Establishment And Validation Of Screening System For Genetic Suppressors Of Essential Genes In Saccharomyces Cerevisiae

Genetic studies have found that the lethal phenotype caused by certain necessary gene mutation can be saved by another gene mutation or overexpression,which is called a suppressor.Genetic suppressor screening plays an important role in genetic engineering research,metabolic pathway regulation,geneticbreeding and cancer drug target identification.Saccharomyces cerevisiae is a single-celled model organism,and its genetic research provides important insights for the basic and applied research of eukaryotes.In this study,Saccharomyces cerevisiae was used as a model to establish an efficient screening system for genetic suppressors of essential genes and to validate its application in the identification of chemotherapeutic drug resistance genes.In this study,the temperature-sensitive mutants rba50ts and gpn2ts of the essential genes RBA50 and GPN2 of Saccharomyces cerevisiae were selected as examples,to establish an efficient screening system for genetic suppressors of essential genes by random mutation and high copy library screening.In addition,in this study,mec1 mutant of the yeast model of tumor suppressor gene ATM/ATR was used to screen camptothecin(CPT)resistance genes,and to verify application of this screening system in the identification of chemotherapeutic drug tolerance genes.On the one hand,the random mutation was used to screen rba50ts reverse strains and gene mutation information was identified by whole genome resequencing.A total of 22 rba50ts candidate genomic suppressors were identified by this screening method.The rba50ts-ygk3 G115R strain was constructed by homologous recombination,and it was successfully verified that ygk3 G115R could recover the growth defect of rba50ts,ygk3 G115R is a genomic suppressor of mutant gene rba50.At the same time,the high copy plasmid library was used to screen gpn2's reverse strains,and 31 dosage suppressors were successfully identified,all of which could suppress the growth defects of gpn2ts.The results show that the essential gene genetic suppressor screening system can effectively enrich suppressors.On the other hand,the yeast model and method for screening chemotherapeutic drug resistance genes were established by using genetic suppressor screening system.The essential gene MEC1 of Saccharomyces cerevisiae is a homologous gene of tumor suppressor gene ATM/ATR.Its knockout leads to a lethal phenotype,which can be suppress by SML1 knockout.This study constructed mec1? strain as a tumor yeast model by this suppression phenomenon,and mec1? was sensitive to chemotherapeutic drug CPT.In this study,26 potential drug resistance genes were identified by genomic suppressor screening,among which Top1 is a known drug target for CPT.The top1 R576G mutants obtained in this study increased the drug resistance of mec1?,which confirmed that the drug target mutation is a mechanisms of CPT drug resistance,and further proved that this screening system has important valuable application in the identification of chemotherapeutic drug tolerance genes.In this study,a screening system for genetic suppressors of essential genes was established by random mutation and high copy library screening using temperature sensitive mutants rba50ts and gpn2ts of Saccharomyces cerevisiae.It has been proved that the screening system can effectively enrich suppressors and has a valuable application in the identification of chemotherapeutic drug resistance genes.In addition,this screening system can obtain several genes related to the function of the target gene at one time,which can not only save exploration time,but also provide clues for related research.