Research On GPD1 Gene Disruption And GPD2 Gene Sliencing In Saccharomyces

In Saccharomyces cerevisiae(S.cerevisiae),Glycerin metabolic pathways and ethanol metabolic pathways are closely related.GPDH plays a central role in glycerol metabolism,and GPDH directly affects the levels of glycerol synthesis.Glycerol fermentation commonly regulated by GPD1 gene and GPD2 gene.There is high homologous recombination efficiency in S.cerevisiae.This homologous recombination can occur with only 30?45 bp homologous region.LoxP-kanMX-LoxP and Cre/LoxP system which based on PCR amplification is a efficient and accurate technique for deleting GPD1 gene.5' UTR is the key factor of the direct regulation of gene expression in eucaryon.The secondary structure of 5'UTR could block gene translation process,and accelerate the degradation of mRNA.PCR amplification primer of knock-out component R1-R2 designed by LoxP-kanMX-LoxP gene sequence and GPD1 upstream and downstream sequence.In order to acquire knock-out component R1-R2,PCR amplification be used in this research with plasmid pUG6 as a PCR template.After R1-R2 integrated into S.cerevisiae Y01,gene mutant strain S.cerevisiae GY01 can be obtained.The experimental results showed,Glycerol production reached the maximum decrease of 18.25%when fermentation 48h in the S.cerevisiae GY01.Recombined strain GY01 has more higher ethanol production(14.23%)compared with the original strain.Based on 5' UTR antisense RNA silencing principle,antisense gene silencing expression vector pUPT-SGPD2 was built.pUPT-SGPD2 digested by restriction enzyme Kpn I,and then linearized pUPT-SGPD2 integrated into S.cerevisiae GY01.Recombined strain S.cerevisiae SGY01 will be obtained.In recombined strain S.cerevisiae SGY01,Glycerol production reached the maximum decrease of 4.18%compared with the S.cerevisiae GY01.And S.cerevisiae GY01 with 14.23%ethanol production higher than S.cerevisiae GY01.As mentioned above,LoxP-kanMX-LoxP gene sequence and Cre/LoxP disruption system is an effictive way for genetic modification of homologous recombination.Cre/LoxP system breaks through difficulties of genetic engineering in S.cerevisiae.And this research was also showed that antisense RNA complementary of the 5'UTR of GPD2 mRNA could inhibit the gene transcription of GPD2.