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黄颡鱼生长抑素基因的克隆及表达谱分析
梁宏伟1,2, 曹 磊1,3, 李 忠,等1,3
1.中国水产科学研究院 长江水产研究所;2.淡水水产健康养殖湖北省协同创新中心;3.华中农业大学 水产学院
摘要:
【目的】克隆黄颡鱼生长抑素(Somatostatin,SS)基因cDNA全长序列,分析其在黄颡鱼胚胎发育、胚后发育阶段及成鱼各个组织中的表达规律。【方法】以黄颡鱼脑组织为材料,根据瓦氏黄颡鱼和斑点叉尾鮰SS基因保守区设计1对引物用于扩增SS基因中间保守区序列,再根据中间保守区序列设计2对引物,分别用于扩增5′端和3′端序列,将扩增得到的中间保守区序列、5′端和3′端序列拼接后得到黄颡鱼SS基因的cDNA全长序列,并对其进行生物信息学分析。用NCBI的Protein Blast对黄颡鱼与其他物种SS基因编码的氨基酸序列同源性进行比较,并构建系统发育树。用实时荧光定量RT-PCR检测SS基因在胚胎发育时期和胚后发育阶段(1~20 d)的表达特征。采用半定量RT-PCR检测SS基因在黄颡鱼脑、心脏、肌肉、胃、肝脏、肾脏、鳃、脾脏、性腺等组织中的表达特征。【结果】黄颡鱼SS基因cDNA全长694 bp,其中5′端非翻译区139 bp,3′端非翻译区211 bp,开放阅读框为345 bp,编码114个氨基酸。黄颡鱼与瓦氏黄颡鱼SS基因编码氨基酸同源性最高,为98%。系统发育树结果显示,黄颡鱼与同属于鲇形目的瓦氏黄颡鱼和斑点叉尾鮰聚为一支。SS基因在黄颡鱼受精卵时期就有表达,并持续至胚胎孵化出膜,且在心跳期和出膜期的表达量显著高于受精卵时期(P<0.05);在出膜后的1~20 d,SS基因在黄颡鱼中稳定表达;在成鱼的各个组织中,SS基因只在脑组织中特异性表达。【结论】初步探明了SS基因在黄颡鱼胚胎发育、胚后发育阶段及成鱼各组织中的表达规律,推测其可能在黄颡鱼胚胎发育中发挥着重要的生理功能。
关键词:  黄颡鱼  生长抑素  基因克隆  表达特征
DOI:
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基金项目:国家“863”计划项目(2011AA100401);国家水产种质资源平台项目
Cloning and expression analysis of somatostatin gene in Pelteobagrus fulvidraco
LIANG Hong-wei,CAO Lei,LI Zhong,et al
Abstract:
【Objective】The objectives of this experiment were to clone somatostatinSS) gene sequence of Pelteobagrus fulvidraco and analyze the sequence and its expression characterization.【Method】A pair of primers were designed based on conserved domain of SS gene of Pelteobagrus vachellii and Ictalurus punctatus.The conserved domain of SS gene was cloned from Pelteobagrus fulvidraco brain total RNA by PCR method.Then two pairs of specific primers based on their middle conserved sequences were used to obtain 5′and 3′end sequences.The full sequence of SS cDNA of Pelteobagrus fulvidraco was obtained by combining the middle conserved fragment with 5′and 3′end sequences.The homologous analysis was performed based on encoding amino acid sequences of Pelteobagrus fulvidraco and other fishes using online Protein Blast,and Phylogenetic tree was constructed by MEGA 5.0.Real-time PCR was used to analyze the expression patterns during the embryonic development stage and postembryonic development stage of SS gene (1-20 d).Distribution and expression levels of SS gene in tissues including brain,heart,muscle,stomach,liver,kidney,gill,spleen and gonad were analyzed by semi-quantitative RT-PCR.【Result】The full length of SS cDNA in Pelteobagrus fulvidraco was 694 bp,containing 139 bp 5′-untranslated region and 211 bp 3′-untranslated region.The length of open reading frame (ORF) was 345 bp encoding 114 amino acids and had high homology with Pelteobagrus vachellii (98%).The phylogenetic analysis showed that Pelteobagrus fulvidraco,Pelteobagrus vachellii and Ictalurus punctatus were from one group.The somatostatin gene was expressed in the whole embryonic development stage and postembryonic development of 20-day old juveniles.The expression levels in the heart beat stage and hatch out stage were significantly higher than in fertilized egg (P<0.05).SS mRNA was only detected in brain of adult Pelteobagrus fulvidraco.【Conclusion】The expression patterns of somatostatin gene were preliminarily proved and somatostatin gene may play an important role in postembryonic development of Pelteobagrus fulvidraco.
Key words:  Pelteobagrus fulvidraco  somatostatin  gene clone  expression characterization