本文選題：豬 + 骨骼肌��； 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：豬肉是人類獲取動(dòng)物蛋白的重要來(lái)源,備受研究者關(guān)注。同時(shí),基于豬在生理、解剖、病理、基因組等與人的相似性,可以作為研究肥胖、肌肉萎縮、糖尿病、高血壓等人類健康相關(guān)疾病的模型。因此,對(duì)與肌肉生長(zhǎng)發(fā)育相關(guān)基因的研究對(duì)于肉質(zhì)的生產(chǎn)、提高和人類健康相關(guān)的醫(yī)學(xué)都具有重要意義。我們把在大白和梅山豬胚胎期65天、出生后3天、60天、120天等四個(gè)不同發(fā)育時(shí)期的背最長(zhǎng)肌中的差異表達(dá),參與多種基本生命活動(dòng)調(diào)節(jié)的ROCK基因確定為候選基因,對(duì)其進(jìn)行功能和調(diào)控機(jī)制的研究。主要研究結(jié)果如下:1、利用定量PCR的方法,用PPIA、HPRT和eEF-1γ作為內(nèi)參基因分別檢測(cè)了2月齡大白豬不同組織中ROCK1,ROCK2基因的表達(dá)情況。2、構(gòu)建了豬ROCK1,ROCK2基因啟動(dòng)子系列缺失的雙熒光報(bào)告載體ROCK1-P0-P11,ROCK2-1F-7F分別轉(zhuǎn)染PK和C2C12細(xì)胞系。熒光活性檢測(cè)發(fā)現(xiàn)在兩種細(xì)胞系中活性趨勢(shì)類似。根據(jù)ROCK1各個(gè)缺失片段雙熒光活性的變化,推斷-744/-402 bp是ROCK1基因的核心啟動(dòng)子區(qū)域;ROCK2中,對(duì)熒光活性影響較大的片段進(jìn)行分析后,推斷ROCK1-7-F(+37—+175bp)區(qū)域?qū)OCK2基因的啟動(dòng)子活性發(fā)揮重要作用。3、對(duì)ROCK1-P5區(qū)域內(nèi)潛在的Sp1結(jié)合位點(diǎn)進(jìn)行定點(diǎn)突變后,熒光活性顯著下降(p0.05);ROCK1-P5分別與不同量的Sp1真核過(guò)表達(dá)載體共轉(zhuǎn)染,發(fā)現(xiàn)Sp1可以促進(jìn)ROCK1-P5活性,這種促進(jìn)作用轉(zhuǎn)染Sp1的量存在正相關(guān)性。EMSA、DNA pull down共同驗(yàn)證了Sp1蛋白可以與ROCK1核心啟動(dòng)子區(qū)域內(nèi)三個(gè)Sp1結(jié)合體外位點(diǎn),并且各位點(diǎn)的結(jié)合能力存在著差異。ChIP實(shí)驗(yàn)驗(yàn)證了Sp1,而非Sp3,與豬ROCK1啟動(dòng)子的體內(nèi)結(jié)合。4、在PK和C3H10T1/2中,共轉(zhuǎn)染C/EBPα真核表達(dá)載體,發(fā)現(xiàn)ROCK2-2B-F,3-F,4-F,7-F片段的活性顯著升高,將這些序列細(xì)化缺失,進(jìn)行熒光活性測(cè)定,發(fā)現(xiàn)PK細(xì)胞中,ROCK2-2B-F/5F這段區(qū)域活性極低,推測(cè)ROCK2-7-F區(qū)域?yàn)楹诵膯?dòng)子。ROCK2-7-F中C/EBPα結(jié)合位點(diǎn)定點(diǎn)突變后熒光活性顯下降。隨后應(yīng)用EMSA實(shí)驗(yàn)進(jìn)一步證明C/EBPα蛋白能與啟動(dòng)子上C/EBPα結(jié)合位點(diǎn)體外結(jié)合。5、過(guò)表達(dá)Sp1時(shí),ROCK1啟動(dòng)子活性升高,而抑制時(shí)則下降;熒光定量PCR和western blot結(jié)果顯示,Sp1促進(jìn)ROCK1在轉(zhuǎn)錄和翻譯水平的表達(dá);同時(shí),Sp1過(guò)表達(dá)后Myod,Myog,MyHC在mRNA水平的表達(dá)上升。而C/EBPα過(guò)表達(dá),促進(jìn)ROCK2轉(zhuǎn)錄活性,同時(shí)熒光定量PCR和western blot結(jié)果顯示,C/EBPα可以刺激ROCK2基因的轉(zhuǎn)錄和翻譯。6、共轉(zhuǎn)染小鼠ROCK1基因3’UTR區(qū)域的熒光活性載體與人工合成的miRNA模擬片段,發(fā)現(xiàn)miR-33-5p,miR-376c-3p分別與ROCK1基因的3’UTR結(jié)合。轉(zhuǎn)染C2C12細(xì)胞后,發(fā)現(xiàn)這兩個(gè)miRNA對(duì)ROCK1轉(zhuǎn)錄水平的影響不明顯,而對(duì)蛋白水平的影響比較顯著。7、miR-142-3p在C2C12細(xì)胞分化過(guò)程中(2day、5day、8day)表達(dá)量不斷下降,而在增殖期(0day)表達(dá)量低于分化初期(2day),高于分化末期(8day);ROCK2在分化時(shí)期(2day、5day、8day)表達(dá)量不斷升高,增殖期表達(dá)量最低。8、共轉(zhuǎn)染小鼠ROCK2基因3’UTR區(qū)域的熒光活性載體與人工合成的miRNA,miRNA inhibitor模擬片段,發(fā)現(xiàn)miR-142-3p可以與ROCK2基因的3’UTR結(jié)合。轉(zhuǎn)染C2C12細(xì)胞后,發(fā)現(xiàn)miR-142-3p對(duì)ROCK2在轉(zhuǎn)錄水平和蛋白水平的影響比較顯著。
[Abstract]:The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . The results were as follows : 1 . It was found that Sp1 - 7 - F ( + 37 - + 175bp ) region plays an important role in the production , improvement and human health of two - month - old pigs . The results showed that the expression of C / EBP 偽 in the region of C2C12 cell was significantly lower than that at the early stage of differentiation ( 2 day , 5 day , 8 day ) .
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S828

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