黑曲霉黑色素辐射防护作用的研究

发布时间：2018-06-25 23:24

本文选题：黑曲霉 + 黑色素　；参考：《兰州大学》2017年硕士论文

【摘要】：目的:为了研究黑色素在~(12)C~(6+)离子束和X射线的辐射条件下对黑曲霉孢子的防护机制,通过电离辐射以及自由基直接处理不同孢子,探讨黑色素对不同孢子存活率的影响。在此基础上,通过研究辐射以及自由基环境下不同颜色孢子细胞内活性氧含量、孢子膜通透性及细胞膜脂质过氧化的差异,以及黑色素的自由基清除作用,证明黑色素保护孢子免受辐射损伤的作用机制。材料与方法:以黑曲霉黑色孢子CON,颜色突变体MUT和黑色素合成被抑制的DM孢子为研究对象,首先用~(12)C~(6+)离子束和X射线对三种孢子进行梯度剂量的辐照,另外又采用3%H_2O_2和0.5M DPPH自由基直接处理三种孢子,采用平板涂布法检测不同孢子存活率,对孢子进行DCFH-DA和PI染色,利用荧光显微镜观察染色并统计染色率,检测孢子内活性氧的含量变化和孢子膜通透性的改变,采用TBA法检测电离辐射和自由基对孢子膜脂质过氧化的损伤;利用紫外分光光度计和扫描电镜,观察H_2O_2对孢子壁的分解作用;最后,提取两种孢子的色素,用酶标仪检测色素对不同自由基的清除率。结果:经过~(12)C~(6+)离子束和X射线对三种孢子的梯度剂量辐照表明,电离辐射对孢子的损伤程度与辐照剂量呈正向相关,在同等辐照条件下,黑色CON孢子的各项损伤程度均低于MUT和DM孢子,而同种孢子在相同辐照剂量下,~(12)C~(6+)离子束对孢子的损伤更为严重;用3%H_2O_2和0.5M DPPH自由基直接处理三种孢子实验结果表明:孢子的损伤程度与自由基的处理时间呈正向相关关系,在同等自由基处理条件下,黑色CON孢子的各项损伤程度均低于MUT和DM孢子;最后,黑色素对自由基的体外清除试验结果显示:黑色素能够有效的清除羟基自由基,DPPH以及超氧自由基,清除能力为DPPH羟基自由基超氧自由基。结论:1黑曲霉中的黑色素能够保证其孢子在辐射环境中成为优势种群,减轻不同辐射对它的损伤程度,具有一定的辐射抗性;2黑曲霉黑色素能够保护孢子,减少环境中不同自由基对孢子的损伤程度;3黑色素能够有效的清除羟基自由基,DPPH以及超氧自由基;4黑曲霉黑色素可以通过清除自由基实现其辐射防护作用。
[Abstract]:Aim: to study the protective mechanism of melanin against Aspergillus Niger spores under ~ (12) C ~ (6) ion beam and X-ray radiation, and to investigate the effect of melanin on the survival rate of different spores by ionizing radiation and free radical. On this basis, the difference of active oxygen species content, membrane permeability and membrane lipid peroxidation in different color spore cells under radiation and free radical conditions were studied, as well as the scavenging effect of melanin on free radical. The mechanism of melanin protecting spores from radiation damage was proved. Materials and methods: three spores of Aspergillus Niger, the color mutants mut and DM spores, which were inhibited by melanin synthesis, were first irradiated with ~ (12) C ~ (6) ion beam and X ray. In addition, three kinds of spores were directly treated with the free radicals of 3 and 0.5M DPPH, the survival rates of different spores were detected by plate coating method, the spores were stained with DCFH-DA and Pi, and the staining rates were observed and counted by fluorescence microscope. To detect the changes of active oxygen species in spores and the permeability of spore membrane, to detect the damage of ionizing radiation and free radicals to the lipid peroxidation of spores membrane by TBA method, and to use ultraviolet spectrophotometer and scanning electron microscope, The decomposition of the spore wall was observed. Finally, the pigments of the two spores were extracted, and the scavenging rates of the pigments to different free radicals were detected by the enzyme standard instrument. Results: the gradient dose of ~ (12) C ~ (6) ion beam and X ray irradiation on three spores showed that the degree of damage to spores by ionizing radiation was positively correlated with the radiation dose, and under the same irradiation conditions, The damage degree of black Con spores was lower than that of mut and DM spores, and the damage to spores of the same spores was more serious under the same irradiation dose of ~ (12) C ~ (6) ion beam. The results of direct treatment of three spores with three kinds of spore free radicals: the degree of spores damage is positively correlated with the treatment time of free radicals, and under the same conditions of free radical treatment, The damage degree of black Con spores was lower than that of mut and DM spores. Finally, the results showed that melanin could effectively scavenge hydroxyl radical DPPH and superoxide free radical. The scavenging ability was DPPH hydroxyl radical superoxide radical. Conclusion the melanin in Aspergillus Niger can ensure that the spores of Aspergillus Niger can become the dominant population in the radiation environment, and reduce the damage degree caused by different radiation. The melanin of Aspergillus Niger with certain radiation resistance can protect the spores. Reducing the damage degree of different free radicals to spores can effectively scavenge the hydroxyl radical DPPH and the superoxide free radical Aspergillus Niger melanin can achieve its radiation protection by scavenging free radicals.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R818

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