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

The high throughput screening of mutagens based on the recombinant yEGFP yeast cells regulated by RNR3

目的用RNR3调控的全发光酵母细胞高通量筛选化学诱变原。方法以含有酵母嗜好遗传密码子的绿色荧光蛋白(yEGFP)报告载体为基本框架,用PCR方法从酵母(W303-1A)基因组扩增RNR3启动子,将其插入到yEGFP的上游,从而构建成RNR3调控的yEGFP酵母报告载体。用醋酸锂方法将载体转化于酵母细胞,构建成RNR3调控的yEGFP发光酵母细胞,可在96孔板中对不同浓度的化学诱变原进行筛选,具有快速、方便和高通量特点。结果对数种不同的DNA损伤药物研究结果表明,DNA烷化剂(甲磺酸甲脂)、DNA断裂剂(顺铂)、DNA结合剂(放线菌素D)和DNA合成酶抑制剂(5-氟尿嘧啶和羟基脲)可诱导RNR3的表达,而苯丁酸氮芥和4-硝基-N-氧化喹啉由于细胞的毒性的作用,未使RNR3发生表达。结论某些与致癌性有关的DNA损伤化学物能诱导RNR3表达,故该发光酵母细胞可用于对某些致癌物的高通量筛选。

Objective To use the whole yeast fluorescent cell regulated by RNR3 promoter for high-throughput screening of chemical mutagens. Methods Based on the basic frame of the reporter vector carrying the gene encoding a green fluorescent protein （yEGFP） optimised for yeast, the RNR3 promoter amplified by PCR from W303-1A yeast genosome was inserted into the yEGFP upstream of this vector to yield the yEGFP yeast reporter vector under the control of the RNR3 promoter. This vector was transformed into the yeast cell by the lithium acetate method to construct the fluorescent yeast strain regulated by RNR3 promoter. This yeast cell could be used to screen the activities of chemical mutagens. The assay was performed on 96-well microplates in the presence of different concentrations of the chemical mutagens, which had the characteristics of rapid, convenience and highthroughpnt. Results The results showed that various drugs that caused DNA damage effectively induced RNR3 expression, such as alkylating agents （methyl methanesulfonate ）, cleavage of DNA （cisplatin）, interealator （aetinomycin D） and inhibitors of polymerases （5-Fluorouracil and hydroxyurea） , while chlorambucil and 4- nitroquinoline-N-oxide did not lead to the expression of RNR3 because of their toxicity to the yeast cell. Conclusion Some of DNA damage chemicals related to carcinogenesis induce RNR3, and therefore this fluorescent yeast can be used to highthroughput screening for some of chemical carcinogen.