Preliminary Study On Transport Of T7 Tanscripts In Saccharomyces Cerevisiae Using HIV-REV-RRE Elements

The T7 transcription system consists of T7 RNA polymerase(T7 RNAP)and T7 promoter,and it is one of the most widely used systems for the efficient expression of proteins in prokaryotes.However,the applications of this system in eukaryotes are quite limited,particularly in Saccharomyces cerevisiae,which is an important industrial microorganism and model system.T7 transcripts without a 5' cap structure cannot always be translated into corresponding proteins in Saccharomyces cerevisiae based on all previous researches.In this study,Rev-RRE,an important regulatory element of HIV-1 retrovirus,was introduced into Saccharomyces cerevisiae.Based on the demonstration of its application in Saccharomyces cerevisiae,efficient genetic elements and operating tools were used to construct and optimize T7 transcription system.Finally,protein expression with enhanced green fluorescent protein(EGFP)as a reporter gene was achieved.Although only a part of the Saccharomyces cerevisiae cells successfully expressed EGFP,it was shown that the Rev-RRE element played a certain role in promoting the trans-nuclear membrane transport of T7 transcription products.The experimental content of this topic includes the following three parts:1.BY4741(Ty4::PCYC1-NLS-T7RNAP-TCYC1)expressing T7 RNAP was successfully constructed,and the construction of BY4741(Ty4::PCYC1-NLS-T7RNAP-TCYC1,pUG-PT7-EGFP-TT7)was completed by transferring self-replicating plasmid carrying T7 promoter and EGFP as reporter gene.Fluorescence microscopy revealed that the latter yeast cells do not have green fluorescence,but the cells were transcribed with a large amount of EGFP mRNA by RT-PCR.Compared with the strain BY4741(pUG-PMet-EGFP-TCYC1),the EGFP mRNA content in the BY4741(Ty4::PCYC1-NLS-T7RNAP-TCYC1,pUG-PT7-EGFP-TT7)wasapproximately 14 times higher than that of BY4741(pUG-PMet-EGFP-TCYC1),which indicated that T7 RNAP exhibited strong transcriptional capabilities in Saccharomyces cerevisiae.2.The recombinant plasmids pESC-Pgal-Rev-EGFP-TCYC1 and pESC-Pgal-NLS-Rev-EGFP-TCYC1 were constructed and transformed into Saccharomyces cerevisiae INVSC1.Confocal microscopy showed Rev protein have a nuclear localization function.Then the yeast strain INVSCI(HO::Pgal-Rev-T ADH1)expressing the Rev protein wassuccessfully constructed and plasmids with EGFP or Fmi as reporter gene were transferred into this strain respectively.According to the results of average fluorescence intensity and the activity of Fmi,a conclusion was reached that Rev protein could bind to the RRE element in Saccharomyces cerevisiae and compete with original spliceosome in Saccharomyces cerevisiae.Finally,the regulation rate of Rev-RRE element on EGFP protein expression fluctuated around 0.55 by changing the galactose concentration in the medium,which showed that the Rev protein molecule bound to RNA with RRE sequence and reached saturation.3.The construction of Saccharomyces cerevisiae strain BY4741(Ty4::PCYC1-NLS-T7RNAP-TCYC1;HO::Pgal-Rev-TADH1)integrating the Rev and T7 RNAP protein genes on the genome was performed by using the knockout plasmid pSH47.Then T7 expression plasmids with the RRE,pUG-PT7-EGFP-RRE-TT7 and pESC-T7RNAP-PT7-Rev-EGFP-RRE-TT7 were transformed into Saccharomyces cerevisiae,respectively.The HIV-1 Rev-RRE element was successfully applied to the transnuclear membrane transport of T7 transcription products.Confocal microscopy showed that some Saccharomyces cerevisiae cells could see green fluorescence,indicating that the HIV-1 Rev-RRE element played a role in the transport of T7 transcription products in Saccharomyces cerevisiae.