RNR3启动子调控的红绿荧光蛋白双信号发光酵母细胞对化学诱变原的筛选

doi:10.3969/j.issn.1004-616x.2019.04.003

癌变·畸变·突变 ›› 2019, Vol. 31 ›› Issue (4): 276-282.doi: 10.3969/j.issn.1004-616x.2019.04.003

RNR3启动子调控的红绿荧光蛋白双信号发光酵母细胞对化学诱变原的筛选

姚佳^1,2, 刘星¹, 卢光玉¹, 朱方渝¹, 吴倩倩³, 李湘鸣¹

1. 扬州大学医学院, 江苏扬州 225001;
2. 扬州市卫生监督所, 江苏扬州 225002;
3. 苏州市相城人民医院, 江苏苏州 215100

收稿日期:2019-02-17 修回日期:2019-06-18 出版日期:2019-07-30 发布日期:2019-08-23
通讯作者: 李湘鸣,E-mail:847264344@qq.com E-mail:847264344@qq.com
作者简介:姚佳,E-mail:ajia0922@163.com。
基金资助:
国家自然科学基金资助（81241099）

Screening of chemical mutagens based on RNR3 romoter-regulated red-green fluorescent protein dual signals in yeast cells

YAO Jia^1,2, LIU Xing¹, LU Guangyu¹, ZHU Fangyu¹, WU Qianqian³, LI Xiangming¹

1. Medical College of Yangzhou University, Yangzhou 225001;
2. Yangzhou Health Supervision Institute, Yangzhou 225002;
3. Xiangcheng People's Hospital, Suzhou 215100, Jiangsu, China

Received:2019-02-17 Revised:2019-06-18 Online:2019-07-30 Published:2019-08-23

摘要/Abstract

摘要： 目的：构建RNR3启动子调控的红绿荧光蛋白双信号发光酵母细胞，用于快速筛选化学诱变原。方法：用PCR方法YIPlac204TC-NLS-DsRed-Express-2扩增DsRed-Express-2红色荧光蛋白，将其插入到pGPD载体，构建成GPD启动子驱动的红色荧光蛋白酵母报告载体（pGPD-DsRed-Express2），用醋酸锂方法将其转化于RNR3启动子调控的绿色荧光蛋白（yEGFP）发光酵母细胞（W303-1A/RNR3-yEGFP），从而构建成红、绿荧光蛋白双信发光酵母细胞。红色荧光蛋白（DsRed-Express-2）信号可用于校正细胞数和状态不同对yEGFP发光的影响。用不同诱变原分别作用于该发光酵母细胞，于第0、4、8、12、16和20小时，用黑色96孔酶标板分别测红、绿荧光蛋白的发光强度，用相对荧光强度（yEGFP/DsRed-Express2）描述诱变原的剂量效应关系。结果：在与DNA发生结合的化合物中，放线菌素D和溴乙锭均呈阴性；在与DNA发生烷基化的化合物中，甲磺酸甲脂（MMS）和瘤可宁呈阳性结果，而丝裂霉素C呈阴性结果；在使DNA发生断裂的诱变原中，顺铂、博来霉素和福来霉素呈阴性，而4-硝基-N-氧化喹啉（4-NQO）呈阳性结果；在抑制DNA合成酶或拓扑异构酶的诱变原中，5-氟尿嘧啶呈阳性结果，而羟基脲、喜树碱和阿非迪霉素均呈阴性结果；非基因毒性化合物秋水仙碱、刀豆氨酸和四环素均呈阴性。结论：该重组发光酵母细胞（W303-1A/yEGFP/DsRed-Express2）可作为传统致突变评价方法的补充，具有快速、方便和高通量特点。

关键词: 酵母细胞, 诱变原, 绿色荧光蛋白, 红色荧光蛋白

Abstract: OBJECTIVE:To construct red-green fluorescent protein dual signals which are regulated by RNR3 promoter in yeast cells for rapid screening of chemical mutagens. METHODS:DsRed-Express-2 red fluorescent protein was amplified by PCR from YIPlac204TC-NLS-DsRed-Express-2 and inserted into pGPD vector to construct a yeast red fluorescent protein reporter vector (pGPD-DsRed-Express2),which was driven by the GPD promoter. The method of lithium acetate was used to transform the vector into green fluorescent protein (yEGFP) cells (W303-1A/RNR3-yEGFP),which was regulated by the RNR3 promoter. Thus,red and green fluorescent protein dual signal fluorescing yeast cells were constructed. Red fluorescent protein (DsRed-Express-2) signal was used to normalize the effect of different cell numbers and the "states" on the fluorescence of yEGFP. Fluorescent intensity of red and green fluorescent proteins was measured in 96-well black microplate at 0,4,8,12,16 and 20 hours after the cells were treated with different mutagens. The relative fluorescence intensity (yEGFP/DsRed-Express2) was used to describe the dose-effect relationship between the mutagens and the cells. RESULTS:Actinomycin D and ethidium bromide exhibited negative results among DNA intercalating compounds;methyl mesylate (MMS) and chlorambucil were positive among DNA-alkylating compounds,while mitomycin C was negative;cisplatin,bleomycin and fleomycin were negative among DNA cleaving agents,while 4-nitro-N-oxyquinoline (4-NQO) was negative. Among the inhibitors of polymerases or topoisomerases,5-fluorouracil appeared positive,while hydroxyurea,camptothecin and aphidicolin were negative. As for non-genotoxic compounds such as colchicine,concanavaline and tetracycline,all of them exhibited negative results. CONCLUSION:The recombinant dual-signal fluorescent yeast cells (W303-1A/yEGFP/DsRed-Express2) can possibly be used as a complement to the traditional mutagenic assays,and have the characteristics of fast,convenience and high throughput.

Key words: yeast cells, mutagens, green fluorescent protein, red fluorescent protein

中图分类号:

R114

姚佳, 刘星, 卢光玉, 朱方渝, 吴倩倩, 李湘鸣. RNR3启动子调控的红绿荧光蛋白双信号发光酵母细胞对化学诱变原的筛选[J]. 癌变·畸变·突变, 2019, 31(4): 276-282.

YAO Jia, LIU Xing, LU Guangyu, ZHU Fangyu, WU Qianqian, LI Xiangming. Screening of chemical mutagens based on RNR3 romoter-regulated red-green fluorescent protein dual signals in yeast cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2019, 31(4): 276-282.

参考文献

[1] 王磊, 罗方妮, 谢云斌, 等. RNR3调控的重组绿色荧光蛋白酵母细胞对化学诱变原的高通量筛选[J]. 毒理学杂志, 2013, 27(4):252-257.
[2] GRIFFITH K L, WOLF R E Jr. Measuring beta-galactosidase activity in Bacteria:cell growth, permeabilization, and enzyme assays in 96-well arrays[J]. Biochem Biophys Res Commun, 2002, 290(1):397-402.
[3] LU Y X, TIAN Y J, WANG R K, et al. Dual fluorescent protein-based bioassay system for the detection of genotoxic chemical substances in Saccharomyces cerevisiae[J]. Toxicol Mech Methods, 2015, 25(9):698-707.
[4] 刘波, 金建玲, 张辉, 等. Ames测试不确定性分析[J]. 应用与环境生物学报, 2007, 13(5):726-730.
[5] 潘峰, 王万峰, 时蕾. Umu测试法及其在环境科学中的应用[J]. 安徽农业科学, 2007, 35(8):2208-2210, 2267.
[6] 李文学, 陈丽萍, 肖勇梅, 等. 代谢活化系统在人细胞转化模型中的应用[J]. 中山大学学报:医学科学版, 2009, 30(2):224-227, 240.
[7] 杨淋清, 纪卫东, 马儒林, 等. 慢病毒介导的RNA干扰技术建立hPARP-1缺陷细胞株[J]. 中华预防医学杂志, 2008, 42(6):461-463.
[8] 马儒林, 庞雅琴, 李文学, 等. 癌基因高表达人上皮细胞转化模型的构建及应用[J]. 中华预防医学杂志, 2008, 42(6):395-399.
[9] 庞雅琴, 陈雯. 体外细胞转化试验研究进展[J]. 癌变·畸变·突变, 2007, 19(6):511-514.
[10] 马礼金, 黄自然. 外源基因在酵母中表达[J]. 华南农业大学学报, 1997, 18(1):114-119.
[11] 洪洄, 王冬梅. 应用酵母进行基因表达的研究进展[J]. 生物学通报, 1999, 34(7):12-14.
[12] DIAO Z Y, YE T M, CAO P, et al. Expression, purification, and characterization of recombinant human soluble BAFF secreted from the yeast Pichia pastoris[J]. Protein Expr Purif, 2007, 54(1):11-17.
[13] FOSS E J. Tof1p regulates DNA damage responses during S phase in Saccharomyces cerevisiae[J]. Genetics, 2001, 157(2):567-577.
[14] YAO R J, ZHANG Z, AN X X, et al. Subcellular localization of yeast ribonucleotide reductase regulated by the DNA replication and damage checkpoint pathways[J]. Proc Natl Acad Sci USA, 2003, 100(11):6628-6633.
[15] LIU Y, DAI H, XIAO W. UAS(MAG1), a yeast Cis-acting element that regulates the expression of MAG1, is located within the protein coding region of DDI1[J]. Mol Gen Genet, 1997, 255(5):533-542.
[16] MERCIER G, DENIS Y, MARC P, et al. Transcriptional induction of repair genes during slowing of replication in irradiated Saccharomyces cerevisiae[J]. Mutat Res, 2001, 487(3/4):157-172.
[17] PRAKASH S, PRAKASH L. Nucleotide excision repair in yeast[J]. Mutat Res, 2000, 451(1/2):13-24.
[18] DUNAWAY S, WALWORTH N C. Assaying the DNA damage checkpoint in fission yeast[J]. Methods, 2004, 33(3):260-263.
[19] DOMKIN V, THELANDER L, CHABES A. Yeast DNA damage-inducible Rnr3 has a very low catalytic activity strongly stimulated after the formation of a cross-talking Rnr1/Rnr3 complex[J]. J Biol Chem, 2002, 277(21):18574-18578.
[20] YAGLE K, MCENTEE K. The DNA damage-inducible gene DIN1 of Saccharomyces cerevisiae encodes a regulatory subunit of ribonucleotide reductase and is identical to RNR3[J]. Mol Cell Biol, 1990, 10(10):5553-5557.
[21] 陈绘丽, 杨频. 错配核酸的研究进展[J]. 化学进展, 2002, 14(2):133-140.
[22] 李元杰, 徐方. 哺乳类细胞DNA复制后修复的研究[J]. 宁夏医学院学报, 2007, 29(1):100-102.
[23] ZHU Y, XIAO W. Differential regulation of two closely clustered yeast genes, MAG1 and DDI1, by cell-cycle checkpoints[J]. Nucleic Acids Res, 1998, 26(23):5402-5408.
[24] MCNABB D S, REED R, MARCINIAK R A. Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae[J]. Eukaryotic Cell, 2005, 4(9):1539-1549.
[25] LIU X M, KRAMER J A, SWAFFIELD J C, et al. Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins[J]. Mutat Res, 2008, 653(1/2):63-69.
[26] AFANASSIEV V, SEFTON M, ANANTACHAIYONG T, et al. Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances[J]. Mutat Res, 2000, 464(2):297-308.
[27] IARC/NCI/EPA Working Group. Cellular and molecular mechanisms of cell transformation and standardization of transformation assays of established cell lines for the prediction of carcinogenic chemicals:overview and recommended protocols[J]. Cancer Res, 1985, 45(5):2395-2399.

RNR3启动子调控的红绿荧光蛋白双信号发光酵母细胞对化学诱变原的筛选

Screening of chemical mutagens based on RNR3 romoter-regulated red-green fluorescent protein dual signals in yeast cells

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 8

编辑推荐

Metrics

本文评价

[1]	杨少哲, 石海军, 褚欣然, 周小玲, 孙平楠. 一种简便的高通量细胞内GFP报告基因检测方法[J]. 癌变·畸变·突变, 2015, 27(5): 394-398,403.
[2]	王瑞鲲，葛宜枝，陆益新，吴倩倩，李湘鸣*. 筛选化学诱变原的RNR3调控重组Lac Z 基因酵母细胞的建立[J]. 癌变·畸变·突变, 2014, 26(3): 213-217.
[3]	金彩霞1;2;李文林1;李元杰2;徐方2;胡以平1. 胰岛素启动子在肝干细胞分化研究中的应用[J]. , 2010, 22(3): 171-174.
[4]	李　蕊/宋旭红/刘戈飞/梁　斌/谢健平/杜　昆/张巧霞/黄东阳. 人神经母细胞瘤NRDR新剪接亚型A2的亚细胞定位[J]. , 2007, 19(3): 215-219.
[5]	朱　吉；李文林；金彩霞；李建秀；胡以平. 绿色荧光蛋白多克隆抗体的制备[J]. , 2007, 19(1): 76-078.
[6]	金彩霞1,2/李文林1/朱　吉1/张　南1/田　棣1/胡以平1,. 胰十二指肠同源盒基因-1启动子在肝干细胞分化研究中的应用[J]. , 2006, 18(5): 337-339.
[7]	杨兴坤;黄天华;谢庆东;熊小芳;吴丛梅. 脂质体介导的精原细胞基因转移[J]. , 2006, 18(4): 277-280.
[8]	何志颖,江千里, 姚玉成,温丽敏,李建秀, 王新民, 胡以平,王健民. RFP标记的YCD基因在HeLa细胞中的表达及其介导的细胞毒性[J]. , 2004, 16(5): 272-275.