瓦氏黄颡鱼生长激素基因克隆及其组织特异性表达分析

Cloning and differential expression of growth hormone in Pelteobagrus vachelli

生长激素(growth hormone,GH)对脊椎动物的生长发育及代谢具有重要作用。采用RT-PCR和RACE技术,克隆了瓦氏黄颡鱼垂体GH cDNA全长序列,应用real-time qPCR法对不同组织中GH mRNA的表达进行检测。序列分析表明,GH cDNA(GenBank登录号:GU395549)序列全长1 203 bp,其5'端非编码区77 bp、3'端非编码区523 bp,开放阅读框(open reading frame,ORF)603 bp,由此推导GH前体蛋白由200个氨基酸组成。同源性比较结果表明,瓦氏黄颡鱼与同目鱼类的GH编码序列同源性较高,与哺乳类和鸟类的同源性较低。Real-time qPCR结果显示,GH mRNA在垂体中的表达量最高,其次是脑、肌肉、肝脏、脂肪组织、胃、脾脏、卵巢或精巢,而在肾脏、心脏、鳃和肠中没有明显的表达。实验结果表明,GH基因在瓦氏黄颡鱼组织中广泛表达,提示GH可能以旁分泌或自分泌的方式对其生长和繁殖发挥重要作用。

Growth hormone（GH） is a pluripotent hormone produced by the pituitary gland in teleosts as in other vertebrates.It plays important roles in the growth,development and metabolism of vertebrates.In this study,to characterize GH in fish and to better understand the potential applications of GH in fish cul-ture,we used reverse transcription-ploymerase chain reaction（RT-PCR） and rapid amplification of cDNA ends（RACE） to obtain the full-length cDNA sequence encoding GH from the pituitary of darkbarbel cat-fish Pelteobagrus vachelli（P.vachelli）,a freshwater fish of high commercial value cultured in China.GH precursor cDNA（GenBank：GU395549） consists of 1 203 bp in size with an open-reading frame（ORF） encoding 200 amino acid,a 77 bp 5＇-untranslated region and a 523 bp 3＇-untranslated region.The four Cys residues are located at conserved positions（Cys71,Cys173,Cys190,Cys198）,and one possible site for N-glycosylation（Asn-Xaa-Ser/Thr,motif） is present at Asn197.Based on multi-sequence alignments and phylogenetic analysis,the P.vachelli GH precursor shared a higher degree of homology（averaged 95%） with other species of fish belonging to the Order Siluriformes,but lower levels of homology between P.vachelli and the mammalian（averaged 34%） and bird（averaged 41%）.The above results reveal that GH is conserved during evolution and is suitable for phylogenetic relationship analysis of Siluriformes.By real-time quantitative PCR,tissue distribution profile showed that GH mRNA signals were all observed in pituitary,brain,muscle,liver,adipose tissue,stomach,spleen,ovary and testis.GH mRNA in pituitary was the most abundant and no significant GH mRNA expression was detected in kidney,heart,gill and intestines.The expression patterns of P.vachelli and previously studied animal GH suggest that GH through autocrine or paracrine pathway exerted its possible important local physiological function during development and growth of P.vachelli.These findings will contribute to the understanding of the evolution of GH and provide some basic information about the characterization of GH in P.vachelli.