本文選題：黑曲霉 + 黑色素�。� 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：目的:為了研究黑色素在~(12)C~(6+)離子束和X射線的輻射條件下對(duì)黑曲霉孢子的防護(hù)機(jī)制,通過電離輻射以及自由基直接處理不同孢子,探討黑色素對(duì)不同孢子存活率的影響。在此基礎(chǔ)上,通過研究輻射以及自由基環(huán)境下不同顏色孢子細(xì)胞內(nèi)活性氧含量、孢子膜通透性及細(xì)胞膜脂質(zhì)過氧化的差異,以及黑色素的自由基清除作用,證明黑色素保護(hù)孢子免受輻射損傷的作用機(jī)制。材料與方法:以黑曲霉黑色孢子CON,顏色突變體MUT和黑色素合成被抑制的DM孢子為研究對(duì)象,首先用~(12)C~(6+)離子束和X射線對(duì)三種孢子進(jìn)行梯度劑量的輻照,另外又采用3%H_2O_2和0.5M DPPH自由基直接處理三種孢子,采用平板涂布法檢測(cè)不同孢子存活率,對(duì)孢子進(jìn)行DCFH-DA和PI染色,利用熒光顯微鏡觀察染色并統(tǒng)計(jì)染色率,檢測(cè)孢子內(nèi)活性氧的含量變化和孢子膜通透性的改變,采用TBA法檢測(cè)電離輻射和自由基對(duì)孢子膜脂質(zhì)過氧化的損傷;利用紫外分光光度計(jì)和掃描電鏡,觀察H_2O_2對(duì)孢子壁的分解作用;最后,提取兩種孢子的色素,用酶標(biāo)儀檢測(cè)色素對(duì)不同自由基的清除率。結(jié)果:經(jīng)過~(12)C~(6+)離子束和X射線對(duì)三種孢子的梯度劑量輻照表明,電離輻射對(duì)孢子的損傷程度與輻照劑量呈正向相關(guān),在同等輻照條件下,黑色CON孢子的各項(xiàng)損傷程度均低于MUT和DM孢子,而同種孢子在相同輻照劑量下,~(12)C~(6+)離子束對(duì)孢子的損傷更為嚴(yán)重;用3%H_2O_2和0.5M DPPH自由基直接處理三種孢子實(shí)驗(yàn)結(jié)果表明:孢子的損傷程度與自由基的處理時(shí)間呈正向相關(guān)關(guān)系,在同等自由基處理?xiàng)l件下,黑色CON孢子的各項(xiàng)損傷程度均低于MUT和DM孢子;最后,黑色素對(duì)自由基的體外清除試驗(yàn)結(jié)果顯示:黑色素能夠有效的清除羥基自由基,DPPH以及超氧自由基,清除能力為DPPH羥基自由基超氧自由基。結(jié)論:1黑曲霉中的黑色素能夠保證其孢子在輻射環(huán)境中成為優(yōu)勢(shì)種群,減輕不同輻射對(duì)它的損傷程度,具有一定的輻射抗性;2黑曲霉黑色素能夠保護(hù)孢子,減少環(huán)境中不同自由基對(duì)孢子的損傷程度;3黑色素能夠有效的清除羥基自由基,DPPH以及超氧自由基;4黑曲霉黑色素可以通過清除自由基實(shí)現(xiàn)其輻射防護(hù)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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R818

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