發(fā)布時間：2018-01-10 18:05

  本文關(guān)鍵詞：根際土壤—微生物—蜈蚣草系統(tǒng)中砷的形態(tài)轉(zhuǎn)化與解毒機制　出處：《南京大學》2017年博士論文　論文類型：學位論文

  更多相關(guān)文章： 蜈蚣草 砷 As~Ⅲ氧化菌 As~Ⅴ還原菌 As~Ⅲ外排

【摘要】：砷是自然界廣泛存在的一種有毒類金屬,屬國際癌癥研究組織最早確認的Ⅰ類致癌物之一。砷超積累植物蜈蚣草的發(fā)現(xiàn)為砷污染土壤修復提供了可能,目前關(guān)于蜈蚣草的研究已從實驗室階段拓展到了野外應用。盡管蜈蚣草具備超強的砷抗性和富集能力,其具體的機制和影響因素仍不明確。在考察蜈蚣草根際砷轉(zhuǎn)化、植物砷吸收和解毒的過程中,微生物的作用是不容忽視的影響因素之一。然而,目前對蜈蚣草根際菌和內(nèi)生菌的研究局限于部分可培養(yǎng)菌株和微生物自身的抗砷特性方面,對這些微生物在蜈蚣草根際及植物組織中的砷轉(zhuǎn)化和植物砷吸收的意義未見深入報道。基于此,本課題從根際土壤-微生物-蜈蚣草系統(tǒng)入手,主要分析了砷的形態(tài)轉(zhuǎn)化、解毒機制及其影響因素,重點考察微生物介導的As~Ⅲ氧化、As~Ⅴ還原和As~Ⅲ甲基化作用及其對蜈蚣草砷吸收和解毒的影響。本課題可為更好理解蜈蚣草高效砷吸收、解毒和實現(xiàn)微生物-植物聯(lián)合修復砷污染土壤提供參考。本課題的主要內(nèi)容如下:1)研究了外加50 mg kg~(-1)As~Ⅴ和/或1.5%PR對3種農(nóng)田土壤中蜈蚣草根際砷氧化還原微生物的群落結(jié)構(gòu)和功能基因多樣性及其與砷賦存形態(tài)的關(guān)系�；�16SrRNA、aroA-like和arsC基因的qPCR分析,發(fā)現(xiàn)砷脅迫顯著降低了 16SrRNA的基因豐度,但顯著增加了aroA-like和arsC的基因豐度。外加PR時,砷脅迫對16S rRNA的抑制效應得到緩解,aroA-like和arsC的相對基因頻率也降到起始水平。土壤中areA-like基因豐度高達arsC基因的50多倍,且前者的生物多樣性也比后者高。冗余度分析表明,As~Ⅴ濃度和DOC是影響基因豐度的主要因素,而土壤pH、可利用態(tài)鈣、磷和Asv濃度是導致aroA-like基因產(chǎn)生多樣性的主要因素。2)研究了蜈蚣草組織提取液(未處理或經(jīng)表面滅菌)對100μgL~(-1) As~Ⅲ或As~Ⅴ的轉(zhuǎn)化情況。結(jié)果發(fā)現(xiàn),添加了經(jīng)表面滅菌處理的蜈蚣草根組織提取液使45%As~Ⅲ發(fā)生了氧化,而未經(jīng)表面滅菌的根組織提取液氧化能力更強,達73%;此外,在蜈蚣草提取液的其他處理組中都檢測到了 Asv還原,達3.7%-24%。然而,過濾或熱處理后溶液中并未觀察到砷的轉(zhuǎn)化現(xiàn)象。以上結(jié)論說明1)混合液中只有微生物在砷形態(tài)轉(zhuǎn)化中發(fā)揮了作用;2)根組織提取液與砷混合液中的Asm氧化可能主要受As~Ⅲ氧化菌調(diào)控,而根狀莖和羽葉中的As~Ⅴ還原主要受As~Ⅴ還原菌調(diào)控;3)根際菌和內(nèi)生菌都在蜈蚣草根際圈和組織中的砷轉(zhuǎn)化過程中發(fā)揮著重要作用。3)研究了無菌條件和37.5 mg kg~(-1) As~Ⅴ脅迫下外加砷氧化還原菌對蜈蚣草根際砷行為及植物砷吸收的影響。結(jié)果表明,接種微生物強化了蜈蚣草對砷、磷的吸收,分別高達47%和69%,而蜈蚣草生物量增加了 20%-74%。此外,在對照組無菌培養(yǎng)基中的砷形態(tài)以As~Ⅲ為主(95%),接種了還原菌的根際砷形態(tài)全為As~Ⅲ,但接種了氧化菌的根際As~Ⅴ比例高達95%。以上結(jié)論說明1)與普通植物類似,在高砷脅迫時蜈蚣草根部也存在明顯的As~Ⅲ外排現(xiàn)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驅(qū)動力。4)研究了 0、50和200 mg kg~(-1) As~Ⅴ對蜈蚣草根際砷的甲基化作用及植物生長和砷富集的影響。種植3個月和6個月后,發(fā)現(xiàn)植物組土壤砷濃度降低了 7.9%-58%,而對照組土壤總砷濃度也降低了 4.0%-31%,且砷流失量與arsM基因豐度的增加呈較好的線性關(guān)系(r0.78,p= 0.0045-0.2115)。此外,與對照組相比,50mgkg~(-1)As~Ⅴ可促進蜈蚣草生長,但200 mgkg~(-1) As~Ⅴ處理使蜈蚣草羽葉葉緣出現(xiàn)了毒害癥狀,該部位的砷濃度達內(nèi)部組織的2.26倍。掃描電鏡-能譜面掃分析顯示,蜈蚣草羽葉砷的轉(zhuǎn)運為葉脈→葉緣。掃描電鏡-能譜線掃分析還發(fā)現(xiàn),蜈蚣草羽葉硅與砷的相對含量存在一定的相關(guān)性(r = 0.49,p0.05)。以上結(jié)論說明1)土壤砷流失可能與水的淋溶、植物吸收和微生物甲基化有關(guān);2)砷從葉脈到葉緣的再轉(zhuǎn)運可能是降低砷負擔的一種有效途徑;3)砷在蜈蚣草羽葉的轉(zhuǎn)運和解毒可能與硅轉(zhuǎn)運通道或硅的參與有關(guān)。
[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.

【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：X53;X17

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