基于蛋白组学和同位素分馏对小球藻吸收氨氮机制的研究

发布时间：2018-01-03 23:24

本文关键词：基于蛋白组学和同位素分馏对小球藻吸收氨氮机制的研究　出处：《湘潭大学》2015年硕士论文　论文类型：学位论文

【摘要】：作为水环境中最常见的污染物,氨氮易引起富营养化现象等一系列环境问题的发生,我国“十二五”期间已经明确提出氨氮减排10%的目标。藻类具有生长迅速以及高效去除氨氮的能力,并且在消减氨氮的同时,可作为鱼类等水生生物的饵料以及再生能源的制备。因此,开发基于藻类的生物技术消减氨氮,是污水厂实现氨氮达标排放、减轻氨氮对水体自然生态环境危害程度的现实需要,也为氨氮转化为有价值的生物资源提供理论和技术支持。利用氨氮作为唯一氮源的微生物能够区分15N和14N,这种选择性分馏为胞外不同氨氮水平在生物体内吸收转化提供重要证据,而蛋白组学技术可揭示此吸收过程中参与氨氮代谢相关蛋白的差异。研究表明,藻类对氨氮的去除主要是通过氨氮的吸收,然而与氨氮吸收代谢相关的氮同位素效应,特别是不同氨氮浓度水平对藻类吸收氨氮的影响机制,包括藻体中氨氮的分馏效应和参与氨氮代谢的相关基因和酶,到目前还没有充分揭示。为此,本文以小球藻(Chlorella vulgaris)为代表,以氨氮为唯一氮源,利用15N分馏机制和蛋白组学研究了不同氨氮浓度水平对藻类吸收氨氮的差异,以及在此过程中参与氨氮代谢的相关基因和酶类,并探索了藻类对氨氮的吸收机制。论文获得了以下研究结论:(1)在4-50 mg/L初始氨氮浓度条件下,小球藻生长速率呈现先上升后下降的趋势,且在10 mg/L时达到最大(4.86×105cells/(mL?d))。小球藻对氨氮的去除效率随初始氨氮浓度的升高而降低,实验六天后氨氮的去除率由100%降至0.4%。小球藻对氨氮的吸收速率随着初始氨氮浓度的升高呈现先升高后下降的趋势,且在10 mg/L时达到最大(2.05×10-10mg/(cell?d))。因此,在10 mg/L氨氮浓度条件下,小球藻具有最大的生长速率以及氨氮吸收速率。叶绿素含量随着氨氮浓度的升高也呈现相同的趋势,且在10 mg/L时叶绿素a(6.49 mg/L)和叶绿素b(6.31 mg/L)均达到最大。通过不同氨氮浓度和碳源浓度共同作用对小球藻生长、氨氮吸收量和碳源吸收量的影响表明,随着碳源浓度的升高,小球藻生物量整体呈上升趋势,且在40 mg/L时达最大(12.21×105 cells/(mL?d))。且相同氨氮浓度下,碳源浓度高于20 mg/L时氨氮去除率均可达100%,对碳源的吸收量整体呈上升趋势,在40 mg/L达到最大(3.94 mg)。(2)藻细胞内氮同位素分馏程度取决于环境中氨氮的浓度。在10 mg/L氨氮浓度条件下,培养液和藻细胞的15N的丰度分别为-3.61±0.06‰和-1.99±0.05‰,富集系数为-1.63±0.06‰。而在4 mg/L氨氮浓度条件下,其培养液和藻细胞的15N的丰度分别为-3.50±0.08‰和-1.13±0.06‰,富集系数为-2.37±0.08‰。说明10mg/L氨氮条件下,小球藻对氨氮具有更明显的分馏效应。此外通过生物转化率的比较发现,在10 mg/L氨氮浓度条件下小球藻对氨氮的转化率(47.4%)明显高于4 mg/L(7.0%),也即外部的氨氮转化为细胞内含氮化合物或生物量的程度更显著。(3)蛋白组学研究结果发现,小球藻在10 mg/L氨氮条件相对4 mg/L氨氮有162个蛋白斑点表现出不同程度的上调和下调,其中有92个斑点具有显著性的差异表达(n≥3,p0.05)。通过飞行时间质谱鉴定出有31个蛋白分别参与了光合作用、糖代谢、三羧酸(TCA)循环、氨基酸代谢和蛋白质合成等等。其中氨基酸代谢是与氮代谢直接相关的代谢过程,尤其是谷氨酸代谢是其余氨基酸代谢以及TCA循环等的中间代谢过程。谷氨酰胺合成酶(GS)作为氨氮在细胞内的初级转化酶,可迅速将氨氮转化为谷氨酰胺和谷氨酸。二维凝胶双向电泳发现GS蛋白在10 mg/L氨氮条件上调了6.44倍,并且通过实时荧光聚合酶链式反应(RT-qPCR)验证其GS基因表达倍数上调了18.03倍。说明此酶在促进藻细胞生长以及对氨氮的吸收利用发挥了极其重要的作用。
[Abstract]:As one of the most common pollutants in water environment, ammonia is easy to cause a series of environmental problems such as eutrophication phenomenon occurred in China during the "12th Five-Year" has clearly put forward the ammonia emission reduction target of 10%. With the rapid growth of algae and high ammonia nitrogen removal ability, and in the reduction of ammonia nitrogen at the same time, can be used as fish and other aquatic organisms the feed and the preparation of renewable energy. Therefore, the development of algae biotechnology is based on ammonia abatement, ammonia emission standards of sewage treatment plant, reduce the need for ammonia water natural ecological environment harm degree of reality, but also provide theoretical and technical support for ammonia nitrogen into valuable biological resources. By using ammonia as the sole nitrogen source microorganisms are able to distinguish between 15N and 14N, the selective fractionation for different extracellular level of ammonia in vivo transformation provides important evidence and technology group can expose protein The difference of ammonia nitrogen metabolism related proteins involved in the absorption process is shown in this. The study showed that the algae removal of ammonia is mainly by ammonia absorption, but absorption of nitrogen isotope effect and ammonia metabolism, especially the different concentration of ammonia absorption level influence mechanism of nitrogen by algae, including ammonia nitrogen fractionation in algae and related genes in ammonia metabolism and enzyme, has not been fully revealed. Therefore, this paper (Chlorella vulgaris) as the representative of Chlorella, using ammonia nitrogen as the sole nitrogen source, the difference by 15N fractionation mechanism and proteomics study on the different level of ammonia nitrogen concentration of ammonia nitrogen absorbed on algae, ammonia metabolism and related genes in this process and enzymes, and to explore the absorption mechanism of algae on ammonia nitrogen. The research obtained the following conclusions: (1) in the 4-50 mg/L initial concentration condition, the growth rate of Chlorella First increased and then decreased, and reached the maximum at 10 mg/L (4.86 * 105cells/ (mL? D)). The removal efficiency of ammonia nitrogen on Chlorella decreased with increasing initial concentration of ammonia nitrogen, six days after the experiment the removal rate of ammonia nitrogen decreased from 100% to 0.4%. on the absorption rate of Chlorella with the initial concentration of ammonia nitrogen appeareddecreasing first increased and then decreased, and reached the maximum at 10 mg/L (2.05 * 10-10mg/ (cell? D)). Therefore, in 10 mg/L ammonia nitrogen concentration, the growth rate of Chlorella has the largest absorption rate of ammonia nitrogen and chlorophyll content. With the increase of ammonia concentration also showed the same trend, and in the 10 mg/L chlorophyll a (6.49 mg/L) and chlorophyll b (6.31 mg/L) reached the maximum. On Chlorella Growth through interaction between different ammonia concentration and concentration of carbon source, nitrogen uptake and carbon absorption effects show that with carbon source concentration Increase the overall Chlorella biomass increased, and reached the maximum at 40 mg/L (12.21 x 105 cells/ (mL? D)). And the same concentration of ammonia, carbon source concentration is higher than 20 mg/L ammonia nitrogen removal rate could reach 100%. The overall uptake of carbon source showed an upward trend, reaching a maximum at 40 mg/L (3.94 mg). (2) the concentration of intracellular nitrogen isotope fractionation degree depends on the environment. In 10 mg/L ammonia nitrogen concentration, the abundance of culture medium and cell 15N were -3.61 + 0.06 and -1.99 + 0.05%%, enrichment coefficient of -1.63 + 0.06 per thousand in 4. Mg/L ammonia concentration under the condition of the medium and cell abundance of 15N were -3.50 + 0.08 and -1.13 + 0.06 per thousand per thousand, enrichment coefficient is -2.37 + 0.08. 10mg/L% ammonia nitrogen conditions, Chlorella fractionation effect is more obvious. In addition the conversion rate of ammonia nitrogen by biological comparison found in 10 mg/L ammonia nitrogen Concentration of ammonia conversion rate of Chlorella (47.4%) was significantly higher than that of 4 mg/L (7%), namely the external ammonia nitrogen into nitrogen or cell biomass containing more significant degree. (3) proteomics research results showed that Chlorella 162 protein spots showed different degrees of up and down in 10 mg/L 4 mg/L ammonia ammonia condition is relatively, which has 92 differentially expressed spots with significant (n = 3, P0.05). By the time of flight mass spectrometry identified 31 proteins were involved in photosynthesis, sugar metabolism, three carboxylic acid (TCA) cycle, amino acid metabolism and protein synthesis. The metabolism of amino acid metabolism the process is directly related to nitrogen metabolism, especially glutamate metabolism is intermediate metabolic processes in other amino acid metabolism and TCA cycle. Glutamine synthetase (GS) as the primary enzyme of ammonia nitrogen in cells, can be quickly Ammonia is converted to glutamine and glutamate. Two dimensional gel electrophoresis showed that GS protein in 10 mg/L ammonia nitrogen conditions raised 6.44 times, and by real-time polymerase chain reaction (RT-qPCR) to verify the GS gene expression ratio raised 18.03 times. This enzyme plays an extremely important role in promoting the growth of algae and the absorption of ammonia use.

【学位授予单位】：湘潭大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X52;X173

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