草鱼ghrelin基因的分子克隆与组织分布及其摄食调控作用分析

Molecular cloning,tissue distribution and food intake regulatory effects of ghrelin in grass carp(Ctenopharyngodon idellus)

ghrelin是一种在脊椎动物摄食调节过程中起重要作用的脑肠肽,具有明显的摄食促进作用。实验利用同源克隆技术获得了草鱼ghrelin基因的cDNA序列和DNA序列,其中cDNA序列全长506 bp,包括90 bp的5′端非编码区(5′-untranslated region,5′UTR),312 bp的开放阅读框(open reading frame,ORF),以及104 bp的3′端非编码区(3′-untranslated region,3′UTR)。开放阅读框编码的103个氨基酸的ghrelin前体肽,经剪切加工后形成含有19个氨基酸的成熟肽。氨基酸序列分析结果显示,草鱼ghrelin与硬骨鱼类ghrelin相似度最高,而与其他脊椎动物相似度较低,同时草鱼ghrelin成熟肽N端的"活性中心"(active core)为鲤科鱼类中常见的GTSF形式。与大多数硬骨鱼类的ghrelin基因结构相同,草鱼ghrelin基因也包括4个外显子和3个内含子。荧光定量PCR检测到ghrelin mRNA大量分布于草鱼的前肠和脾,脑、肾、肝、肌肉、皮和鳔等组织也有ghrelin mRNA分布。草鱼脑和肠中的ghrelin表达水平在摄食后下降,随着饥饿时间的延长表达水平逐步升高,最后维持在较高水平,表明ghrelin作为摄食启动信号对草鱼的摄食活动起到了促进作用。

Ghrelin is one of vital brain-gut peptides involved in feeding regulation in vertebrates,and is well known for its appetite-stimulating capacity.Nucleotide sequences of ghrelin gene were identified both at DNA and cDNA levels,using homology-based cloning techniques from grass carp（Ctenopharyngodon idellus）.The 506 bp full-length ghrelin cDNA consists of a 90 bp 5′-untranslated region,a 312 bp open reading frame and a 104 bp 3′-untranslated region.The 103-amino acid prepropeptide encoded by open reading frame turns into a mature peptide with 19 amino acids after posttranslational modification.putative amino acid sequence analysis among vertebrates indicated that grass carp ghrelin showed the highest sequence homology with teleosts,and lower homology with other vertebrates.Comparison of the mature amino acid sequences revealed a conserved biological active core of ghrelin in grass carp.Genomic organization of grass carp ghrelin was composed of four exons and three introns,which was similarto that of most of teleostean species.qPCR results revealed that ghrelin was highly expressed in the fore-gut and spleen with much lower levels of expression in other tissues including brain,kidney,liver,muscle,skin and air bladder.The expression pattern that decreased in a short period of time after satiation,increased thereafter as starved time went on and kept at a high level implicated ghrelin in the orexigenic effect,as a signal of meal initiation,on short-term dietary intake in grass carp.