The root-associated type II methanotrophs significantly contribute to CH₄ oxidation-dependent N₂ fixation. However, it is unclear whether type I methanotrophs are involved in CH₄ oxidation and N₂ fixation, especially in natural wetlands. So far, limited attention has given to root-associated active microorganisms. Here, metatranscriptomic analysis of root-associated microbes has been proposed to reveal the aerobic methanotrophs contributing to CH₄ and nitrogen cycles in the roots of Phragmites australis grown in a natural wetland. Results showed Methylocystaceae (type II methanotrophs) and Methylococcaceae (type I methanotrophs) as major taxa (relative abundance, 14%) at transcription level. However, based on 16S rRNA gene sequencing, contribution of these taxa was < 1% at DNA level. Genes encoding methane monooxygenase (enzyme responsible for the first step of CH₄ oxidation) were detected in Methylomonas (pmoCBA) and Methylosinus (mmoXYZCB). Furthermore, genes related to methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase were also detected in Methyosinus and Methylomonas, while mcrA gene was observed in Methanospirillum and Methanofollis. Moreover, nitrogenase structural genes, such as nifHDK, were found in Methylosinus (Methylocystaceae) and Methylomonas (Methylococcaceae). Minor nitrogenase genes were detected in Cyanothece, Lyngbya, Pelobacter and Smithella of Cyanobacteriaceae family. In addition, N₂ fixing activity of P. australis was determined by analyzing the natural abundance of δ¹⁵N from June to August. The N₂ fixing activity of P. australis increased in presence of CH₄ in root system under ¹⁵N-N₂ feeding. Metatranscriptomic analysis revealed that not only type II methanotrophs, but also type I methanotrophs oxidize CH₄ and fix N₂.

根相关的 II 型嗜甲烷菌显著促进 CH₄ 氧化依赖性 N₂ 固定。然而，目前尚不清楚 I 型嗜甲烷菌是否参与 CH₄ 氧化和 N₂ 固定，尤其是在自然湿地中。到目前为止，对根相关活性微生物的关注有限。在这里，已经提出了根相关微生物的元转录组学分析，以揭示在天然湿地中生长的芦苇根中促进 CH₄ 和氮循环的好氧嗜甲烷菌。结果显示甲基球菌科 （II 型甲烷菌） 和甲基球菌科 （I 型甲烷菌） 在转录水平上是主要分类群 （相对丰度，14%）。然而，基于 16S rRNA 基因测序，这些分类群在 DNA 水平上的贡献为 < 1%。在甲基单胞菌 （pmoCBA） 和甲基肌苷 （mmoXYZCB） 中检测到编码甲烷单加氧酶 （负责 CH₄ 氧化第一步的酶） 的基因。此外，在 Methyosinus 和 Methylomonas 中也检测到与甲醇脱氢酶、甲醛脱氢酶和甲酸盐脱氢酶相关的基因，而在 Methanospirillum 和 Methanofollis 中观察到 mcrA 基因。此外，在 Methylosinus （甲基球菌科） 和 Methylomonas （甲基球菌科） 中发现了 nifHDK 等固氮酶结构基因。在蓝杆菌科的 Cyanothece、Lyngbya、Pelobacter 和 Smithella 中检测到少量固氮酶基因。此外，P 的 N₂ 固定活性。 通过分析 6 月至 8 月δ¹⁵N 的自然丰度来确定南方。在 ¹⁵N-N₂ 喂食下，根系中存在 CH₄ 时，P. australis 的 N₂ 固定活性增加。宏转录组学分析显示，不仅 II 型嗜甲烷菌，而且 I 型嗜甲烷菌也会氧化 CH₄ 并固定 N₂。