大刺鳅生长激素基因克隆与表达分析  

Cloning and expression analysis of growth hormone gene in Mastacembelus armatus

作　　者：钟东明 黄小琪 何佩莹 周惠强[1] 张明清[1] 舒琥[1] ZHONG Dong-ming;HUANG Xiao-qi;HE Pei-ying;ZHOU Hui-qiang;ZHANG Ming-qing;SHU Hu(School of Life Science,Guangzhou University,Guangzhou 510006,China)

机构地区：[1]广州大学生命科学学院

出　　处：《南方农业学报》2020年第1期194-201,共8页Journal of Southern Agriculture

基　　金：中国—东盟海上合作基金项目(外财函〔2017〕513号);广东省海洋渔业科技攻关与研发项目(A201601A05号);广东省海洋和渔业发展专项(2017A0007号)~~

摘　　要：【目的】揭示生长激素(GH)是否能以旁分泌或自分泌方式对大刺鳅(Mastacembelus armatus)的生长和发育起作用,为其生长调节研究积累基础数据。【方法】采用RACE克隆大刺鳅GH基因cDNA序列,并通过实时荧光定量PCR对大刺鳅不同组织及胚胎不同发育时期GH基因的表达情况进行分析。【结果】大刺鳅GH基因c DNA序列全长815 bp,其中,5’端非编码区长33 bp,3’端非编码区长167 bp,开放阅读框(ORF)615 bp,共编码204个氨基酸,GenBank登录号MH885947;大刺鳅GH氨基酸序列的前17个氨基酸残基组成信号肽,也存在4个保守的半胱氨酸残基(69Cys、177Cys、194Cys和202Cys)。大刺鳅GH蛋白分子量为23007.33 Da,理论等电点(pI)为6.90,其氨基酸组成中以亮氨酸(Leu)的含量最高(15.7%),丝氨酸(Ser)次之(14.2%),色氨酸(Trp)含量最低(0.5%)。大刺鳅GH氨基酸序列与斑鳜(Siniperca scherzeri)、花鲈(Lateolabrax maculatus)和黄鳝(Monopterus albus)的同源性较高,对应的同源性分别为95.59%、95.07%和92.16%;在基于GH氨基酸序列同源性构建的系统发育进化树中,大刺鳅与同属于合鳃目的黄鳝聚为一支,亲缘关系最近。GH基因在大刺鳅不同组织中均有表达,其中以脑组织中的相对表达量最高,而肠道中的相对表达量最低;GH基因在大刺鳅胚胎各发育时期也均有表达,其中以囊胚期的相对表达量最高,而出膜期的相对表达量最低。【结论】大刺鳅GH基因具有高度的保守区域,在各组织中广泛表达,尤其在脑组织中高表达,说明GH是以自分泌或旁分泌方式对大刺鳅生长发挥重要作用,且在胚胎发育过程中对细胞分化具有促进作用。【Objective】To investigate whether growth hormone(GH)played a paracrine or autocrine role in the growth and development of Mastacembelus armatus,which could provide the essential basic information for growth-related research of the M.armatus.【Method】In this experiment,the full-length c DNA sequence encoding GH of the M.armatus was cloned through the RACE techniques.Real-time fluorescence quantitative PCR was used to analyze the GH gene expression in different tissues and at different embryo development stages.【Result】The full length cDNA of GH gene was815 bp with 33 bp 5’-untranslated region,167 bp 3’-untranslated region,and 615 bp open reading frame(ORF).It encoded 204 amino acids(GenBank number:MH885947).The first 17 amino acid residues of GH amino acid sequence formed signal peptide,and there were also 4 conserved cysteine residues(69 Cys,177 Cys,194 Cys and202 Cys).The molecular weight of GH protein was 23007.33 Da,and the theoretical isoelectric point(pI)was 6.90.Among the amino acid components,content of leucine(Leu)was the highest(15.7%),content of serine(Ser)was the second(14.2%),and content of tryptophan(Trp)was the lowest(0.5%).The GH amino acid sequence of the M.armatus had high homology with Siniperca scherzeri,Lateolabrax maculatus,and Monopterus albus,with corresponding homology of 95.59%,95.07%and92.16%,respectively.On the phylogenetic tree based on GH amino acid sequence homology,M.armatus and the M.albuswere clustered into one branch,their genetic relation was the closest.The results of RT-qPCR showed GH mRNA was expressed in different tissues andat all embryonic stagesof the M.armatus,that the expression of GH mRNA was the highest in the brain tissue and the lowest in intestine.During the embryonic development period,the expression of GH mRNA was the highest at blastocyst stage and the lowest at membrane production stage.【Conclusion】The GH gene of M.armatusalso has a highly conserved region and is widely expressed in various tissues,especially in brain tissues.It is suggested

关 键 词：大刺鳅 生长激素(GH) 基因克隆 保守区域 系统发育进化树 表达分析 

分 类 号：S965.199[农业科学—水产养殖]