盐芥响应重金属铬胁迫的蛋白质组学研究

发布时间：2018-07-13 20:27

【摘要】：近年来,重金属污染情况日趋严重,铬成为污染我国土壤主要的重金属之一,严重影响植物的生长发育,尤其是对农作物生产造成较大的损害,同时,铬也严重危害人类的生命健康,如铬可以通过食物链中营养级传递进入机体,并且含量逐渐累积。尽管人们尝试用多种不同的方法对重金属污染进行治理,但是收效甚微,这已经成为全中国,乃至全世界范围的一个难题。人们经过不断的摸索和筛选,发现盐芥能够更好地作为植物模型进行相关的实验研究,因为盐芥在抵抗干旱、高盐及低温等恶劣环境的过程中,表现出较强的耐受能力,并且形成了特定的生态反应机制来响应这些逆境胁迫。为了深入地对恶劣的环境下植物产生的不良影响进行研究,蛋白质组学技术通过对植物在不同条件下表达所产生的蛋白质进行分析,进而探究其不同的反应机制,将加深人们对这些微观分子水平的认识和理解,并且为进一步提高植物在不同环境下的生长能力,如抗逆能力等,提供了理论上的指导。本文的实验材料——盐芥为山东生态型,用重金属铬处理盐芥幼苗,通过分析盐芥生理生化指标的变化情况和盐芥叶片的比较蛋白质组学研究,揭示重金属铬胁迫下盐芥的应答反应机制。主要的研究结果如下:1.铬胁迫对盐芥生长和生理指标的影响待盐芥长至6周龄时,用不同浓度的K2Cr2O7溶液处理盐芥幼苗,发现250mg/L的K2Cr2O7溶液处理的盐芥表现出明显的叶片枯萎,但是依然可以生存,故在后续的实验中采用250 mg/L的K2Cr2O7溶液作为铬胁迫处理条件。用浓度为250 mg/L的K2Cr2O7溶液浇灌幼苗,并分别于0 h、1 h、6 h、24 h、48 h时,观察盐芥幼苗的形态变化,并对铬胁迫处理前后盐芥叶片中铬含量、相对电导率(REL)、脯氨酸(Proline)含量等生理指标进行检测和比较分析。经过铬胁迫处理48 h后的盐芥叶片出现明显的枯萎现象;铬在盐芥叶片中的含量随着处理时间的增加而逐渐增加,在48 h时达到最大(343.0 mg/kg);盐芥叶片的相对电导率(REL)和对照组相比,虽然出现了升高,但是并没有明显的变化;盐芥中脯氨酸的含量呈现先下降后上升的趋势,处理6 h后脯氨酸的含量明显低于对照组(p0.05),处理48 h后,脯氨酸的含量达到最高(p0.01)。实验结果表明,随着铬胁迫处理时间的延长,铬在盐芥植株内逐渐积累,盐芥可能通过保护叶片的细胞结构不受到破坏和升高叶片中的脯氨酸含量,响应铬胁迫引起的盐芥叶片水分胁迫。2.铬胁迫下盐芥叶片的蛋白质组学分析铬胁迫处理前后的盐芥叶片总蛋白经过2-DE分离,可以重复检测到大约1200个蛋白点,利用Image Master 2D Platinum7.0软件对铬胁迫处理前后的盐芥叶片总蛋白的电泳胶图进行分析,发现61个蛋白点同时满足蛋白表达量差异倍数Ratio≥1.5和Anova p0.05,其中有18个蛋白点的表达量在1 h、6 h、24 h、48 h均下调,有7个蛋白点的表达量在1 h、6 h、24 h、48 h均上调,有18个蛋白点的表达量在1 h、6 h、24 h、48 h的变化不规律,有的时间点表达量上调,有的时间点表达量下调。通过MALDI-TOF/TOF分析,最后确认了43个蛋白的种类、等电点、分子量等身份,占总蛋白数的70.5%。经过数据库查询和软件分析,预测这些差异蛋白可能参与的生物学过程,并根据其功能的不同分为7类,第一类:参与蛋白质的合成、折叠和降解过程(23%);第二类:参与光合作用和构成叶绿体组织(18.5%);第三类:参与细胞内氧化还原反应与平衡(18.5%);第四类:参与糖类、脂质等代谢过程(16%);第五类:参与胁迫和防御反应(14%);第六类:参与RNA调节和转运(5%);第七类:参与质子运输过程(5%)。这些蛋白中有一部分已经被证实为逆境胁迫响应蛋白,其中有一半以上的蛋白参与蛋白质合成折叠和降解、光合作用和叶绿体组织、氧化还原反应和平衡、物质代谢(糖类、脂质等)和胁迫与防御反应,表明以上生物学过程的相关蛋白可能在盐芥响应铬胁迫中发挥关键性的作用。
[Abstract]:In recent years, the pollution of heavy metals has become more and more serious. Chromium has become one of the major heavy metals in the soil of our country. It seriously affects the growth and development of plants, especially for the production of crops. At the same time, chromium also seriously endangers human life and health, such as chromium can pass through the food chain and pass into the body, and the content of chromium can be carried out. Gradually, although people try to use a variety of different methods to treat heavy metal pollution, but little efficiency, it has become a problem in China, and even the world. In the process of drought, high salt and low temperature, it shows strong tolerance and forms a specific ecological response mechanism to respond to these stresses. In order to study the adverse effects of plants in harsh environment, proteomics technology expresses the eggs produced by plants under different conditions. The analysis of white matter and further explore its different reaction mechanisms will deepen people's understanding and understanding of these micromolecular levels, and provide theoretical guidance for further improving the growth capacity of plants in different environments, such as the ability to resist, and so on. The experimental material of this paper, salt mustard, is ecotype in Shandong, with heavy metal chromium. By analyzing the changes of physiological and biochemical indexes of salt mustard and comparative proteomics of salt mustard leaves, the response mechanism of salt mustard under heavy metal chromium stress was revealed. The main results were as follows: 1. the effects of chromium stress on the growth and physiological indexes of salt mustard were long to 6 weeks of age in salt mustard with different concentrations of K2Cr2O7 The solution treated salt mustard seedlings, found that the salt mustard treated by 250mg/L K2Cr2O7 solution showed obvious leaf blight, but still could survive, so 250 mg/L K2Cr2O7 solution was used as the condition of chromium stress in subsequent experiments. The seedlings were irrigated with a K2Cr2O7 solution of 250 mg/L, and were observed respectively at 0 h, 1 h, 6 h, 24 h, 48 h. The morphological changes of the seedlings of salt mustard were detected and compared with the contents of chromium, relative conductivity (REL) and proline (Proline) in the leaves of salt mustard before and after CR stress treatment. The leaves of salt mustard after 48 h treated with chromium stress showed obvious wilt, and the content of chromium in the leaves of salt mustard increased with the increase of treatment time. The maximum (343 mg/kg) was reached at 48 h, and the relative conductivity (REL) of the leaf of salt mustard was higher than that of the control group, but there was no obvious change; the proline content in the salt mustard decreased first and then increased, and the proline content was significantly lower than that of the control group (P0.05) after 6 h treatment, and the proline was treated after 48 h. The experimental results showed that with the prolongation of the treatment time of chromium stress, chromium was accumulated in the plant of salt mustard. The salt mustard could not be destroyed by the cell structure of the leaves and increased the proline content in the leaves, and the water stress of salt mustard leaves caused by chromium stress in the leaves of salt mustard under.2. stress in response to chromium stress. In white matter analysis, the total protein of leaf mustard leaves before and after chromium stress treatment was separated by 2-DE, and about 1200 protein points could be repeated. Image Master 2D Platinum7.0 software was used to analyze the gel electrophoresis of total protein of leaf mustard leaves before and after CR stress treatment. The results showed that 61 protein points met the difference of protein expression of R. Atio > 1.5 and Anova P0.05, of which 18 protein points are expressed in 1 h, 6 h, 24 h, 48 h, and 7 protein points are up regulated in 1 h, 6 h, 24 h, 48 h. In the end, the species of 43 proteins, isoelectric points, molecular weight and other identities were identified, and the 70.5%. of the total protein number was analyzed by database query and software analysis to predict the possible biological processes of these proteins. According to their different functions, they were divided into 7 categories: the first class: the synthesis, folding and degradation process of the protein and protein (23%); the second category: Participating in photosynthesis and forming chloroplast tissue (18.5%); third types: involved in intracellular redox reaction and balance (18.5%); Fourth: participation in carbohydrates, lipids and other metabolic processes (16%); fifth: participation in stress and defense responses (14%); sixth: participation in RNA regulation and transport (5%); seventh: participation in proton transport process (5%). These proteins include Some of these proteins have been identified as stress response proteins, with more than half of the proteins involved in protein synthesis and degradation, photosynthesis and chloroplast tissue, redox reaction and balance, material metabolism (carbohydrates, lipids, etc.) and stress and defense responses, indicating that the related proteins in the biological process may respond to chromium in salt mustard. Play a key role in coercion.
【学位授予单位】：阜阳师范学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X53;Q945.78

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