| 引用本文: | 梁冬冬,范兆飞,邹玉霞,谭训刚,吴志昊,焦 爽,李 军,尤 锋.牙鲆17β-HSD1基因克隆及其表达调控的初步研究[J].海洋科学,2017,41(9):65-73. |
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| 牙鲆17β-HSD1基因克隆及其表达调控的初步研究 |
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梁冬冬1,2,3, 范兆飞1,2,3, 邹玉霞1,2, 谭训刚1,2, 吴志昊1,2, 焦 爽1,2, 李 军1,2, 尤 锋1,2
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1.中国科学院 海洋研究所 中国科学院实验海洋生物学重点实验室;2.青岛海洋科学与技术国家实验室实验海洋生物学与生物技术实验室;3.中国科学院大学
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| 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-PCR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。 |
| 关键词: 牙鲆(Paralichthys olivaceus) 17β-羟类固醇脱氢酶1 ORF 克隆 表达 调控 |
| DOI：10.11759/hykx 20170303001 |
| 分类号: |
| 基金项目:国家自然科学基金资助项目(41276171、31502156); 青岛海洋科学与技术国家实验室鳌山科技创新计划项目(2015ASKJ02); 鲆鲽类产业技术体系课题(NYCYTX-50-G03); 国家水产种质资源共享服务平台(2017DKA30470) |
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| Molecular characterization, expression, and regulation of 17β-HSD1 in the olive flounder Paralichthys olivaceus |
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LIANG Dong-dong,FAN Zhao-fei,ZOU Yu-xia,TAN Xun-gang,WU Zhi-hao,JIAO Shuang,LI Jun,YOU Feng
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| Abstract: |
| 17β-Hydroxysteroid dehydrogenase 1 (17β-HSD1) is an important enzyme as it is involved in the synthesis 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 of 17β-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 300 μmol/L cAMP (P < 0.05). Moreover, transfecting the cultured primary testis cells with 2 and 3 μg NR5a2 and 1 μg NR0b1 significantly downregulated its expression, whereas it was significantly upregulated upon treatment with 2 μg NR0b1 (P < 0.05), and all these regulations were dose-dependent. This study indicates that 17β-HSD1 is involved in flounder gonadal differentiation, and its expression is respectively regulated by cAMP, NR5a2, and NR0b1. These results may provide useful information for the study of fish gonadal differentiation. |
| Key words: Paralichthys olivaceus 17β-HSD1 ORF clone expression regulation |
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