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Vpliv alelov gena MKT1 na odpornost kvasovke Saccharomyces cerevisiae proti kofeinu in rapamicinu
Kogovšek, Peter (Author), Petrovič, Uroš (Mentor) More about this mentor... This link opens in a new window

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Abstract
Namen naloge je bil primerjava funkcije alelov gena MKT1 v različnih genetskih ozadjih kvasovke Saccharomyces cerevisiae. Gen MKT1 vpliva na procesiranje več različnih RNA molekul, s čimer sodeluje v več celičnih procesih. Izbrali smo dvajset segregant križanja dveh izbranih sevov kvasovke S. cerevisiae, ki so imele isti alel gena MKT1. Z metodo CRISPR-Cas9 smo najprej izvedli delecijo gena MKT1 v vseh segregantah, nato pa smo v genom segregant vstavili alel gena MKT1, ki izhaja iz drugega starševskega seva teh segregant. Želeli smo ugotoviti razliko v rasti med variantami z različnimi aleli na gojiščih, ki vsebujejo kemikalije, ki aktivirajo signalno pot TOR. Zato smo variante segregant nacepili na plošče s kofeinom in rapamicinom in jih medsebojno primerjali po odpornosti. Pri vseh pogojih so variante z aleloma MKT1BY in mkt1Δ rasle enako, iz česar sklepamo, da je Mkt1BY nefunkcionalen protein. Na ploščah s kofeinom pri polovici sevov ni bilo razlik v rasti med variantami alelov, pri drugi polovici pa je varianta z alelom MKT1AWRI rasla slabše od drugih dveh. Iz tega smo postavili hipotezo, da na odpornost proti kofeinu gen MKT1 vpliva skupaj s še enim genom. Na gojišču z rapamicinom so se segregante glede na rast razdelile v tri skupine. Iz tega sklepamo, da je odpornost proti rapamicinu kompleksna oziroma poligenska lastnost. Da bi ugotovili, kateri in koliko genov še sodeluje v odpornosti proti rapamicinu in kofeinu, bi morali sekvencirati genome segregant in primerjati razporeditev SNP med njimi.

Language:Slovenian
Keywords:kvasovka, Saccharomyces cerevisiae, CRISPR-Cas9 sistem, zamenjava alelov, gen MKT1, sev AWRI1631, sevBY4741, redčitvena linija, kofein, rapamicin
Work type:Master's thesis/paper (mb22)
Organization:BF - Biotechnical Faculty
Year:2019
COBISS.SI-ID:5119055 Link is opened in a new window
Views:51
Downloads:28
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Secondary language

Language:English
Title:Influence of different MKT1 alleles on caffeine and rapamycin resistance in yeast Saccharomyces cerevisiae
Abstract:
The aim of the study was to compare the function of the MKT1 gene alleles in strains of the yeast Saccharomyces cerevisiae with different genetic backgrounds. MKT1 gene influences processes of several RNA molecules and through this affects several cellular processes. We chose 20 segregants that were obtained from a cross between two strains of yeast S. cerevisiae, which all share the same allele of gene MKT1. Using CRISPR-Cas9 method we first deleted the MKT1 gene in these segregants, and in the next step inserted the MKT1 allele from the other parental strain. We wanted to determine the difference in growth between variants with different MKT1 alleles on media with added chemicals that activate the TOR signalling pathway. In order to achieve this, we cultivated the three different variants of each segregant on in the presence of caffeine or rapamycin and compared their resistance. The variants with the MKT1BY and mkt1Δ alleles grew comparably in all conditions. From this result we concluded that the Mkt1BY protein variant is non-functional. On the medium with caffeine there was no difference in the growth between segregants in half of the segregants, but in the other half the variant with the MKT1AWRI allele was less resistant to caffeine. From this result we hypothesized that MKT1 gene influences caffeine resistance in combination with another gene. On the medium with rapamycin the segregants were grouped into 3 different groups. From this result we concluded that the resistance against rapamycin is a complex, i.e. polygenic trait. To determine which and how many genes influence resistance against rapamycin and caffeine, the segregants’ genomes should be sequenced and the SNP distribution among segregants analyzed.

Keywords:yeast, Saccharomyces cerevisiae, CRISPR-Cas9 system, allele swap, MKT1 gene, AWRI1631 strain, BY4741 strain, dillution assay, caffeine, rapamycin

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