粗糙脉孢菌过氧化氢酶基因转录调控机制的研究

发布时间：2018-04-08 08:39

  本文选题：粗糙脉孢菌　切入点：过氧化氢酶　出处：《中国农业大学》2016年博士论文

【摘要】：基因表达的精确调控对于生物体的正常发育至关重要,同时也是生物体应对胁迫刺激的基础。粗糙脉孢菌含有3种过氧化氢酶(Catalase):Cat-1、Cat-2、Cat-3。其中,Cat-3的作用最为重要,可保护菌丝体抵抗H2O2胁迫并可有效调控粗糙脉孢菌菌丝的生长发育。cat-3的表达水平在不同的时空条件下受到严格控制,该基因可作为一个良好的实例来研究基因调控机制。在研究中,我们构建了异染色质组装缺陷突变体dim-5KO、hpoKO、H3K9Q、H3K9L、H3K9R以及cul4KO、dcaf26KO、dim-7KO菌株,这些突变体表现出较强的H2O2抗性；与之对应,突变体中cat-3基因高水平表达。进一步的实验分析发现cat-3基因上游存在5kb的AT-rich序列,在该区域中H3K9me3修饰以及HP1蛋白高水平富集。这些结果表明cat-3基因上游区域可形成异染色质并参与抑制cat-3基因的表达。同时,在cat-3启动子及其ORF区域分布高水平的H3ac修饰,dim-5KO突变体及H2O2处理后的野生型菌株中H3ac修饰水平增加。此外,Western blot及酶谱检测分析显示在dim-5KO等突变体中,Cat-3蛋白条带电泳迁移位置发生变化,暗示在该蛋白上化学修饰可能发生了变化。除cat-3之外,在以上突变体中cat-1和cat-2的表达水平也是升高的。为了进一步研究cat-3基因表达与其上游的异染色质区域之间的直接关系,我们构建了cat-3上游区段的敲除菌株cat-3Δ5-3、cat-3Δ3-1、cat-3Δ5。这些上游区段缺失菌株具有较强的H202抗性。与之对应,在这些菌株中cat-3的表达水平显著增高。较之cat-3A3-1与cat-3A5突变体,cat-3A5-3突变体中cat-3的表达量要更高一些。更为有趣的是在zat-3A5与dhn-5K0等异染色质组装缺陷菌株的双突变体中,cat-3的表达水平提升幅度更高(高于其中任一单突变体)。同时,在cat-3△5-3、cat-3Δ3-1、cat-3Δ5突变体中RNAPo Ⅱ的募集水平显著增加。其中,cat-3A5-3突变体中的增加幅度最大。表明cat-3上游5kb基因间区在基因的转录调控过程中起抑制作用。与dim-5K0等突变体不同,cat-3Δ5-3、 cat-3Δ3-1, cat-3Δ5菌株中Cat-3蛋白的电泳迁移位置与野生型菌株基本相似。综上所述,本研究运用遗传学、分子生物学的技术手段对粗糙脉孢菌过氧化氢酶catalase的转录调控机制进行了探索。这些结果为粗糙脉孢菌及其它物种中相关基因转录调控机制的研究提供了支持与参考。
[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，

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