洋底深部真菌反硝化产N2新途径及原位作用模拟

Fungi are the main eukaryotes in deep subseafloor biosphere and an important part of the food chain in the subseafloor ecosystem. Due to the late start of its research and the limited pure culture strains, little is known about the diversity, distribution and the role in driving element cycle of deep subseafloor fungi. Our recent study found that a strain of Aspergillus species inhabiting at a depth of 1.6 km below seafloor has the ability to denitrify and produce N2 under anaerobic conditions, which is completely different from the currently known denitrifying process of fungi with N2O as the end product. However, the denitrification mechanism of N2 production by this strain is not clear, and whether there are more denitrifying fungi and their roles in the deep subseafloor nitrogen cycle remains unknown. This project intends to analyze the denitrification mechanism of Aspergillus sp. in deep subseafloor by means of multi-dimensional omics and gene editing technology. The diversity of denitrifying fungi in deep subseafloor was elucidated by measuring the denitrification ability of 27 deep subseafloor fungal species (69 strains). The sediment samples containing denitrifying fungi were cultured in simulated in-situ environment to investigate the role of denitrifying fungi in driving nitrogen cycle in deep subseafloor environment. The implementation of this project is expected to reveal not only new denitrifying fungi and mechanisms, but also new mechanisms for fungi to adapt to the extreme subseafloor environment, so as to propose a new direction for the study of fungi driving the nitrogen cycle in the earth.

真菌是洋底深部生物圈的主要真核生物，是洋底生态系统食物链的重要一环。由于对其研究起步较晚且纯培养菌株有限，人们对洋底深部真菌多样性、分布及其在驱动元素循环中的作用知之甚少。我们近期研究发现，一株栖息于洋底1.6km深的曲霉具有在厌氧条件下反硝化产N2能力，完全不同于目前已知的以N2O为终产物的真菌反硝化过程，但目前尚不清楚该菌株反硝化产N2机理，也不了解洋底深部是否存在更多的反硝化真菌及其在氮循环中的作用。本项目拟运用多维组学、基因编辑等手段，解析洋底深部曲霉的反硝化作用机理；通过检测已获得的27种69株洋底深部真菌的反硝化能力，阐明洋底深部反硝化真菌的多样性；采用模拟原位环境培养含反硝化真菌的洋底沉积物样品，探究反硝化真菌在驱动洋底深部氮循环中的作用。该项目的实施，不仅有望揭示新的反硝化真菌类群及机理，还可能诠释真菌适应洋底极端环境的新机制，从而提出真菌驱动地球氮循环的研究新方向。

运用同位素示踪、生化和组学以及基因沉默等检测分析手段，研究了从2km深的洋底沉积物样品中分离获得的真菌的反硝化途径及其应对厌氧环境的分子机制，为深入了解深部生物圈生命及其在驱动元素生物地球化学循环中的作用提供新见解。主要结果如下：.（1）发现并证明埋藏于1.3~2.5km深洋底沉积物中的真菌（19种40株）普遍具有厌氧反硝化作用、DNRA作用以及硝化作用，无厌氧氨氧化作用，而且这些真菌的氮转化能力与其所处沉积层深度和地质环境有关；（2）证明从2.4km深洋底沉积物中分离获得的聚多曲霉29R-4-F02具有完全反硝化产N2途径；（3）完成了对菌株29R-4-F02基因组的测序、组装和注释，探究了该菌在不同氮源培养基中的转录组谱，鉴定了参与反硝化的基因，包括dNar、nirK/nirD、P450nor，但未发现Nos基因；（4）证明菌株29R-4-F02能够在厌氧、高压（35Mpa）环境下正常生长，并发挥反硝化作用；（5）解析了洋底深部真菌在厌氧环境中的生长、发育、代谢及产能机理，发现真菌通过多种机制应对厌氧环境，包括：无法完成有性生殖、增加菌丝中线粒体的数量、提高乙醇和氨基酸合成代谢、分泌胞外多糖等。 .总之，我们的研究表明，真菌拥有独特的遗传基础和进化机制，丰富的生理与代谢多样性，能够在厌氧、高压的含煤洋底沉积环境中存活数千万年之久，并在驱动厌氧生态系统的元素循环中发挥重要作用。

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