斑鳢生长激素基因的克隆及表达分析  

Gene Cloning and Expression Analysis of Growth Hormone Gene from Blotched Snakehead(Channa maculata)

作　　者：高丹丹 欧密 吴燕铎 陈昆慈[1] 刘海洋 罗青[1] 赵建[1,2] GAO Dan-Dan;OU Mi;WU Yan-Duo;CHEN Kun-Ci;LIU Hai-Yang;LUO Qing;ZHAO Jian(Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences/Key Laboratory of Tropical and Subtropical Fishery Resources Application and Cultivation,Ministry of Agriculture and Rural Affairs,Guangzhou 510380,China;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]中国水产科学研究院珠江水产研究所/农业农村部热带亚热带水产种质资源利用与养殖重点实验室,广州510380 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《农业生物技术学报》2021年第12期2328-2341,共14页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2018YFD0901201);现代农业产业技术体系建设专项资金(CARS-46);中国水产科学研究院珠江水产研究所自主科技创新项目(ZC-2019-11);以农产品为单元的广东省现代农业产业技术体系创新团队建设项目(2020KJ150);中国-东盟海上合作基金资助(CAMC-2018F)。

摘　　要：生长激素(growth hormone, GH)是垂体分泌的多肽,在硬骨鱼类的发育和体细胞的生长过程中具有重要的调节作用。为探究生长激素在斑鳢(Channa maculata)雌、雄生长差异中的作用及其与性激素的相互作用机制,本研究克隆了斑鳢GH基因(GenBank No. MW574005)序列,分析了其表达模式及对外源性类固醇激素的响应。结果表明,斑鳢GH基因cDNA序列全长882 bp,开放阅读框(ORF)为639 bp,共编码212个氨基酸。斑鳢GH基因序列长1 604 bp,含有5个外显子和4个内含子。在5’端上游鉴定到一段长1 199 bp的侧翼序列,该序列含有前B细胞白血病转录因子3 (pre-B-cell leukemia transcription factor3, PBX3)、信号转导和转录激活因子1 (signal transducers and activators of transcription 1, STA1)和叉头转录因子O3 (forkhead box O3, FOXO3)等多种转录因子结合位点。组织表达分析显示,GH在斑鳢垂体中高表达,且在雄性中的表达极显著高于雌性(P<0.01);不同发育时期表达分析显示,GH基因在365 d的雄性斑鳢垂体中表达量最高,且极显著高于同时期雌性斑鳢(P<0.01)。经ELISA检测验证,各发育时期内,雄性个体血清中的GH含量均高于同时期雌性个体。激素诱导实验显示,17α-乙炔基雌二醇(17α-ethynylestradiol, EE2)抑制雌、雄斑鳢垂体中GH基因的表达;17α-甲基睾丸酮(17α-methyltestosterone, MT)促进雄鱼垂体中GH基因的表达,但降低雌鱼垂体中GH基因的表达。该研究为进一步探究斑鳢雌雄生长二态性机制和生长调控的分子机理研究提供参考。Growth hormone(GH) is a polypeptide secreted by pituitary, which plays an important role in the development of teleost and the growth of somatic cells. In order to explore the role of GH in the growth dimorphism between male and female blotched snakehead(Channa maculata) and the mechanism of its interaction with sex hormones, in this study, GH gene(GenBank No. MW574005) of blotched snakehead was cloned and its expression pattern and response to exogenous steroid hormones were analyzed. The results showed that, the cDNA sequence of blotched snakehead GH gene was 882 bp in length, with an open reading frame(ORF) of 639 bp, encoding 212 amino acids. The length of GH sequence of blotched snakehead was1 604 bp and contained 5 exons and 4 introns. A 1 199 bp flanking sequence was identified at the upstream of 5’-terminal, which contains pre-B-cell leukemia transcription factor 3(PBX3), signal transducers and activators of transcription 1(STA1), forkhead box O3(FOXO3) and other transcription factors binding sites.Tissues expression analysis showed that GH was highly expressed in the pituitary of blotched snakehead, and the expression in male blotched snakehead was extremely significant higher than that in female(P<0.01);Expression analysis in different developmental stages showed that GH gene expression in the pituitary of male blotched snakehead was the highest at 365 d post hatching, which was extremely significant higher than that in female(P<0.01). The results of ELISA showed that the serum GH content in male blotched snakehead was higher than that in female in each developmental period. Hormone induction experiment showed that 17α-ethynylestradiol(EE2) inhibited GH gene expression in female and male blotched snakehead;17α-methyltestosterone(MT) promoted GH gene expression in male pituitary, but decreased GH gene expression in female pituitary. This study provides the reference for further exploring the growth dimorphism mechanism of male and female blotched snakehead and the molecular mechanism of growth regula

关 键 词：斑鳢 生长激素(GH) 基因克隆 表达分析 

分 类 号：S917.4[农业科学—水产科学]