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[1]盛冠一,诸葛斌,宗红,等.高灵敏度铜抗性酿酒酵母表达系统的构建与应用[J].应用与环境生物学报,2014,20(03):357-362.[doi:10.3724/SP.J.1145.2014.12033]
　SHENG Guanyi,ZHUGE Bin,ZONG Hong,et al.Construction and application of the high sensitivity expression system of copper-resistant Saccharomyces cerevisiae[J].Chinese Journal of Applied & Environmental Biology,2014,20(03):357-362.[doi:10.3724/SP.J.1145.2014.12033]
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高灵敏度铜抗性酿酒酵母表达系统的构建与应用()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
20卷
期数:
2014年03期
页码:
357-362
栏目:
研究论文
出版日期:
2014-06-25

文章信息/Info

Title:
Construction and application of the high sensitivity expression system of copper-resistant Saccharomyces cerevisiae
作者:
盛冠一; 诸葛斌; 宗红; 陆信曜; 方慧英; 宋健; 诸葛健
1江南大学工业生物技术教育部重点实验室 无锡 214122 2江南大学生物工程学院工业微生物研究中心 无锡 214122 3江南大学化学与材料工程学院 无锡 214122
Author(s):
SHENG Guanyi;  ZHUGE Bin;  ZONG Hong;  LU Xinyao;  FANG Huiying;  SONG Jian;  ZHUGE Jian
1Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China 2School of Biotechnology, Research Centre of Industrial Microbiology, Jiangnan University, Wuxi 214122, China 3School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China
关键词:
酿酒酵母; PCR-mediated Technique; cup1; 铜抗性; 绿色荧光蛋白
Keywords:
Saccharomyces cerevisiae;  PCR-mediated Technique;  cup1;  copper-resistance;  gfp
分类号:
Q939.97 : Q786
DOI:
10.3724/SP.J.1145.2014.12033
文献标志码:
A
摘要:
利用PCR-mediated Technique对酿酒酵母W303-1A金属硫蛋白基因（cup1）进行敲除，获得一株铜离子敏感型酿酒酵母（Saccharomyces cerevisiae W303-1A cup1D），其对Cu2+抑制浓度由1.2 mmol/L降为0.08 mmol/L；以pYX212载体为基本构架，以S. cerevisiae W303-1A cup1D为宿主菌，构建了以cup1作为筛选标记的pYX212M表达系统，并成功表达了绿色荧光蛋白基因（gfp），从而实现了高灵敏度铜抗性酿酒酵母表达系统的构建与应用. 本研究构建的高灵敏度铜抗性酿酒酵母表达系统不仅廉价、安全，而且丰富了酵母的表达系统，同时对微生物的铜抗性机理及生物修复功能的研究具有指导意义.
Abstract:
This study was aimed to construct a high sensitivity expression system of copper-resistant Saccharomyces cerevisiae. With the PCR-mediated technique which allows single-step deletion of chromosomal gene to knock out the cup1 gene of S. cerevisiae W303-1A, the lowest inhibition concentration of Cu2+ was determined by gradient dilution. The construction of the expression system pYX212M used pYX212 as the basic frame, cup1 as the selective gene and the S. cerevisiae W303-1A cup1D as the host. The pYX212MGFP was constructed, transforming S. cerevisiae W303-1A cup1D to observe whether the strain displayed green fluorescence. A copper-sensitive S. cerevisiae W303-1A cup1D was obtained and its lowest inhibition concentration of Cu2+ was 1.2 mmol/L compared with 0.08 mmol/L of S. cerevisiae W303-1A. The expression system pYX212M was achieved. The green fluorescence was seen under the microscope by transforming pYX212MGFP into the S. cerevisiae W303-1A cup1D, demonstrating the successful expression of gfp. The results suggested that the high sensitivity expression system of copper-resistant S. cerevisiae can be constructed. It not only enriches the expression system of S. cerevisiae, but also provides guidance in the research of the copper resistance mechanisms and the bioremediation function of the microorganism.

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备注/Memo

备注/Memo:
国家高技术研究发展计划（“863”计划）项目（2012AA021201，2011AA02A207）资助
更新日期/Last Update: 2014-07-01