| 青鱼ERRα的克隆、序列分析、组织表达及其对不同EDCs暴露的响应 |
| 摘要点击 2110 全文点击 2100 投稿时间：2007-11-20 修订日期：2008-01-24 |
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| 中文关键词 内分泌干扰物 雌激素相关受体 青鱼 实时定量PCR |
| 英文关键词 endocrine disrupting chemicals estrogen-related receptor α (ERRα) medaka Q-RT-PCR |
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| 中文摘要 |
| 内分泌干扰物质(EDCs)可以通过多种通道影响鱼类的生长、发育和繁殖, 而雌激素相关受体(ERR)是一类目前没有引起足够重视的潜在致毒信号通道. 本研究克隆了青鱼(Oryzias latipes)ERRα mRNA全序列, 并通过实时定量RT-PCR方法对其在不同组织中的表达和不同EDCs暴露下的响应进行了分析.发现青鱼ERRα与其它脊椎动物ERRα氨基酸序列有较高的同源性,特别是DNA结合结构域(DBD)在从鱼类到哺乳类动物的进化过程中高度保守, 配体结合结构域(LBD)序列与哺乳动物的LBD有66.4%～67.0%的序列相同. 青鱼ERRα与青鱼雌激素受体ERα、ERβ和雄激素受体ARα、ARβ的DBD氨基酸数相同, 且有较高序列相似性, 但LBD的长度和序列差异较大. 青鱼ERRα基因有5个外显子组成,位于第14号染色体. 青鱼ERRα基因在各组织中广泛表达,其中在性腺、脑、脾脏、眼和肠中表达较高,且在雌雄性腺中差异表达,表明其在雌雄性别分化和性腺发育中发挥调控作用. 暴露200 ng/L炔雌醇(EE2)、200 ng/L雌酮(E1)、200 ng/L己烯雌酚(DES)、 100 μg/L阿特拉津(AT)和200 ng/L雌二醇(E2)后, 青鱼精巢中ERRα mRNA水平分别显著下降至对照组的0.54、 0.56、 0.61、 0.63和0.65倍(p<0.05), 但暴露1 μg/L三丁基锡和1 μg/L三苯基锡后上升至对照组的1.34和1.35倍(p>0.05),表明ERRα可能参与外源EDCs影响鱼类性别分化和性腺发育的过程. 此外,在青鱼ERRα基因上游没有发现类似哺乳动物ERRα基因上游的类固醇激素反应元件半位点, 说明鱼类ERRα基因的调控模式与哺乳类存在差异. |
| 英文摘要 |
| Endocrine disrupting chemicals (EDCs) can bind or block nuclear receptors in the body and subsequently affect growth, development and reproduction of fish. Estrogen-related receptors (ERRs), belonging to the nuclear receptor superfamily, have been implicated in diverse physiological processes in estrogen signal pathway in mammals, while little is known about them in fishes. Complete mRNA sequence of ERRα from medaka (Oryzias latipes) was cloned, and the sequence is similar to those of other vertebrates, especially that the DNA-binding domain (DBD) of ERRα is highly conserved among the vertebrates (97.4%-100% sequence identities) and the ligand-binding domain (LBD) of medaka ERRα is 66.4%-67.0% sequence identities with those of mammals. The DBD of medaka ERRα is of the same length and has high sequence identity with those of estrogen receptor (ERα and ERβ) and androgen receptor (ARα and ARβ) of medaka, but much difference was found between the LBD of medaka ERRα with those of ERα, ERβ, ARα and ARβ. ERRα gene is located in chromosome 14 and is consisted of 5 exons. The expressions of ERRα in different tissues and the transcriptional responses of ERRα in testis of medaka exposed differential EDCs were studied by quantitative real-time RT-PCR. ERRα is expressed at apparently high levels in gonad, brain, eye, spleen and intestine, though it was broadly expressed in tissues. Significant transcriptional difference was found between testis and ovary, implying ERRα would be involved in sex differentiation and gonad development in fish. After 3 weeks exposure of medaka to 200 ng/L ethynylestradiol (EE2), 200 ng/L estrone (E1), 200 ng/L diethylstilbestrol (DES), 100 μg/L atrazine (AT) and 200 ng/L 17β-estradiol (E2), transcripts of ERRα were significantly decreased to 0.54, 0.56, 0.61, 0.63 and 0.65 of control (p< 0.05) in the testes, respectively. And those in the 1 μg/L tributyltin (TBT) and 1 μg/L triphenyltin (TPT) exposure groups were up-regulated to 1.34 and 1.35 folds of control (p>0.05), respectively. These results suggested that ERRα would take actions in the disruption of sex differentiation and gonad development in fish by EDCs. In addition, no multiple steroid hormone-response element half-sites was found in medaka, which were reported in the upstream of ERRα gene in mammals, indicating there would be different regulation patters of ERRα between teleost and mammal. |
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