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题名: 水稻土厌氧微生物对硒氧离子还原以及纳米化研究
作者: 鲍鹏
学位类别: 博士后
答辩日期: 2014-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 朱永官
关键词: 硒还原,兼性厌氧梭菌,二氧化碳固定,硝酸盐还原,硒还原酶,兼性厌氧芽孢杆菌,基因组测序,纳米硒,肿瘤 ; selenium reduction, facultative anaerobic Clostridium sp, carbon dioxide fixation, nitrate reduction, selenium reductase, facultative anaerobic Bacillus sp, genome sequencing, nano selenium, tumor
学位专业: 环境科学
中文摘要:     硒是一种多变价的元素,是人体必需的微量元素之一。研究表明,硒是生物体内多种蛋白质和酶的重要组分,被认为是一种参与生命活动的重要微量元素。环境中过高或过低的硒分布均会产生不同的生态效应。许多种微生物均可在硒的形态转化中发挥重要作用,微生物被确认能够独立完成硒的地球化学循环,但微生物以何种途径和机制参与调控硒的地球化学循环,以及相应的影响因素作用的机制仍未得到清晰的阐明。在水稻种植中实现土壤无机硒向有机硒的转化,提高稻米中硒含量, 是改善人体硒营养和促进人体健康的重要途径。目前缺乏水稻土中与元素转化归趋密切相关的微生物对亚硒酸盐的还原作用的机理性解释。 纳米硒能够被生物体迅速吸收,提高机体的免疫能力,同时避免了硒所带来的副作用,是已发现的急性毒性最低的补硒制剂。纳米硒的高效低毒为硒制剂的临床抗肿瘤应用展开了更加广阔的前景。生物大分子通常用来控制合成纳米硒粒子。微生物来源的蛋白在控制合成纳米硒工艺上具有很好的应用潜力。 本课题一方面的研究目的是分离纯化水稻土中硒还原厌氧微生物,探讨二氧化碳的固定和硝酸盐还原对其还原硒氧离子的影响效应, 以及常见的环境影响条件对硒还原的作用。另一方面着重研究厌氧微生物细胞内参与调控形成纳米硒颗粒的蛋白,以及利用这些蛋白控制合成的纳米硒粒子的抗肿瘤作用。主要结果如下:
    1. 从水稻土中分离鉴定一株梭菌属菌株BXM为兼性厌氧菌,无鞭毛,不移动,产芽孢,革兰氏阳性,梭形菌。菌体长约2-3 μm宽约0.3-0.5 μm。基因组G+C摩尔百分含量为26.3 mol%。主要脂肪酸成分为十六碳饱和脂肪酸C16:0 (44.7%),反异式十五碳饱和脂肪酸C15:0 anteiso(18.7%)。与Clostridium sulfidigenes SGB2T的DNA-DNA相似性为36%。菌株BXM生长的温度范围为21-45oC,pH范围为6.0-7.6, 最适pH 7.0。可以利用酵母膏,乳酸,葡萄糖为碳源, 具有还原硫,亚硒酸盐,硒酸盐的能力。菌株BXM能够以氢气为能源,固定二氧化碳产生乙酸达2.7 mmol l-1。元素分析表明Clostridium sp. BXM将硒酸根,亚硝酸根还原生成红色非晶体单质硒。菌株Clostridium sp. BXM表现出产乙酸菌的特性。菌株Clostridium sp. BXM可以在厌氧和好氧条件下还原硒酸盐和亚硒酸盐,菌株Clostridium sp. BXM还原亚硒酸盐
的能力随温度的升高而升高;体系中二氧化碳的存在可以促进硒氧离子的还原;硝酸盐抑制菌株对硒氧离子的还原能力;本研究中二氧化碳固定和硝酸盐还原能够相互促进。
    2. 经过多个指标的对比确证菌株ZYKT是芽孢杆菌属中一全新的菌种, 且为少数的几种厌氧芽孢杆菌。定名为Bacillus oryziterrae (=CGMCC 1.5179T =DSMZ 26460T)。ZYKT具有在厌氧条件下还原硝酸根成为亚硝酸根和铵的能力。在厌氧条件下可将亚硒酸还原成为单质硒,并且在细胞内形成纳米硒颗粒。对ZYKT基因组信息分析结果表明其具有硝酸盐还原酶和亚硝酸还原酶基因, 这与其还原硝酸盐能力相符合。包括一种呼吸型硝酸盐还原酶基因(NarG, NarH, NarI和NarJ),一种细胞周质空间硝酸盐还原酶基因(NapA, NapB和NapD),和一种依赖于甲酸盐的亚硝酸还原酶基因(nrfA)和含铜离子的亚硝酸还原酶基因(nirK)。以及两种DMSO还原酶基因。经过蛋白鉴定结果与基因组信息相结合,我们初步确定菌株ZYKT调控纳米硒颗粒形成的蛋白为一种310bp的核糖体蛋白。由菌株ZYKT还原产生的纳米硒对非小细胞肺癌细胞H157具有显著的抑制作用。
英文摘要:     Selenium is an element of changing values is one of the essential trace elements. Studies have shown that selenium is an important component of various proteins and enzymes, and is considered as an important trace element involved in the life activity. Too high or too low selenium in the environment will result in different ecological effect. Many species of microorganisms play important role in the transformation of selenium, and microorganisms was confirmed control selenium biogeochemical cycle independently, but microbial ways and mechanisms involved in the regulation of selenium geochemical cycles, and the corresponding impact factors mechanisms have not yet been clearly elucidated. Increasing the conversion of organic selenium selenium content in rice is an important way to improve the human selenium nutrition and promote human health. Current lack of paddy soil and fate is closely related elements into microbial reduction of selenite on the machine rational explanation.
    Efficiency and low toxicity nano-selenium selenium application for clinical anti-tumor agents launched a broader outlook. Biological macromolecules are usually used to control the synthesis of nano selenium particles. Microbial protein source has a good potential in the controlled synthesis of nano-selenium process. The purpose of this aspect of the issue is separation and purification of selenium reduction anaerobic microorganisms from paddy soil, explore the effects of reduction of carbon dioxide fixation and reduction of nitrate on selenium oxygen ions reduction. On the other hand focuses on proteins involved in the regulation of anaerobic microbial cells forming nano-selenium particles, and the use of anti-tumor effect of these proteins control the synthesis of nano-selenium particles.
    1. Separated and identified a paddy soil Clostridium strains BXM a facultative anaerobes, no flagella, non-motility, spore-forming, gram-positive, spindle-shaped bacteria. Cell is about 2-3 μm width 0.3-0.5 μm. Genomic mol% G+C content is 26.3 mol%. The main fatty acid composition of sixteen carbon saturated fatty acid C16: 0 (44.7%), anti-carbon saturated fatty fifteen different type C15: 0 anteiso (18.7%). And Clostridium sulfidigenes SGB2T DNA-DNA similarity of 36%. Growth of strain BXM temperature range 21-45oC, pH range 6.0-7.6, and the optimum pH 7.0. Can use yeast extract, lactic acid, glucose as carbon source, reduce sulfur, selenite, selenate capabilities. Elemental analysis showed that Clostridium sp. BXM reduce selenate,nitrite reduction to form a red amorphous elemental selenium. Clostridium sp. Strain BXM exhibit characteristic has acetogenic bacteria. Strains BXM can reduce selenite and selenite under anaerobic and aerobic conditions, the ability to reduce selenite increases with temperature rise.
    2. Through comparison of various aspects, ZYK are confirmed as a new species of Bacillus, and a small number of several anaerobic Bacillus. Named Bacillus oryziterrae (= CGMCC 1.5179T = DSMZ 26460T). ZYKT reduce nitrate under anaerobic conditions to nitrite and ammonium. Under anaerobic conditions can be reduce selenite into elemental selenium and form nano particles in the cell. ZYKT genomic information analysis showed that it has nitrate reductase and nitrite reductase gene. Including a respiratory type of nitrate reductase gene (NarG, NarH, NarI and NarJ), a cellular periplasmic space nitrate reductase gene (NapA, NapB and NapD), and a formate-dependent nitrite reductase gene (nrfA) and copper ions nitrite reductase gene (nirK). Two types of DMSO reductase gene. After protein identification results with a combination of genomic information, we initially identified protein from ZYKT regulating nano-selenium particles formed as a 310 bp ribosomal protein. Nano-selenium produced by ZYKT shown significantly inhibition to non-small cell lung cancer H157 cells.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34113
Appears in Collections:中澳联合土壤环境研究室_学位论文

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Recommended Citation:
鲍鹏. 水稻土厌氧微生物对硒氧离子还原以及纳米化研究[D]. 北京. 中国科学院研究生院. 2014.
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