Enriching plant microbiota for a metagenomic library construction

• Published in: Environmental microbiology 2008-10, Vol.10 (10), p.2684-2691
• Main Authors: Wang, Hao-Xin, Geng, Zhao-Liang, Zeng, Ying, Shen, Yue-Mao
• Format: Article
• Language: English
• Subjects: Actinobacteria; Bacteria - growth & development; Bacteria - isolation & purification; Biodiversity; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Ribosomal - chemistry; DNA, Ribosomal - genetics; Gene Library; Genes, rRNA; Genotype; Molecular Sequence Data; Phylogeny; Plants - microbiology; Proteobacteria; Ribotyping; RNA, Bacterial - genetics; RNA, Ribosomal, 16S - genetics; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid
• ISSN: 1462-2912; 1462-2920
• DOI: 10.1111/j.1462-2920.2008.01689.x

Plant microbiota (the microorganisms that live in any associations with plant tissues) represents a rather unexplored area of metagenomic research compared with soils and oceans. Constructing a metagenomic library for plant microbiota is technically challenging. Using all the biomass without pre-enrichment could lead to vast proportions of the host plant DNA in the metagenomic library, doubtless obliterating the microbial contribution. Therefore, the first and essential step is to enrich for the constituent microorganisms from plant tissues. Here, a strong enrichment for plant microbiota was achieved by coupling SDS (sodium dodecyl sulfate) with NaCl, creating a predominantly microbial metagenomic library that contains 88% bacterial inserts. 16S rDNA sequence analysis revealed that the metagenomic DNA of enrichments originates from very diverse microorganisms. At least 74 distinct ribotypes (at a 97% threshold) from seven different bacterial phyla were identified and mainly distributed among Actinobacteria and Proteobacteria. Additionally, a simplified version of Amplified Ribosomal DNA Restriction Analysis (ARDRA) was developed for a quick and efficient assessment of the enriching procedures. This work opens further insight into the great biotechnical potential of plant microbiota, holding more potential for drug discovery through a metagenomic strategy, and paving the way for recovery and biochemical characterization of functional gene repertoire from plant microbiota.