| 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 |
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| 引用本文： | 唐修峰,秦华,匡璐,王欣欣,宋玉翔,高豪,刘林梦,任一,单军,张焕朝,王保战.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[J].土壤学报,2021,58(5):1311-1321. |
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| 作者姓名： | 唐修峰 秦华 匡璐 王欣欣 宋玉翔 高豪 刘林梦 任一 单军 张焕朝 王保战 |
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| 作者单位： | 南京林业大学,浙江农林大学,中国科学院南京土壤研究所,中国科学院南京土壤研究所,中国科学院南京土壤研究所,上海美吉生物医药科技有限公司,上海美吉生物医药科技有限公司,上海美吉生物医药科技有限公司,中国科学院南京土壤研究所,南京林业大学,中国科学院南京土壤所 |
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| 基金项目： | 浙江农林大学省部共建亚热带森林培育国家重点实验室开放基金（KF2017-02）和国家自然科学基金项目 |
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| 摘 要： | 全球30%以上陆地面积是酸性土壤(pH5.5),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(qPCR)、凝胶电泳半定量和宏基因组测序等技术研究土壤中氨氧化古菌(Ammonia-oxidizing archaea,AOA)、氨氧化细菌(Ammonia-oxidizing bacteria,AOB)和Comammox的相对丰度以及群落组成特征。研究发现AOA和AOB amoA基因丰度分别为2.61×106 copies·g~(-1)和1.45×106copies·g~(-1);而comammoxamoA基因qPCR结果存在显著的非特异性扩增,导致其丰度被高估,而经凝胶电泳半定量矫正后,约为(1.38～1.47)×106copies·g~(-1),该结果和土壤宏基因测序揭示的comammox相对丰度基本吻合。此外,宏基因组分析发现经典嗜酸group1.1a-associated仅占AOA总类群的12%,而group1.1b则占88%,尽管目前仍未有嗜酸group 1.1b AOA纯菌株的报道。AOB主要类群为Nitrosospira(约64%),而Nitrosomonas约占36%。Comammox主要类群为clade B(约64%),而clade A仅占36%且均隶属于clade A.1亚枝,这暗示clade B与已报道的嗜中性comammox clade A纯菌株有极大的生理代谢差异。总之,本研究提供了综合利用qPCR、半定量和宏基因组分析土壤氨氧化微生物群落的策略,并建议优化comammox的qPCR引物,同时本研究系统分析了酸性马尾松林土壤中氨氧化微生物的相对丰度和群落组成特征。
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| 关 键 词： | 酸性森林土壤 氨氧化微生物 comammox amoA基因 宏基因组学 |
| 收稿时间： | 2020/2/2 0:00:00 |
| 修稿时间： | 2020/4/8 0:00:00 |
Metagenomics Based Study on Community Characteristics of Ammonia-Oxidizing Microorganisms in Acid Forest Soil |
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| TANG Xiufeng,QIN Hu,KUANG Lu,WANG Xinxin,SONG Yuxiang,GAO Hao,LIU Linmeng,REN Yi,SHAN Jun,ZHANG Huanchao,WANG Baozhan.Metagenomics Based Study on Community Characteristics of Ammonia-Oxidizing Microorganisms in Acid Forest Soil[J].Acta Pedologica Sinica,2021,58(5):1311-1321. |
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| Authors: | TANG Xiufeng QIN Hu KUANG Lu WANG Xinxin SONG Yuxiang GAO Hao LIU Linmeng REN Yi SHAN Jun ZHANG Huanchao WANG Baozhan |
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| Institution: | Nanjing forestry university,Zhejiang A&F University,Nanjing institute of Soil Science, Chinese Academy of Sciences,Nanjing institute of Soil Science, Chinese Academy of Sciences,Nanjing institute of Soil Science, Chinese Academy of Sciences,Shanghai Majorbio Bio-pharm Biotechnology Co.,Ltd,Shanghai Majorbio Bio-pharm Biotechnology Co.,Ltd,Shanghai Majorbio Bio-pharm Biotechnology Co.,Ltd,Nanjing institute of Soil Science, Chinese Academy of Sciences,Nanjing forestry university,Nanjing institute of Soil Science, Chinese Academy of Sciences and Nanjing Agricultural University |
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| Abstract: | 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. |
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| Keywords: | Acidic forest soil Ammonia-oxidizing microorganisms Comammox amoA gene Metagenomics |
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