荷那龙罗非鱼ghrelin cDNA的克隆及表达特征

cDNA Cloning and Expression Characterization of Ghrelin of Zanzibar tilapia,Oreochromis hornorum

以荷那龙罗非鱼胃组织中提取的总RNA为模板,采用RT-PCR方法克隆出荷那龙罗非鱼ghrelin前体基因的cDNA全序列。所获得的ghrelin cDNA总长为833 bp,包括97 bp的5′非编码区,412 bp的3′非编码区和324 bp的开放阅读框,编码107个氨基酸残基。核苷酸序列同源性分析显示,荷那龙罗非鱼ghrelin与莫桑比克罗非鱼和尼罗罗非鱼ghrelin序列的相似性达99.0%以上。采用半定量RT-PCR方法检测雌、雄荷那龙罗非鱼胃、肌肉和垂体等多个组织中ghrelin基因的表达,结果显示,荷那龙罗非鱼ghrelin基因有广泛的组织分布,ghrelin基因在雌、雄鱼胃中的表达量均最高,在其他组织的表达量存在雌雄个体差异。雌鱼肌肉中ghrelin基因的表达量明显高于雄鱼,是雄鱼的132倍。试验鱼饥饿4周后,采用荧光定量RT-PCR方法检测胃中ghrelin基因表达量变化,结果表明:饥饿组较对照组ghrelin mRNA表达量有增加,饥饿组个体中ghrelin基因表达量最高的个体的表达量比对照组中表达量最低的个体增加了47.3倍。

Ghrelin precursor cDNA was identified from the stomach of Zanzibar tilapia,Oreochromis hornorum by RT-PCR.The total sequence of ghrelin was 833 bp in length,consisting of 97 bp of 5′-untranslated region（UTR）,412 bp of 3′-UTR and 324 bp open reading frame（ORF）encoding 107-amino-acids prepro-ghrelin.The nucleotide sequence identities among Zanzibar tilapia and other tilapia are above 99.0%.The expression of ghrelin genes in stomach,muscle and pituitary and some other tissues in male and female Zanzibar tilapia were detected by RT-PCR.The results showed that ghrelin was widely distributed in Zanzibar tilapia tissues.High expression levels were detected in stomaches of both female and male individuals,but there were different expression levels in other tissues detected between females and males.The expression level of ghrelin in female muscle was 132 times higher than in male＇s.The effect of fast on ghrelin expression in Zanzibar tilapia was also investigated using relative RT-PCR.The results indicated that the expression level of ghrelin in stomach increased in the fasting individual much more than in the normal feeding individual.The highest ghrelin expression level in fasting individual was 47.3 times than the lowest in normal ones.