Mechanistic insights into induction of vitellogenin gene expression by estrogens in Sydney rock oysters, Saccostrea glomerata.
Mechanistic insights into induction of vitellogenin gene expression by estrogens in Sydney rock oysters, Saccostrea glomerata.
复制标题
DOI：
10.1016/j.aquatox.2016.02.023
复制
发表时间：
2016-05
影响因子：
4.5
通讯作者：
T. Tran;G. MacFarlane;R. Kong;W. O'Connor;R. M. K. Yu
中科院分区：
文献类型：
--
作者：
T. Tran;G. MacFarlane;R. Kong;W. O'Connor;R. M. K. Yu
牡蛎等海洋软体动物通过诱导蛋黄蛋白前体卵黄蛋白原 (Vtg) 对雌激素化合物做出反应，从而利用雌激素污染的生物标志物。尽管有这种应用，但雌激素发挥作用诱导软体动物卵黄发生的精确分子机制尚不清楚。作为解决这个问题的第一步，我们从悉尼岩牡蛎 Saccostrea glomerata (sgVtg) 中克隆了编码 Vtg 的基因。使用根据 Genbank 中提供的部分 sgVtgcDNA 序列设计的引物，通过 5'- 和 3'-RACE 获得了 8498 bp 的全长 sgVtgcDNA。 sgVtg 的开放阅读框 (ORF) 被确定为 7980 bp，明显长于其他牡蛎物种的直系同源物。其推导的蛋白质序列在 N 端和 C 端区域与其他软体动物 Vtgs 具有最高的同源性。 sgVtg的全长基因组DNA序列是通过基因组PCR和基因组步移分别针对基因体和侧翼区域获得的。基因组序列跨度为 20 kb，由 30 个外显子和 29 个内含子组成。计算机分析确定了启动子区域中的三个紧密间隔的半雌激素响应元件 (ERE) 和转录起始位点下游 62 bp 处的 210 bp CpG 岛。在体外（外植体）和体内（罐）暴露于 17β-雌二醇 (E2) 后，在卵巢中观察到 sgVtgmRNA 表达的上调。值得注意的是，体外雌激素受体（ER）拮抗剂治疗消除了上调，表明转录激活需要雌激素依赖性受体。使用体内暴露卵巢的亚硫酸氢盐基因组测序分析 5' CpG 岛的 DNA 甲基化。在对照和 E2 暴露的牡蛎中，CpG 岛被发现低甲基化（含有 0-3% 甲基胞嘧啶）。然而，没有观察到甲基化的显着差异或甲基化与sgVtg表达水平之间的任何相关性。总体而言，这些结果支持含有 ERE 的启动子和雌激素激活受体可能参与海洋软体动物的雌激素信号传导。
Marine molluscs, such as oysters, respond to estrogenic compounds with the induction of the egg yolk protein precursor, vitellogenin (Vtg), availing a biomarker for estrogenic pollution. Despite this application, the precise molecular mechanism through which estrogens exert their action to induce molluscan vitellogenesis is unknown. As a first step to address this question, we cloned a gene encoding Vtg from the Sydney rock oysterSaccostrea glomerata(sgVtg). Using primers designed from a partialsgVtgcDNA sequence available in Genbank, a full-lengthsgVtgcDNA of 8498 bp was obtained by 5′- and 3′-RACE. The open reading frame (ORF) ofsgVtgwas determined to be 7980 bp, which is substantially longer than the orthologs of other oyster species. Its deduced protein sequence shares the highest homology at the N- and C-terminal regions with other molluscan Vtgs. The full-length genomic DNA sequence of sgVtg was obtained by genomic PCR and genome walking targeting the gene body and flanking regions, respectively. The genomic sequence spans 20 kb and consists of 30 exons and 29 introns. Computer analysis identified three closely spaced half-estrogen responsive elements (EREs) in the promoter region and a 210-bp CpG island 62 bp downstream of the transcription start site. Upregulation ofsgVtgmRNA expression was observed in the ovaries followingin vitro(explants) andin vivo(tank) exposure to 17β-estradiol (E2). Notably, treatment with an estrogen receptor (ER) antagonistin vitroabolished the upregulation, suggesting a requirement for an estrogen-dependent receptor for transcriptional activation. DNA methylation of the 5′ CpG island was analysed using bisulfite genomic sequencing of thein vivoexposed ovaries. The CpG island was found to be hypomethylated (with 0–3% methylcytosines) in both control and E2-exposed oysters. However, no significant differential methylation or any correlation between methylation andsgVtgexpression levels was observed. Overall, the results support the possible involvement of an ERE-containing promoter and an estrogen-activated receptor in estrogen signalling in marine molluscs.