Isolation Of Germination Mutants In Saccharomyces Cerevisiae

Yeast cells undergo meiosis and form spores under nutritional starvation. The spore wall has 4 layers: mannan, β-glucan, chitosan and dityrosine layer. Spores can be used as microencapsule by using its character of spore wall for immobilization of endogenous enzymes, or be made resemble chitosan beads by genetic disruption for immobilization of exogenous enzymes through chemical ways. However, spores start to germinate when treated with nutrient which leads a detriment of spore structure and its application. The germination process is wildly believed to be divided into 2 stages, and induced the first stage by glucose, but the mechanism is not clear yet. Therefore in order to get germination defective strains to be used in industrial application, and also for the purpose of analyzing the mechanism of spore germination, chemical mutagenesis was taken against Saccharomyces cerevisiae.EMS was used as mutagen for yeast cells. Moreover, defective strains were isolated from Saccharomyces cerevisiae through resistance of outer spore layer to ether. 4 mutants were found: AN120-m5,AN120-m8,AN120-m10,AN120-m17. Except that AN120-m5 was slightly deficient in cell growth after verifying, these 4 mutant can normally sporulate and grow well. Specifully, These 4 mutants exhibited severe germination defect in liquid medium and on solid medium, however only 1% spores of AN120-m10 germinated in liquid medium, besides, single spore of AN120-m5 cannot germinate on solid medium completely. Next whether a single gene was mutated in the strains has been verified. Hybrid cells of AN120-m5 cannot be collected because of its defect on solid medium, thus we can only mate the other 3 mutant haploid cell with wild type haploid cell after 3 generations, and spores of wild type exhibited a germination eudismic ratio of 2:2 compared with mutant spores, this suggested a great possibility that these mutants were single gene mutants. The actin localization was also verified in the germination process, compared with wild type the research found that two mutants exhibited different patterns: aggregate pattern maintained and no aggregate pattern, this suggested mutation was involved in the actin localization.Four mutants isolated in the research were assumed as single gene mutated strains, which laid a foundation in confirming the mutated gene in the next screening. After verification at least 3 genes were mutated, this facilitates the analyzing of germination mechanism and constructing strains that completely don’t germinate which can be used as microencapsule for industrial application.