| 题名 | 丛枝菌根真菌耐砷机制研究 |
| 作者 | 孙玉青 |
| 学位类别 | 博士 |
| 答辩日期 | 2016-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 陈保冬 |
| 关键词 | AM真菌，砷形态，砷还原，砷甲基化，基因功能验证 arbuscular mycorrhizal fungi, arsenic speciation, arsenic reductase, arsenite methyltransferase gene, gene function verification |
| 其他题名 | Studies on the Arsenic Tolerance of Arbuscular Mycorrhizal Fungi |
| 学位专业 | 生态学 |
| 中文摘要 | 丛枝菌根（arbuscular mycorrhiza，AM）真菌是一类广泛存在于陆地生态系统并与大多数植物形成共生关系的土壤真菌。菌根共生体在植物适应重金属污染环境中的作用得到越来越多的关注。很多研究表明菌根共生体能够增强植物对砷（As）污染的耐受能力。在 As污染环境中，AM真菌能够影响宿主植物对As的吸收和累积，并能够影响植物体内 As的形态转化。然而，有关AM真菌吸收转化砷的生理和分子机制仍不清楚，也极少有研究关注 AM真菌自身对于As的耐受性。此外，在逆境胁迫下，AM真菌生长发育受到抑制，AM真菌多样性降低，但在长期高砷污染环境中，有关 AM真菌群落结构以及优势种属的研究亦少见报道。 本论文调查了湖南省雄黄矿地区长期高砷污染环境中AM真菌的多样性，探明了高砷污染环境中AM真菌的优势种属以及各采样点中的共有种属，分析了影响 AM真菌群落结构的主要土壤环境因子。研究了根生囊霉属（Rhizophagus）AM真菌 Rhizophagus irregularis DAOM197198对 As的吸收及转化，从分子层面揭示了 AM真菌耐受砷的机理。主要研究内容及结果归纳如下： （1）采用 454-焦磷酸测序的方法调查研究了湖南省雄黄矿地区 AM真菌的多样性。在雄黄矿地区长期砷污染的土壤中，AM真菌仍具有较高的多样性。共检测到 11个属的 AM真菌，在所有样点中都出现的属为球囊霉属（Glomus），根生囊霉属（Rhizophagus）以及近明囊霉属（Claroideoglomus）。其中，Glomus为优势属。冗余分析（RDA）表明，影响雄黄矿地区 AM真菌群落结构的主要环境因子为土壤 pH，其次是土壤镉（Cd）浓度和总砷（T-As）浓度。 （2）采用双重无菌培养体系研究了 AM真菌根外菌丝对砷的吸收及转化。研究发现，AM真菌根外菌丝具有将 As(Ⅴ)还原为 As(Ⅲ)的能力，并且能够将As(Ⅲ)转化为二甲基砷（DMA），在此过程中产生中间产物单甲基砷（MMA）。菌根室固体培养基中加入 10 mg/kg Na3AsO4•12H2O处理，培养16周后，菌丝室液体培养基以及菌丝中均检测到了 As(Ⅴ)、As(Ⅲ)及DMA，菌丝内的总砷浓度达到 3009.9 μg/kg。菌丝室中加入 200 μg/kg Na3AsO4•12H2O处理12 h后，菌丝室中开始出现少量 DMA，处理 108 h后，菌丝室中 DMA在总 As中所占比例达到 29.8%。菌丝灭活对照组以及无菌丝的对照组的液体培养基中始终只有 As(Ⅴ)一种砷形态。在菌丝室中加入 200 μg/kg Na3AsO4•12H2O时，菌丝内砷形态的变化更为快速。处理 1 h后菌丝内即检测到了 As(Ⅲ)与 As(Ⅴ)两种形态，其浓度分 别为：138.7 μg/kg、156.4 μg/kg。处理 12 h后，菌丝内共检测到了4种形态的砷：As(Ⅲ)、As(Ⅴ)、MMA、DMA，且菌丝中的总 As浓度达到 1813.9 μg/kg。从48h开始菌丝内只有As(Ⅴ)一种形态。灭活后的菌丝体内自始至终均只有 As(Ⅴ)一种形态，最大浓度为 603.4 μg/kg。这些结果表明：AM真菌菌丝具有砷的还原能力以及甲基化砷的能力；同时，AM真菌根外菌丝对砷具有较强吸附能力，即使已经灭活的菌丝，仍然能够吸附较多的砷。 （3）在 AM真菌Rhizophagus irregularis DAOM197198菌株中，克隆得到砷的还原酶基因（RirrarsC）以及砷的甲基转移酶基因（RirrarsM）。经过异源表达分析，RirrarsC、RirrarsM基因均具有相应的功能。砷还原缺陷型大肠杆菌WC3110在表达了 RirrarsC基因后，对 As(Ⅴ)的还原能力提高了 33.7%。砷敏感型大肠杆菌 AW3110在表达了 RirrarsM基因后能够将 As(Ⅲ)转化为 DMA，中间产物为 MMA。这一结果揭示了 AM真菌耐受砷的分子机理。 本研究通过高通量测序技术探究了长期砷污染环境中AM真菌的多样性，了解了适应高砷污染环境的 AM真菌种属以及影响AM真菌群落结构的环境因子。利用双重无菌体系证明了AM真菌菌丝具有砷的还原、甲基化能力，并通过分子克隆的手段获得了AM真菌菌丝中砷的还原酶及甲基转移酶相关功能基因，从生理和分子角度揭示了AM真菌耐受砷的机理，深化了对菌根共生体参与砷的迁移、转化等过程的认识，为利用菌根修复砷污染土壤提供了理论依据。 |
| 英文摘要 | Arbuscular mycorrhizal fungi (AMF) are ubiquitous soil fungi that can form symbiotic associations with the majority of the land plant families. It has been well demonstrated that the mycorrhizal symbioses play important roles in plant adaptation to heavy metal contaminated environments. AMF can affect the arsenic (As) uptake and accumulation, even influence the As speciation in the host plants under As contaminations. However, the underlying mechanism for As translocation and transformation by AM fungi has not been resolved yet. On the other hand, the growth and development of AMF could be restrained, and the AMF diversity would decrease under the environmental stresses, but there is rare report on the AMF community structure and the dominant AMF species in the long-term heavily As contaminated environments. In the present study, we investigated the AMF biodiversity in the Realgar mining area in Hunan province, China, and revealed the key influencing factors for AMF community structure. By using an in vitro culture system, we studied the As uptake and transformation by AMF, and further uncovered the molecular mechanisms involved in the As tolerance of AMF. The main results are summarized as follows: (1) The AMF biodiversity in the Realgar mining area in Hunan province was investigated using 454-pyrosequencing technique. The results indicated that the AMF diversity is relatively high even in such long-term contaminated soils. A total of 11 AMF genera were identified, among which Glomus, Rhizophagus, and Claroideoglomus clarodeum were detected in all sampling sites, and Glomus was the dominant genus. Redundancy analysis indicated that soil pH, Cd concentrations and total As were the main factors influencing AMF community structure in the Realgar mining area. (2) In an in vitro culture system for AM fungi, we investigated the As transformation by the extraradical mycelium. The results indicated that the AM hyphae could not only reduce arsenate (As(Ⅴ)) into arsenite (As(Ⅲ)), but also methylate the As(Ⅲ), and monomethylated arsenic (MMA) was produced as the intermediate in the process. When the solid M medium in the root compartment was treated by 10 mg/kg Na3AsO4•12H2O for 16 weeks, the As species in the liquid M medium in the hyphal compartment and in the mycelium were As(Ⅴ)、As(Ⅲ) and DMA, and the total As concentration in the mycelium reached 3009.9 μg/kg. When the hyphal compartment was treated with 200 μg/kg Na3AsO4•12H2O, small amount of DMA was detected in the liquid M medium after 12 hours. From then on, DMA began to accumulate, and after 108 hours, the DMA accumulated up to 29.8% in the total As concentration. By contrast, there was only As(Ⅴ) in the liquid M medium in the control which the mycelium was inactivated by 2% formalin solution, or in absence of mycelium. On the other hand, the As speciation in the mycelium changed more quickly, As(Ⅲ) and As(Ⅴ) were detected in the mycelium after 1 hour, and the concentration was 138.7 μg/kg and 156.4 μg/kg respectively. There were four As species in the mycelium after 12 hour: As(Ⅲ)、As(Ⅴ)、MMA、DMA, and the total As concentration was 1813.9 μg/kg. Only As(Ⅴ) was detected from 48 hour to 108 hour. The As species in the inactivated mycelium was As(Ⅴ) from the beginning to the end,and the highest concentration was 603.4 μg/kg. These results strongly supported that the AMF hyphae can reduce As(Ⅴ) to As (Ⅲ), and methylate As(Ⅲ) to MMA and DMA. Meanwhile the AMF hyphae showed a strong adsorption ability to As, even if it has been inactivated. (3) An arsenate reductase gene (RirrarsC) and arsenite methyltransferase gene (RirrarsM) were identified in Rhizophagus irregularis DAOM197198 strain. The RirrarsC and RirrarsM genes were cloned and gene functions were verified by heterologous expression in the reductase gene promoter defect strain and an As-sensitive strain of E.coli. The reduction ability was increased by 33.71% when the defective E.coli WC3110 expressed RirrarsC gene. The sensitive E.coli AW3110 could methylate the trivalent arsenic into dimethylarsine after expression of RirrarsM gene, and the intermediate was MMA. The study thus uncovered the molecular mechanism of the As tolerance of AMF. In summary, the study revealed the molecular diversity of AMF in the long-term As contaminated environment, and uncovered the molecular mechanism involved in the As tolerance of AMF. It has provided deeper insight into the As migration,transformation, accumulation, detoxification and resistance in the AM associations, and also set theoretical basis for application of AMF for remediation of As contaminated soils. |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/36959]  |
| 专题 | 生态环境研究中心_城市与区域生态国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 孙玉青. 丛枝菌根真菌耐砷机制研究[D]. 北京. 中国科学院研究生院. 2016. |
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