半滑舌鳎新型膜孕激素受体基因分子特征及其在卵巢发育过程的作用  

Molecular characterization of the novel membrane progestin receptor gene and its role during ovarian development in the half smooth tongue sole Cynoglossus semilaevis Günther

作　　者：柳学周 史宝 李晓晓[1,2,3] 徐永江 王珊珊 李晓妮 

机构地区：[1]农业部海洋渔业可持续发展重点实验室 [2]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,山东青岛266071 [3]上海海洋大学水产与生命学院,上海201306

出　　处：《中国水产科学》2015年第4期608-619,共12页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(31201982);国家鲆鲽类产业技术体系项目(CARS-50);山东省优秀中青年科学家科研奖励基金项目(BS2013SW042);留学人员科技活动项目择优资助经费项目

摘　　要：为进一步提高半滑舌鳎(Cynoglossus semilaevis Günther)人工育苗技术水平,对半滑舌鳎新型膜孕激素受体(m PR-like)基因进行研究。采用同源克隆和末端快速扩增(RACE)方法,获得全长为2 002 bp的半滑舌鳎新型膜孕激素受体的c DNA序列;二级结构分析表明,该蛋白存在7个跨膜区域;三级结构分析表明该蛋白有多个结合位点。使用MEGA4.0临位相联法和Clustal X方法对m PR-like的氨基酸序列进行聚类分析和序列相似度分析,发现半滑舌鳎m PR-like与青鳉(Oryzias latipes)和三刺鱼(Gasterosteus aculeayus)的m PR-like聚为一支,相似度分别为68%和72%。应用实时定量PCR方法分析了m PR-like m RNA表达情况,发现m PR-like m RNA在性成熟雌性半滑舌鳎不同组织表达具有广泛性,但表达量存在差异,在脑、卵巢、心脏、鳃、脾和胃等组织表达丰富;在不同发育阶段卵母细胞中,m PR-like m RNA表达水平从II时相卵母细胞到V时相卵母细胞持续升高,而在VI时相卵母细胞的表达水平显著下降(P<0.05);在半滑舌鳎繁殖周期的脑和卵巢组织中,m PR-like m RNA的表达水平从性腺发育II期到V期持续升高,并且在V期达到最高峰(P<0.05),VI期表达量开始下降;在半滑舌鳎繁殖周期的垂体组织中,不同繁殖期m PR-like m RNA表达水平变化幅度不大,但在性腺发育V期时垂体中的表达量显著高于其他繁殖期(P<0.05)。放射免疫测定的结果表明,半滑舌鳎雌鱼血清中孕酮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IV期含量迅速升高并在V期达到峰值。该基因在半滑舌鳎多种组织中的表达特征,预示其具有多种生理学作用;在卵母细胞和繁殖周期脑–垂体–卵巢的表达特征表明其参与卵母细胞成熟过程和生殖调控。To improve techniques of artificial breeding, this research focused on the novel membrane progestin receptor （mPRL） of half smooth tongue sole Cynoglossus semilaevis Gunther. The full-length cDNA encoding mPRL was cloned from the half smooth tongue sole by means of homology cloning and RACE PCR analyses. The complete cDNA sequence of mPRL （GenBank accession number： KF277065） was 2 002 bp in length, consisting of a 5′ untranslated region （UTR） of 163 bp, a coding sequence of 1 056 bp, and a 3′UTR of 783 bp. The molecular weight of mPRL was 17.62 kD. Structural analysis of the translated cDNA suggested that it encoded membrane protein with seven trans-membrane domains. Tertiary structure of the mPRL protein showed that it had several binding sites. The rooted phy-logenetic tree was constructed using the neighbor-joining method on MEGA4.0 for comparative analysis of half smooth tongue sole mPRL and selected vertebrate sequences. The similarity （%, identity） of half smooth tongue sole mPRL in comparison with other representative sequences was analyzed using Clustal X. The results indicated that the half smooth tongue sole mPRL was clustered with mPRL of other fish. Half smooth tongue sole had 68% identity with Japanese medaka （Oryzias latipes） and 72% with three-spined stickleback （Gasterosteus aculeatus）. The mPRL mRNA expression in half smooth tongue sole was detected by quantitative real-time PCR. And found to be widely, although not homogeneously expressed. mPRL transcripts were highly abundant in the brain, ovary, heart, gill, spleen, and stomach. In addition, positive signals were obtained for mPRL mRNA in the pituitary gland, muscle, and intestine, al-though the resulting transcript levels were lower than in the ovary. Representative sections of ovaries showed the mor-phological characteristics during development, which was divided into five stages. The expression level of mPRL mRNA at various stages of oogenensis in the sole showed that the level of mPRL mRNA in the brain and ova

关 键 词：半滑舌鳎 新型膜孕激素受体 基因克隆 表达 孕酮激素 

分 类 号：Q959[生物学—动物学] S917[农业科学—水产科学]