斑鳢ghr基因克隆、表达分析及性类固醇激素诱导的响应  

Cloning and Expression Analysis of the ghr Gene in Channa maculata and Its Response to Sexual Steroid Hormone Induction

作　　者：吴玉霞 张啸天 张扬 赵建[1,2] 罗青[1] 刘海洋 费树站 陈昆慈[1,2] 欧密 WU Yu-Xia;ZHANG Xiao-Tian;ZHANG Yang;ZHAO Jian;LUO Qing;LIU Hai-Yang;FEI Shu-Zhan;CHEN Kun-Ci;OU Mi(Pearl River Fisheries Research Institute/Key Laboratory of Tropical and Subtropical Fishery Resources Application and Cultivation,Ministry of Agriculture and Rural Affairs,Chinese Academy of Fishery Sciences,Guangzhou 510380,China;College of Fisheries and Life Sciences,Shanghai Ocean University,Shanghai 201306,China;School of Life and Health Sciences,Hunan University of Science and Technology,Xiangtan 411201,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 [3]湖南科技大学生命科学与健康学院,湘潭411201

出　　处：《农业生物技术学报》2024年第6期1371-1384,共14页Journal of Agricultural Biotechnology

基　　金：财政部和农业农村部:国家现代农业产业技术体系(CARS-46);国家自然科学基金面上项目(32373127);广东省基础与应用基础研究基金(2021A1515012075);广州市科技计划(202201010120);中国水产科学研究院基本科研业务费(2023XT0202,2023TD37);广东省淡水水产种质资源库运行项目(2022-SBH-00-001)。

摘　　要：生长激素(growth hormone, GH)-胰岛素样生长因子(insulin-like growth, IGF)轴是调控鱼类生长的主要内分泌轴线。为探讨生长激素受体(growth hormone receptor, GHR)在斑鳢(Channa maculata)雌、雄生长差异中的调控机制,本研究克隆了斑鳢ghr基因序列,分析了基因结构,解析了ghr在成鱼不同组织和不同发育时期肝脏中的表达模式及其对外源性类固醇激素刺激的响应表达。结果表明,斑鳢ghr基因cDNA序列全长2 602 bp,包括100 bp 5’-UTR,1 872 bp开放阅读框,编码623个氨基酸,630 bp 3’-UTR。斑鳢ghr基因组序列长18 118 bp,包含9个外显子和8个内含子,在5’-端上游鉴定到899 bp的侧翼序列,经分析发现,ghr 5’-端侧翼序列含有雄激素受体(androgen receptor, AR)、含溴结构域蛋白4 (bromodomaincontaining protein 4, BRD4)和抑制元素-1-沉默转录因子(repressor element-1-silencing transcription factor,REST)等多种转录因子结合位点。成鱼组织表达分析证实ghr在斑鳢肝脏中高表达,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表明,ghr基因在365 d的雄性斑鳢肝脏中表达量最高,且极显著高于同时期雌性斑鳢(P<0.01)。激素诱导实验显示,长期注射17α-乙炔基雌二醇(17α-ethynylestradiol, EE2)会抑制雌、雄斑鳢ghr基因的表达;长期注射17α-甲基睾丸酮(17α-methyltestosterone, MT)会抑制雌性斑鳢ghr的表达,但在一定时间内会促进雄性斑鳢ghr的表达。以上结果表明,GHR可能在斑鳢雌雄生长二态性中发挥作用,其表达受性类固醇激素调控,本研究为进一步探究GHR调控斑鳢生长发育的分子机理研究提供参考。The growth hormone(GH)-insulin-like growth factor(IGF)axis is the main endocrine axis that regulates fish growth.In order to explore the regulatory mechanism of growth hormone receptor(GHR)in the growth difference between female and male blotched snakehead(Channa maculata),the ghr gene in C.maculata was cloned,gene structure was analyzed,and the expression patterns of ghr in different adult tissues and liver at different developmental stages were clarified,meanwhile,its response to exogenous sex steroid hormones were also analyzed.The results showed that 2602 bp cDNA sequence of the ghr gene in C.maculata was obtained,including 100 bp 5'-UTR,1872 bp ORF,encoding 623 amino acids,and 630 bp 3'-UTR.The genome sequence of ghr gene in C.maculata was 18118 bp long,including 9 exons and 8 introns.899 bp 5'-flanking sequence was identified,which contained androgen receptor(AR),bromomain-containing protein 4(BRD4)and suppressor element-1-silencing transcription factor(REST)and other transcription factor binding sites.ghr was highly expressed in the liver of adult C.maculata,and its expression in males was significantly higher than that in females(P<0.01).The expression analysis of ghr gene at different developmental stages displayed that the highest expression of ghr gene was found in the liver of 365 d male C.maculata,which was significantly higher than that in females at the same period(P<0.01).Hormone induction experiments showed that long-term injection of 17α-ethynylestradiol(EE2)inhibited ghr expression in male and female C.maculata,long-term injection of 17α-methyltestosterone(MT)inhibited ghr expression in female C.maculata,but promoted ghr expression in males within a certain period of time.The results demonstrate that GHR might play a key role in the growth difference between females and males and it is also regulated by sex steroid hormones in C.maculata.This study provides the reference for further exploring the molecular mechanism of growth regulation in blotched snakehead.

关 键 词：斑鳢 生长激素受体基因(ghr) 基因克隆 表达分析 激素诱导 

分 类 号：S917.4[农业科学—水产科学]