酵母绿色荧光蛋白报告载体的构建及其在过氧化物酶体研究中的应用

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

摘要:

以载体pYES2为基础,构建了酵母表达载体pYES2G,该载体含有融合了过氧化物酶体定位信号1(PTS1)的绿色荧光蛋白报告分子GFP-SKL编码基因,该基因以酵母TEF1启动子启动。pYES2转化研究表明,在野生型酵母INVScl中,GFP-SKL蛋白在细胞中呈点状聚集,而在酵母PEX5p缺陷菌株ATCC4003603中,荧光为弥散状,证明报告分子GFP-SKL可通过PEX5p蛋白有效定位到过氧化物酶体。在载体pYES2G的多克隆位点分别连入酵母及产黄青霉PEX5p编码基因得到载体pYES2G/ScPEX5和pYES2G/PcPEX5,转化酵母ATCC4003603,荧光均呈聚集状,证明外源PEX5p基因的表达恢复了缺陷菌株的功能。pYES2G载体为真菌过氧化物酶体相关基因的功能研究提供了直观有效的方法。

Abstract:

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 β-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.