大刺鳅生长分化因子2(GDF2)基因克隆及表达分析

Molecular Characteristics and Expression Analysis of GDF2 Gene in Mastacembelus armatus

研究大刺鳅生长分化因子2(growth differentiation factor 2,GDF2)基因与大刺鳅生长发育之间的关系。利用RT-PCR对大刺鳅生长发育相关基因GDF2进行了克隆并对其进行生物信息学分析;通过实时荧光定量PCR研究大刺鳅GDF2基因在肝、性腺、脑等9种组织中的表达情况;通过注射促黄体素释放激素(LHRH-A2)、绒毛膜促性腺激素(HCG)以及地欧酮(DOM)三种激素,研究大刺鳅GDF2基因在肝组织中表达量的变化。结果表明:大刺鳅GDF2基因cDNA长2053 bp,其中5’非编码区序列长145 bp,3’非编码区序列长645 bp,编码区(ORF)序列长1263 bp,编码420个氨基酸的多肽序列,通过生物信息学分析得出GDF2蛋白属于膜外蛋白,蛋白预测理论等电点(p I)为5.38,分子量(Mw)为47.97 kDa,存在潜在N-糖基化位点5个,在线分析软件预测GDF2氨基酸序列N端为信号肽序列。系统进化树分析表明,大刺鳅GDF2与黄鳝GDF2聚为一支,亲缘关系最为接近。实时荧光定量PCR结果显示GDF2基因在肝组织中表达量最高,其次为心、鳃、肠、脾,在脑组织中的表达量最低。LHRH-A2、DOM、HCG三种外源激素注射后,对大刺鳅GDF2 mRNA在肝组织中表达均具有一定的促进作用。研究结果表明GDF2基因主要参与大刺鳅的肝脏组织生长发育调控,基因表达量还受不同类型催产激素的诱导的影响。

The aim of this study was to investigate the relationship between growth differentiation factor 2(GDF2)gene and the growth and development of Mastacembelus armatus.The growth and development related gene GDF2 of M.armatus was cloned by PCR and analyzed by bioinformatics.The length cDNA of GDF2 gene from M.armatus was 2053 bp,the 5'non-coding region is 145 bp,the 3'non-coding region is 645 bp,the open reading frame(ORF)is 1263 bp,and the polypeptide sequence encoding 420 amino acids.According to the bioinformatics analysis,the GDF2 gene belongs to the outer membrane protein,and the isoelectric point(p I)of the protein prediction theory is 5.38,and the molecular weight(MW)is 47.97 kDa.There are 5 potential n-glycosylation sites.The cutting site of GDF2 amino acid sequence is between 24 cysteine(Cys)and 25 lysine(Lys).Phylogenetic tree analysis showed that GDF2 of Mastacembelus armatus and Monopterus albus had the highest similarity in amino acid sequence and the closest genetic relationship.The expression of GDF2 gene was highest in liver tissue,followed by heart,Gills,intestine and spleen,and lowest in brain tissue.After injection of Luteinizing Hormone Releasing Hormone A2(LHRH-A2),domperidone(DOM)and Chorionic Gonadotrophin(HCG)hormones,the expression of GDF2 mRNA in liver of M.armatus was significantly promoted.In conclusion,the expression of GDF2 gene is mainly involved in the regulation of liver growth and development of M.armatus,and the gene expression was also affected by the induction of exogenous hormones.