Abstract：
The Chinese alligator (Alligator sinensis) is a freshwater crocodilian endemic to China. So far, the endocrine regulation of feeding and growth in Chinese alligator is poorly understood. In this study, the molecular structure and tissue expression profiles of ghrelin and its receptor GHSR in the Chinese alligator were characterized for the first time. The full-length cDNA of ghrelin was 1770 bp, including a 37 bp 5 \'-UTR (untranslated region), a 435 bp ORF (open reading frame) and a 1298 bp 3 \'-UTR. The ORF encodes a ghrelin precursor, which consists of 145 amino acid residues, including a signal peptide with 52 amino acid residues at the N-terminus, a mature peptide with 28 amino acid residues, and a possibly obestain at the C-terminus. The full-length cDNA of GHSR was 3961 bp, including a 5\'-UTR of 375-bp, an ORF of 1059-bp and a 3\' -UTR of 2527-bp. The ORF encodes a protein of 352 amino acid residues containing seven transmembrane domains, with multiple N glycosylation modification sites and conserved cysteine residue sites. The active core \"GSSF\" of Chinese alligator ghrelin was identical to that of mammals and birds, and the ghrelin binding site of GHSR was similar to that of mammals. The amino acid sequences of both ghrelin and GHSR share high identity with American alligator (Alligator mississippiensis) and birds. Ghrelin was highly expressed in cerebrum, mesencephalon, hypothalamus and multiple peripheral tissues, including lung, stomach and intestine, suggesting that it could play functions in paracrine and/or autocrine manners in addition to endocrine manner. GHSR expression level was higher in hypothalamus, epencephalon and medulla oblongata, and moderate in multiple peripheral tissues including lung, kindey, stomach and oviduct, implicating that ghrelin/GHSR system may participate in the regulation of energy balance, food intake, water and mineral balance, gastrointestinal motility, gastric acid secretion and reproduction. During hibernation, the expression of ghrelin and GHSR in the brain was significantly increased, while ghrelin was significantly decreased in heart, liver, lung, stomach, pancreas and ovary, and GHSR was significantly decreased in heart, liver, spleen, lung, kindey, stomach, ovary and oviduct. These temporal changes in ghrelin and GHSR expression could facilitate the physiological adaption to the hibernation of Chinese alligator. Our study could provide basic data for further studies on the regulation of feeding, physiological metabolism and reproduction of Chinese alligator, which could also be useful for the improvement of artificial breeding of this endangered species.
摘要：
扬子鳄（Alligatorsinensis）是中国特有的淡水鳄鱼。到目前为止,对扬子鳄摄食和生长的内分泌调节知之甚少。在这项研究中,首次对扬子鳄ghrelin及其受体GHSR的分子结构和组织表达谱进行了表征。ghrelin的cDNA全长为1770bp,包括一个37bp的5'-UTR（非翻译区），435bpORF(开放阅读框)和1298bp3'-UTR。ORF编码生长素释放肽前体,由145个氨基酸残基组成，包括在N末端具有52个氨基酸残基的信号肽，具有28个氨基酸残基的成熟肽,在C端可能有一个obestain。GHSR的全长cDNA为3961bp，包括375-bp的5'-UTR，ORF为1059-bp，3'-UTR为2527-bp。ORF编码含有7个跨膜结构域的352个氨基酸残基的蛋白质,具有多个N糖基化修饰位点和保守的半胱氨酸残基位点。扬子鳄ghrelin的活性核心“GSSF”与哺乳动物和鸟类相同，GHSR的ghrelin结合位点与哺乳动物相似。ghrelin和GHSR的氨基酸序列与美国短吻鳄（Missippiensis）和鸟类具有很高的同一性。Ghrelin在大脑中高表达，中脑,下丘脑和多个外周组织，包括肺,胃和肠，这表明它除了内分泌外，还可以在旁分泌和/或自分泌方式中发挥作用。GHSR在下丘脑的表达水平较高，上脑和延髓,在包括肺在内的多个外周组织中中等，Kindey,胃和输卵管,暗示ghrelin/GHSR系统可能参与能量平衡的调节，食物摄入量,水和矿物质平衡，胃肠蠕动,胃酸分泌和繁殖。在冬眠期间,ghrelin和GHSR在脑中的表达显著增加，虽然ghrelin在心脏中显著减少，肝脏,肺，胃,胰腺和卵巢,心脏的GHSR显著下降，肝脏,脾,脾肺，Kindey,胃,卵巢和输卵管。这些ghrelin和GHSR表达的时间变化可以促进扬子鳄对冬眠的生理适应。本研究可为进一步研究摄食调控提供基础数据,扬子鳄的生理代谢和繁殖，这也可能有助于改善这种濒危物种的人工繁殖。
