摘要
为了获得海水青鳉Oryzias melastigma雌激素受体的基因信息及其表达特性,采用RACE-PCR技术和实时荧光定量PCR方法,进行了雌激素受体基因的克隆、表达特性分析及其在17α-炔雌醇(EE2)暴露下的基因响应研究。结果表明:海水青鳉雌激素受体基因与其他脊椎动物雌激素受体基因同源性较高,特别是DNA结合结构域(DBD)在进化上高度保守,配体结合结构域(LBD)次之;OM-ERα/β1/β2在所检测的10种组织中均有表达,其中在青鳉肝脏、性腺、脾脏和肠中表达量较高;OM-ERα在肝脏中的表达和OM-ERβ1/β2在性腺中的表达,雌雄间存在显著性差异(P<0.05);在EE2浓度为5、50、500ng/L的水体中暴露96 h后,海水青鳉仔鱼中的分子标志物卵黄蛋白原基因(vtg1)被显著诱导(P<0.05),且呈浓度依赖关系;中浓度(50 ng/L)的EE2诱导仔鱼OM-ERβ1基因表达,OM-ERβ2对EE2暴露呈现低浓度诱导、高浓度抑制的趋势;攻毒试验结果表明,OM-ERα和vtg1之间存在一种正相关关系,暗示EE2可能主要通过ERα调控vtg1基因的表达;EE2攻毒下,β亚型存在一种倒U型的剂量-反应关系。研究表明:克隆获得的海水青鳉3个雌激素受体亚型基因OM-ERα/β1/β2,为后续开展海水模式鱼类雌激素受体相关研究提供了基础信息;OM-ERα/β1/β2的组织表达特性及EE2对仔鱼OM-ERα/β1/β2表达的不同调控,显示出海水青鳉3种ER亚型基因存在着不同的生理功能,且其对环境污染物的应答模式不同。
In this study,gene clone,m RNA expression pattern and gene response in normal and 17α-ethynylestradiol（ EE2）-exposed conditions were carried out in marine medaka Oryzias melastigma using RACE-PCR and RTqPCR methods in order to obtain the gene information of estrogen receptors and to investigate their expression characterization in marine medaka. The results showed that there were high homology in estrogen receptors between marine medaka and other vertebrates,specially in the six domain of OM-ERα/β1/β2,the most evolutionarily conserved domain being DNA binding domain（ DBD）,followed by ligand binding domain（ LBD）. OM-ERα/β1/β2 were widely expressed in the ten tested tissues,highly expressed in liver,gonad,spleen and intestine. There were gender differences in the expression of liver OM-ERα and gonad OM-ERβ1/β2. The biomarker gene vtg1 m RNA was significantly induced in the larvae exposed to 5,50 and 500 ng/L of EE2 in a dose-dependent manner（ P 0.05）,with significant positive relationship between vtg1 and OM-ERα,indicating that OM-ERα is the crucial subtype involved in the regulation of vtg1. In larvae,OM-ERβ1 was positively response to 50 ng/L EE2,and there was a trend that OM-ERβ2 was induced by low dose EE2,and inhibited by high dose（ 500 ng/L） of EE2,with an inverted-U typed relationship between the dose and response. The expression patterns of OM-ERα/β1/β2 and their distinct responsiveness to EE2 exposure revealed that there were different physiological functions among the three estrogen receptors. The findings provide basic information with further endocrine disruption-related investigations.
作者
张民
安哲
彭会
ZHANG Min;AN Zhe;PENG Hui(State Key Laboratory of Marine Environmental Science, Xiamen 361102, China;College of Ocean and Earth Science, Xiamen Lniversity, Xia-men 361102, China)
出处
《大连海洋大学学报》
CAS
CSCD
北大核心
2018年第2期158-168,共11页
Journal of Dalian Ocean University
基金
国家自然科学基金资助项目(41276102)
