
大菱鲆神经肽Y基因克隆及其在工厂化养殖饥饿-投喂策略中的表达特征分析
中国工程科学 ›› 2014, Vol. 16 ›› Issue (9) : 42-49.
大菱鲆神经肽Y基因克隆及其在工厂化养殖饥饿-投喂策略中的表达特征分析
cDNA Cloning of Neuropeptide Y gene of turbot Scophthalmus maximus L. and its expression analysis of the strategy of starvation and feeding in industrial culture
Han Jian1,2、Liu Bin2、Jiang Zhiqiang1、Mu Xiaosheng3、Lei Jilin1,2
本文通过逆转录聚合酶链式反应(RT-PCR)和末端快速扩增技术(RACE)克隆了大菱鲆神经肽Y(NPY)基因全长为729 bp 的互补脱氧核糖核酸(cDNA)序列,其中5′非翻译区(5′-UTR)为70 bp,3′非翻译区(3′-UTR)为359 bp,开放阅读框(ORF)为300 bp。ORF编码含有99 个氨基酸的蛋白质多肽,N端28 个氨基酸为信号肽,信号肽后是由36 个氨基酸组成的NPY成熟肽。针对大菱鲆NPY蛋白质与其他物种的同源性进行比较,结果显示大菱鲆NPY与其他物种的同源性在53.9 %~98 %,特别是与同属于鲆鲽鱼类的牙鲆和美洲拟鲽的同源性高达97 %~98 %,表现出很高的保守性。采用实时定量PCR对大菱鲆NPY基因的表达特征进行分析,结果表明,大菱鲆NPY基因在神经组织和外围组织中都有表达,其中在下丘脑中的表达量最高。饥饿实验中,随着饥饿的进行,NPY的表达量显著变化(P<0.05),呈现先升高再降低,再显著升高并维持一段高水平表达后,再次下降的趋势,饥饿后再投喂会使NPY的表达量升高,并且饥饿时间越长,再投喂后NPY的表达水平越高,但是再投喂2 h 后表达量还会再次下降至相似的水平,这都预示着NPY是一种摄食刺激因子,并且是一种短期的调控机制。
In this study, the full length of turbot NPY cDNA was cloned using RT-PCR and rapid-amplification of cDNA ends (RACE). Turbot NPY cDNA is 729bp with a 5´-untranslated region (UTR), 135bp 3´-UTR and 299bp ORF. The open reading frame encodes a 99 amino acid NPY. The first 28 amino acids constitute the signal sequence followed by the 36 amino acid mature peptide. Turbot NPY share a 53.9–98.0% identity with NPY from other fish, especially, Turbot NPY share a 97–98.0% identity with NPY from other flounder (Japanese flounder, winter flounder).Using real-time PCR to analysis NPY expression in turbot. Tissue distribution studies show that NPY mRNA is expressed in brain, in particular the hypothalamic, and in peripheral tissues. During food deprivation, the NPY undergo significant change in expression (P<0.05), with NPY levels being elevated first, then decreased, and being significantly increased, maintain a high level of expression, then decreased again during food deprivation. Expression level was significantly higher with refeeding after fasting than the normal feeding, the longer of food deprivation, the higher of expression level. But the expression level of 2h after refeeding will fall again to a similar level. Our results suggest that NPY is conserved peptides that might be involved in the regulation of feeding and other physiological functions in turbot.
Turbot (Scophthalmus maximus L.) Neuropeptide Y Starvation Feeding Differential expression
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