Study On The Actinobacteria Biodiversity Of Han-xing Iron Mine Area By Culture-independed Methods

Extraction methods of metagenomic DNA of 3 kinds of different samples from seven sampling sites of Han-Xing Iron Mine were first explored and established preliminary. The results showed that: the microorganism in ore-dressing waste water and mine drainage is too little to extract the metagenomic DNA directly after millipore filtration, but the metagenomic DNA could be extracted efficiently after enriched by different concentrations of modified M5'broth and iron-extract broth; the samples from raw ore and tailings contain a lot of heavy metal ions, especially iron ions, which will not only affect the activity of lysozyme, but also affect the efficiency of PCR amplification subsequently, therefore, metal ions and the inhibition effects should be removed by using high concentrations of TE buffer before DNA extraction; SDS-high-salt method were used for the metagenomic DNA extraction; differential sediment pre-treament was adopted before extracting the metagenomic DNA of sediment samples directly. The above methods are fit for the corresponding kind of samples, and the DNAs obtained meet the needs of PCR.Actinobacteria diversity of Han-Xing iron mine area samples were studied by PCR- TGGE and clone library of 23S rDNA specific indel sequences. The results of PCR-TGGE showed that actinobacteria groups of the samples are less, only 1 to 4 major bands. However, the 23S rDNA specific indel sequences clone libraries showed much higher diversity, which indicated that the PCR-TGGE method is only fit for analysing the major group(s) of the samples, but not sensity to the minor group(s). Sequence determination of positive clones and diversity analysis of different samples showed that: the groups in different kinds of samples from the same location are not complete same and the same group(s) also exist(s) in different samples from the various sampling sites. The major group in most samples has higher homology with Streptomyces members. Some other groups were also found, they shared higher homology with Nocardia, Nocardioides, Microbacterium, Kribbella, Clavibacter, Rhodococcus, Actinosynema, Leifsonia and Oerskovia members respectively. However, all the sequences obtained have lower homologies with the known sequences, sharing the highest similarities between 78.9%~98.5%. More comprehensive and accurate information on actinobacteria diversity could be obtained by the combination of two methods.