银鲫crh基因的克隆、组织表达谱及其对摄食的影响

Cloning, tissue expression profiling, functional characterization of corticotropin-releasing hormone (crh) on feeding in gibel carp (Carassius auratus gibelio)

关键词:  银鲫  crh基因  RT-qPCR  基因表达  摄食调控

Key words:  Carassius auratus gibelio  crh gene  RT-qPCR  Gene expression  Feeding regulation

摘要:

为研究crh (Corticotropin-releasing hormone)基因对鱼类摄食的调控作用，首次克隆了银鲫(Carassius auratus gibelio) crh基因cDNA全长序列。运用实时荧光定量PCR(RT-qPCR)技术检测crh基因mRNA在不同组织的表达及餐前、餐后和禁食对其表达的影响。结果显示，银鲫crh基因的cDNA序列全长为920 bp，其中，5-UTR为53 bp，3-UTR为378 bp，开放阅读框(Open reading frame, ORF)为489 bp。推导银鲫crh基因的ORF区编码蛋白由162个氨基酸组成，其中，含有11个氨基酸的保守区，24个氨基酸的信号肽，41个氨基酸的成熟肽。氨基酸序列的多重比较分析显示，银鲫crh基因与金鱼(Carassius auratus)的同源性为99%，与鲤(Cyprinus carpio)的同源性为96%。荧光定量PCR表达分析显示，银鲫crh基因在下丘脑中的表达量最高，其次是端脑和心脏。crh基因的表达量在餐前与餐后没有显著变化(P>0.05)，在禁食第5天、第7天出现极显著降低(P<0.01)，而在复投喂后第9天、第11天出现极显著升高(P<0.01)。以上结果显示，crh基因在银鲫摄食调控方面可能扮演着重要角色。

Abstract:

To elaborate the mechanism of corticotropin-releasing hormone (crh) on feeding in gibel carp (Carassius auratus gibelio), we cloned and identified the full-length cDNA sequence of crh for the first time. We also employed real-time quantitative PCR (RT-PCR) to explored crh mRNAon distribution in different tissues and investigated the expression of crh mRNA during postprandial and fasting. Here, a 920 bp full-length of crh gene cDNA was obtained, including a 53 bp of 5-UTR, a 378 bp of 3-UTR, and a 489 bp of an open reading frame (ORF). The ORF of the gibel carp crh gene contains 162 bp nucleotides, including an 11-amino acid conserved region, a 24-amino acid signal peptide, and a 41-amino acid mature peptide. The amino acid sequence of crh shares a high level of similarity with teleost CRH (99% with Carassius auratus and 96% with Cyprinus carpio). The expression of gibel carp crh was observed in almost all tissues, with the highest expression detected in the hypothalamus, followed by the myelencephalon and heart. There was no significant change in crh gene expression in the fed group compared with the fasted group in gibel carp hypothalamus after a meal (P>0.05). However, the expression level of the crh gene showed a significant decrease after fasting on day 5 and day 7 (P<0.01), a significant increase of the crh gene was observed after refeeding on day 9 and day 11. The results suggest that the crh gene may play a critical role in feeding regulation in gibel carp.