亚硝酸盐添加对土壤硝化和反硝化基因转录活性及N_(2)O排放的影响

Effects of Nitrite Addition on Transcription Activity of Nitrification and Denitrification Functional Genes and N2O Emission in Soil

设施菜田土壤氧化亚氮(N_(2)O)脉冲式排放期间通常伴随着亚硝酸盐( NO_(2)^(-))的大量积累,为揭示NO_(2)^(-)对设施菜田土壤N_(2)O排放的影响机制,以两种典型蔬菜种植区土壤(碱性土壤/酸性土壤)为研究对象,通过室内培养试验,对比厌氧和好氧培养条件下添加NO_(2)^(-)后两种土壤无机氮转化与N_(2)O、氮气(N_(2))和二氧化碳(CO_(2))等气体排放,以及氨氧化单加氧酶α亚基调控基因(amoA)、亚硝酸盐还原酶调控基因(nirK和nirS,统称nir)和N_(2)O还原酶调控基因(nosZ)的丰度和转录情况。结果显示:受pH等环境因素影响,土壤中 NO_(2)^(-)含量并不一定与N_(2)O排放之间存在相关性,但添加NO_(2)^(-)的处理显著增加了两种土壤的N_(2)O排放量和N_(2)O/(N_(2)O+N_(2))指数(IN_(2)O)(P<0.05)。碱性土壤中,60 mg·kg^(-1)外源 NO_(2)^(-)对土壤CO_(2)排放无明显抑制作用,厌氧培养条件下nirK基因、好氧培养条件下amoA和nirS基因均出现了添加 NO_(2)^(-)后转录拷贝数显著高于空白处理的现象,而nosZ基因无此现象。酸性土壤中,amoA转录活性整体较低,好氧空白处理时nirS基因转录拷贝数随培养时间的延长而增加(P<0.05);60 mg·kg^(-1)外源NO_(2)^(-)明显降低了酸性土壤的CO_(2)排放量、相关基因的丰度及转录拷贝数。上述结果显示,土壤中积累的NO_(2)^(-)会通过诱导nir基因转录与N_(2)O还原酶竞争电子和抑制N_(2)O还原酶活性等途径,增加土壤的IN_(2)O,影响有氧条件下N_(2)O的排放途径,研究结果将为探索设施菜田土壤氮素高效利用和N_(2)O减排提供科学依据。

【Objective】Studies relating soil N availability to N_(2)O emissions commonly focus on NO_(3)^(-) and in some cases NH_(4)^(+).Thus,less effort has been devoted to measuring soil NO_(2)^(-) despite its role as a central substrate in N_(2)O production.【Method】In this study,two typical greenhouse vegetable soils(alkaline vs.acid soil)were selected to explore the influencing mechanism of NO_(2)^(-) on N_(2)O emission.Also,its association with the inorganic nitrogen transformation processes,gaseous emission(N_(2)O,N_(2),CO_(2)),and the abundances and transcription copies of functional genes(amoA,nirK,nirS and nosZ)under anaerobic(0%O_(2))and aerobic(21%O_(2))conditions through in-lab incubation and real-time quantitative polymerase chain reaction(qPCR).【Result】The natural accumulation and tolerance of 2 NO-were higher in alkaline soil than in acidic soil.With respect to pH,the relative concentration of 2 NO^(-)_(2)in soil did not correlate with N_(2)O emissions.However,the addition of NO_(2)^(-) significantly increased the N_(2)O emission and N_(2)O/(N_(2)O+N_(2))index(IN_(2)O)of the two soils(P<0.05),and decreased the N_(2)emission in both soils under anaerobic conditions(50.9%and 94.2%in alkaline and acidic soils,respectively).In the alkaline soil,exogenous NO_(2)^(-) at 60 mg·kg^(–1)had no significant inhibition effect on soil CO_(2)emission,and the transcription copies of nirK gene at 16 h under anaerobic incubation,amoA gene at 16 h and nirS gene at 84 h under aerobic incubation were significantly higher than that of control check(N0),but nosZ gene had no such phenomenon.In acid soil,the overall gene and transcription activity of amoA was low,and the transcription copies of the nirS gene increased with the increase of incubation time in aerobic N0 treatment(P<0.05).Exogenous NO_(2)^(-) at 60 mg·kg^(–1)significantly reduced the CO_(2)emission,and the abundance and transcription copies of related genes in the acid soil.Oxygen significantly reduced the transcription copies of denitrification functional genes in both soils,and nirK was more sensitive.Compared with the N0 treatment under anaerobic incubation,the transcription copies of nirK,nirS and nosZ in alkaline soil were reduced by 97.3%,74.5%and 89.0%,respectively,at 16 h under aerobic incubation.The variation trend of the denitrification genes transcription copies in both soils under aerobic conditions was different.In the alkaline soil,the transcription copies of denitrification functional genes were significantly decreased with the increase in incubation time(P<0.05).In the acidic soil,only nirK transcription copies decreased significantly with the increase of incubation time(P<0.05)in N0 treatment under aerobic conditions,while nirS and nosZ transcription copies increased,or decreased first and then increased,respectively.【Conclusion】The accumulation of NO_(2)^(-) in soils will increase soil IN_(2)O and affect the N_(2)O emission pathway by inducing nir gene transcription to compete for electrons with N_(2)O reductase and inhibiting N_(2)O reductase activity.These results provide a scientific basis for exploring the efficient utilization of soil nitrogen and N_(2)O reduction in greenhouse vegetable soils.