阿勒泰大尾羊FABP4、ADIPOQ和CFD基因分子克隆及其组织差异性表达的研究

发布时间：2018-06-28 09:22

本文选题：阿勒泰羊 + 饥饿与非饥饿模型　；参考：《石河子大学》2015年硕士论文

【摘要】：目的:阿勒泰羊是一种脂臀型绵羊品种,其独特的尾脂在维持机体正常的新陈代谢和体温过程中扮演了重要的角色,主要通过尾部脂肪沉积与代谢消长规律来适应恶劣的环境以维持机体正常的新陈代谢和体温,是研究绵羊尾脂沉积与代谢机制的理想模型。近年来的研究表明,FABP4、ADIPOQ和CFD基因参与葡萄糖和脂肪酸代谢以及脂肪细胞分化等生理过程,初步推测其与脂肪沉积代谢密切相关。本文以阿勒泰羊为研究对象,探讨了FABP4、ADIPOQ、CFD基因对阿勒泰羊尾脂沉积与代谢的影响。方法:1、阿勒泰羊饥饿与非饥饿模型的构建。模拟阿勒泰羊在自然生理状态下尾脂沉积与代谢两种极端状态,即在夏秋季节牧草丰茂时脂肪沉积、春冬季节牧草匮乏时尾脂分解代谢的方式,对阿勒泰羊进行了饥饿试验,构建了阿勒泰羊饥饿与非饥饿状态下尾脂沉积与代谢的动物模型。2、以阿勒泰羊尾脂组织c DNA为模板,通过RT-PCR方法克隆获得FABP4、ADIPOQ和CFD基因完整的CDS区序列。3、以阿勒泰羊心脏、肝脏、脾脏、肺脏、肾脏、肌肉、肠系膜脂肪、尾脂等主要组织c DNA为模板,采用半定量RT-PCR方法获得FABP4、ADIPOQ和CFD基因在阿勒泰羊不同组织中的表达谱。4、应用实时荧光定量PCR技术检测了FABP4、ADIPOQ和CFD基因在阿勒泰羊饥饿与非饥饿模型尾脂组织中的表达变化情况。结果:1、通过2周逐步限饲和4周完全禁食(期间自由饮水)后,饥饿模型阿勒泰羊精神上表现出焦躁不安、采食欲望强烈;其臀尾表现出“瘪瘦”表型,臀尾部皮肤松弛、软塌,臀部尾脂体积减小了1/3以上;体重显著下降;尾脂脂肪细胞体积变小,呈扁平的梭形。2、克隆得到绵羊FABP4、ADIPOQ和CFD基因完整的CDS序列,分别编码132、239、261个氨基酸。绵羊FABP4核苷酸序列及氨基酸序列与山羊、牛、牦牛、马鹿等反刍动物对应序列的同源性均高达90%以上;绵羊ADIPOQ基因的核苷酸序列与牛、小鼠、大鼠、野猪、人等对应序列的同源性在75%以上,其中与牛对应序列的同源性高达85%以上;绵羊CFD基因核苷酸序列及其编码的氨基酸序列与反刍动物牛的同源性均高达96.9%以上。3、半定量RT-PCR检测结果表明,FABP4基因在阿勒泰羊心脏、肺脏、肌肉、肠系膜脂肪、尾脂组织中均有表达,但在肠系膜脂肪和尾脂中高丰度表达;ADIPOQ基因在尾脂组织中特异性高表达;CFD基因在心脏、肝脏、脾脏、肺脏、肌肉、尾脂中均表达,但在尾脂中高丰度表达。4、实时荧光定量PCR结果表明,阿勒泰羊FABP4基因在非饥饿组尾脂中的表达量略高于饥饿组尾脂,但二者差异不显著(P0.05);ADIPOQ、CFD基因则结果相反,阿勒泰羊ADIPOQ和CFD基因在饥饿组尾脂中的表达量显著高于在饥饿组尾脂中的表达(P0.01)。结论:上述结果表明,FABP4、ADIPOQ、CFD基因在阿勒泰羊尾脂沉积与代谢过程中具有一定的调控作用,可以作为哺乳动物脂肪组织发育调控过程中重要的候选功能基因。
[Abstract]:Objective: Altay Sheep is a kind of fat-buttock sheep breed. Its unique tail fat plays an important role in maintaining normal metabolism and body temperature. It is an ideal model to study the mechanism of tail fat deposition and metabolism in sheep, mainly through the regulation of tail fat deposition and metabolism to adapt to bad environment to maintain normal metabolism and body temperature. Recent studies have shown that FABP4ADIPOQ and CFD genes are involved in glucose and fatty acid metabolism and adipocyte differentiation. In this paper, the effect of FABP4 ADIPOQN CFD gene on fat deposition and metabolism of tail fat in Altay sheep was studied. Methods: 1, the model of starvation and non-starvation in Altay sheep. Two extreme states of tail fat deposition and metabolism were simulated in Altay sheep under natural physiological conditions, that is, fat deposition during summer and autumn, and catabolism of tail fat in spring and winter when forage was scarce. The animal model of tail lipid deposition and metabolism under starvation and non-starvation in Altay sheep was constructed. The complete CDS sequence of FABP4ADIPOQ and CFD gene was cloned by RT-PCR from the cDNAs of Altay sheep tail fat tissue. The CDS region of FABP4ADIPOQ and CFD gene was cloned from the heart and liver of Altay sheep. The main tissues of spleen, lung, kidney, muscle, mesenteric fat, tail fat, etc. The expression profiles of FABP4AADIPOQ and CFD genes in different tissues of Altay sheep were obtained by semi-quantitative RT-PCR. The expression of FABP4AADIPOQ and CFD genes in tail fat tissue of Altay sheep model of starvation and non-starvation was detected by real-time fluorescent quantitative PCR. The results showed that after 2 weeks of diet restriction and 4 weeks of complete fasting (free drinking), Altay sheep showed mental agitation and strong appetite for food intake, and its buttocks and tails showed "punk" phenotype and loose skin of buttocks and tails. After soft collapse, the volume of fat in the buttocks decreased by more than one third, the weight of fat cells decreased significantly, and the fat cells of tail fat became smaller, showing flattened fusiform. The complete CDS sequences of FABP4ADIPOQ and CFD genes were cloned and encoded 132239261 amino acids, respectively. The nucleotide sequence and amino acid sequence of sheep FABP4 were more than 90% homology with goat, cow, yak, red deer and other ruminants, and the nucleotide sequence of sheep ADIPOQ gene was higher than that of cattle, mice, rats, wild boar, etc. The homology of human equivalent sequence is more than 75%, and the homology with cattle is more than 85%. The nucleotide sequence of sheep CFD gene and the amino acid sequence encoded by CFD gene were above 96.9%. The results of semi-quantitative RT-PCR showed that FABP4 gene was found in the heart, lung, muscle and mesenteric fat of Altay sheep. The high abundance of ADIPOQ gene was expressed in mesenteric fat and tail fat, and the expression of CFD gene in heart, liver, spleen, lung, muscle and tail fat was also found in heart, liver, spleen, lung, muscle and tail fat. The results of real-time fluorescence quantitative PCR showed that the expression of FABP4 gene in non-starvation group was slightly higher than that in hungry group, but there was no significant difference between them (P0.05). The expression of ADIPOQ and CFD genes in the tail fat of the hungry group was significantly higher than that in the starved group (P0.01). Conclusion: these results suggest that FABP4 ADIPOQN CFD gene may play an important role in the deposition and metabolism of tail fat in Altay, and it can be used as an important candidate gene in the regulation of adipose tissue development in mammals.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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