磁螺菌Magnetospirillum gryphiswaldense MSR-1中OxyR同源蛋白的功能分析

发布时间：2018-06-16 01:11

本文选题：Magnetospirillum + gryphiswaldense　；参考：《中国农业大学》2017年博士论文

【摘要】：趋磁细菌是能在胞内合成特殊细胞器—磁小体,并沿磁力线排列和运动细菌的总称。磁小体由四氧化三铁或四硫化三铁纳米磁颗粒及其外包被的生物膜组成,粒径分布在30～120 nm之间。由于趋磁细菌细胞结构和基因组构成简单,是研究生物矿化的模式生物。目前趋磁细菌的研究主要集中于磁小体合成机制,尤其是磁小体岛基因的研究,岛外基因功能及调控机制的研究较少。前期的实验证据表明磁小体的合成与氧化还原密切相关。本工作以格瑞菲斯瓦尔德磁螺菌(Magnetospirillum gryphiswaldense MSR-1)为材料,研究两个oxyR同源基因的功能及调控机制。实验室在前期工作中已经构建了基因oxyR-Like的缺失突变和互补菌株,通过对其一系列表型的测定,发现其磁小体合成严重受阻,并利用表达谱测定了突变株中基因的总体表达情况,但OxyR-Like的调控机制并没有被阐明。本文利用凝胶阻滞(EMSA)和DNaseI足迹实验深入的研究了 OxyR-Like的调控机制。结果表明,OxyR-Like可以结合MGMSRv-2_0006基因,pdhA基因簇以及mcpA基因的启动子区。利用DNaseI足迹实验找到了 OxyR-Like结合的DNA保守序列5'-ATN{3}AN{5}TTATCA-3'。利用qRT-PCR检测,发现突变株中所有与三羧酸循环相关的基因表达量均下调,暗示在突变株中能量代谢受到抑制。oxyR-4250基因是MSR-1中另外一个oxyR同源基因。本文构建了 oxyR-4250的缺失突变株和互补菌株,并对其表型进行了测定。结果表明,oxyR-4250缺失突变株生长缓慢,完全失去磁感应能力,且铁吸收能力相比野生型菌株下降五倍。通过透射电镜对突变株进行观察,发现在突变株中磁小体数量变少,粒径变小,并且不再成链排列。EMSA实验结果表明,OxyR-4250可以调控ahpC和sodB基因的表达,证明OxyR-4250在MSR-1中可以调控抗氧化基因的表达。但在MSR-1中OxyR-4250并不调控katE,katG等在大肠杆菌中受OxyR调控的基因,说明在MSR-1中其他抗氧化调控机制的存在。通过在EMSA结合体系中加入还原剂,证明了 OxyR-4250可以响应环境中的氧化还原信号,从而改变其DNA结合能力。但EMSA实验并未发现OxyR-4250可以结合磁小体岛基因簇的启动子区,说明OxyR-4250不能直接调控磁小体的合成。DNase I足迹实验证明OxyR-4250结合DNA的保守序列为5'-TCGATTTNTNTNANNANNANAACTCNT-3'。定点突变后的 EMSA 实验结果证明 OxyR-4250蛋白中212Cys在其结合DNA过程中起关键作用。综上所述,本论文首次阐明了磁螺菌MSR-1中两个oxyR同源基因的功能,明确了 OxyR-Like 调控能量代谢相关的基因并与磁小体合成相关;明确了 OxyR-4250 在 MSR-1 中不同于大肠杆菌中的抗氧化调控机制,为胞内氧化还原的调控与磁小体形成直接相关提供了证据。
[Abstract]:Magnetotactic bacteria are the general names of bacteria which can synthesize special organelles-magnetic bodies and arrange and move along the magnetic line of force. The magnetic bodies are composed of Fe _ 2O _ 3 or Fe _ 2O _ 4 nanoparticles and their biofilms. The particle size is between 30 ~ 120 nm. Because of the simple cell structure and genome composition of magnetotactic bacteria, it is a model organism to study biomineralization. At present, the research of magnetotactic bacteria is mainly focused on the mechanism of magnetosome synthesis, especially on the gene of magnetic body island. Previous experimental evidence shows that the synthesis of magnetic bodies is closely related to redox. In this work, the function and regulatory mechanism of two oxyR homologous genes were studied using Magnetospirillum gryphiswaldense MSR-1 as a material. The deletion and complementary strains of oxyR-Like gene have been constructed in our laboratory. Through a series of phenotypic tests, we found that the magnetic body synthesis was seriously blocked, and the expression profile was used to determine the overall expression of genes in the mutant strain. However, the regulatory mechanism of OxyR-Like has not been clarified. In this paper, the regulatory mechanism of OxyR-Like was studied by gel blocking EMSA) and DNase I footprint experiments. The results showed that OxyR-Like could bind MGMSRv-20006 gene to PDHA gene cluster and promoter region of mcpA gene. The OxyR-Like binding DNA conserved sequence 5- ATN {3} an {5} TTATCA-3 was found by using DNase I footprint test. By using qRT-PCR, we found that all the genes related to tricarboxylic acid cycle were down-regulated, suggesting that the energy metabolism of the mutant was inhibited. OxyR-4250 gene was another oxyR homologous gene in MSR-1. The deletion mutant and complementary strain of oxyR-4250 were constructed and their phenotypes were determined. The results showed that the mutant strain was slow to grow and completely lost its magnetic induction ability, and its iron absorption capacity was five times lower than that of the wild-type strain. The results of transmission electron microscopy (TEM) showed that the number of magnetic corpuscles became smaller and the particle size became smaller, and the results of EMSA experiment showed that OxyR-4250 could regulate the expression of ahpC and sodB genes in the mutant, and the results of EMSA experiment showed that OxyR-4250 could regulate the expression of ahpC and sodB genes. It is suggested that OxyR-4250 can regulate the expression of antioxidant genes in MSR-1. However, OxyR-4250 did not regulate the OxyR regulated genes in Escherichia coli such as katEkatG in MSR-1, indicating the existence of other antioxidant regulatory mechanisms in MSR-1. It is proved that OxyR-4250 can respond to the redox signal in the environment and change its DNA binding ability by adding reducing agent in EMSA binding system. However, the EMSA experiment did not find that OxyR-4250 could bind to the promoter region of the magnetic body island gene cluster, which indicated that OxyR-4250 could not directly regulate the synthesis of magnetic bodies. DNase I footprint experiments showed that the conserved sequence of OxyR-4250 binding DNA was 5- TCGATTTNTNANNANAACTCNT-3. The EMSA results of site-directed mutation showed that 212Cys in OxyR-4250 protein played a key role in its binding to DNA. To sum up, the function of two oxyR homologous genes in magnetic spirilli MSR-1 was first elucidated, and the genes related to the regulation of energy metabolism by OxyR-Like were identified and related to the synthesis of magnetic bodies. The antioxidant regulation mechanism of OxyR-4250 in MSR-1 was clarified, which provided evidence for direct correlation between intracellular redox regulation and magnetic body formation.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q78

【相似文献】