The Effect Of Ras/PKA On The Longevity Of Saccharomyces Cerevisiae

Longevity and aging are important branches of medicine and developmental biology.Aging can lead to the loss of related functions of the body and make it more vulnerable to disease.The disadvantages of using advanced mammals to study the mechanisms of aging are long research periods and high research costs.As a eukaryotic organism,Saccharomyces cerevisiae has the same cell and tissue structure as higher mammalian cells.Because yeast has a wide range of specific molecular tools and resources,the use of yeast as a biological model to study the complex molecular processes of multicellular organisms such as humans provides a good basis for biological research.The Ras/PKA pathway is highly conserved in all eukaryotes and in cellular processes associated with apoptosis and senescence.Ras/PKA pathway is the main mediator of caloric restriction-dependent stress resistance and life extension,and the regulation of Ras/PKA pathway affects cell viability and apoptosis under the conditions of natural gradual acidification and acid stress of culture medium.Downregulation of Ras/PKA activates the anti-stress pathway,which slows yeast aging by undoing this ability to inhibit damage repair.The down-regulation of Ras/PKA activates the anti-stress pathway,which can slow down the aging of yeast by increasing the sensitivity of cells to stress,regulating some genes involved in oxidative repair and detoxification,and enhancing the ability of cell damage repair.So it provides important reference for the study of human life span.In this study,the CRISPR/Cas9 was used to edit the genes of S.cerevisiae CYR1,TFS1,SDC25,CDC25,RAS1 and PDE2,and the following results were obtained:1.The CRISPR/Cas9 technology was used to design and successfully construct the editing plasmids that successfully knocked out the targeted genes CYR1,TFS1,SDC25 and CDC25 in S.cerevisiae,so as to achieve the targeted gene deletion in S.cerevisiae.2.The CRISPR/Cas9 technology was used to design and successfully construct the targeted gene of targeted mutant S.cerevisiae,in which both the strong promoter and theediting plasmid of the gene to be enhanced were inserted,the gene RAS1 was mutated and the gene PDE2 was enhanced,and the targeted gene editing of S.cerevisiae was realized.Not only increased the copy number of PDE2 gene,but also increased the expression of PDE2 by inserting PGK1 p promoter.3.The genes of CYR1,TFS1,SDC25,CDC25,RAS1 and PDE2 in the genome of strain W303 of S.cerevisiae were superposition edited by CRISPR/Cas9 technology,and the mutant strain w303-v that down-regulated the Ras/PKA pathway was obtained.4.The chronological life span,replicative life span and growth curve of S.cerevisiae strains W303 and W303-V were measured respectively.The results showed that the mutant strain W303-V had fast cell proliferation,and the chronological life span and replicative life span were 1.75 and 1.78 times longer than the original strain.