Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper
Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper
复制标题
雌雄同体红斑石斑鱼卵巢芳香酶(Cyp19a1a)的表达模式、细胞定位及启动子活性分析
DOI：
10.1016/j.mce.2009.04.003
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发表时间：
2009-08-13
影响因子：
4.1
通讯作者：
Gui, Jian-Fang
中科院分区：
文献类型：
--
作者：
Huang, Wei;Zhou, Li;Gui, Jian-Fang
芳香化酶在性腺的性别分化中起着关键作用。本研究克隆了赤点石斑鱼卵巢芳香化酶基因的全长cDNA，并制备了相应的抗EaCyp 19 a1 a抗血清。Western blot和免疫荧光研究揭示了EaCyp 19 a1 a在成体卵巢中的特异性表达模式及其在人工性逆转过程中的动态表达变化。EaCyp 19 a1 a在卵母细胞周围的滤泡层的滤泡细胞中表达，因为在卵巢细胞中观察到强烈的EaCyp 19 a1 a免疫荧光。在人工性逆转过程中，EaCyp 19 a1 a的表达从雌性到雄性显著下降，睾丸组织中几乎没有观察到任何阳性信号。随后，我们克隆并测序了EaCyp 19 a1 a启动子的T侧翼序列，共1967 bp，并显示了一些转录因子的潜在结合位点，如SOX 5、加塔基因家族、CREB、AP 1、FOXL 1、C/EBP、ARE和SF-1。此外，我们制备了一系列5'缺失启动子构建体，并进行了EaCyp 19 a1 a启动子活性的体外荧光素酶测定。结果表明，CREB调控区-1010 ~-898可能是EaCyp 19 a1 a启动子的主要顺式作用元件，而调控区-1216 ~-1010的元件加塔和SOX 5可能是抑制元件。值得注意的是，我们发现了一个共同的保守序列区的鱼类卵巢型芳香化酶启动子的同源性从93%到34%。TATA box、SF-1、SOX 5和CREB等基序在该区域存在，并且在大多数鱼类中具有保守性。(C)2009爱思唯尔爱尔兰有限公司保留所有权利。
Aromatase plays a key role in sex differentiation of gonads. In this study, we cloned the full-length cDNA of ovarian aromatase from protogynous hermaphrodite red-spotted grouper (Epinephelus akaara), and prepared the corresponding anti-EaCyp19a1a antiserum. Western blot and immunofluorescence studies revealed ovary-specific expression pattern of EaCyp19a1a in adults and its dynamic expression change during artificial sex reversal. EaCyp19a1a was expressed by follicular cells of follicular layer around oocytes because strong EaCyp19a1a immunofluorescence was observed in the cells of ovaries. During artificial sex reversal, EaCyp19a1a expression dropped significantly from female to male, and almost no any positive EaCyp19a1a signal was observed in testicular tissues. Then, we cloned and sequenced a total of 1967 bp T-flanking sequence of EaCyp19a1a promoter, and showed a number of potential binding sites for some transcriptional factors, such as SOX5, GATA gene family, CREB, AP1, FOXL1, C/EBP, ARE and SF-1. Moreover, we prepared a series of 5' deletion promoter constructs and performed in vitro luciferase assays of EaCyp19a1a promoter activities. The data indicated that the CREB regulation region from -1010 to -898 might be a major cis-acting element to EaCyp19a1a promoter, whereas the elements GATA and SOX5 in the region from -1216 to -1010 might be suppression elements. Significantly, we found a common conserved sequence region in the fish ovary-type aromatase promoters with identities from 93% to 34%. And, the motifs of TATA box, SF-1, SOX5, and CREB existed in the region and were conserved among the most of fish species. (C) 2009 Elsevier Ireland Ltd. All rights reserved.