technique, gene expression levels of selected genes were determined by Quantitative RT- PCR. The genes were selected from microarray data according to their high up- or downregulation levels. Highy upregulated genes HSP12, FMP45, HSP26, HXK1 and highly downregulated genes PHO84, NSR1, ZRT1 were chosen for gene expression analysis by qRT-PCR. The qRT-PCR experiments were performed under control and stress conditions for '905', 'F1' 'R625' and 'P8' strains. The expression profile of 'F1' was normalized to that of the wild type laboratory strain '905', and the expression profile of 'P8' was normalized to that of the wild type industrial strain 'R625'. In conclusion, it was found that freeze-thaw stress causes significant changes in gene expression patterns of both laboratory and industrial strains. In addition, some differences were also found between gene expression level results of microarray and qRT-PCR analyses. This indicates the importance of verifying DNA microarray results by qRT-PCR, as qRT-PCR is a more sensitive method.