基于宏基因组学的酸性森林土壤氨氧化微生物群落特征研究

全球30％以上陆地面积是酸性土壤(pH<5.5),而酸性土壤中氨氧化微生物群落特征研究是破译其硝化过程微生物学机理的基础.尤其随着完全硝化微生物(Complete ammonia oxidizer,comammox)的发现,亟需重新认知酸性土壤中氨氧化微生物类群.以酸性马尾松林为研究对象,综合利用荧光定量PCR(qPC...

Metagenomics Based Study on Community Characteristics of Ammonia-Oxidizing Microorganisms in Acid Forest Soil

[Objective] The acidic forest soils are characterized by low pH and depletion of NH₃ substrate, which are considered to be the major factors shaping ammonia-oxidizing communities (AOA, AOB and comammox) in soils. However, so far little is known about the relationship between absolute and relative numbers of the three kinds of ammonia oxidizers in acidic forest soils, especially due to the absence of reliable real-time quantitative PCR (qPCR) primers for the newly identified comammox Nitrospira. Here this study aims to develop a strategy to determine abundance of comammox Nitrospira, and to investigate community structures of AOA, AOB and comammox in the acidic forest soils. [Method] Absolute abundances of amoA genes of AOA, AOB and comammox in soils were measured with the aid of qPCR. However, Ntsp-amoA-162F/359R and comaB-244F/659R, the two primers for comammox amoA genes would trigger significant non-specific amplification, and hence overestimation of the abundance of comammox. Therefore, qPCR data of comammox amoA genes needs to be calibrated with the semi-quantitative method based on agarose gel electrophoresis of the qPCR products, and by the metagenomic sequencing of the total soil DNA. Furthermore, community compositions of AOA, AOB and comammox in the acidic forest soils were also characterized with the aid of metagenomic sequencing. [Result] The qPCR demonstrate that the amoA genes of AOA and AOB were 2.61×10⁶ and 1.45×10⁶ copies·g^-1, respectively in abundance, while the qPCR of comammox amoA gene exhibits significant non-specific amplification, and the calibrated data show that comammox amoA gene, was about 1.38×10⁶ ~1.47×10⁶ copies·g^-1 in abundance. Furthermore, Group 1.1b was the predominant one, accounting for ~88.07% of AOA, while the classical 1.1a-associated acidophilic group accounted only for ~11.93%. Of AOB, Nitrosospira was the major group, accounting for ~63.96%, while Nitrosomonas made up ~36.04% of the total AOB gene sequence. Clade B was the major group, accounting for ~63.39% of the comammox, while Clade A accounted only for ~36.61% of total comammox amoA gene sequence and exclusively fell into Sub-clade A.1. [Conclusion] In this study, a strategy for determining the absolute abundance of comammox amoA genes in soil was developed based on comprehensive utilization of the techniques of qPCR, semi-quantitation and metagenomic analysis. In the acidic forest soils studied the ratio of comammox to AOA and AOB in amoA gene abundance is found to be ~0.55 and ~0.98, respectively. Group 1.1b, Nitrosospira and Clade B is the major group of AOA, AOB and comammox, respectively, in the soils, and the potential molecular mechanisms underpinning the adaption of these ammonia oxidizers to the environments low in pH and short of NH₃ substrates warrant further physio-biochemical studies of pure cultures.