陈美玲,刘琳,杨志兵,武佳韵,黄儒强,李赞,凌源智,曾健辉,马广智.利用唐鱼雌激素受体构建环境雌激素重组酵母测评系统的研究[J].环境科学学报,2015,35(1):317-323
利用唐鱼雌激素受体构建环境雌激素重组酵母测评系统的研究
- Recombinant yeast assay system for environmental estrogens mediated by estrogen receptor from Tanichthys albonubes
- 基金项目：广东省科技计划项目(No.20120401)
- 陈美玲
- 华南师范大学生命科学学院, 广州 510631
- 刘琳
- 华南师范大学生命科学学院, 广州 510631
- 黄儒强
- 华南师范大学生命科学学院, 广州 510631
- 李赞
- 华南师范大学生命科学学院, 广州 510631
- 凌源智
- 华南师范大学生命科学学院, 广州 510631
- 曾健辉
- 华南师范大学生命科学学院, 广州 510631
- 马广智
- 1. 华南师范大学生命科学学院, 广州 510631;2. 广东科学技术职业学院, 珠海 519090
- 摘要：利用唐鱼雌激素受体基因片段构建重组酵母,用以筛选环境雌激素类化学物质.实验先将唐鱼雌激素受体terα基因片段插入载体pGADT7中构建表达质粒pGADT7/TERα,同时将雌激素效应元件(ere)片段插入pMP206载体中构建报告质粒pMP206/ERE-LacZ;然后把表达质粒和报告质粒共转化到酵母AH109中,经筛选成功构建了由唐鱼雌激素受体调控的表达lacZ基因的重组酵母AHpTERα/ERE.结果表明,所构建的重组酵母AHpTERα/ERE在不同浓度17β-雌二醇(E2)的诱导下,β-半乳糖苷酶的活性呈现出明显的剂量-效应关系,其EC50为(0.521±0.700) nmol·L-1.与DMSO对照组相比,重组酵母在17β-雌二醇诱导下有明显的β-半乳糖苷酶活性增强的现象.在不同浓度17α-乙炔基雌二醇(EE2)、壬基酚(NP)及其混合物、双酚A(BPA)、17β-雌二醇(E2)(阳性对照)的诱导下,重组酵母均呈现出剂量-效应关系,且灵敏度大小为E2 >EE2 >NP>BPA.结果表明,本研究成功构建了重组基因酵母测评系统,初步判定该重组酵母可应用于环境雌激素的筛选.
- Abstract：Yeast with recombinant estrogen receptor fragment of Tanichthys albonubes (terα) was used to screen the environmental estrogen-like compounds. terα and estrogen responsive element (ere) were cloned into pGADT7 and pMP206 respectively to obtain receptor (pGADT7/terα) and reporter (pMP206/ERE-LacZ) constructs. The recombinant yeast AHpTERα/ERE was obtained by co-transformation of the receptor and reporter constructs, in which the expression of lacZ is regulated by the activity of terα. The assay sensitivity was evaluated by different concentrations of 17β-estradiol (E2) treatment. The results showed that the activity of beta-galactosidase induced by E2 was dose-dependent, and was significantly higher than that of DMSO control. The EC50 of E2 was (0.521±0.700) nmol·L-1. The assay system is also sensitive to other estrogen compounds like 17α-ethynylestradiol (EE2), Bisphenol A (BPA) and nonylphenol (NP) technical mixtures dose-dependently, and the sensitivity of different chemicals ranked as E2 > EE2 >NP >BPA. The results showed that recombinant yeast assay system for environmental estrogens could be applied to screen the estrogens-like compounds in the environment.
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