|本期目录/Table of Contents|

[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]
点击复制

高灵敏度铜抗性酿酒酵母表达系统的构建与应用()
分享到：

《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.

参考文献/References:

1 Sherman Fred. Getting started with yeast [J]. Method Enzymol, 2002, 350: 3-41
2 Fan LP, Li AL. Huo GC. Development review on food grade selection marker in lactic acid bacteria [J]. Food Sci, 2006, 27 (9): 264-267
3 Nuria RI, Milan V. Advances in the structure and chemistry of metallothioneins [J]. J Inorg Biochem, 2002, 88 (3-4): 388-396
4 王昊, 林飞, 柴智丽, 周水英. 金属硫蛋白的应用研究进展[J]. 中国实用医刊, 2013, 8 (40): 109-111 [Wang H, Lin F, Chai ZL, Zhou SY. Research progresses of applied metallothioneins. Chin J Prac Med, 2013, 8 (40): 109-111]
5 张保林, 卢景雰, 王文清, 任宏伟, 茹炳根 金属硫蛋白抗自由基损伤研究[J]. 生物物理学报, 1992, 8 (3): 539-543 [Zhang BL, Wang WQ, Ren HW, Lu JF, Ru BG. Role of metallothionein protection against radiation-induced oxidative stress [J]. Acta Bioch Bioph Sin, 1992, 8 (3): 539-543]
6 苗兰兰, 张东杰, 王颖. 复合诱变高产金属硫蛋白酵母菌株的筛选[J]. 食品科学, 2013, 34 (19): 261-264 [Miao LL, Zhang DJ, Wang Y. Combined mutation screening of high-activity Saccharomyces cerevisiae strain capable of producing metallothioneins [J]. Food Sci, 2013, 34 (19): 261-264]
7 Freitas JD, Wintz H, Kim JH, Poynton H, Fox T. Yeast, a model organism for iron and copper metabolism studies [J]. Biometals, 2003, 16 (1): 185-197
8 Thomas JC, Davies EC, Malick FK, Endreszl C,Williams CR, Abbas M, Petrella S, Swisher K, Perron M, Edwards R, Ostenkowski P, Urbanczyk N, Wilesend WN, Murray KS. Yeast metallothionein in transgenic tobacco promotes copper uptake from contaminated soils [J]. Biotechnol Prog, 2003, 19 (2): 273-280
9 姜伯乐, 吴圣进, 何勇强. 酵母耐铜基因CUP1改良荧光假单胞菌耐铜性研究初报[J]. 中国农学通报, 2007, 23 (9): 96-99 [Jiang BL, Wu SJ, He YQ. Preliminary report on copper resistence of Pseudomonas fluorescens modified with yeast cup1 gene [J]. Chin Agric Sci Bull, 2007, 23 (9): 96-99]
10 郜瑞莹, 王建龙. 酿酒酵母生物吸附铜离子的动力学及吸附平衡研究[J]. 应用与环境生物学报, 2007, 13 (6): 848-852 [Gao RY, Wang JL. Kinetics and equilibrium of Cu2+ biosorption by dried biomass of Saccharomyces cerevisiae [J]. Chin J Appl Environ Biol, 2007, 13 (6): 848-852]
11 Welch J, Fogel S, Buchman C, Karin M. The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein [J]. EMBO J, 1989, 8 (1): 255-260
12 Koller A, Valesco J, Subramani M. The CUP1 promoter of Saccharomyces cerevisiae is inducible by copper in Pichia pastoris [J]. Yeast, 2000, 16: 651-656
13 Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington J. Crystal structure of the aequorea victoria green fluorescent protein [J]. Sci, 1996, 273 (5280): 1392-1395
14 范晓静, 邱思鑫, 吴小平, 洪永聪, 蔡学清, 胡方平. 绿色荧光蛋白基因标记内生枯草芽孢杆菌[J]. 应用与环境生物学报, 2007, 13 (4): 530-534 [Fan XJ, Qiu SX, Wu XP, Hong YZ, Cai XQ, Hu FP. Endophytic Bacillus subtilis strain BS- 2 labeled with green fluorescent protein gene [J]. Chin J Appl Environ Biol, 2007, 13 (4): 530-534]
15 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Short Protocols in Molecular Biology [M]. New York: Greene Pub. Associates and Wiley-Interscience, 2002
16 王萍, 厉荣玉, 董群. 白假丝酵母菌DNA提取方法的研究[J]. 热带医学杂志, 2008, 12 (8): 1222-1224 [Wang P, Li RY, Dong Q. A method of DNA extraction from Saccharomyces albicans [J]. J Trop Med, 2008, 12 (8): 1222-1224]
17 Longtine MS, Mckenzie A, Demarini DJ, Shan NG,.Wach A, Brachat A, Philippsen P, Pringle JR. Additional modules for versatile and economical PCR-based gene depletion and modification in Saccharomyces cerevisiae [J]. Yeast, 1998, 14: 953-961

相似文献/References:

[1]郜瑞莹,王建龙.酿酒酵母生物吸附Cu2+的动力学及吸附平衡研究[J].应用与环境生物学报,2007,13(06):848.
　GAO Ruiying,et al..Kinetics and Equilibrium of Cu2+ Biosorption by Dried Biomass of Saccharomyces cerevisia[J].Chinese Journal of Applied & Environmental Biology,2007,13(03):848.
[2]李岩,王海燕,张义正.甘薯蔗糖转运蛋白IbSUT1x在酵母细胞中的定位[J].应用与环境生物学报,2010,16(06):798.[doi:10.3724/SP.J.1145.2010.00798]
　LI Yan,WANG Haiyan,ZHANG Yizheng.Localization of IbSUT1x Protein from Ipomoea batatas (L.) Lam in Yeast Cells[J].Chinese Journal of Applied & Environmental Biology,2010,16(03):798.[doi:10.3724/SP.J.1145.2010.00798]
[3]李宇浩,靳艳玲,龙飞,等.降粘酶在新鲜木薯发酵生产高浓度乙醇中的应用[J].应用与环境生物学报,2013,19(03):501.[doi:10.3724/SP.J.1145.2013.00501]
　LI Yuhao,JIN Yanling,LONG Fei,et al.Using Viscosity Reducing Enzyme as Annexing Agent in the Very High Gravity Ethanol Fermentation with Fresh Cassava[J].Chinese Journal of Applied & Environmental Biology,2013,19(03):501.[doi:10.3724/SP.J.1145.2013.00501]
[4]柯崇榕,吴毕莎,邵庆伟,等.酿酒酵母PDC1基因过表达菌株的构建[J].应用与环境生物学报,2013,19(04):704.[doi:10.3724/SP.J.1145.2013.00704]
　KE Chongrong,WU Bisha,SHAO Qingwei,et al.Construction of Saccharomyces cescerevisiae Mutant with Overexpression of PDC1 Gene[J].Chinese Journal of Applied & Environmental Biology,2013,19(03):704.[doi:10.3724/SP.J.1145.2013.00704]
[5]周利,汤岳琴,孙照勇,等.基于连续发酵驯化的高耐盐性酿酒酵母的育种[J].应用与环境生物学报,2014,20(03):360.[doi:10.3724/SP.J.1145.2014.11032]
　ZHOU Li,TANG Yueqin,SUN Zhaoyong,et al.Breeding of high salt-tolerant Saccharomyces cerevisiae strains based on continuous ethanol fermentation[J].Chinese Journal of Applied & Environmental Biology,2014,20(03):360.[doi:10.3724/SP.J.1145.2014.11032]
[6]张明明 万青青 张克俞 熊 亮 白凤武,赵心清**.过表达分支酸歧化酶编码基因ARO7对酿酒酵母抑制物耐受性的影响*[J].应用与环境生物学报,2016,22(02):201.[doi:10.3724/SP.J.1145.2015.09013]
　ZHANG Mingming,WAN Qingqing,ZHANG Keyu,et al.Effect of overexpression of chorismate mutase encoding gene ARO7 on theinhibitor tolerance of Saccharomyces cerevisiae[J].Chinese Journal of Applied & Environmental Biology,2016,22(03):201.[doi:10.3724/SP.J.1145.2015.09013]
[7]谷鸿宇,诸葛斌,方慧英,等.渗透压调控启动子表达木糖醇脱氢酶基因XYL2对重组酿酒酵母木糖代谢的影响[J].应用与环境生物学报,2016,22(06):1122.[doi:10.3724/SP.J.1145.2016.01035]
　GU Hongyu,ZHUGE Bin,et al.Effect of controlled overexpression of XYL2 coding for xyltiol dehydrogenase by osmo-responsive promoters on xylose metabolism in Saccharomyces cerevisiae[J].Chinese Journal of Applied & Environmental Biology,2016,22(03):1122.[doi:10.3724/SP.J.1145.2016.01035]
[8]张影,苟敏,孙照勇,等.混合糖发酵条件下甲酸抑制木糖发酵的机制[J].应用与环境生物学报,2017,23(06):990.[doi:10.3724/SP.J.1145.2017.02011]
　ZHANG Ying,GOU Min,SUN Zhaoyong,et al.The inhibitory mechanism of action of formic acid on xylose fermentation during mixed sugar fermentation[J].Chinese Journal of Applied & Environmental Biology,2017,23(03):990.[doi:10.3724/SP.J.1145.2017.02011]
[9]缪晡,苟敏,陈栋,等.不同糖发酵条件下酿酒酵母组成型启动子和诱导型启动子评价[J].应用与环境生物学报,2019,25(05):1185.[doi:10.19675/j.cnki.1006-687x.2019.07015]
　MIAO Pu,GOU Min,CHEN Dong & TANG Yueqin,et al.Genome-wide evaluation of constitutive and inducible promotors in Saccharomyces cerevisiae utilizing different carbon sources[J].Chinese Journal of Applied & Environmental Biology,2019,25(03):1185.[doi:10.19675/j.cnki.1006-687x.2019.07015]
[10]代勇华 刘盼婷 宋超 胡国全** 何明雄**.酿酒酵母在糖质燃料乙醇中的发酵工艺优化[J].应用与环境生物学报,2022,28(03):1.[doi:10.19675/j.cnki.1006-687x.2021.02051]
　DAI Yonghua,LIU Panting,et al.Optimization of fermentation process for saccharide fuel-ethanol production by Saccharomyces cerevisiae[J].Chinese Journal of Applied & Environmental Biology,2022,28(03):1.[doi:10.19675/j.cnki.1006-687x.2021.02051]

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