本文選題：粗糙脈孢菌　切入點：過氧化氫酶　出處：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：基因表達的精確調(diào)控對于生物體的正常發(fā)育至關(guān)重要,同時也是生物體應(yīng)對脅迫刺激的基礎(chǔ)。粗糙脈孢菌含有3種過氧化氫酶(Catalase):Cat-1、Cat-2、Cat-3。其中,Cat-3的作用最為重要,可保護菌絲體抵抗H2O2脅迫并可有效調(diào)控粗糙脈孢菌菌絲的生長發(fā)育。cat-3的表達水平在不同的時空條件下受到嚴格控制,該基因可作為一個良好的實例來研究基因調(diào)控機制。在研究中,我們構(gòu)建了異染色質(zhì)組裝缺陷突變體dim-5KO、hpoKO、H3K9Q、H3K9L、H3K9R以及cul4KO、dcaf26KO、dim-7KO菌株,這些突變體表現(xiàn)出較強的H2O2抗性；與之對應(yīng),突變體中cat-3基因高水平表達。進一步的實驗分析發(fā)現(xiàn)cat-3基因上游存在5kb的AT-rich序列,在該區(qū)域中H3K9me3修飾以及HP1蛋白高水平富集。這些結(jié)果表明cat-3基因上游區(qū)域可形成異染色質(zhì)并參與抑制cat-3基因的表達。同時,在cat-3啟動子及其ORF區(qū)域分布高水平的H3ac修飾,dim-5KO突變體及H2O2處理后的野生型菌株中H3ac修飾水平增加。此外,Western blot及酶譜檢測分析顯示在dim-5KO等突變體中,Cat-3蛋白條帶電泳遷移位置發(fā)生變化,暗示在該蛋白上化學(xué)修飾可能發(fā)生了變化。除cat-3之外,在以上突變體中cat-1和cat-2的表達水平也是升高的。為了進一步研究cat-3基因表達與其上游的異染色質(zhì)區(qū)域之間的直接關(guān)系,我們構(gòu)建了cat-3上游區(qū)段的敲除菌株cat-3Δ5-3、cat-3Δ3-1、cat-3Δ5。這些上游區(qū)段缺失菌株具有較強的H202抗性。與之對應(yīng),在這些菌株中cat-3的表達水平顯著增高。較之cat-3A3-1與cat-3A5突變體,cat-3A5-3突變體中cat-3的表達量要更高一些。更為有趣的是在zat-3A5與dhn-5K0等異染色質(zhì)組裝缺陷菌株的雙突變體中,cat-3的表達水平提升幅度更高(高于其中任一單突變體)。同時,在cat-3△5-3、cat-3Δ3-1、cat-3Δ5突變體中RNAPo Ⅱ的募集水平顯著增加。其中,cat-3A5-3突變體中的增加幅度最大。表明cat-3上游5kb基因間區(qū)在基因的轉(zhuǎn)錄調(diào)控過程中起抑制作用。與dim-5K0等突變體不同,cat-3Δ5-3、 cat-3Δ3-1, cat-3Δ5菌株中Cat-3蛋白的電泳遷移位置與野生型菌株基本相似。綜上所述,本研究運用遺傳學(xué)、分子生物學(xué)的技術(shù)手段對粗糙脈孢菌過氧化氫酶catalase的轉(zhuǎn)錄調(diào)控機制進行了探索。這些結(jié)果為粗糙脈孢菌及其它物種中相關(guān)基因轉(zhuǎn)錄調(diào)控機制的研究提供了支持與參考。
[Abstract]:The precise regulation of gene expression is very important for the normal development of organisms, and also the basis for organisms to deal with stress stimuli.C. crassa contains three catalase species: Cat-1, Cat-2Cat-3.Cat-3 plays the most important role in protecting mycelium against H2O2 stress and can effectively regulate the growth and development of mycelium. The expression level of cat-3 is strictly controlled under different time and space conditions.This gene can be used as a good example to study the mechanism of gene regulation.Further experimental analysis showed that the AT-rich sequence of 5kb existed in the upstream of cat-3 gene, and H3K9me3 modification and high level enrichment of HP1 protein were found in this region.These results suggest that the upstream region of cat-3 gene can form heterochromatin and participate in inhibiting the expression of cat-3 gene.At the same time, the level of H3ac modification was increased in the wild type strain treated with cat-3 promoter and its ORF with high level of H3ac modified dim-5KO mutant and treated with H2O2.In addition, Western blot and zymogram analysis showed that the electrophoretic migration sites of cat-3 protein bands in dim-5KO and other mutants changed, suggesting that the chemical modification on the protein might have changed.In addition to cat-3, the expression of cat-1 and cat-2 was also increased in the above mutants.In order to further study the direct relationship between the expression of cat-3 gene and the heterochromatin region in the upstream region of cat-3, we constructed the knockout strain cat-3 螖 5-3cat-3 螖 3-1cat-3 螖 5.These upstream deletion strains had strong resistance to H 202.Correspondingly, the expression of cat-3 in these strains was significantly increased.The expression of cat-3 was higher than that in the mutants of cat-3A3-1 and cat-3A5.What is more interesting is that the expression of cat-3 in the double mutants with heterochromatin assembly defects such as zat-3A5 and dhn-5K0 is higher than that in any of the single mutants.At the same time, the recruitment level of RNAPo 鈪，

本文編號：1720874