【作者】 李东伟；

【导师】 章孝荣； 刘洪瑜；

【作者基本信息】 安徽农业大学 ， 动物遗传育种与繁殖， 2016， 博士

【摘要】 肉牛业是现代畜牧业生产的一个重要组成部分。在肉牛业中,高档牛肉生产所占的比例一直是衡量一个国家肉牛业发展水平的一个重要指标。在我国,黄牛品种资源丰富,但由于缺乏长期有效的系统化肉用性能选育,故而牛肉品质参差不齐,优质高档牛肉产量低,主要表现为牛肉肌内脂肪含量少及分布不均匀等脂肪沉积缺陷。因而有必要对其脂肪沉积的规律进行深入研究。microRNA是一类重要的调控型小分子非编码RNA,主要通过与靶基因mRNA配对发挥转录后调控作用,广泛参与哺乳动物生长发育、细胞分化和凋亡等调控。其中,miR-143和miR-378可能对脂肪沉积具有显著的调控作用。为了进一步阐明miR-143和miR-378在肉牛体脂沉积中的功能及作用机制,探索其对肉牛品种选育的科学意义,本项研究开展了以下几个方面的工作:(1)利用Normfinder、GeNorm和ΔCt分析等,筛选了用于miRNA定量PCR分析的最优内参;(2)选取本地黄牛背最长肌肌内脂肪,采用消化法分离培养前体脂肪细胞,并通过诱导成脂、油红O染色、绘制增殖曲线以及脂肪合成标志性基因表达的检测等确认牛前体脂肪细胞系构建成功;(3)采用miR-143和miR-378慢病毒载体稳定转染前体脂肪细胞,经过灭稻瘟素筛选和单细胞克隆扩增,获得miR-143和miR-378稳定表达的细胞株,再通过荧光定量RT-PCR法检测留取miR-143和miR-378稳定表达上调或下调的细胞株;(4)利用生物信息学软件TargetScan和PicTar等预测miR-143和miR-378靶基因,通过GO和KEGG功能性分析富集miR-143和miR-378可能参与的信号通路,对miR-143预测靶基因TGFβ2与MAP2K5以及miR-378预测靶基因CaMKK2进行双荧光素酶报告系统和Western Blot分析;(5)在体外细胞培养条件下,对比分析了miR-143和miR-378表达水平的改变对前体脂肪细胞分化过程中的细胞形态、增殖及迁徙能力等的影响。目前,获得的主要结论如下:(1)利用Normfinder、geNorm和ΔCt分析,确认U6、miR-191、let-7f在组织间的表达稳定性较佳,其中U6和miR-191共同作为内参使用则具有最佳的稳定性。(2)miR-143和miR-378在牛的组织中表达广泛,在脂肪中表达水平不高,随着前体脂肪细胞的成熟分化而表达逐渐增强。(3)成功构建了miR-378慢病毒载体,获得了miR-143和miR-378稳定表达的细胞系。(4)利用现有miRNA靶基因预测数据库和GO/KEGG分析发现,二者潜在靶基因的功能较多表现在细胞生长、增值与死亡调控方面,涉及到的某些信号通路与脂肪细胞成熟分化有联系。利用双荧光素酶报告系统分析,确定miR-143能够靶向调控MAPK信号通路中的MAP2K5和TGFβ2,miR-378能够靶向调控AMPK信号通路中的CaMKK2。(5)通过绘制miR-143和miR-378转染细胞增殖曲线并计算倍增时间,发现miR-143和miR-378过表达细胞增殖速度较慢,而其抑制表达细胞增殖速度较快;分光光度计检测显示,二者的过表达细胞内脂肪含量较高,而抑制表达细胞内脂肪含量较低;荧光定量PCR检测分析,其过表达细胞在成脂分化过程中PPARγ、C/BPβ和ADI等表达增强,而Pref-1表达减弱,靶基因MAP2K5表达增强,而TGFβ2和CAMKK2表达减弱;miR-143和miR-378在转录与翻译水平抑制其靶基因的表达;miR-143和miR-378过表达细胞多处于G0/G1期,细胞迁移能力较强,而抑制表达则S期细胞比例有所增多,细胞迁移能力减弱。本文在前人已有的研究基础之上,通过体外构建牛前体脂肪细胞系并诱导其成熟分化等一系列试验,再次验证了miR-143和miR-378对前体脂肪细胞分化具有促进作用,提出并证实了miR-143和miR-378新的靶基因及可能涉及的信号通路。这为深入理解miR-143和miR-378对牛肌内脂肪沉积的作用机制,以及进一步开展脂肪沉积良好的肉牛新品种培育提供了参考依据。更多还原

【Abstract】 Beef cattle industry is an important component of modern husbandry production.The proportion of high-grade beef production has been an important indicator of beef cattle industry development level for one country.In our country,cattle breed resources are abundant,but due to lack of effective systematic meat quality breeding for a long time,so the beef quality is uneven,and high quality beef production is low,mainly for few of beef intramuscular fat and maldistribution of fatty deposition.Therefore it is necessary to conduct the thorough research to the rule of cattle fatty deposition.miRNAs is a kind of important small non-coding RNA molecules,mainly to regulate the transcription by matching the target gene mRNA,widely participating in mammals growth and development,cell differentiation and apoptosis,etc.miR-143 and miR-378 have significant control effect on the fat deposition based on the foregone scientific researches.This study mainly included the following several parts:(1)Identifying of suitable endogenous control genes for miRNA quantitative real time PCR assays in cattle solid tissues;(2)Constructing beef cattle preadipocytes cell lines successfully;(3)Screening monoclonal cell strains by transfected lentiviral vector for up/down-regulated miR-143 and miR-378;(4)Bioinformatic predicting and verifying the effect of both targeting specific genes;(5)Detecting the action on preadipocytes differentiation,proliferation and migration ability,etc..Currently,the main conclusions were as follows:(1)U6,miR-191 and let-7f were statistically superior to the other endogenous controls in cattle solid tissues;The combined utilization of miR-191 and U6 would be more suitable to as normalizers of miRNA expression levels than individual U6 snRNA or certain a miRNA for miRNA qPCR assays.(2)miR-143 and miR-378 express were extensive and different obviously in cattle tissues or organs by fluorescence quantitative RT-PCR detection;the adipogenesis of preadipocytes cultured in vitro rooted in intramuscular fat isolated from longissimus dorsi of beef cattle,was confirmed by oil red O staining;miR-143 and miR-378 expression levels increased with adipocyte differentiation.(3)The monoclonal cell strains presented higher expression of adipogenesis landmark genes such as PPARγ,C/EBPβ,ADI,SREBP1 c and KLF5,and lower Pref-1 expression for miR-143 and miR-378 stable overexpression by lentiviral vectors transfection,and otherwise cell doubling time and intracellular fat content had varying degrees of increment compared with empty vector transfection or no treatment control group.And that would be inversed when miR-143 and miR-378 stable expression inhibition by lentiviral vectors transfection.Thus these proved that miR-143 and miR-378 could promote preadipocytes differentiation.(4)Following bioinformatic prediction the miR-143 and miR-378 target genes by TargetScan and PicTar methods,GO and KEGG functional analysis showed that miR-143 participates in the regulation of cell growth and proliferation,involving the MAPK and TGFβ signaling;miR-378 works in biological processes of apoptosis,death,and transcription regulation,involving fat cytokine signaling pathways;Through base pairs analysis of miRNAs and candidate target genes,it was said that TGFβ2 and MAP2K5 would be candidate target genes of miR-143 and CaMKK2 would be one of candidate target genes of miR-378.(5)Dual luciferase report system and Western Blot analyses confirmed that the TGFβ2 and MAP2K5 were target genes of miR-143,and CaMKK2 was one target gene of miR-378.The former suggested that miR-143 contributes to preadipocytes differentiation via TGFβ and MAPK signaling pathways possibly and the later suggested that miR-378 played a role in preadipocytes differentiation via AMPK signaling pathway possibly.It was verified that miR-143 and miR-378 may promote preadipocytes differentiation by a series of trials such as constructing preadipocytes cell lines,inducing adipogenesis,and so on,based on the existing research.New traget genes and possible signaling passway involved of miR-143 and miR-378 were proved to a certain degree.The further clarification on functional mechanisms of miR-143 and miR-378 during intramuscular fat deposition would be helpful for new varieties breeding of beef cattle.更多还原