Abstract
Isolation of genes from DNA in soil through culture-independent methods such as PCR and clone library screening is an effective approach to obtain genes or gene products of industrial importance from soil microorganisms, the large majority of which cannot be cultured. It is assumed that the use of a wide variety of soils differing in classification, land use, fertilizer/pesticide management, or soil treatment may enable the isolation of more diverse target genes. To verify this assumption, after incubation with and without addition of naphthalene and fluorene, the diversity of ring-hydoxylating dioxygenase (RHD) genes in a soil with long-term application of manure, a soil with long-term application of TPN/γ-HCH/manure, a coniferous forest soil, and a citrus orchard soil was examined. DNA was directly extracted from the above soils, followed by PCR-T-RFLP analysis targeting RHD genes. As a result, different T-RFLP patterns were observed among the four original untreated soils. Addition of naphthalene and fluorene changed the T-RFLP pattern in each soil, and the difference in the patterns among the four treated soils was more significant. Sequencing of the PCR products obtained from the coniferous forest soil treated with naphthalene and fluorene revealed the amplification of partial RID genes, and phylogenetic analysis based on the deduced amino acid sequences showed their diversity. These results supported the assumption described above.
