【摘要】 本文旨在克隆鲤(Cyprinus carpio)激素敏感性脂肪酶a (hormone-sensitive lipase a, lipea)基因,探究其时空表达特征及其与脂肪沉积的关系,以期为鲤脂肪沉积分子调控机制研究奠定理论基础。本实验采用RACE技术克隆获得鲤lipea基因,其cDNA全长为3379 bp,包括230 bp的5′非翻译区(5′-UTR)、1067 bp的3′非翻译区(3′-UTR)和编码693个氨基酸的开放阅读框。多序列比对显示,鲤lipea与斑马鱼(Danio rerio)、牙鲆(Paralichthys olivaceus)和虹鳟(Oncorhynchusmykiss)的氨基酸序列相似性分别为88.17%、64.94%和63.35%,不同物种间序列差异主要集中在C-端的多肽序列上。蛋白质结构分析显示该蛋白含有3个功能域分别具有脂肪酶活性、乙酰基水解酶活性和水解酶活性。应用实时荧光定量PCR技术分析了lipea基因在鲤各组织、不同发育时期以及脂肪含量极端差异个体的肌肉和脂肪组织中mRNA表达量的变化。结果显示, lipea在各组织中均有表达,腹腔脂肪中相对表达量最高,其次是腹部肌肉,血液中最少。随着胚胎的发育, lipea的表达水平呈现下降趋势, 8细胞期表达量最高,其次是囊胚晚期,开口期最少。lipea表达量与背部肌肉脂肪含量呈正相关,但差异不显著(P>0.05),与腹腔脂肪量呈负相关,且差异极显著性(P<0.01),推测lipea基因对鲤腹腔脂肪沉积可能具有抑制作用。本实验结果可为进一步解析鲤lipea基因的功能和基因调控脂肪沉积分子机制研究提供参考。更多还原

【Abstract】 The objectives of this study were to investigate the expression pattern of lipea gene in different tissues and developmental stages of common carp(Cyprinus carpio), to lay the foundation for studying and understanding the molecular regulation mechanism of fat deposition in common carp. In this study, a full-length cDNA sequence of lipea gene in common carp was cloned by reverse transcription PCR and rapid-amplification of cDNA ends(RACE) technology, and the expression pattern of lipea gene in different tissues of common carp and during its different developmental stages. cDNA of the lipea gene of common carp was 3379 bp, with an open reading frame of 2079 bp, 5? untranslated region was 230 bp, 3? untranslated region was 1067 bp, encoding 693 amino acids.Multiple sequences alignment revealed that the amino acid sequences of lipea have 88.17%, 64.94%, and 63.35%identity with Danio rerio, Paralichthys olivaceus, and Oncorhynchus mykiss, respectively. It was determined that the sequence differences were mainly concentrated on the C-terminal polypeptide sequence between different species, and the structural analysis of proteins showed that the protein contains three functional domains with lipase activity, acetyl hydrolase activity, and hydrolase activity, respectively. Real-time quantitative PCR showed that the lipea gene was expressed in all tested tissues, and the relative expression level was the highest in visceral fat, followed by abdominal muscles, and lowest in the blood. Its expression declined during the development of embryo;it was the highest in the 8-cell stage, followed by the late blastula stage. It was the lowest in the feeding period.The lipea mRNA expression in different fat tissues negatively correlated with adipose tissue in the abdominal cavity; the highest and the lowest expression levels differed significantly(P<0.01). The expression also positively correlated with dorsal muscle but it was not significant(P>0.05). Knowledge of the lipea gene highlights its function and molecular regulation mechanism of fat development in common carp.更多还原