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

Abstract

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

CLC Number:

R114

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.

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

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