鹽芥響應(yīng)重金屬鉻脅迫的蛋白質(zhì)組學(xué)研究
發(fā)布時(shí)間：2018-07-13 20:27
【摘要】：近年來,重金屬污染情況日趨嚴(yán)重,鉻成為污染我國土壤主要的重金屬之一,嚴(yán)重影響植物的生長發(fā)育,尤其是對農(nóng)作物生產(chǎn)造成較大的損害,同時(shí),鉻也嚴(yán)重危害人類的生命健康,如鉻可以通過食物鏈中營養(yǎng)級傳遞進(jìn)入機(jī)體,并且含量逐漸累積。盡管人們嘗試用多種不同的方法對重金屬污染進(jìn)行治理,但是收效甚微,這已經(jīng)成為全中國,乃至全世界范圍的一個(gè)難題。人們經(jīng)過不斷的摸索和篩選,發(fā)現(xiàn)鹽芥能夠更好地作為植物模型進(jìn)行相關(guān)的實(shí)驗(yàn)研究,因?yàn)辂}芥在抵抗干旱、高鹽及低溫等惡劣環(huán)境的過程中,表現(xiàn)出較強(qiáng)的耐受能力,并且形成了特定的生態(tài)反應(yīng)機(jī)制來響應(yīng)這些逆境脅迫。為了深入地對惡劣的環(huán)境下植物產(chǎn)生的不良影響進(jìn)行研究,蛋白質(zhì)組學(xué)技術(shù)通過對植物在不同條件下表達(dá)所產(chǎn)生的蛋白質(zhì)進(jìn)行分析,進(jìn)而探究其不同的反應(yīng)機(jī)制,將加深人們對這些微觀分子水平的認(rèn)識和理解,并且為進(jìn)一步提高植物在不同環(huán)境下的生長能力,如抗逆能力等,提供了理論上的指導(dǎo)。本文的實(shí)驗(yàn)材料——鹽芥為山東生態(tài)型,用重金屬鉻處理鹽芥幼苗,通過分析鹽芥生理生化指標(biāo)的變化情況和鹽芥葉片的比較蛋白質(zhì)組學(xué)研究,揭示重金屬鉻脅迫下鹽芥的應(yīng)答反應(yīng)機(jī)制。主要的研究結(jié)果如下:1.鉻脅迫對鹽芥生長和生理指標(biāo)的影響待鹽芥長至6周齡時(shí),用不同濃度的K2Cr2O7溶液處理鹽芥幼苗,發(fā)現(xiàn)250mg/L的K2Cr2O7溶液處理的鹽芥表現(xiàn)出明顯的葉片枯萎,但是依然可以生存,故在后續(xù)的實(shí)驗(yàn)中采用250 mg/L的K2Cr2O7溶液作為鉻脅迫處理?xiàng)l件。用濃度為250 mg/L的K2Cr2O7溶液澆灌幼苗,并分別于0 h、1 h、6 h、24 h、48 h時(shí),觀察鹽芥幼苗的形態(tài)變化,并對鉻脅迫處理前后鹽芥葉片中鉻含量、相對電導(dǎo)率(REL)、脯氨酸(Proline)含量等生理指標(biāo)進(jìn)行檢測和比較分析。經(jīng)過鉻脅迫處理48 h后的鹽芥葉片出現(xiàn)明顯的枯萎現(xiàn)象;鉻在鹽芥葉片中的含量隨著處理時(shí)間的增加而逐漸增加,在48 h時(shí)達(dá)到最大(343.0 mg/kg);鹽芥葉片的相對電導(dǎo)率(REL)和對照組相比,雖然出現(xiàn)了升高,但是并沒有明顯的變化;鹽芥中脯氨酸的含量呈現(xiàn)先下降后上升的趨勢,處理6 h后脯氨酸的含量明顯低于對照組(p0.05),處理48 h后,脯氨酸的含量達(dá)到最高(p0.01)。實(shí)驗(yàn)結(jié)果表明,隨著鉻脅迫處理時(shí)間的延長,鉻在鹽芥植株內(nèi)逐漸積累,鹽芥可能通過保護(hù)葉片的細(xì)胞結(jié)構(gòu)不受到破壞和升高葉片中的脯氨酸含量,響應(yīng)鉻脅迫引起的鹽芥葉片水分脅迫。2.鉻脅迫下鹽芥葉片的蛋白質(zhì)組學(xué)分析鉻脅迫處理前后的鹽芥葉片總蛋白經(jīng)過2-DE分離,可以重復(fù)檢測到大約1200個(gè)蛋白點(diǎn),利用Image Master 2D Platinum7.0軟件對鉻脅迫處理前后的鹽芥葉片總蛋白的電泳膠圖進(jìn)行分析,發(fā)現(xiàn)61個(gè)蛋白點(diǎn)同時(shí)滿足蛋白表達(dá)量差異倍數(shù)Ratio≥1.5和Anova p0.05,其中有18個(gè)蛋白點(diǎn)的表達(dá)量在1 h、6 h、24 h、48 h均下調(diào),有7個(gè)蛋白點(diǎn)的表達(dá)量在1 h、6 h、24 h、48 h均上調(diào),有18個(gè)蛋白點(diǎn)的表達(dá)量在1 h、6 h、24 h、48 h的變化不規(guī)律,有的時(shí)間點(diǎn)表達(dá)量上調(diào),有的時(shí)間點(diǎn)表達(dá)量下調(diào)。通過MALDI-TOF/TOF分析,最后確認(rèn)了43個(gè)蛋白的種類、等電點(diǎn)、分子量等身份,占總蛋白數(shù)的70.5%。經(jīng)過數(shù)據(jù)庫查詢和軟件分析,預(yù)測這些差異蛋白可能參與的生物學(xué)過程,并根據(jù)其功能的不同分為7類,第一類:參與蛋白質(zhì)的合成、折疊和降解過程(23%);第二類:參與光合作用和構(gòu)成葉綠體組織(18.5%);第三類:參與細(xì)胞內(nèi)氧化還原反應(yīng)與平衡(18.5%);第四類:參與糖類、脂質(zhì)等代謝過程(16%);第五類:參與脅迫和防御反應(yīng)(14%);第六類:參與RNA調(diào)節(jié)和轉(zhuǎn)運(yùn)(5%);第七類:參與質(zhì)子運(yùn)輸過程(5%)。這些蛋白中有一部分已經(jīng)被證實(shí)為逆境脅迫響應(yīng)蛋白,其中有一半以上的蛋白參與蛋白質(zhì)合成折疊和降解、光合作用和葉綠體組織、氧化還原反應(yīng)和平衡、物質(zhì)代謝(糖類、脂質(zhì)等)和脅迫與防御反應(yīng),表明以上生物學(xué)過程的相關(guān)蛋白可能在鹽芥響應(yīng)鉻脅迫中發(fā)揮關(guān)鍵性的作用。
[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.
【學(xué)位授予單位】：阜陽師范學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X53;Q945.78
本文編號：2120643
[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.
【學(xué)位授予單位】：阜陽師范學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X53;Q945.78
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