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砷(As)是一种有毒的类重金属元素,普遍存在于土壤、淡水和海洋生态系统中。含砷化合物的毒性和生物有效性由其形态所决定,对于大多数生物体,亚砷酸盐(As(Ⅲ))的毒性大于砷酸盐(As(Ⅴ)),而无机砷的毒性大于五价甲基砷(包括一甲基砷(Monomethyl arsenic,MMAs)、二甲基砷(Dimethyl arsenic,DMAs)和三甲基砷氧化物(Trimethyl arsenic oxide,TMAsO))、砷胆碱和砷甜菜碱[1]。硅藻是一类重要的水生光合自养微藻,对全球初级生产力贡献约20%,在海洋和淡水生态系统的金属生物地球化学循环中发挥重要作用[2-3]。已有研究发现6种海洋硅藻具有砷转化能力,在胞内将As(Ⅴ)转化为As(Ⅲ),随后生成MMAs和DMAs,其中布氏双尾藻(Ditylum brightwellii)的细胞体积相对较大,表现出较高的砷甲基化效率[4]。淡水硅藻中也存在砷抗性和砷转化功能物种,淡水水华硅藻链状弯壳藻(Achnanthidium minutissimum)能在100 μmol·L−1 As(Ⅴ)和10 μmol·L−1 As(Ⅲ)条件下生长,在河流生物膜中为主要的砷耐受类群[5-6]。A. minutissimum在含1 μmol·L−1 As(Ⅴ)的培养液中能将As(Ⅴ)进行胞内还原后转化为二甲基砷和其他有机砷产物(约占总砷10%),并将胞内As(Ⅲ)排出胞外(约占50%)[6]。然而,不同淡水硅藻对无机砷底物的转化能力和砷抗性差异的研究较为缺乏。
水体中溶解态硅酸盐是硅藻类浮游植物体生长所必需的营养成分[7]。硅藻通过特定的膜蛋白(硅酸盐转运蛋白SITs)能对胞外硅酸盐进行特异性识别和跨膜转运,利用硅合成硅质细胞壁合成和维持细胞代谢[8]。硅酸盐代谢与硅藻多种生理过程关系紧密,包括呼吸和光合作用。硅酸盐的补充能增加硅藻光合作用速率和中心代谢三羧酸循环酶的丰度,并有助于其细胞分裂[9]。在重金属胁迫下(如Cd),硅转运蛋白转录活性显著下调,可能降低溶解性硅酸盐的吸收从而减少细胞壁的生物合成[10]。硅酸盐有效性对海洋硅藻重金属敏感性产生影响,在硅酸盐缺乏的条件下硅藻的Cd、Cu和Pb的耐受性明显降低,而硅酸盐补充后硅藻能通过高表达细胞膜和液泡金属转运蛋白和更高的抗氧化活性,呈现更强的重金属抗性[11-12]。因此,硅酸盐与硅藻重金属抗性关系密切,但硅酸盐有效性对淡水硅藻砷转化的影响及其作用机制尚不清楚。
菱形藻属(Nitzschia)是常见的淡水硅藻属,其繁殖速度快、分布广、易获取且具有重金属抗性等特点,有应用于水体环境中重金属修复的潜力[13]。本研究选取菱形藻属中的针状菱形藻(Nitzschia acicularis)和谷皮菱形藻(Nitzschia palea)为研究对象,探究不同淡水硅藻对无机砷As(Ⅴ)和As(Ⅲ)的转化能力和As(Ⅲ)抗性,以及硅酸盐对硅藻砷转化的影响和此过程中相关基因的转录表达,为探究淡水硅藻参与砷生物地球化学循环及其影响机制提供参考。
淡水硅藻的砷甲基化和砷氧化代谢机制
Metabolic mechanisms of arsenic methylation and oxidation in freshwater diatoms
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摘要: 硅藻砷甲基化和氧化过程在淡水生态系统砷生物地球化学循环中发挥重要作用,并且硅藻的重金属抗性与营养元素硅酸盐有效性紧密相关。然而,不同硅藻对无机砷的转化能力和砷抗性差异,硅酸盐对硅藻砷转化的影响机制尚不清楚。选取2种淡水硅藻,即针状菱形藻(Nitzschia acicularis)和谷皮菱形藻(Nitzschia palea),作为研究对象,探究其砷甲基化和氧化作用,以及硅酸盐影响下砷转化和此过程中的相关基因转录活性。结果表明,硅藻暴露于As(Ⅲ)的10 d培养期内主要产生二甲基砷,针状菱形藻的二甲基砷转化率为5.54%,高于谷皮菱形藻的转化率(0.80%)。谷皮菱形藻的As(Ⅲ)氧化作用比针状菱形藻强,前者和后者的氧化率分别为90.1%和3.2%。2种硅藻砷甲基化和氧化能力的差异表明不同硅藻As(Ⅲ)胁迫下的主要砷抗性策略不同。针状菱形藻在As(Ⅲ)胁迫下显著上调砷甲基转移功能基因(arsM)表达,并显著下调硅酸盐转运基因(sit)表达。这表明硅藻驱动胞内砷甲基化反应和减少硅/砷转运活性,从而有助于胞内砷解毒和减少As(Ⅲ)吸收。硅酸盐添加对针状菱形藻的arsM基因表达没有明显影响,但上调了线粒体核糖体RNA 12S基因表达,同时促进As(Ⅲ)向As(Ⅴ)转化,这表明硅酸盐可促进硅藻砷氧化和呼吸产能活性,有助于增加硅藻对砷的抗性。本研究证实了硅藻间砷甲基化和氧化能力差异,并且硅酸盐是影响其砷转化的重要因素,可为硅藻应用于砷污染水体修复提供参考。Abstract: Arsenic (As) methylation and oxidation processes in freshwater diatoms play an important role in the biogeochemical cycle of As in freshwater ecosystems, and the heavy metal resistance of diatoms is closely related to the availability of nutrient silicates. However, the transformation ability and As resistance of different diatoms to inorganic arsenic vary, and the mechanism of the silicate effect on the As transformation in diatoms are still unclear. In this study, two freshwater diatoms (Nitzschia acicularis and Nitzschia palea) were selected to explore their As methylation and oxidation, as well as the influence of silicate on the As transformation and the transcriptional activity of related genes during this process. The results showed that diatoms exposed to As(III) mainly produced dimethylarsenic (DMAs) during the 10-day culture period mainly produced dimethyl-arsenic, and the conversion rate of DMAs in the Nitzschia acicularis was 5.54%, higher than that in the Nitzschia palea (0.8%). The oxidation of As(III) was stronger in Nitzschia acicularis than in Nitzschia palea with the oxidation rate of 90.1% and 3.2%, respectively. The differences of As methylation and oxidation capacities bwtween the two diatoms indicated that the main arsenic resistance strategies under As(III) stress were different. With exposing to As(III), the expression of arsenic methyl transfer functional gene (arsM) was significantly up-regulated and the expression of silicate transport gene (sit) was significantly down-regulated. This suggested that diatoms drived intracellular arsenic methylation and reduced Si/As transport activity, thereby contributing to intracellular arsenic detoxification and reducing As(III) absorption.The addition of silicate had insignificant effect on transcriptional activity of arsM in Nitzschia acicularis, but up-regulated the express of mitochondrial ribosomal RNA 12S gene, and promoted the conversion of As(III) to As(V). These results indicated that silicate could promote As oxidation and respiratory activity of diatoms, and contribute to the increase of arsenic resistance of diatoms. This study confirmed the differences in As methylation and oxidation capacity among diatoms, and that silicate was an important factor affecting As transformation, wihch can provide theoretical support for the application of diatoms in the remediation of As-contaminated water.
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表 1 实时荧光定量PCR引物序列及反应条件
Table 1. Primers and reaction conditions for real-time quantitative PCR
目的基因 引物名称 引物序列 扩增条件 β-actin act-F CTTGGCTGGTCGTGACTTGA 95 ℃预变性 2 min;
95 ℃ 10 s,
60 ℃ 20 s,
40个循环。act-R ACCATCGGGAAGCTCGAAAG arsM arsM-F GATGGGCTGGTCGTTGGCAT arsM-R TGGGAGCAAGGCAAAAGGCA 12S 12S-F AGGATGCAAGTGTTATCCGGA 12S-R CAATATCTACGCATTTCACCGCT sit sit-F ATCAACGCTACCACCTGCAT sit-R GAGCCTCGTGGGTAACAACA -
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