Research On Regulating Autophagy To Prolong Lifespan Of Saccharomyces Cerevisiae

Autophagy is an intracellular lysosomal-mediated degradation mechanism used for cytoplasmic renewal and metabolism.Autophagy is highly conserved in all eukaryotes,from yeasts to humans,both morphologically and in the composition of the proteins that form the core mechanism of autophagy.Autophagy plays an important role in cell development,immune defense,programmed cell death,tumor suppression and neurodegenerative diseases.However,too much or too little autophagy can be detrimental to cell growth.Therefore,it is important for cells to maintain an appropriate level of autophagy.In this study,the strain of Saccharomyces cerevisiae BY4741 was used as the experimental object,and the TOR1,SCH9,RAS2 gene knockout mutant strains were constructed by Cre-lox P gene editing technology,and the regulation of autophagy intensity was realized by superposition of gene knockout.The relationship between autophagy intensity and cell life was analyzed,and yeast mutant strains with extended life were obtained.The following results were obtained:1.Using the S.cerevisiae BY4741 strain as the starting strain,use Cre-loxp technology to edit the TOR1,SCH9 and RAS2 genes of S.cerevisiae BY4741,PCR amplify the upstream and downstream fragments of the three target genes and perform splicing,and p Easy Blunt zero cloning vector was ligated and transformed into E.coli competent cells.After the plasmid was digested,it was ligated with the p DC10 digested fragment.A recombinant knockout plasmid with three genes was successfully constructed,namely:p Easy B-RAS2::hpt II,p Easy B-SCH9::hpt II and p Easy B-TOR1::hpt II.The homologous recombinant components were amplified by PCR and introduced into S.cerevisiae BY4741,and S.cerevisiae BY4741 ?ras2,S.cerevisiae BY4741 ?sch9,and S.cerevisiae BY4741?tor1 mutant strains were obtained.The plasmid p SH65 was used to remove the resistance expression frame integrated into the genome,and the genes were superimposed and edited to obtain S.cerevisiae BY4741 ?ras2?sch9,S.cerevisiae BY4741 ?ras2?tor1,S.cerevisiae BY4741 ?tor1?sch9 and S.cerevisiae BY4741 ?tor1?sch9?ras2 mutant strains.2.The original strain and seven mutant strains were used as experimental strains,the autophagy detection kit was used to selectively label autophagy vesicles.The staining was observed under a confocal microscope.Using a standard FITC filter for imaging,The laser intensity,voltage,gain and background correction of all the mutant strains were consistent with that of the original strain in imaging.The autophagy intensity was calculated using Ipp6.0,and the autophagy intensity was measured with the area of the measurement object and the average optical density as parameters.According to the analysis of the autophagy intensity results,S.cerevisiae BY4741 ?tor1?sch9?ras2 has the strongest degree of autophagy,and the autophagy degree of all the seven mutant strains is stronger than that of S.cerevisiae BY4741.3.Spread the eight strains on the SDC medium,use the number of CFUs on day 3 as the initial survival period(100% survival period),adjust the dilution and volume according to the mortality rate,and sample every 48 hours for spreading until survival The rate is 1%,based on which the time-series life span of the eight strains is determined.According to the analysis of the chronological lifespan graph,the chronological lifespan of S.cerevisiae BY4741?ras2?sch9,S.cerevisiae BY4741 ?tor1?sch9 and S.cerevisiae BY4741 ?tor1?sch9?ras2mutant strains is reduced;The the chronological lifespan of mutant strains S.cerevisiae BY4741 ?ras2?tor1,S.cerevisiae BY4741 ?sch9,S.cerevisiae BY4741 ?tor1 and S.cerevisiae BY4741 ?ras2 have been extended.The starting strain S.cerevisiae BY4741 has a chronological lifespan of 35 days,the mutant strain S.cerevisiae BY4741 ?ras2?tor1 has a chronological lifespan of 43 days,S.cerevisiae BY4741 ?sch9 has a chronological lifespan of41 days,S.cerevisiae BY4741 ?tor1 has a chronological lifespan of 39 days,and S.cerevisiae BY4741 ?ras2 has a chronological lifespan of 37 days.The chronological lifespan of the mutant strain S.cerevisiae BY4741 ?ras2?tor1 is 1.22 times that of S.cerevisiae BY4741;The chronological lifespan of the mutant strain S.cerevisiae BY4741 ?sch9 is 1.17 times that of S.cerevisiae BY4741;The chronological lifespan of the mutant strain S.cerevisiae BY4741 ?tor1 is 1.11 times that of S.cerevisiae BY4741;The chronological lifespan of the mutant strain S.cerevisiae BY4741 ?ras2 is 1.06 times that of S.cerevisiae BY4741.4.Obtained a mutant strain S.cerevisiae BY4741 ?ras2?tor1 with a chronological lifespan 1.22 times that of the starting strain,and its chronological lifespan and degree of autophagy were significantly improved.By analyzing the relationship between the intensity of autophagy and the chronological lifespan,it is found that within a certain range,with the increase of the autophagy intensity,the chronological lifespan is significantly prolonged,but when the autophagy intensity exceeds a certain limit,the chronological lifespan decreases instead.