花鲈瘦素基因的克隆及低盐度条件下调控表达分析

Cloning and expression analysis of Leptin A gene in seabass Lateolabrax maculatus exposed to low salinity

瘦素(Leptin,Lep)在动物摄食、生长、生殖、免疫、能量平衡等方面具有重要作用,为研究花鲈Lateolabrax maculatus Lep功能,采用c DNA末端快速扩增法(RACE)克隆花鲈Lep A c DNA全长序列。急性低盐度调控试验设置海水组(盐度32)、半海水组(盐度16)和淡水组(盐度0),盐度调控24、48、96、144、192 h后测定花鲈肝脏中Lep A mRNA的表达情况。结果表明:Lep A c DNA全长序列为643 bp,其中开放阅读框483 bp,编码161个氨基酸,其氨基酸序列由信号肽和A、B、C、D 4个α螺旋区域组成;花鲈Lep A氨基酸序列与其他物种同源性较低,但三级结构较为保守;花鲈Lep A主要在肝脏中表达,其次为脑;低盐调控48 h时,半海水组与淡水组Lep A表达无显著性差异(P>0.05),但均显著低于海水组(P<0.05),分别为海水组的33.38%和26.86%。研究表明,急性低盐度调控可降低花鲈Lep A表达水平。

cDNA sequence of Leptin A （LepA） gene, which plays key roles in food intake, growth, reproduction, immunity and energy balance, was cloned by rapid amplification of cDNA ends （RACE） in seabass Lateolabrax maculatus exposed to acute salinity changes： seawater group（a salinity of 32） , semi-seawater group （a salinity of 16） and fresh water group （a salinity of 0）. Hepatic LepA mRNA were determined by qPCR 24, 48, 96, 144, amt 192 h after salinity exposure. It was found that LepA cDNA was 643 bp in length containing a CDs of 161 amino acid residules, a signal peptide, and four （x-helices （A, B, C and D）. Deduced amino acid sequencing showed that the sequence of LepA showed low identity to Leps from other fishes, with conservative three-dimensional struc- ture modeling. LepA was primarily expressed in liver, and a little expression of the LepA was observed in brain. There was significantly less expression of LepA mRNA in seabass in semi-seawater group （accounting for 33.38% of seawater group expression） and fresh water group （accounting for 26.86% of seawater group expression） than that in seawater group at 48 h （P〈0.05）, without significant difference between the semi-seawater group and fresh water group （ P〉0.05 ）. LepA mRNA expression was induced to be decreased in all groups after 96 h. The findings suggested that expression of LepA mRNA in seabass be decreased under acute low salinity regulation.