Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed
Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed
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DOI：
10.1128/aem.00019-16
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发表时间：
2016-02
影响因子：
4.4
通讯作者：
Pia K. Wüst;Heiko Nacke;K. Kaiser;S. Marhan;J. Sikorski;E. Kandeler;R. Daniel;J. Overmann
中科院分区：
文献类型：
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作者：
Pia K. Wüst;Heiko Nacke;K. Kaiser;S. Marhan;J. Sikorski;E. Kandeler;R. Daniel;J. Overmann
摘要现代测序技术可以分别基于土壤微生物群落的DNA和RNA对土壤微生物群落总量和潜在活性进行高分辨率分析。本研究采用定量聚合酶链式反应和454焦磷酸测序的方法，研究了不同提取方法对三种不同土壤类型(瘦素土、冻土和潜育土)中16S rRNA基因和转录本丰度和多样性的影响。核酸的质量和产量因所采用的提取方法和所分析的土壤类型而有很大差异。细菌核糖体含量(16S rRNA转录本与16S rRNA基因的比值)可以作为细菌细胞潜在活性的指标，不同提取方法获得的核酸提取物之间存在2个数量级的差异。根据提取方法的不同，土壤优势类群，特别是放线杆菌和变形杆菌的相对丰度变化高达10倍。通过这种系统的方法，本研究允许根据特定的土壤属性、感兴趣的核酸和目标生物推导出选择适当的提取方案的指南。
ABSTRACT Modern sequencing technologies allow high-resolution analyses of total and potentially active soil microbial communities based on their DNA and RNA, respectively. In the present study, quantitative PCR and 454 pyrosequencing were used to evaluate the effects of different extraction methods on the abundance and diversity of 16S rRNA genes and transcripts recovered from three different types of soils (leptosol, stagnosol, and gleysol). The quality and yield of nucleic acids varied considerably with respect to both the applied extraction method and the analyzed type of soil. The bacterial ribosome content (calculated as the ratio of 16S rRNA transcripts to 16S rRNA genes) can serve as an indicator of the potential activity of bacterial cells and differed by 2 orders of magnitude between nucleic acid extracts obtained by the various extraction methods. Depending on the extraction method, the relative abundances of dominant soil taxa, in particular Actinobacteria and Proteobacteria, varied by a factor of up to 10. Through this systematic approach, the present study allows guidelines to be deduced for the selection of the appropriate extraction protocol according to the specific soil properties, the nucleic acid of interest, and the target organisms.