牙鲆17β-HSD1基因克隆及其表达调控的初步研究

摘要

17β-羟类固醇脱氢酶1(17β-HSD1)的主要作用是将雌酮(El)转化为发挥功能的雌二醇(E2).作者从牙鲆(Paralichthys olivaceus)性腺转录组数据库获得该基因的开放阅读框(ORF)序列,对其进行了验证,并分析了该基因在高温、外源性激素处理条件下性腺分化期性腺组织中的差异表达以及cAMP和转录因子(NR5a2和NR0b1)在精巢原代细胞中对该基因表达的作用.结果显示,牙鲆17β-HSD1基因的ORF为873bp,编码290个氨基酸,与其他鱼类的有很高的相似性.半定量RT-PCR结果表明,该基因在卵巢中高表达,精巢有少量表达,并且在雌性个体的鳃、头肾、肾、脾、胃和肠中也有表达.实时定量RT-P C R结果显示,该基因在卵巢或精巢分化的关键时期表达量较高;在精巢原代培养细胞中,外源信号分子cAMP及转录因子NR5a2可以显著下调17β-HSD1基因的表达(P<0.05),且呈现剂量效应,转录因子NR0b1对该基因的调控也与剂量有关.作者推测牙鲆17β-HSD1基因在性腺分化中起一定的作用,其表达受到调控因子的作用,这些结果将有助于增加对鱼类性腺分化和发育的认识.%17β-Hydroxysteroid dehydrogenase 1 (17β-HSD1) is an important enzyme as it is involved in the syn-thesis of both 17β-estradiol and testosterone. In this study,17β-HSD1 open reading frame sequence was obtained from the olive flounder Paralichthys olivaceus gonadal transcriptome data and verified. Phylogenetic tree analysis showed that flounder 17β-HSD1 was clustered with 17β-HSD1 proteins from fish species such as Oreochromis niloticus and Oryzias latipes. Semiquantitative RT-PCR results indicated the expression of 17β-HSD1 in the ovary was higher than that in the testis, and it was also expressed in the female gill, head kidney, kidney, spleen, stomach, and intestine. Real-time quantitative PCR analysis revealed upregulated expression patterns of17β-HSD1 during the key phases of ovarian and testicular differentiation.17β-HSD1 expression was significantly downregulated in the cultured primary testis cells treated with 75, 150, and 300μmol/L cAMP (P < 0.05). Moreover, transfecting the cul-tured primary testis cells with 2 and 3μg NR5a2 and 1μg NR0b1 significantly downregulated its expression, whereas it was significantly upregulated upon treatment with 2μg NR0b1 (P < 0.05), and all these regulations were dose-dependent. This study indicates that17β-HSD1 is involved in flounder gonadal differentiation, and its expres-sion is respectively regulated by cAMP, NR5a2, and NR0b1. These results may provide useful information for the study of fish gonadal differentiation.