摘要:
目前关于环境雌激素(environmental estrogens,EEs)的效应评估大多数是基于人的雌激素受体(estrogen receptor,ER)的离体检测，缺乏EEs对鱼类ER影响的研究。本研究利用酵母双杂交技术构建模式生物稀有鮈鲫不同ER亚型重组荧光双杂交酵母，用以快速检测EEs的雌激素活性并研究不同ER亚型对17β-雌二醇(E2)敏感度的差异。采用反转录多聚酶链反应法获取稀有鮈鲫ERα、ERβ1和ERβ2的配体结合域(LBD)序列，构建并鉴别诱饵质粒pGBKT7-ERα-LBD、pGBKT7-ERβ1-LBD和pGBKT7-ERβ2-LBD。将诱饵质粒和猎物质粒pGAD424-GRIP1同时转化荧光酵母Y187-Luc，分别构建3株稀有鮈鲫ERα-GRIP1、ERβ1-GRIP1和ERβ2-GRIP1重组荧光双杂交酵母，并考察了E2、双氢睾酮(DHT)、9-顺维甲酸(9-cis RA)、三碘甲状腺原氨酸(T3)和孕酮(PG)对荧光素酶的诱导情况。结果显示：ERα-GRIP1、ERβ1-GRIP1和ERβ2-GRIP1重组荧光双杂交酵母能够专一性地被E2诱导产生荧光素酶，并存在显著的剂量-效应关系，半数效应浓度(EC50)值分别为1.98×10-9、1.77×10-10、和3.52×10-10 mol·L-1，对E2的敏感程度排序为：ERβ1-GRIP1 > ERβ2-GRIP1 > ERα-GRIP1。研究表明，稀有鮈鲫不同ER亚型对E2的响应具有差异，3株重组荧光双杂交酵母不仅可以应用于快速识别内分泌干扰物中的类雌激素物质，分析稀有鮈鲫不同ER亚型对EEs的敏感性，解析雌激素污染物对稀有鮈鲫的作用机制，评估鱼类接触EEs的潜在风险，以期为水质保障和污染治理提供重要依据。
Abstract:
Most of the current evaluations of effects for environmental estrogens (EEs) are based on the in vitro assays regarding with humans ER and lack the effects of EEs on fish ER. In this study, the model organism rare minnow different estrogen receptor (ER) subtypes recombinant fluorescent yeasts were constructed by two-hybrid yeast technique to rapidly detect the estrogenic activity of EEs and investigate the difference in sensitivities between 17β-estradiol (E2) and different ER subtypes. The ligand binding domain (LBD) sequences of rare minnow ERα, ERβ1 and ERβ2 were obtained by reverse transcription polymerase chain reaction, and the bait plasmids pGBKT7-ERα-LBD, pGBKT7-ERβ1-LBD and pGBKT7-ERβ2-LBD were constructed and identified. Then the bait plasmid and prey plasmid pGAD424-GRIP1 were simultaneously transformed into fluorescent yeast Y187-Luc, and three rare minnow recombinant fluorescent two-hybrid yeasts ERα-GRIP1, ERβ1-GRIP1 and ERβ2-GRIP1 were constructed. Finally, the luciferase induced by E2, dihydrotestosterone (DHT), 9-cis retinoic acid (9-cis RA), triiodothyronine (T3) and progesterone (PG) were examined using these yeasts. The results showed that ERα-GRIP1, ERβ1-GRIP1 and ERβ2-GRIP1 recombinant fluorescent two-hybrid yeasts specifically produced luciferase induced by E2 with a significant dose-response relationship and the values of half effect concentration (EC50) were 1.98×10-9, 1.77×10-10 and 3.52×10-10 mol·L-1, respectively. Moreover, the order of sensitivity to E2 was ERβ1-LBD> ERβ2-LBD > ERα-LBD. Our results suggested that different ER subtypes of rare minnow had different responses to E2. Furthermore, three recombinant fluorescent two-hybrid yeasts can be applied to rapidly identify estrogen-like substances of endocrine disruptors, analyze sensitivity between EEs and different ER subtypes of rare minnow, analyze the mechanisms of EEs on rare minnow and evaluate potential risks of fish exposure to EEs in order to provide an important basis for water quality protection and pollution control.
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