The greenhouse gas nitrous oxide (N₂O) is produced in soil as a consequence of complex co-occurring processes conducted by diverse microbial species, including fungi. The fungal p450nor gene encodes a nitric oxide reductase associated with fungal denitrification. We thus hypothesized that p450nor gene expression is a marker for ongoing fungal denitrification. Specific PCR primers and quantitative PCR (qPCR) assays were developed targeting p450nor genes and transcripts. The novel PCR primers successfully amplified p450nor from pure cultures, and were used in an mRNA targeted qPCR to quantify p450nor gene transcription (i.e., gene expression) during denitrification activity in cultures of the fungal model denitrifier Fusarium oxysporum. Gene expression was induced by high (5 mM) and low (0.25 mM) nitrite concentrations. Nitrite stimulated N₂O production rates by F. oxysporum, which correlated well with an up to 70-fold increase in p450nor gene expression during the first 12–24 h of anoxic incubation. The relative p450nor gene peak expression and peak N₂O production rates declined 20- and 2-fold on average, respectively, towards the later phase of incubation (48–120 h). The ¹⁵N site preference of N₂O (SP(N₂O)) was high for F. oxysporum and independent of reaction progress, confirming the fungal origin of N₂O produced. In conclusion, the developed fungal p450nor gene expression assay together with the analysis of SP(N₂O) values provide a basis to improve current tools for the identification of fungal denitrification and/or N₂O production in natural systems like soils.

由于包括真菌在内的多种微生物共同进行的复杂共生过程，土壤中产生了温室气体一氧化二氮（N ₂ O）。真菌p450nor基因编码与真菌反硝化作用相关的一氧化氮还原酶。因此，我们假设p450nor基因表达是正在进行的真菌反硝化的标记。针对p450nor基因和转录本开发了特异性PCR引物和定量PCR（qPCR）分析方法。新型PCR引物成功地从纯培养物中扩增出p450nor，并用于靶向mRNA的qPCR中定量p450nor真菌模型反硝化镰孢镰刀菌培养物中反硝化活性过程中的基因转录（即基因表达）。基因表达是由高（5 mM）和低（0.25 mM）亚硝酸盐浓度诱导的。亚硝酸盐刺激了尖孢镰刀菌的N ₂ O产生速率，这与缺氧孵育的前12–24小时内p450nor基因表达增加多达70倍相关。相对于p450nor基因的峰值表达和峰值N ₂ O产生率，分别向孵化的后期（48–120小时）分别下降了20倍和2倍。在¹⁵ N中的n个站点偏好₂ O（SP（N ₂O））对于尖孢镰刀菌而言很高，并且与反应进程无关，这证实了所产生的N ₂ O的真菌起源。总之，已开发的真菌p450nor基因表达分析以及SP（N ₂ O）值分析为改善当前用于鉴定土壤等自然系统中真菌反硝化和/或N ₂ O产生的工具提供了基础。