圆斑星鲽促性腺激素释放激素基因克隆及表达特性  

Cloning and expression characteristics of gonadotropin releasing hormone genes in spotted halibut Verasper variegatus

作　　者：柳学周[1,2] 徐永江[1,2] 廖梅杰[1,2] 潘传燕[3] 王妍妍[1,2] 

机构地区：[1]中国水产科学研究院黄海水产研究所,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 [2]农业部海洋渔业可持续发展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]中国海洋大学水产学院,山东青岛266003

出　　处：《中国水产科学》2013年第1期12-24,共13页Journal of Fishery Sciences of China

基　　金：国家鲆鲽类产业技术体系项目(CARS-50);国家863计划项目(2012AA10A413);中央级公益性事业单位基本科研业务费项目(20603022012022);国家留学人员科技活动项目择优资助经费资助项目

摘　　要：采用RACE技术,在圆斑星鲽(Verasper variegatus)脑中克隆到3种GnRH基因的cDNA序列:cGnRH-Ⅱ、sGnRH和sbGnRH。每种GnRH都包括1个信号肽、1个Gly-Lys-Arg连接序列和1个GnRH相关肽。其中,cGnRH-Ⅱ的cDNA全长为568 bp,编码85个氨基酸,ORF为255 bp,5′UTR为141 bp,3′UTR为169 bp。sGnRH的cDNA全长为457 bp,编码90个氨基酸,ORF为270 bp,5′UTR为41 bp,3′UTR为143 bp。sbGnRH的cDNA全长为381 bp,编码98个氨基酸,ORF为294 bp,5′UTR为48 bp,3′UTR为36 bp。分析了3种GnRH基因的编码氨基酸序列与其他脊椎动物的同源性,圆斑星鲽GnRH与鲽形目鱼类氨基酸同源性最高,其次为鲈形目鱼类。对3种GnRH基因的系统进化分析表明,圆斑星鲽GnRH基因与其他鲽形目鱼类亲缘关系最近,其次为鲈形目、鲑形目和鳗鲡目鱼类。荧光定量PCR分析表明,3种GnRH基因都在脑中表现出最高表达水平且具有性别特异性表达模式:雌性中不同组织的GnRH mRNA表达水平都相应高于雄性。组织表达分析表明,cGnRH-Ⅱ的mRNA仅在脑中表达,而sbGnRH在各个组织都有表达,sGnRH仅在脑、垂体和性腺中表达。脑中sbGnRH mRNA的表达水平在卵巢成熟过程中变化显著(P<0.05),而其他两种GnRH的mRNA表达水平变化不显著(P>0.05)。本研究首次在圆斑星鲽脑中克隆到3种GnRH基因,其组织和季节表达水平变化表明sbGnRH可能是圆斑星鲽生殖调控的关键GnRH类型,本结果可为圆斑星鲽生殖调控机制和人工繁育技术研究提供理论支撑。We cloned and sequenced the gonadotropin-releasing hormone（GnRH） genes from spotted halibut（Verasper variegatus）by isolating their cDNAs.This species expressed three molecular forms of GnRH in the brain： chicken type GnRH-II（cGnRH-II）,seabream type GnRH（sbGnRH）,and salmon type GnRH（sGnRH）. Each GnRH cDNA encoded a signal peptide（SP）,GnRH,and a GnRH-associated peptide（GAP）,which was connected to GnRH by a Gly–Lys–Arg sequence.The cGnRH-II consisted of 85 amino acid residues,and the cGnRH-II cDNA was 568 bp in length,consisting of a 141 bp 5′UTR,a 255 bp open reading frame（ORF）,and a 169 bp 3′UTR.The sGnRH consisted of 90 amino acid residues and was 457 bp in length.It consisted of a 41 bp 5′UTR,a 270 bp ORF,and a 143 bp 3′UTR.The sbGnRH precursor consisted of 98 amino acid residues,and the cDNA was 381 bp in length and consisted of a 48 bp 5′UTR,a 294 bp ORF,and a 36 bp 3′UTR. Homology analysis of the deduced acid residues of the GnRH precursors revealed that the three spotted halibut GnRH genes had the highest identity with homologs from pleuronectiformes,followed by perciformes,salmoniformes, and anguilliformes.Phylogenetic analysis indicated that the multiple molecular forms of spotted halibut GnRHs shared the closest evolutionary relationship with counterparts from pleuronectiformes.The tissue and sex specific expression of these three genes was determined using real time PCR.Expression was highest in the brain for both sexes.cGnRH-II was detected exclusively in the brain whereas sbGnRH was expressed globally in both sexes.sGnRH was detected in the pituitary and gonad（in addition to the brain tissue） in females,but not in the gonad of males.The expression pattern of all three GnRH genes was sex-specific with females having higher mRNA levels than males.We evaluated the changes in GnRH mRNA expression in the female brain during ovary maturation.sbGnRH mRNA levels increased significantly（P0.05） after August（vitellogenic stage）,peaked in December（pre

关 键 词：圆斑星鲽 GNRH 分子克隆 表达特性 

分 类 号：S917[农业科学—水产科学]