Cloning and sequence analysis of sheep (Ovis aires) resistin gene (RETN) and its expression pattern in starving model.

Resistin (RETN) is a specifically produced hormone by fat cells, which plays an important role in glucose and lipid metabolism and also is one of popular candidate genes in animal fat deposition and metabolism. Aiming at study the expression pattern of RETN gene in rump-fat deposition and metabolism of sheep (Ovis aires), long fragment PCR, bioinformatics method and semi-quantitative RT-PCR were used to Online system: http://www.jabiotech.org 农 业 生 物 技 术 学 报 Journal of Agricultural Biotechnology 2015, 23(8): 981~990 DOI: 10.3969/j.issn.1674-7968.2015.08.001 农业生物技术学报 Journal of Agricultural Biotechnology 绵羊(Ovis aires)脂尾(臀)性状是一种耐逆性 状。一些脂肉型绵羊品种可在水草丰美的季节大 量囤积臀(尾)脂肪,以供牧草匮乏、天寒地冻之时 “保命”所需。阿勒泰羊就是这类绵羊品种的典型 代表,其臀脂的重量最高可达胴体总重的 25%(马 桢等, 2012)。这种高脂肉率品种售卖时不仅经济 效益差,也不符合人类健康饮食的需求。随着牧 区定居点的兴建以及牧草种植配套技术的推广, 传统的受限于季节与营养水平波动的放牧饲养模 式正在向集约舍饲的现代化饲养模式转变,阿勒 泰尾(臀)脂的“保命”作用已微不足道(王世银等, 2013;张伟等, 2013;甘尚权等, 2013)。因此,破解 调控绵羊尾(臀)脂沉积与代谢性状的关键基因,继 而通过转基因或基因组编辑技术改良高脂绵羊品 种,培育低脂绵羊新品系是当前绵羊育种工作者 亟待研究的科学问题 (梁耀伟等, 2013; Ragkos, Abas, 2015)。 脂肪不仅是胴体的重要组成成分,还参与动物 机体众多生理生化反应,为动物生命活动提供能量 保证。脂肪组织由脂肪细胞这一基础物质聚集而 成,而脂肪细胞由起源于中胚层的间充质干细胞逐 步分化而最终形成成熟的脂肪细胞 (Otto, Lane, 2005)。其间经历了间充质干细胞发育阶段、脂肪 母细胞发育阶段、前脂肪细胞发育阶段、不成熟脂 肪细胞发育阶段以及成熟脂肪细胞发育阶段等历 程(Miura, 2015)。其中前脂肪细胞发育阶段在整 个脂肪发育阶段尤为重要,其在众多转录因子调控 下 (Auwerx et al., 2003; Li et al., 1998; Yin et al., 2006),激活脂肪组织相关基因,并在这些基因的顺 序性调控下,经一系列复杂的步骤分化为成熟脂肪 细胞。目前,脂肪研究大多集中在以小鼠(Mus mus⁃ culus) 3T3-L1前脂肪细胞为模式细胞系的脂肪细 胞分化与调控研究领域,并且已被证实可以调控脂 肪细胞分化的转录因子数目众多(Issemann, Green, 1990; Tontonoz et al., 1993; Linhart et al., 2001; Seale et al., 2007),其中转录因子过氧化物酶体增 殖物激活受体γ (peroxisome proliferators-activated receptorγ , PPARγ )与CCAAT/增强子结合蛋白-α基 因(CCAAT/enhancer-binding protein α , C/EBP α)是 控制脂肪细胞分化的主效因子 (Wu et al., 1995; 1999)。此外,脂肪细胞还可以分泌一些其他的细 胞因子及激素,如抵抗素 (resistin, RETN)、瘦素 (leptin)、脂联素(adiponectin)、内脂素(visfatin)和肿 瘤坏死因子α(TNF-α)等也在不同程度地与脂肪细 胞的分化活动相关联。 RETN基因是美国科学家Steppan等(2001b)发 现的一种由脂肪细胞所特异分泌的活性多肽激 素,由于其与胰岛素抗性具有某种关联性,故被命 名为抵抗素。现已证明人类(Homo sapiens)、小鼠 和大鼠 (Rattus norvegicus)基因组中均有 RETN基 因,且编码区序列的同源性较高(Shuldiner et al., 2001; Steppan et al., 2001b)。小鼠RETN mRNA由 591个碱基组成,编码 114个氨基酸,而人RETN则 编码 108个氨基酸 (Chung et al., 2014)。RETN基 因属于富含半胱氨酸的分泌型蛋白质抵抗素样分 子(resistin-like molecules, RELMs)家族中的一员, amplify the full-length sequences, analyze the sequences and detect gene expression profiling of sheep RETN gene, respectively. Fluorescence quantitative Realtime PCR (qRTPCR) method was used to analyze the expression change of RETN gene between starving and its control groups based on starving model in Altay sheep. The results of cloning and sequence analysis showed that RETN gene encoded 109 amino acids (GenBank No. KJ704841), which was different from the published RETN sequence in GenBank database. RETN mRNA expression profiling showed that RETN expressed in liver, spleen, lung and rump fat differently, especially highly in the liver, significantly higher than that of other groups (P<0.01), which indicated that RETN gene might play important physiological and biochemical functions in fat synthesis and metabolism in the liver. The result of qRTPCR showed that RETN mRNA significantly differentially expressed in between starving group and its control groups, with a expression level in persistent starvation group of 5.2 times than that of control group, suggesting that RETN gene might play an important role in fat mobilization. The above findings provide fundamental data for further study on RETN biological function in fat-rump sheep.