GFP遗传改造四膜虫细胞的构建及其对重金属污染的荧光响应

摘要

为了获得可用于检测环境中多种重金属污染的遗传改造生物细胞,将具有重金属诱导特性的金属硫蛋白基因MTT1启动子和外源报告基因绿色荧光蛋白gfp的融合序列,通过基因枪法转染嗜热四膜虫细胞.经同源重组和巴龙霉素抗性筛选,获得稳定的GFP遗传改造四膜虫细胞株.该细胞株经Cd2+诱导,MTT1启动子可启动外源基因gfp正确表达,在蓝光激发下可发绿色荧光.GFP遗传改造四膜虫细胞对重金属离子的荧光响应效应由大到小依次为Cd2+,Cr6+,Hg2+;响应浓度范围和具有剂量-效应关系的浓度范围由大到小依次为Cd2+,Hg2+,Cr6+;最低检测限由大到小依次为Cr6+,Hg2+,Cd2+,而对Cu2+无荧光响应效应.因此,GFP遗传改造四膜虫可作为环境中Cd2+、Hg2+和Cr6+污染的生物检测细胞.%To obtain genetically modified cells which can be used to detect a variety of environ-mental heavy metals pollution,the fusion gene containing heavy metals induced metallothionein gene MTT1 promoter and exogenous gfp reporter gene was transfected into Tetrahymena ther-mophilia by biolistic bombardment.By homologous recombination under increasing paromomy-cin,the genetically modified Tetrahymena with GFP was constructed.With treatment of Cd2 + , the MTT1 promoter can initiate the correct expression of exogenous gfp gene of the genetically modified Tetrahymena in which green fluorescence can be induced by blue light excitation.The fluorescence response of the genetically modified Tetrahymena with GFP to heavy metals was in order of Cd2 + ,Cr6 + and Hg2 + .And the concentration ranges within which fluorescence was in-duced and with dose-response relationship were both in order of Cd2 + ,Hg2 + and Cr6 + .The de-tection limit of heavy metals was in order of Cr6 + ,Hg2 + and Cd2 + .However,the genetically modified Tetrahymena with GFP did not respond to copper.The results showed that the geneti-cally modified Tetrahymena with GFP can be used for detecting Cd2 + ,Hg2 + and Cr6 + pollution in environment.