半滑舌鳎促黄体激素基因克隆和表达分析及其血清浓度测定  

Full length cDNA cloning and expression of luteinizing hormone (LH) and which serum concentration was measured in half smooth tongue sole Cynoglossus semilaevis Günther

作　　者：柳学周[1] 史宝[1] 王珊珊[1] 徐永江[1] 李晓晓[1] 

机构地区：[1]中国水产科学研究院黄海水产研究所,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,山东青岛266071

出　　处：《中国工程科学》2014年第9期50-60,共11页Strategic Study of CAE

基　　金：现代农业产业技术体系建设专项资金(CARS-50);国家自然科学基金项目(31201982);国家国际科技合作专项项目(2013DFA31410);山东省优秀中青年科学家科研奖励基金项目(BS2013SW042);2012年度留学人员科技活动项目择优资助经费资助

摘　　要：利用RACE技术首次克隆了半滑舌鳎脑垂体中促黄体激素(LH)基因cDNA全长序列。该基因全长670 bp,开放阅读框为477 bp,编码158个氨基酸。与其他脊椎动物的LH成熟肽氨基酸序列同源性比较表明:半滑舌鳎LH与鲽形目和鲈形目同源性高58%～68%。另外,半滑舌鳎LH含有12个保守的半胱氨酸残基和1个N-糖基化位点(19～21 NQT)。实时荧光定量组织表达分析表明,LH mRNA在脑、垂体、卵巢等组织中表达,垂体中表达量最丰富,其他组织尤以脑、性腺表达量较高,推测半滑舌鳎LH可能具有广泛的垂体外生理功能;实时荧光定量PCR方法对繁殖周期雌性半滑舌鳎LH表达水平进行测定,结果表明,LH mRNA在Ⅱ～Ⅵ各繁殖周期的脑、垂体、卵巢3种组织都有表达,但表达水平有差异,在Ⅳ、V期表达量最丰富,说明LH主要促进卵母细胞最终成熟及排卵。利用125I标记的放射免疫测定(RIA)技术检测繁殖周期半滑舌鳎血清LH浓度,结果表明LH在V期含量最丰富,也说明LH主要促进卵母细胞最终成熟及排卵。The full length cDNA of LH has been cloned from pituitary of half smooth tongue sole C. Gunther by using RACE technology for the first time. The cDNA of LH is 670 bp,and the open reading frame is 477 bp encoding peptides of 158 amino acid. Multiple amino acid se-quences alignments were performed among vertebrates,and the results showed that tongue sole LH mature peptide was the most similar to their orthologs of Pleuronectiformes and Perciformes which was 58%~68%identity. And 12 conserved cysteine residues and 1 N-linked glycosyl-ation site were found in the deduced amino acid sequence. Real-time PCR analysis showed that LH mRNA was expressed in all twelve tissues including brain,pituitary,ovary and so on. The highest expression level was in pituitary. And the stronger amplification signals were detected in the brain and gonad than all other non-pituitary tissues. It suggested that LH may had the more physiological functions because LH mRNA of tongue sole expressed widely in non-pitu-itary tissues. Quantitative real-time PCR was used for analyzing the expression of female C. semilaevis LH mRNA in the reproductive cycle. The results showed that LH mRNA was ex-pressed in all three tissues including brain,pituitary and ovary from the stage Ⅱto stage Ⅵ, and the highest expression level appeared on the stageⅣ and stageⅤwhich suggested that the main function of LH is to promote eggs finally mature and discharge. Radio immunoassay （RIA）marked by 125I was used to detect the concentration of plasma LH in C.semilaevis. The re-sults showed that the biggest plasma LH concentration appareed on the stage of Ⅴwhich also suggested that the main function of LH was to promote eggs finally mature and discharge.

关 键 词：半滑舌鳎 促黄体激素(LH) CDNA克隆 实时荧光定量PCR 放射免疫测定 

分 类 号：S96[农业科学—水产养殖]