条石鲷mPRα基因的cDNA克隆和表达模式分析  

CLONING AND EXPRESSION OF MEMBRANE PROGESTIN RECEPTOR(mPRα) IN ROCK BREAM OPLEGNATHUS FASCIATUS

作　　者：史宝[1] 柳学周[1] 陈圣毅[1,2] 徐永江[1] 臧坤[1,2] 李晓妮[1] 

机构地区：[1]农业部海洋渔业可持续发展重点实验室青岛市海水鱼类种子工程与生物技术重点实验室中国水产科学研究院黄海水产研究所,青岛266071 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《海洋与湖沼》2015年第3期642-650,共9页Oceanologia Et Limnologia Sinica

基　　金：国家高技术研究发展计划项目;2012AA10A413号;国家自然科学基金项目;31201982号;国家鲆鲽类产业技术体系;CARS-50号;山东省优秀中青年科学家科研奖励基金项目;BS2013SW042号

摘　　要：采用同源性克隆和末端快速扩增(RACE)方法,首次获得全长为1222bp的条石鲷膜孕激素受体α(mPRα)的cDNA序列。其开放阅读框为1060bp,编码了含353个氨基酸的蛋白,N端前16个氨基酸为信号肽。氨基酸序列比较分析发现其存在7个跨膜区域。其预测的蛋白质三级结构中存在着多个蛋白结合位点。同源性比较和进化分析表明,条石鲷mPRα与鲈形目鱼类mPRα聚为一支,进化关系较近,相似度达到95%以上。实时荧光定量PCR方法检测mPRα mRNA表达情况:发现mPRα mRNA在性成熟雌性条石鲷各组织均有表达,并在脑、垂体和卵巢组织中的表达较丰富。在条石鲷繁殖周期的脑和垂体组织中,mPRα mRNA的表达水平在卵巢发育的Ⅳ期达到最大值;在繁殖周期的卵巢组织中,mPRα mRNA的表达水平从卵巢发育Ⅲ期到Ⅴ期持续升高并且在Ⅴ期达到最大值(P<0.05)。条石鲷雌鱼血清中雌二醇激素的含量变化在整个繁殖周期中差异显著(P<0.05),在性腺发育Ⅲ期含量迅速升高并在Ⅳ期达到峰值。本研究为条石鲷卵巢成熟调控机制研究提供了基础参考资料。A full-length c DNA sequence of mPRα was cloned first time from the ovary of Oplegnathus fasciatus by homology cloning and RACE techniques. The mPRα c DNA sequence is 1222 bp and its open reading frame is 1060 bp, encoding a precursor protein of 353 amino acids preceded by a signal peptide of 16 amino acids residues. Sequence alignment of the mPRα and amino acid of other species suggested that it had seven transmembrance domains. Tertiary structure of mPRα protein showed that it had several binding sites. Sequences comparison and phylogenetic analysis showed the mPRα clusters with perciformes in more than 95% identity. The mPRα mRNA expression of sexually matured rock bream was detected by quantitative real-time PCR(q RT-PCR) method. Tissue expression analysis showed that mPRα mRNA was expressed widely in the fish, and mPRα transcripts were highly abundant in brain, pituitary, and ovary. Expression level of mPRα mRNA in brain and pituitary of reproductive cycle showed that mPRα mRNA were maximum at stageⅤ. And the expression level of mPRα mRNA in the reproductive cycle showed that it significantly increased from Ⅳovarian development stage to stageⅤ(P<0.05), then peaked in the stage Ⅴ. Changes in serum estrogen values of reproductive cycle indicated that plasma estrogen values changed dramatically(P<0.05), and the estrogen values increased rapidly and reached maximumat stage Ⅳ. The research may provide a reference for understanding ovarian maturation regulation of O. fasciatus.

关 键 词：条石鲷 膜孕激素受体 基因克隆 表达 雌激素 

分 类 号：S917.4[农业科学—水产科学]