根际土壤—微生物—蜈蚣草系统中砷的形态转化与解毒机制

发布时间：2018-01-10 18:05

本文关键词：根际土壤—微生物—蜈蚣草系统中砷的形态转化与解毒机制　出处：《南京大学》2017年博士论文　论文类型：学位论文

【摘要】：砷是自然界广泛存在的一种有毒类金属,属国际癌症研究组织最早确认的Ⅰ类致癌物之一。砷超积累植物蜈蚣草的发现为砷污染土壤修复提供了可能,目前关于蜈蚣草的研究已从实验室阶段拓展到了野外应用。尽管蜈蚣草具备超强的砷抗性和富集能力,其具体的机制和影响因素仍不明确。在考察蜈蚣草根际砷转化、植物砷吸收和解毒的过程中,微生物的作用是不容忽视的影响因素之一。然而,目前对蜈蚣草根际菌和内生菌的研究局限于部分可培养菌株和微生物自身的抗砷特性方面,对这些微生物在蜈蚣草根际及植物组织中的砷转化和植物砷吸收的意义未见深入报道。基于此,本课题从根际土壤-微生物-蜈蚣草系统入手,主要分析了砷的形态转化、解毒机制及其影响因素,重点考察微生物介导的As~Ⅲ氧化、As~Ⅴ还原和As~Ⅲ甲基化作用及其对蜈蚣草砷吸收和解毒的影响。本课题可为更好理解蜈蚣草高效砷吸收、解毒和实现微生物-植物联合修复砷污染土壤提供参考。本课题的主要内容如下:1)研究了外加50 mg kg~(-1)As~Ⅴ和/或1.5%PR对3种农田土壤中蜈蚣草根际砷氧化还原微生物的群落结构和功能基因多样性及其与砷赋存形态的关系。基于16SrRNA、aroA-like和arsC基因的qPCR分析,发现砷胁迫显著降低了 16SrRNA的基因丰度,但显著增加了aroA-like和arsC的基因丰度。外加PR时,砷胁迫对16S rRNA的抑制效应得到缓解,aroA-like和arsC的相对基因频率也降到起始水平。土壤中areA-like基因丰度高达arsC基因的50多倍,且前者的生物多样性也比后者高。冗余度分析表明,As~Ⅴ浓度和DOC是影响基因丰度的主要因素,而土壤pH、可利用态钙、磷和Asv浓度是导致aroA-like基因产生多样性的主要因素。2)研究了蜈蚣草组织提取液(未处理或经表面灭菌)对100μgL~(-1) As~Ⅲ或As~Ⅴ的转化情况。结果发现,添加了经表面灭菌处理的蜈蚣草根组织提取液使45%As~Ⅲ发生了氧化,而未经表面灭菌的根组织提取液氧化能力更强,达73%;此外,在蜈蚣草提取液的其他处理组中都检测到了 Asv还原,达3.7%-24%。然而,过滤或热处理后溶液中并未观察到砷的转化现象。以上结论说明1)混合液中只有微生物在砷形态转化中发挥了作用;2)根组织提取液与砷混合液中的Asm氧化可能主要受As~Ⅲ氧化菌调控,而根状茎和羽叶中的As~Ⅴ还原主要受As~Ⅴ还原菌调控;3)根际菌和内生菌都在蜈蚣草根际圈和组织中的砷转化过程中发挥着重要作用。3)研究了无菌条件和37.5 mg kg~(-1) As~Ⅴ胁迫下外加砷氧化还原菌对蜈蚣草根际砷行为及植物砷吸收的影响。结果表明,接种微生物强化了蜈蚣草对砷、磷的吸收,分别高达47%和69%,而蜈蚣草生物量增加了 20%-74%。此外,在对照组无菌培养基中的砷形态以As~Ⅲ为主(95%),接种了还原菌的根际砷形态全为As~Ⅲ,但接种了氧化菌的根际As~Ⅴ比例高达95%。以上结论说明1)与普通植物类似,在高砷胁迫时蜈蚣草根部也存在明显的As~Ⅲ外排现象以降低砷毒性;2)As~Ⅲ氧化菌是蜈蚣草高效砷富集的重要驱动力。4)研究了 0、50和200 mg kg~(-1) As~Ⅴ对蜈蚣草根际砷的甲基化作用及植物生长和砷富集的影响。种植3个月和6个月后,发现植物组土壤砷浓度降低了 7.9%-58%,而对照组土壤总砷浓度也降低了 4.0%-31%,且砷流失量与arsM基因丰度的增加呈较好的线性关系(r0.78,p= 0.0045-0.2115)。此外,与对照组相比,50mgkg~(-1)As~Ⅴ可促进蜈蚣草生长,但200 mgkg~(-1) As~Ⅴ处理使蜈蚣草羽叶叶缘出现了毒害症状,该部位的砷浓度达内部组织的2.26倍。扫描电镜-能谱面扫分析显示,蜈蚣草羽叶砷的转运为叶脉→叶缘。扫描电镜-能谱线扫分析还发现,蜈蚣草羽叶硅与砷的相对含量存在一定的相关性(r = 0.49,p0.05)。以上结论说明1)土壤砷流失可能与水的淋溶、植物吸收和微生物甲基化有关;2)砷从叶脉到叶缘的再转运可能是降低砷负担的一种有效途径;3)砷在蜈蚣草羽叶的转运和解毒可能与硅转运通道或硅的参与有关。
[Abstract]:Arsenic is a toxic metal widely exist in nature, is the international organization for cancer research's first confirmed class I carcinogen. Found arsenic hyperaccumulator Pteris vittata has provided the possibility for the arsenic contaminated soil remediation, the current research on the laboratory stage from Pteris vittata extended to the field. Although with arsenic resistant super pterisvittata the enrichment ability and its mechanism, and the influence of factors is still not clear. In the study of transformation in rhizosphere of plants of Pteris vittata arsenic, arsenic uptake and detoxification, the role of microorganisms is one of the factors that can not be ignored. However, current research on the rhizosphere bacteria of Pteris vittata and endophytes are confined to the part of hand cultivation of arsenic resistant strains and microbial characteristics of its own, in-depth coverage of these microorganisms in the rhizosphere of Pteris vittata and plant tissue and plant transformation of arsenic in arsenic absorption was not significant. Based on this, this lesson Starting with the problems from the rhizosphere soil microorganism of Pteris vittata system, mainly analyzes the transformation of arsenic speciation, detoxification mechanism and its influence factors, As~ III focuses on microbial mediated oxidation, As~ reduction and As~ III V methylation and the absorption of arsenic detoxification and Pteris vittata. This topic for a better understanding of pterisvittata efficiency arsenic absorption, provide a reference implementation of detoxification and micro phyto combined remediation of arsenic contaminated soil. The main contents of this thesis are as follows: 1) were studied with 50 mg kg~ (-1) As~ diversity V and / or 1.5%PR genes of 3 kinds of community structure and function of Pteris vittata rhizosphere oxidation of arsenic in farmland soil microbial reduction and its relationship with the arsenic speciation analysis. Based on 16SrRNA, aroA-like and arsC gene of qPCR, found that arsenic stress significantly reduced the abundance of 16SrRNA gene, but significantly increased the gene abundance of aroA-like and arsC. With PR, arsenic The stress of the inhibitory effect of 16S on rRNA in remission, relative gene frequency of aroA-like and arsC also dropped to initial level. ArsC gene areA-like gene abundance in soil up to 50 times, and the biodiversity is also higher than the latter. The analysis shows that the redundancy, As~ V and DOC concentration are the main factors influencing gene abundance however, soil pH, available calcium, phosphorus and Asv concentration are the main factors leading to aroA-like gene diversity.2) of extract of Pteris vittata (untreated or by tissue surface sterilization) in 100 gL~ (-1) As~ III or As~ V conversion condition. The results showed that adding the surface sterilization of centipede extract the grassroots 45%As~ III oxidation occurs, without root tissue surface sterilization, extract the stronger oxidation ability, up to 73%; in addition, the extraction of other liquid in the treatment group were detected Asv in Pteris vittata, up to 3.7%-24%. However, after heat treatment or filtration solution was not observed in transformed arsenic. It concluded that 1) in the mixed solution of microorganisms in arsenic speciation only played a role; 2) root extract Asm oxidation mixture with arsenic in As~ III oxidizing bacteria was mainly affected by the regulation, and the rhizome and adding As~ V reduction is mainly controlled by As~ V reducing bacteria control; 3) plays an important role in.3 rhizosphere bacteria and endophytic bacteria in the rhizosphere of Pteris vittata and the arsenic transformation process of aseptic conditions) and 37.5 mg kg~ (-1) and the influence of arsenic redox bacteria on Pteris vittata roots when arsenic behavior and plant arsenic absorption As~ V stress. The results showed that microbial inoculation enhanced the absorption of phosphorus on arsenic pterisvittata, respectively, up 47% and 69%, while vittata increased 20%-74%. in control group in sterile culture medium with arsenic As~ III (95%), Inoculated rhizosphere arsenic reducing bacteria for As~ III, but inoculated rhizosphere As~ V ratio oxidizing bacteria 95%. above conclusion 1) similar to ordinary plants in high arsenic stress when the centipede roots are obviously As~ III efflux phenomenon to reduce arsenic toxicity; 2) As~ 3 oxidizing bacteria is an important driving force of.4 high arsenic) of Pteris vittata 0,50 and 200 mg kg~ (-1) and As~ methylation in plants of Pteris vittata rhizosphere arsenic V effect of growth and enrichment. The planting of 3 and 6 months after the discovery of a plant soil arsenic concentration decreased by 7.9%-58%, but also reduce the 4.0%-31% concentration of control group total arsenic and arsenic of soil loss and the abundance of arsM gene showed a good linear relationship (r0.78, p=, 0.0045-0.2115). In addition, compared with the control group, 50mgkg~ (-1) As~ V can promote the growth of Pteris vittata, but the 200 mgkg~ (-1) As~ treatment. The pterisvittata V leaves The edge of the toxic symptom, 2.26 times the concentration of arsenic in the parts of the internal organization of the surface. The scanning electron microscope scan analysis showed that arsenic transport. Vittata vein and leaf margin. Scanning electron microscopy and energy spectrum analysis and also found that there is a correlation between the relative content of silicon and arsenic. Vittata (r = 0.49, P0.05). The above conclusion 1) soil arsenic leaching and water erosion, plant uptake and microbial methylation; 2) translocation to arsenic from the leaf leaf margin may be an effective way to reduce arsenic burden; 3). The transport of arsenic in Pteris vittata and detoxification and silicon transport channel or silicon concerned.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X53;X17

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