[Construction of yeast expression vector containing GFP-SKL reporter gene and its function in study of peroxisome]

Peroxisomes are important subcellular organelles that are present in almost all eukaryotic cells. They are involved in a variety of metabolic functions include fatty acid beta-oxidation, H2O2-based respiration and so on. The last step of penicillin biosynthetic is also located in peroxisome in Penicillium chrysogenum. Peroxisome biogenesis has been well elucidated in Saccharomyces cerevisiae and a lot of yeast peroxisome-deficient strains were available to validate the functions of peroxisome genes from other organisms. On the base of vector pYES2, the yeast expression vector pYES2G was constructed, which containing GFP-SKL reporter gene that fused the peroxisomal targeting signal 1 (PTS1) and used TEF1 as a promotor. Cells of INVScl transformed with vector pYES2G displayed a punctate fluorescence pattern; while transformants of ATCC4003603 (a pex5-deficient yeast strain) with pYES2G showed a diffuse fluorescence pattern, which indicated that GFP-SKL can be localized in peroxisome effectively by PEX5p. Furthemore, the plasmids of pYES2G/ScPEX5 and pYES2G/PcPEX5 were created by cloning PEX5p encoding genes of S. cerevisiae and P. chrysogenum into the multiple cloning site of pYES2G, and then transformed into the yeast strain ATCC4003603, respectively. Both transformants showed punctate fluorescence patterns, which suggested ATCC4003603 was complemented by the foreign ScPEX5p and PcPEX5p. The plasmid pYES2G provides a visible and effective method for studying the functions of fungal peroxisome related genes.