转MT工程菌的构建及其对重金属的抗性研究

利用重叠延伸PCR技术克隆金属硫蛋白(MT)和绿色荧光蛋白(GFP)基因片段,并将两基因融合连接构建重组表达载体,采用氯化锂法转化毕赤酵母,获得工程菌株.荧光显微镜观察发现,工程菌在蓝光激发下发出绿色荧光,说明GFP基因被正确表达.在培养基中加入一定浓度的铜(1.0 mmol/L,1.5 mmol/L)、铬(150 μmol/L,200 μmol/L)、镉(120 μmol/L,140 μmol/L)、砷(40 μmol/L,60 μmol/L)化合物后,对照菌生长抑制,转基因菌株长势明显好于对照菌,表现出对金属离子的耐受性,说明工程菌过表达MT能够增强宿主对重金属离子的耐受性,提高菌株耐污能力,在微生物法净化重金属废水中具有一定优势.

Engineered Yeast Cells Displaying Metallothionein Enhance Self Tolerance to Environmental Heavy Metals

The metallothionein(MT) gene and green fluorescent protein(GFP) gene were cloned,modified and fused by gene splicing by overlap extension PCR technique,and then one recombinant expression vector was constructed and transformed into P.pastoris.The engineered yeast cells could emit the green fluorescence under a fluorescence microscope,indicating the correct expression of MT-GFP fusion gene in yeast cells.On YPD medium containing respectively copper(1.0 mmol/L,1.5 mmol/L),cadmium(120 μmol/L,140 μmol/L),chromium(150 μmol/L,200 μmol/L) and arsenic(40 μmol/L,60 μmol/L) ions,the engineered yeast strain cells could grow and divide to form some colonies,but the control strains without exogenous MT gene can not grow normally to form a clear colony,especially after exposed to higher concentrations of the metals.The results showed that transgenic yeast cells expressed the metallothionein gene increased the self tolerance to environmental heavy metals,which might be a useful material for wastewater treatment.