Dokument

Summary:
Metatranscriptomics is a state-of-the-art technique to elucidate the functional activities of microbial communities, but its application is still limited to marine microbial assemblages. In my PhD project, we established the complete approach of soil metatranscriptomics, involving RNA extraction, cDNA library preparation by random priming, 454-pyrosequencing, and bioinformatic data analysis. The approach was tested on microbial communities in the oxic surface layer and the anoxic bulk soil of flooded rice paddy soil microcosms. Total RNA was recovered in high integrity and purity by low-pH phenol extraction (pH 5.0) followed by Q-Sepharose column chromatography. We were able to enrich mRNA sequences up to 50-70% in the metatranscriptome libraries using Ribo-Zero™ rRNA removal kit (Meta-Bacteria). All the 454 reads obtained were preprocessed prior to data analysis to minimize sequence ambiguities. A total of 10,000 SSU-ribotags (total RNA) were analyzed to elucidate community composition in the oxic and anoxic zones at three different incubation time points (25, 45 and 90 days after transplantation of rice seedlings). Additionally, about 45,000 and 12,000 mRNA-tags (enriched mRNA) were obtained for the analysis of functional activities in, respectively, the oxic and anoxic zone of 90-day-old rice microcosms. SSU-ribotag data analysis revealed no major temporal changes in community composition except for Geobacter, Clostridia and methanogens in the anoxic bulk soil. However, the taxonomic composition of microbial communities was clearly distinct between the oxic and anoxic zones, with cyanobacteria being the dominant group in the surface layer. Although mRNA-tags related to basic cellular functions were most abundant in both mRNA datasets, the expression of specific functions in response to different oxygen conditions was observed such as, for example, methane oxidation in the oxic zone and methanogenesis in the anoxic zone. Our metatranscriptomic approach provides a means to analyze the composition and functional gene expression of complex soil microbial communities while avoiding the limitations of PCR-based approaches.

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