Transformation of yeast Saccharomyces cerevisiae with plasmid containing growth inhibitor resistance gene YAP1

Neki od spojeva koji nastaju kiselinskom predobradom lignoceluloznih materijala inhibiraju rast kvasca Saccharomyces cerevisiae jer povećavaju količinu reaktivnih kisikovih spojeva u stanici. Gen YAP1 kodira za transkripcijski faktor koji u prisutnosti reaktivnih kisikovih spojeva povećava razinu ekspresije mnogih gena čiji proteinski produkti sudjeluju u smanjenju oksidativnog stresa. Cilj ovog rada bio je transformirati odabrane diploidne sojeve kvasca S. cerevisiae, nastale križanjem prirodnih izolata iz vina (E5) i cvijeta kaktusa Opunitha megacantha (2421), plazmidom pSP-YAP1 koji sadrži gen YAP1. Prvo je restrikcijskom analizom provjerena struktura plazmida pSP-YAP1, nakon čega su transfomirani homozigotni diploidni sojevi E5 i 2421 te heterozigotni diploidni soj H2 nastao križanjem haploidnih sojeva E5 i 2421.Some compounds that are by-products of the acidic hydrolysis of a raw lignocellulosic material inhibit the growth of the yeast Saccharomyces cerevisiae due to the accumulation of reactive oxygen species in the cell. YAP1 gene encodes transcription factor that, in the presence of reactive oxygen species, increases the expression of genes that encode proteins involved in the response to oxidative stress. The aim of this work was to transform selected diploid S. cerevisiae strains that were constructed by mating of E5, a natural isolate from wine, and 2421, an isolate from the flower of the cactus Opunitha megacantha, with the plasmid pSP-YAP1 containing the YAP1 gene. First, the structure of the plasmid pSP-YAP1 was confirmed by restriction mapping and then homozygote diploid strains E5 and 2421 and heterozygote diploid H2 strain were transformed successfully