草鱼Isthmin-1基因序列分析及转录水平的营养调控

SEQUENCE ANALYSIS AND NUTRITIONAL REGULATION OF ISTHMIN-1 IN GRASS CARP(CTENOPHARYNGODON IDELLA)

为探讨Isthmin-1(Ism-1)在草鱼糖和脂类代谢中的作用,研究采用RT-PCR技术克隆草鱼Ism-1的开放阅读框(ORF),生物信息学分析Ism-1及其编码的氨基酸序列,RT-qPCR技术检测Ism-1在草鱼各组织中的分布特点,并在细胞和活体水平上分析不同营养条件下Ism-1 mRNA的表达变化。结果显示,成功克隆草鱼Ism-1的ORF区。序列分析表明,草鱼Ism-1基因开放读码框为1380 bp,编码459个氨基酸,预测该蛋白相对分子量为50.96 kD。氨基酸多序列比对和系统进化树分析显示,草鱼Ism-1与黑头软口鲦(Pimephales promelas)的进化关系最近(氨基酸相似度为96.51%)。Ism-1在草鱼各组织中均有表达,在红肌中表达量最高,其次是鳃、脑和白肌等组织。饥饿再投喂实验结果表明,饥饿14d后肝胰脏中Ism-1的表达量显著上调(P<0.05),恢复投喂后表达量降低,但仍显著高于对照组(P<0.05),白肌中Ism-1的表达量在饥饿和再投喂后均显著增加(P<0.05)。腹腔注射不同浓度的胰高血糖素显著下调草鱼肝胰脏中Ism-1的mRNA水平(P<0.05),油酸孵育草鱼原代肝细胞24h时,Ism-1的表达量显著减少(P<0.001)。结果提示,草鱼Ism-1可能参与机体糖和脂类的代谢过程调控,这为后续研究Ism-1的功能提供了基础数据。

In order to explore the role of Isthmin-1(Ism-1)in glucose and lipid metabolism of grass carp,we cloned the open reading frame(ORF)of Ism-1 by RT-PCR,and the sequence was analyzed by bioinformatics technology.The distribution characteristics of Ism-1 in different tissues of grass carp were detected by RT-qPCR,and the expression of Ism-1 under different nutritional conditions were analyzed in vitro and in vivo.In this study,ORF region of Ism-1 was successfully cloned.Sequence analysis showed that the ORF region of Ism-1 was 1380 bp,encoding 459 amino acids,and the relative molecular weight of protein was 50.96 kD.Amino acid multiple sequence alignment and phylogenetic tree analysis showed that the evolutionary relationship of Ism-1 between grass carp and fathead minnow was the closest(amino acid similarity was up to 96.51%).Ism-1 was widely expressed in multiple tissues,and had higher expression in red muscle,next were gill,brain and white muscle of grass carp.The fasting and refeeding experiment indicated the expression of Ism-1 in hepatopancreas was significantly upregulated after 14 days of starvation treatment(P<0.05),and the expression decreased after refeeding,but was still significantly higher than that of control group(P<0.05).The expression of Ism-1 in white muscle was significantly up-regulated after starvation and refeeding(P<0.05).The results of intraperitoneal injection of glucagon experiment eventuated that the expression of Ism-1 in hepatopancreas was significantly downregulated compared with control group(P<0.05).In addition,the expression of Ism-1 was significantly decreased(P<0.001)in hepatocytes after treatment with 80μg/mL oleic acid for 24h.Overall,this research confirmed Ism-1 may be involved in the regulation of glucose and lipid metabolism of grass carp,which would provide essential data for the following study of the function of Ism-1.