【作者】 薛亮；

【导师】 江泽平；

【作者基本信息】 中国林业科学研究院 ， 生态学， 2013， 博士

【摘要】 近年来，由于对锑矿开采缺乏有效的规划与管理，以及对锑的不合理使用，致使我国土壤、水体及空气中锑含量急剧上升，对人类及整个生态系统的健康构成严重威胁。植物修复作为一种经济、高效且相对安全的环境修复技术具有广阔的应用前景。然而，由于锑修复植物材料的缺乏，加之对锑的植物毒性效应以及植物的耐锑机制不明确，致使锑污染治理过程中植物修复技术的应用于受到极大限制。因此，本研究通过在锑矿矿区开展植物对重金属的积累特征分析，评价其用于植物修复的潜力。并以筛选获得的耐锑植物芒为研究材料，开展植物在锑胁迫下的生理生化及蛋白质组学响应研究，深入了解锑的植物毒性效应及植物的耐锑机理，为获取理想的锑修复植物材料提供有效方法。主要研究结果如下：（1）锑矿矿区植物重金属积累特征研究。本研究通过在湖南省锡矿山锑矿矿区开展植物和土壤的实地调查，分析锑矿区植物不同组织重金属的积累特征。在调查的10种草本植物中，芒（Miscanthus sinensis）和白茅（Imperata cylindrica）具有汞（Hg）和镉（Cd）的植物萃取潜力，美洲商陆（Phytolacca americana）具有Cd和铅（Pb）的植物稳定潜力，苎麻（Boehmeria longispica）对砷（As）和锑（Sb）表现出较强的转移能力，繁穗苋（Amaranthus paniculatus）和芒可应用于Sb的植物稳定，狗牙根（Cynodon dactylon）可分别应用于As和Sb的植物稳定。其中，芒作为一种潜在的能源植物在修复锑污染的同时带来巨大的经济价值，是一种较为理想的锑修复植物材料。此外，本研究中发现的这些锑富集植物可用于植物耐锑机理的研究。（2）两种生态型芒对锑的积累特征及生长响应比较。矿区和非矿区生态型芒根部和叶片中的锑含量均随锑处理浓度增加而极显著上升。两种生态型芒对锑的富集主要位于根部，锑的转移系数范围为0.13~0.18。两种生态型芒不同组织对锑的积累量差异不显著，表明其在对锑的富集能力方面并无较大差异。锑的加入对不同生态型芒的生长、生物量均造成显著抑制，其中矿区生态型芒的苗高和根长较对照的减少率低于非矿区生态型芒；矿区生态型芒地上部分干重和地下部分干重较对照的下降量低于非矿区生态型芒，表明矿区生态型芒对锑的耐受性高于非矿区生态型芒。（3）锑胁迫下两种生态型芒的活性氧及渗透调节机制研究。锑胁迫会使两种芒叶片MDA含量增高，表明脂质过氧化反应加剧。其中，非矿区生态型芒MDA积累的幅度大于矿区生态型芒。同时，在芒对体内活性氧的调节过程中，矿区生态型芒SOD、CAT和POD活性随胁迫浓度增大而显著升高，而非矿区生态型芒POD活性被抑制，且除CAT活性外，SOD活性均低于矿区生态型芒。此外，在芒的渗透调节过程中，可溶性糖、脯氨酸含量均随胁迫浓度增大而显著升高，其中非矿区可溶性糖和脯氨酸含量增值均低于矿区生态型芒。结果表明：矿区生态型芒在锑胁迫下通过较高的抗氧化酶活性和渗透调节物质来增强其对锑的耐受性。（4）锑胁迫下矿区生态型芒的差异蛋白质组学研究。本研究通过运用双向电泳结合质谱鉴定技术，开展矿区生态型芒在锑胁迫下的蛋白质组学响应研究。通过分别开展芒叶片及根部在锑胁迫后差异蛋白组学研究，共获得48个差异蛋白质点，共成功鉴定了其中的31个差异蛋白，主要涉及自由氧调节、能量代谢、信号转导、细胞分裂及细胞结构稳定、蛋白折叠及组装和转录。此外，在本研究中发现一些差异蛋白在锑处理后均上调或诱导表达。其中包括：活性氧调节相关蛋白、伽马响应蛋白、CBS结构域蛋白、锌指蛋白、蛋白丝氨酸／苏氨酸激酶、热休克蛋白Hsp90、ACC氧化酶的同源蛋白、病程相关蛋白PR10等。这些蛋白可能在增强芒对锑的耐受性方面发挥重要作用，其中的一部分蛋白已被证实与植物环境胁迫的耐性相关。（5）锑对植物的毒性危害。通过开展芒在锑胁迫下的蛋白质组学响应研究发现，大量与活性氧调节相关的蛋白质被检测到。如：过氧化物酶Peroxidase73-like、过氧化物还原酶、抗坏血酸过氧化物酶、谷胱甘肽S-转移酶、氧化还原酶活性蛋白C-signal等。本研究结果从分子水平阐明氧化胁迫在锑对植物的毒害过程中起到主要作用。更多还原

【Abstract】 In recent years, due to the lack of effective planning and management of the antimonymining activities and irrational use of antimony, the antimony content in the soil, water and airrose sharply, which posed a serious threat to the health of humans and the entire ecosystem.Phytoremediation as an economic, efficient and relatively safe environmental remediationtechnology has broad application prospects. However, the lack of plant materials for antimonyphytoremediation and undefined mechanism about the antimony phytotoxicity andtolerance inplants made the phytoremediation technology for antimony pollution management extremelyrestricted. Therefore, the current research was carried out to analyze the characteristics ofheavy metal accumulation in plants through the antimony mining areas to assess the potentialsof phytoremediaiton in these plants. Besides, antimony-tolerant plant Miscanthus sinensis wasused to study plant physiology, biochemistry and proteomics response under antimony stresswhich provided an effective method for the understanding of the mechanism of antimonyphytotoxicity and tolerance in plant, which provide an effective method for obtaining idealplant material for antimony phytoremediation.The major results are summarized as follows:(1) Research on the characteristics of heavy metal accumulation in plants grown on anantimony mining areas. A field survey of plants and associated soil in an antimony mining areaof Xikuangshan, Hunan Province, China was conducted to identify species that accumulateheavy metals in their tissues. Of the10herbaceous plants, Miscanthus sinensis and Imperatacylindrica exhibited Hg and Cd phytoextraction potentials. Boehmeria longispica was capableof accumulating Sb and As in its shoots. Moreover, Phytolacca americana could be used forthe phytostabilization of Pb and Cd, Amaranthus paniculatus and Miscanthus sinensis could beused for Sb stabilization, Cynodon dactylon had considerable potential for As and Sbstabilization. Additionally, Miscanthus sinensis considered as a potential energy plant couldbring amount of economic value during its Sb phytoremediation, recognized as a ideal material for Sb phytoremediation. Besides, all these Sb accumulation plants could be used for the studyon plant tolerant mechanism of Sb.(2) Comparison of characteristics in antimony accumulation and growth responses underantimony stress between two ecotypes of Miscanthus sinensis. The content of antimonyconcentration in mine and non-mine eco-Miscanthus sinensis roots and leaves increasedsignificantly with increasing concentration in treatments. Antimony in these two plants weremainly accumulated in its roots with transfer coefficient ranges from0.13to0.18. Differentorganizations of the two ecotypes was not significant different in antimony accumulation,indicating that there were not big difference in capacity of antimony uptake. Antimony addedon different ecotypes Miscanthus sinensis caused significant inhibition on its growth andbiomass. Besides the reduction rate of seedling height and root length in mine eco-Miscanthussinensis compared with the control was lower than the non-mine eco-Miscanthus sinensis.Meanwhile, the dry weight of eco-Miscanthus sinensis shoot and root decline less than thenon-mine eco-Miscanthus sinensis compared with the control, which indicate that the mineeco-Miscanthus sinensis possessed a higher antimony tolerance than the non-mineeco-Miscanthus sinensis.(3) Reactive oxygen and osmotic adjustment mechanism of the two eco-Miscanthussinensis under antimony stress. The antimony stress cause a increase content of MDA in botheco-Miscanthus sinensis leaves, which indicated that lipid peroxidation were aggravated.Among them, the MDA accumulation in non-mining eco-Miscanthus sinensis were greaterthan the mining. Meanwile, as to the adjustment process of ROS regulation, SOD, CAT andPOD activities in mining eco-Miscanthus sinensis rose significantly higher while the PODactivities in non-mining eco-Miscanthus sinensis were inhibited.Except for CAT, SODactivities in non-mining eco-Miscanthus sinensis were lower than the mine one. As to theosmotic adjustment process, soluble sugar and proline content increased significantly with theincreased antiomny concentration in both tow ecotypes. Among them, the increasing rate ofsoluble sugar and proline contentin non-mining eco-Miscanthus sinensis were lower than mining one. The results showed that mining eco-Miscanthus sinensis enhanced its antimonytolerance through higher antioxidant enzyme activities and osmotic adjustment matters.(4) Comparative proteomics research on the mining eco-Miscanthus sinensis under antimonystress. In this study, two-dimensional electrophoresis combined with mass spectrometricidentification technology were carry out to analyze proteomic response of miningeco-Miscanthus sinensis to antimony Stress. Comparative proteomics research were carried inboth leaves and roots under antimony stress. A total of48different protein spots were gainedand31spots were successfully identified. The results showed that these proteins were mainlyinvolved in free oxygen regulation, energy metabolism, signal transduction, cell division andcell structure stability, Protein folding and assembling, and transcription. In addition, severaldifferent proteins were upregulated or induced by the antimony stress including active oxygenregulator protein, gamma response proteins, CBS domain protein, zinc finger protein, proteinserine/threonine kinase, heat shock protein Hsp90, ACC oxidase homolog,pathogenesis-related protein PR10. These proteins might playe important roles in enhancingthe antimony tolerance, part of which had been identified to be associated with plantenvironmental stress tolerance.(5) Phytoxicity of Antimony. A large number of active oxygen regulator proteins weredetected through the proteomic study on Miscanthus sinensis responses to antimony stress,including Peroxidase Peroxidase73-like peroxide reductase, ascorbate peroxidase, glutathioneS-transferase enzyme, C-signal protein with oxidoreductase activity. The results clarified thatoxidative stress play a major role in antimony phytotoxicity at the molecular level.更多还原