Study On The Copper-resistance Of Saccharomvces Cerevisiae

Saccharomyces cerevisiae, as an eukaryotic model organism, has extenxive applicationsin all kinds of biological research. However, some heterologous resistance genes andauxotroph are always utilized as the selective maker gene at present in the expression systemof S cerevisiae, which not only costs a lot but has a negative effect on cell. So the relativelycheap selective maker gene is desirable for researchers.Copper is a essential trace element in Scerevisiae, but this matal iron can be toxic to the cell when present in excess and the cup1gene and ccc2gene are pretty key in copper-resistance mechanisms. The copper-resistanceseletive maker gene is ideal beacause it is cheap, high-sensitive,and not harmful to the cell,but there is a few bit reports concerned about it.This study was aimed to construct a highsensitive expression system of copper-resistance S. cerevisiae, expressed heterologous proteinby it and pratly investigated the copper-resistance mechanisms.(1) To construct the high-sensitive copper-resistance S. cerevisiae, the transcription levelof copper-resistance gene cup1and ccc2was firstly investigated. The results revealed that asthe copper concentration rised, the transcription level of two genes presnted first increasedand then decreased trend. Both reached the highest in0.6mM Cu2+compared with thetranscription level in0mM Cu2+and both elavated3times,1.3times, respectively.Meanwhile, the transcription level of cup1gene was two times than its of ccc2gene at thesame copper stress concentrations. It was supposed that the increased transcription level ofcup1is more important to resist high concentrations of extracellular copper.(2) Sucessfully construct the high-sensitive copper-resistance S. cerevisiae. S. cerevisiaeW303-1A cup1and S. cerevisiae W303-1A ccc2were obtained by knocking out the cup1gene and ccc2gene using the PCR-mediated Technique. S. cerevisiae W303-1A cup1lowestinhibition concentration of Cu2+was declined from1.2mM to0.08mM, decreased by93%. S.cerevisiae W303-1A ccc2lowest inhibition concentration of Cu2+was declined from1.2mM to0.8mM, decreased by33.3%. S. cerevisiae W303-1A cup1is more sensitive tocopper iron than S. cerevisiae W303-1A ccc2. Also, It was supposed that cup1gene plays arelatively more important role in copper-resistance mechanisms and the role ofmetallothionein chelation of copper ion was more key than increase of copper iontransportation. S. cerevisiae W303-1A cup1, as the host in this research was obtained andthere is no big difference in terms of colony morphology and fermention performance.(3) Sucessfully construct a high sensitive expression system of copper-resistance S.cerevisiae. Using S. cerevisiae W303-1A cup1as host, pYX212as basic frame, cup1as theselective gene,0.3mM Cu2+SC as screening medium, an expression system pYX212M wasconstructed.The probability of positive transformers was80%above and plasmid stability ofpYX212M averaged88%in0.3mM Cu2+SC medium. gfp was successfully expressed by thisexpression system.The constructions of this paved the way for the applications of cheapselective makers.