水分变化对关中塿土硝化与反硝化关键功能基因转录水平的调控

Cultivated soils are a major source of nitrous oxide, which has become the third most important greenhouse gas contributing to global warming. N2O is produced biologically in soils during nitrification and denitrification. Soil redox conditions changes after rainfall and irrigation that affects the potential of nitrification and denitrification. These two processes can be identified by functional genes. The transcriptional activities of amoA and nirK/nirS gene could represent the functions of nitrification and denitrification, respectively; the transcription of nosZ gene controls N2O reduction process, which is thought to be extraordinarily sensitive to the environmental changes. In this project, the simulated irrigation experiments will be settled in early spring and midsummer to have different soil temperature. The water content along the 0-20 cm soil profile will be controlled by irrigating with 2 mmol/L NH4NO3 as substrate. Glucose (10 mmol/L) will be included as a carbon substrate treatment. Soil RNA will be extracted from different soil layers (0-5 cm, 5-10 cm, 10-15 cm and 15-20 cm) at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h after irrigation. Expression of amoA, nirK, nirS and nosZ gene will be quantified by real-time PCR; community succession and the dominant populations will be investigated by denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing. The objectives of this project are to explore the regulation of soil moisture on the activity and the community succession of nitrifying and denitrifying microorganisms; to clarify the contribution of soil moisture on nitrification and denitrification separately; to reveal how sensitive are the nitrifying and the denitrifying microbes to environmental temperature; and to confirm the regulation of organic carbon availability on nitrification and denitrification.

农田土壤是氧化亚氮的主要排放源之一。硝化和反硝化作用是土壤中参与N2O排放的主要生物学过程。降雨或灌水后短期内由于氧化还原条件发生改变，影响土壤中硝化与反硝化作用潜势。通过amoA、nirK或nirS基因转录活性的研究可分别表征硝化和反硝化作用的能力；nosZ基因转录水平控制着氧化亚氮还原为N2的过程，其对环境因素响应敏感。本项目拟通过田间模拟灌溉试验，在控制0-20 cm土柱剖面含水量、灌水后0-72 h内的不同采样时间，分别在早春和仲夏设置温度变化处理，在NH4NO3作为底物的条件下设置添加葡萄糖处理，采样提取土壤RNA，采用定量PCR、DGGE和高通量测序技术，对关键功能基因表达量及多样性进行分析，明确土壤水分含量与微生物功能活性及群落结构演替特征的关系，阐明土壤水分变化对硝化和反硝化作用的贡献，揭示硝化、反硝化微生物对环境温度的敏感性，验证有机碳源对硝化与反硝化过程的调控能力。

氮素循环是农田养分转换的重要途径之一。硝化和反硝化作用是土壤中参与氮素循环的主要生物学过程。降雨或灌水后短期内由于土壤氧化还原条件发生改变，影响土壤中硝化与反硝化作用潜势。本项目通过控制水分条件的土壤培养实验，结合氧气浓度和添加碳源处理，监测培养过程中土壤NO3-、NO2-和NH4+等无机氮素含量；提取土壤总RNA，使用荧光定量PCR技术分析硝化、反硝化作用关键功能基因细菌amoA、古菌amoA、nirK、nirS和nosZ等转录水平丰度差异；并挑选样品通过基于转录水平的16S扩增子高通量测序技术或宏转录组测序技术，得到各处理样品中具有功能活性的微生物群落组成。研究发现：.（1）土壤水分含量显著影响无机氮的转化过程。低水分条件下无机氮转化速率总体降低；随水分含量增加，硝化和反硝化作用速率都有所增加。好氧培养条件下，随水分含量的增加各功能基因丰度均有不同程度的增加；氨氧化细菌功能基因比氨氧化古菌功能基因对水分的响应更灵敏；nirK和nosZ基因对对水分的响应比nirS基因更灵敏；硝化过程相关微生物群落结构受氧气影响较大，而反硝化过程相关微生物群落结构受氧气影响较小；不同硝化、反硝化微生物种群对土壤水分的响应敏感性具有明显差异。.（2）好氧条件下土壤水分通过调节通气孔气度（土壤中含氧量）影响无机氮转化过程，而厌氧条件下反应了土壤水分对硝化、反硝化过程的直接影响。本研究结果表明土壤水分除依靠调节土壤孔隙中氧气含量控制无机氮转化过程之外，水分本身对硝化与反硝化过程也具有重要的调控作用。.（3）碳源添加显著提高了硝化、反硝化微生物转录活性，尤其是氨氧化细菌的转录活性，从而抑制了氨氧化古菌的转录活性。添加碳源后，氨氧化细菌成为硝化过程乃至无机氮素转化过程的主要贡献者。.本研究阐明了土壤水分变化对硝化和反硝化作用的贡献，揭示了硝化、反硝化微生物对氧气的敏感性，验证了有机碳源对硝化与反硝化过程的调控能力。研究结果为理解降雨或灌溉条件下土壤氮素循环微生物学机理提供理论依据。