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    氟苯尼考联合铜环境胁迫对土壤固氮菌成膜能力及相关基因表达的研究

    Biofilm-forming capacity of soil nitrogen-fixing bacteria and related gene expression under florfenicol combined with copper environmental stress

      摘要
    • 摘要:
      目的  研究氟苯尼考和铜(Cu)残留对土壤固氮菌的生态毒性效应，为评价兽药的环境风险提供依据。
      方法  对从花生根际土壤中分离出的一株优势固氮菌RpEC2071进行氟苯尼考和铜胁迫处理，试验设置空白组(0 μg/mL)、氟苯尼考添加组(40 μg/mL)、Cu添加组(200 μg/mL)和混合组(氟苯尼考40 μg/mL+Cu 200 μg/mL)，并于给药后采样。采用苯酚硫酸法和96孔法研究氟苯尼考和Cu单独或联合处理对固氮菌胞外多糖产生及生物膜形成能力的影响。采用RT-qPCR测定分析固氮菌生物膜及固氮相关基因的mRNA表达水平。
      结果  氟苯尼考和Cu单独胁迫会促进生物膜的形成，二者生物膜形成能力约为空白组的2倍；混合胁迫会抑制生物膜的形成，空白组生物膜形成能力是其3.1倍。胞外多糖分泌结果与生物膜形成能力测定结果基本一致。生物膜相关基因与氮代谢调控基因呈显著正相关，氟苯尼考和Cu的添加改变了fliQ、nifH和nnrR等基因的表达水平，且二者联合处理后会对其表达分别产生协同或拮抗作用。
      结论  氟苯尼考与Cu的单独或联合胁迫影响固氮菌RpEC2071中生物膜相关基因表达，进而对其生物膜形成能力产生影响。预示了以氟苯尼考和Cu为例的兽药及重金属残留胁迫会对土壤固氮菌产生潜在的生态毒性效应，从而破坏土壤中固氮生态系统。

       

      Abstract:
      Objective  The ecotoxic effects of florfenicol and copper (Cu) residues on soil nitrogen-fixing bacteria were studied to provide a basis for evaluating the environmental risks of veterinary drugs.
      Method  A dominant nitrogen-fixing bacterium RpEC2071 was isolated from peanut root enclosure and treated under florfenicol and Cu stress. We set the blank group (0 μg/mL), florfenicol group (40 μg/mL), Cu group (200 μg/mL) and mixed group (flufenicol 40 μg/mL, Cu 200 μg/mL), and collected samples at multiple time points after dosing. The phenol-sulfuric acid method and 96-well microplate method were used to study the effects of florfenicol and Cu alone or in combination on the production of extracellular polysaccharides and biofilm formation of nitrogen-fixing bacterium. RT-qPCR was used to determine nitrogen-fixing bacterium biofilm formation and the mRNA expression levels of nitrogen fixation-related genes.
      Result  Florfenicol and Cu alone promoted the formation of biofilms, and the biofilm formation capacity of both was about twice that of the blank group. Under mixed stress, the biofilm formation was inhibited, and the biofilm formation capacity of the blank group was 3.1 times that of it. The results of extracellular polysaccharide secretion were basically consistent with the results of the determination of biofilm formation ability. Biofilm-related genes were significantly positively correlated with nitrogen metabolism regulatory genes, and the addition of florfenicol and Cu changed the expression levels of genes such as fliQ,ntrX andnnrR, and it would produce synergistic or antagonistic effects after the combination treatment of florfenicol and Cu.
      Conclusion  The individual or combined stress of florfenicol and Cu affects the expression of biofilm-related genes in nitrogen-fixing bacteria RpEC2071, which in turn affects its ability to form biofilm. This research reveals the ecotoxic potential of veterinary drugs and heavy metal residue stresses on soil nitrogen-fixing bacteria, using florfenicol and Cu as examples. As a result, this can lead to the impairment of the nitrogen-fixing ecosystem in soil.

       

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