GFP工程酵母菌的构建及其对Cu2+的荧光响应

从酿酒酵母基因组DNA中克隆到金属硫蛋白启动子(PCUP1)片段,将绿色荧光蛋白(GFP)基因置于PCUP1的调控下,构建重组质粒pCUP9K-GFP,并通过氯化锂法转化毕赤酵母,获得工程菌株。工程菌细胞及其发酵液中可检出GFP荧光,表明PCUP1能启动外源基因GFP转录,使工程菌表达并分泌GFP。研究发现,工程菌培养液中分别加入10μmol/L的铜、铬、镉和砷离子后,铜处理组GFP荧光强度明显增加,其余三种离子对工程菌荧光强度影响不大;用铜离子诱导后,工程菌发酵上清液的荧光强度明显增强,并与铜离子浓度(0～1mmol/L)呈正相关。研究表明,该工程菌中启动子PCUP1受铜离子诱导,GFP的表达对铜离子具有剂量依赖性,在一定浓度范围内,GFP荧光强度与铜离子浓度呈正相关。

Construction of an Engineered Yeast with Green Fluorescent Protein Gene and Its Fluorescence in Response to Copper Ion

The encoding region of green fluorescent protein(GFP) gene and the promoter region of S.cerevisiae metallothionein(CUP1) gene were amplified to construct a recombinant expression vector pCUP9K-GFP.By using lithium chloride transformation method,the recombinant expression vector was transformed into P.pastoris GS115 cells to create a yeast strain.Green fluorescent protein can be detected in engineered yeast cells and in their fermentation supernatant,indicating that engineered yeast cells could express GFP normally and then secrete GFP to the extracellular medium.The relative fluorescent intensity of the culture supernatant increased in a concentration-dependent manner after engineered yeast exposed to copper ions in a concentration ranging from 5μmol/L to 1mmol/L.However,the fluorescence of engineered yeast did not respond to chromium,cadmium and arsenic ions.This results suggest that CUP1 promoter can be induced specifically by copper ions,which may be used for the monitoring of environmental copper contamination.