Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • For functional metagenomics based gene screening the DNA

    2022-08-05

    For functional metagenomics-based gene screening, the DNA library construction approach is widely used . Fosmid and cosmid libraries are usually used to explore novel genes. Lee et al. reported that a halotolerant and protease-resistant α-galactosidase was identified from the gut metagenome of , a polyphagous insect, using a fosmid library with 92,000 individual fosmid clones. However, the technique is time consuming and depends on the screening platform and probability. Metagenomic sequencing is a powerful alternative to functional gene isolation for comprehending complex microbial gene diversity . In this study, 55.68 Gb of sequence was obtained from paddy soil that had a long-term application of organic compost using the Illumina Genome Analyzer. After deep sequencing of soil DNA, several putative open reading frames (ORFs) were identified and annotated. Gene Ontology (GO), Metagenomics RAST (MG-RAST), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) of soil metagenomes were further used for the analysis of functional profiles and metabolic pathways. To verify the identified ORFs whether functional genes can be obtained, expressed and characterized from the soil metagenome, database search and ORF prediction with over 250 Mesoridazine in length provided information for the putative glycoside hydrolase genes. A putative gene encoding α-galactosidase with a low identical match and e-value, and high query coverage was synthesized and overexpressed in . Then, the activity and characterization of α-galactosidase were determined and described. Materials and methods
    Results
    Discussion High-throughput sequencing through NGS, which can provide a large amount of information at the sequence level, has been applied in various metagenomes, such as agricultural soil, insects, and the human gut, to interpret the microbial diversity directly 6, 7, 11, 23. In this study, an in-depth analysis of paddy soil microbial sequencing was conducted with 55.68 Gb sequences using the Illumina Genome Analyzer, and the metagenome sequences were submitted to the NCBI database. The total bases and predicted genes of the soil metagenome were 12,000- and 397-fold greater than that of E. coli K12 with 4.64 Mb and 4498 genes (24). The sequences of the soil metagenome relating to Proteobacteria were determined to be the most abundant Eubacteria (Fig. 1), corresponding to previous characterizations of the different microbial consortia through 16S rRNA analysis 4, 5. At the genus level, a large number of reads (2.4%) associated with Candidatus Solibacter, a member of the phylum Acidobacteria, were recognized, followed by Candidatus Koribacter, Geobacter, Streptomyces, Bradyrhizobium, and Bacillus. Candidatus Solibacter and Candidatus Koribacter are usually predominant in contaminated sites (25). In paddy soil with long-term application of organic compost, they could play a crucial role in decomposing organic matter for the carbon cycle. The relative abundance of the main nitrogen-fixing bacterial genera including Bradyrhizobium, Bacillus, Burkholderia, Pseudomonas, Rhizobium, Acinetobacter, Mesorhizobium, Paenibacillus, Achromobacter, Agrobacterium, and Stenotrophomonas was also observed in the metagenome (26). In addition, all sequences of viruses were derived from unclassified species with 0.05%. In Eukaryota, fungi are the most dominant category, the largest phylum of which is Ascomycota (0.3%) (27). However, 0.15% of fungi belonged to unclassified species in the metagenomic analysis. With respect to the functional profiles, GO, MG-RAST, and KEGG were performed (Fig. 2). The significantly enriched GO terms were cellular, metabolic, and single-organism of the biological process as well as cell, cell part, and membrane of the cellular component together with binding and catalytic activity of the molecular function. These remarkable terms were the same as the soil rhizosphere microbial communities of the transgenic soybean line ZUTS31 (26). This indicated that these GO terms were essential and abundant in the microbial structure of agricultural soil. According to functional annotation using MG-RAST, sequences corresponding to carbohydrates were dominant. Of the identified sequences, genes involved in one-carbon, di-carbon, and oligosaccharide metabolisms such as decarboxylase, UDP-glucose 4-epimerase, and galactosidase, which provide energy for supporting life, were relatively abundant (28). Moreover, several CO2 fixation genes associated with the Calvin–Benson cycle and photorespiration could be identified and may be derived from Cyanobacteria. Nitrogen cycling, including nitrogen fixation, nitrification, and denitrification, is essential for limiting the nutrient supply for microbiological processes and rice production (29). In this study, FixA and FixB required for free-living microaerophilic nitrogen fixation in Bradyrhizobium could be found based on the KEGG pathway (30). On the other hand, the functional genes of ammonia mono-oxygenase, nitrite oxidoreductase, nitrate reductase, and nitrite reductase were used as molecular markers to examine the dynamics of nitrifier and denitrifier communities (29). However, only nitrate and nitrite reductases were exhibited in the paddy soil metagenome. This suggested that spatial and geochemical factors could result in the uneven distribution of ammonium oxidizers (31). Furthermore, a few sequences related to infectious diseases corresponding to amoebiasis, toxoplasmosis, African trypanosomiasis, Chagas disease, leishmaniasis, and malaria were detected. Nevertheless, a complete pathway of infectious diseases was absent.