猪ROCK1和ROCK2基因转录调控机制研究

发布时间：2018-05-31 15:54

本文选题：猪 + 骨骼肌　；参考：《华中农业大学》2016年博士论文

【摘要】：猪肉是人类获取动物蛋白的重要来源,备受研究者关注。同时,基于猪在生理、解剖、病理、基因组等与人的相似性,可以作为研究肥胖、肌肉萎缩、糖尿病、高血压等人类健康相关疾病的模型。因此,对与肌肉生长发育相关基因的研究对于肉质的生产、提高和人类健康相关的医学都具有重要意义。我们把在大白和梅山猪胚胎期65天、出生后3天、60天、120天等四个不同发育时期的背最长肌中的差异表达,参与多种基本生命活动调节的ROCK基因确定为候选基因,对其进行功能和调控机制的研究。主要研究结果如下:1、利用定量PCR的方法,用PPIA、HPRT和eEF-1γ作为内参基因分别检测了2月龄大白猪不同组织中ROCK1,ROCK2基因的表达情况。2、构建了猪ROCK1,ROCK2基因启动子系列缺失的双荧光报告载体ROCK1-P0-P11,ROCK2-1F-7F分别转染PK和C2C12细胞系。荧光活性检测发现在两种细胞系中活性趋势类似。根据ROCK1各个缺失片段双荧光活性的变化,推断-744/-402 bp是ROCK1基因的核心启动子区域;ROCK2中,对荧光活性影响较大的片段进行分析后,推断ROCK1-7-F(+37—+175bp)区域对ROCK2基因的启动子活性发挥重要作用。3、对ROCK1-P5区域内潜在的Sp1结合位点进行定点突变后,荧光活性显著下降(p0.05);ROCK1-P5分别与不同量的Sp1真核过表达载体共转染,发现Sp1可以促进ROCK1-P5活性,这种促进作用转染Sp1的量存在正相关性。EMSA、DNA pull down共同验证了Sp1蛋白可以与ROCK1核心启动子区域内三个Sp1结合体外位点,并且各位点的结合能力存在着差异。ChIP实验验证了Sp1,而非Sp3,与猪ROCK1启动子的体内结合。4、在PK和C3H10T1/2中,共转染C/EBPα真核表达载体,发现ROCK2-2B-F,3-F,4-F,7-F片段的活性显著升高,将这些序列细化缺失,进行荧光活性测定,发现PK细胞中,ROCK2-2B-F/5F这段区域活性极低,推测ROCK2-7-F区域为核心启动子。ROCK2-7-F中C/EBPα结合位点定点突变后荧光活性显下降。随后应用EMSA实验进一步证明C/EBPα蛋白能与启动子上C/EBPα结合位点体外结合。5、过表达Sp1时,ROCK1启动子活性升高,而抑制时则下降;荧光定量PCR和western blot结果显示,Sp1促进ROCK1在转录和翻译水平的表达;同时,Sp1过表达后Myod,Myog,MyHC在mRNA水平的表达上升。而C/EBPα过表达,促进ROCK2转录活性,同时荧光定量PCR和western blot结果显示,C/EBPα可以刺激ROCK2基因的转录和翻译。6、共转染小鼠ROCK1基因3’UTR区域的荧光活性载体与人工合成的miRNA模拟片段,发现miR-33-5p,miR-376c-3p分别与ROCK1基因的3’UTR结合。转染C2C12细胞后,发现这两个miRNA对ROCK1转录水平的影响不明显,而对蛋白水平的影响比较显著。7、miR-142-3p在C2C12细胞分化过程中(2day、5day、8day)表达量不断下降,而在增殖期(0day)表达量低于分化初期(2day),高于分化末期(8day);ROCK2在分化时期(2day、5day、8day)表达量不断升高,增殖期表达量最低。8、共转染小鼠ROCK2基因3’UTR区域的荧光活性载体与人工合成的miRNA,miRNA inhibitor模拟片段,发现miR-142-3p可以与ROCK2基因的3’UTR结合。转染C2C12细胞后,发现miR-142-3p对ROCK2在转录水平和蛋白水平的影响比较显著。
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【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S828

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