牙鲆(Paralichthys olivaceus)抗缪勒氏管激素Ⅱ型受体基因(amhr2)表达特征分析及功能初探  

Expression characteristics and function of Anti-Müllerian hormone receptorⅡgene(amhr2)in Paralichthys olivaceus

作　　者：李泽 王丽娟[1,2] 邹聪聪 舒畅 吴志昊 邹玉霞[1,2] 尤锋 LI Ze;WANG Lijuan;ZOU Congcong;SHU Chang;WU Zhihao;ZOU Yuxia;YOU Feng(Chinese Academy of Sciences and Shandong Province Key Laboratory of Experimental Marine Biology,Center for Ocean Mega-Science(Institute of Oceanology,Chinese Academy of Sciences),Qingdao 266071,China;Laboratory for Marine Biology and Biotechnology,Pilot National Laboratory for Marine Science and Technology(Qingdao),Qingdao 266237,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院实验海洋生物学重点实验室,海洋大科学中心(中国科学院海洋研究所),山东省实验海洋生物学重点实验室,山东青岛266071 [2]青岛海洋科学与技术试点国家实验室,海洋生物学与生物技术实验室,山东青岛266237 [3]中国科学院大学,北京100049

出　　处：《热带海洋学报》2024年第1期94-106,共13页Journal of Tropical Oceanography

基　　金：国家重点研发计划(2022YFD2400402,2018YFD0900202);山东省自然科学基金(ZR2022MC026);青岛海洋科学与技术试点国家实验室海洋生物学与生物技术功能实验室青年科学基金项目(YQ2018NO01)。

摘　　要：抗缪勒氏管激素Ⅱ型受体(anti-Müllerian hormone receptorⅡ,Amhr2)是抗缪勒氏管激素(anti-Müllerian hormone,Amh)的特异受体。amhr2基因在鱼类性腺分化和发育中发挥重要作用,更决定了有的鱼种的性别,然而,相关功能研究较为有限。本研究旨在明晰amhr2在我国重要海水养殖鱼类—牙鲆(Paralichthys olivaceus)性腺分化和发育过程中的表达特征,并初步探究其功能。首先,克隆了牙鲆amhr2-CDS序列,共1536bp,编码511个氨基酸。系统发育分析显示牙鲆Amhr2近C端较为保守,与其他硬骨鱼类聚为一枝,并与上游sp1以及下游的prr13和PCBP2在基因组中共定位。蛋白结构分析显示,牙鲆Amhr2包含信号肽、跨膜结构域和保守的酪氨酸蛋白激酶结构域。进而,利用实时荧光定量PCR(q PCR)分析表明,牙鲆amhr2主要表达于性腺,且在精巢中的表达极显著高于卵巢(P<0.01),其在I-V期精巢中持续高表达,而在卵巢中仅在I期高表达,后显著下降(P<0.05)。性腺分化期基因的表达,是对雌核发育组(对照组,20±0.5℃,100%雌性)与雌核发育高温诱导组(HT组,28±0.5℃,100%雄性)鱼苗进行检测的,amhr2在对照组全长(totallength,TL)2cm的实验鱼性腺中表达最高,后逐渐下降;而HT组实验鱼性腺中的表达呈现先上升后下降的趋势,并在精巢开始分化的6cm TL时表达最高。利用Hela细胞进行亚细胞定位分析发现,Amhr2与其配体Amh均定位于细胞质。同时,通过原核表达重组牙鲆Amh,并用其孵育离体性腺组织,q PCR分析显示精巢中amhr2的表达没有显著变化,但卵巢中表达显著上升(P<0.05)。进一步通过双荧光素酶报告分析表明,Amh和Amhr2共转染能够显著抑制雌激素合成的关键芳香化酶基因cyp19a的表达(P<0.01)。综上所述,牙鲆amhr2主要表达于精巢,但在不同性腺发育时期表达不同,且其可能与Amh共同影响cyp19a的转录,对雄性表型形成的启动和性腺发育起作用。Anti-Mullerian hormone receptorⅡ(Amhr2)is the specific receptor of Anti-Mullerian hormone(Amh)in the TGF-βpathway.It is critical for gonadal differentiation and development in fish and determination of the sex in some fish species.However,the expression and function of amhr2 in fish has been poorly studied.To clarify the expression characteristics and function of amhr2,a 1536 bp CDS encoding 511 amino acids,was obtained from Paralichthys olivaceus,an important commercially cultured marine fish in China.The flounder Amhr2 protein was clustered with those from other teleosts,with the highly conservative motifs occurring in the C-terminal region.Gene collinearity analysis revealed that the amhr2 collaborates with specificity protein 1(sp1)upstream,and proline-rich protein 13(prr13)and poly(rC)binding protein 2(PCBP2)downstream of the teleost genome.The predicted flounder Amhr2 protein was composed of a signal peptide,two transmembrane domains,and a conservative tyrosine protein kinase domain.Real-time quantitative PCR(qPCR)results showed that amhr2 was mainly expressed in the adult gonads,with significantly higher expression in the testes than in the ovaries(P<0.01).At stages I-V of the testis,the expression of amhr2 remained at a high level,whereas the expression level in the ovaries was significantly higher at stage I than at stagesⅡ-V(P<0.05).During the flounder gonadal differentiation period,gynogenesis(control,20±0.5℃,100%female)and gynogenesis at high temperature(HT,28±0.5℃,100%male)were used to detect the expression of amhr2.The results showed that the expression of amhr2 in the control group was highest at 2 cm total length(TL)and then decreased continuously,while the expression in the HT group first increased and then decreased.The inflection point of amhr2 expression in the HT group was at 6 cm TL,with the highest level.To investigate the effect of Amh on amhr2,we examined the co-localization of Amhr2 and Amh with Hela cells.Examination of subcellular localization showed that both Amhr2 and Amh

关 键 词：牙鲆(Paralichthys olivaceus) amhr2 性腺分化和发育 基因表达 功能 

分 类 号：Q953.3[生物学—动物学]