光唇鱼(Acrossocheilus fasciatus)Leptin基因克隆及禁食-再投喂对其表达的影响

瘦素(Leptin)是在动物摄食和能量代谢调节中具有重要作用的一种蛋白。为研究光唇鱼(Acrossocheilus fasciatus)中Leptin基因(AfLep)的结构和功能,作者克隆了其c DNA序列全长。结果显示AfLep由1315个核苷酸组成,含1个长度为516bp的开放阅读框,预测编码蛋白由171个氨基酸残基组成并具有长度为20aa的信号肽。系统进化树中AfLep与其他鲤科鱼类的Leptin蛋白聚为一簇,与齐口裂腹鱼(Schizothorax prenanti)进化相关性最高。多重序列比对显示AfLep与齐口裂腹鱼Leptin蛋白相似性最高(86.7%),与鲤科其他鱼类间的相似性均在80%以上。AfLep具有脊椎动物Leptin蛋白的4个保守?螺旋结构,其三级结构与人Leptin相似。AfLep在光唇鱼肝脏中的表达量最高,其次是脑和肾,其他组织中仅有微弱表达。实时荧光定量PCR检测显示禁食后光唇鱼肝脏AfLep表达量降低(P0.05),禁食1d、3d、7d时的表达量分别比与禁食前降低了58.25%、73.82%和92.05%;禁食1d和3d的鱼经再投喂后肝脏AfLep表达量均显著升高(P0.05),但禁食7d的鱼再投喂后AfLep表达量仅略有升高(P0.05)。上述结果表明AfLep参与了光唇鱼的摄食管理和能量代谢调控。

CLONING AND EXPRESSION OF LEPTIN GENE IN ACROSSOCHEILUS FASCIATUS DURING FASTING AND REFEEDING

Leptin plays an important role in regulation of food intake and energy expenditure in animals. To study the structure and function of Leptin gene in commercial fish Acrossocheilus fasciatus, we cloned the cDNA sequence of Leptin gene in A.fasciatus, named AfLep. The full-length of AfLep cDNA sequence was 1315bp, contained an open reading frame (ORF) of 516bp encoding 171 amino acids; and the initiative 20 amino acid consisted of signal peptide. In the phylogenetic tree, AfLep gathered with Leptin of other Cyprinidae fish and was most closely related to Leptin of Schizothorax prenanti. The multiple sequence alignment showed that AfLep shared the highest amino acid sequence identify (86.7%) with Leptin of S. prenanti, and had more than 80% sequence identity to other Cyprinidae fish. The predicted protein of AfLep has four a helices regions, which is conserved in vertebrates. The predicted tertiary structure of AfLep is similar to human Leptin. AfLep gene had the highest expression level in liver, followed by brain and kidney, and weakly expressed in other tissues. Real-time fluorescent quantitative PCR (RT-qPCR) showed that the expression of AfLep decreased significantly in liver after being fasted (P<0.05), and reduced by 58.25%, 73.82%, and 92.05% after fasting 1d, 3d, and 7d, respectively, compared to pre-fasting. When the fish were refed 1d and 3d after fasting, the expression level of AfLep in liver significantly increased (P<0.05), but in the group of fasting 7d the AfLep expression was slightly elevated after refeeding (P>0.05). All these results indicate that AfLep participates in the adjustment of feeding regulation and energy metabolism.