纳米银胁迫下土壤微生物活性与腐皮镰刀菌（Fusarium solani）转录组的响应

发布时间：2018-10-23 18:43

【摘要】：纳米材料的特殊结构使得它们具有许多特异的优良性质,其中纳米银的广谱抗菌性给纳米银的广泛应用开辟了广阔的前景,同样也给土壤环境带来了胁迫风险。本文通过在室内培养条件下,以硝酸银与石墨烯为对照,测定纳米银对土壤呼吸、土壤酶以及土壤微生物群落功能多样性的影响,分析它们对土壤微生物的影响,预估纳米银给土壤生态环境带来的风险;探讨土传真菌病害腐皮镰刀菌(Fusarium solani)生长、细胞结构及转录组对纳米银的响应。得到的主要结果如下:1.纳米银对土壤呼吸强度存在抑制作用,较同浓度硝酸银显著。石墨烯对土壤呼吸无影响。2.纳米银与硝酸银均抑制土壤可培养细菌、真菌、放线菌的数量;100 mg·kg-1和150 mg·kg-1的纳米银与硝酸银处理显著降低了三种土壤微生物的数量。低于100 mg·kg-1的石墨烯处理土壤细菌、真菌、放线菌数量均无显著变化;但1000 mg·kg-1的石墨烯显著增加土壤细菌与真菌的数量。3.纳米银显著抑制土壤脲酶与土壤脱氢酶活性,且抑制作用大于同浓度硝酸银处理,其中纳米银≥10 mg·kg-1时,土壤脲酶、脱氢酶活性均显著降低,但硝酸银达到100mg·kg-1以上这两种酶活性才显著下降;纳米银与硝酸银对土壤过氧化氢酶和碱性磷酸酶活性无影响;石墨烯存在显著抑制了土壤碱性磷酸酶活性。4.纳米银对土壤微生物群落的总体活性、Shannon、Simpson、Mc Intosh指数的负面影响大于硝酸银,两者在150 mg·kg-1处理下均最为显著,石墨烯存在降低了土壤微生物群落功能多样性。5.纳米银对F.solani生长的抑制存在明显的剂量效应,EC50值为3.6 mg·L-1,10mg·L-1的纳米银可完全抑制F.solani的生长。纳米银可破坏F.solani的细胞壁、细胞膜、细胞器等,且在细胞膜上观察到疑似纳米银颗粒。6.基于对纳米银处理后F.solani转录组数据的分析,共得到23791条Unigenes,分别在6 h、12 h、24 h时发现862、867、774个差异表达基因(P0.05)。GO功能富集分析表明纳米银处理后F.solani在蛋白质加工、氧化还原酶活性、分解代谢、细胞成分作用于碳-氮的水解酶活性、结构分子活性、翻译、外部包装结构、RNA结合和前体代谢物与能量的产生等类别表达差异较大。KEGG代谢途径分析表明,纳米银处理后F.solani在转录、翻译、折叠,分选和降解、碳水化合物代谢、能量代谢、信号转导与信号分子和相互作用等途径表达差异较大。7.差异表达基因在能量和物质代谢、信号转导和遗传信息途径的比较集中,主要包括TCA循环、硫代谢、抗氧化酶活性、热激蛋白、蛋白质合成与分解、DNA修复等过程。纳米银可通过扰乱F.solani的能量物质代谢、产生ROS、造成蛋白质甚至DNA损伤杀死F.solani细胞。
[Abstract]:The special structure of nanomaterials makes them have many special and excellent properties. The broad-spectrum antimicrobial properties of nanocrystalline silver have opened up a broad prospect for the wide application of nano-silver, and have also brought about stress risks to the soil environment. In this paper, the effects of silver nitrate and graphene on soil respiration, soil enzymes and the functional diversity of soil microbial communities were determined under indoor culture conditions, and their effects on soil microbes were analyzed. To estimate the risk of nano-silver to soil ecological environment and to study the growth, cell structure and transcriptional response of soil-borne fungal disease Fusarium vulgaris (Fusarium solani) to nano-silver. The main results are as follows: 1. Nano silver inhibited soil respiration, which was more significant than that of silver nitrate at the same concentration. Graphene has no effect on soil respiration. 2. Both silver nanoparticles and silver nitrate inhibited the number of culturable bacteria, fungi and actinomycetes in the soil, and the treatment of 100 mg kg-1 and 150 mg kg-1 of silver nanoparticles and silver nitrate significantly reduced the number of three kinds of soil microbes. The amount of soil bacteria, fungi and actinomycetes treated with graphene less than 100 mg kg-1 had no significant change, but 1000 mg kg-1 of graphene significantly increased the number of soil bacteria and fungi. The activity of soil urease and soil dehydrogenase was significantly inhibited by nano-silver, and the inhibitory effect was greater than that of the same concentration of silver nitrate. When nano-silver was more than 10 mg kg-1, the activity of soil urease and dehydrogenase decreased significantly. However, silver nitrate and silver nitrate had no effect on the activities of catalase and alkaline phosphatase, and graphene significantly inhibited the activity of alkaline phosphatase. The negative effect of silver nanoparticles on the total activity of soil microbial communities was greater than that of silver nitrate, and the existence of graphene decreased the functional diversity of soil microbial communities. The growth of F.solani was inhibited by nano-silver in a dose-dependent manner, and the growth of F.solani was completely inhibited by silver nanoparticles with a EC50 value of 3.6 mg L ~ (-1) ~ (-1) ~ (10 mg 路L ~ (-1). Silver nanoparticles can destroy the cell wall, cell membrane and organelle of F.solani, and the suspected silver nanoparticles can be observed on the cell membrane. 6. 6. Based on the analysis of F.solani transcriptome data after silver nanoparticles treatment, a total of 23791 Unigenes, differentially expressed genes were found at 6 h or 12 h or 24 h. (P0.05). GO functional enrichment analysis showed that F.solani was involved in protein processing and redox enzyme activity after treatment with silver nanoparticles. Catabolism, cellular components acting on carbon-nitrogen hydrolase activity, structural molecular activity, translation, external packaging structure, RNA binding and the production of precursor metabolites and energy were significantly different. KEGG metabolic pathway analysis showed that, There were significant differences in the expression of F.solani in transcription, translation, folding, sorting and degradation, carbohydrate metabolism, energy metabolism, signal transduction and signal molecules and interactions. The differentially expressed genes are concentrated in energy and substance metabolism, signal transduction and genetic information pathway, including TCA cycle, sulfur metabolism, antioxidant enzyme activity, heat shock protein, protein synthesis and decomposition, DNA repair and so on. Silver nanoparticles can damage F.solani cells by disrupting the energy metabolism of F.solani and producing ROS, causing protein and even DNA damage.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S154.3;S432.44

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