Genetic Diversity Of Bacteria Isolated From Sub-alpine Meadow Soil Affected By Snow Cover Thickness And Litter

In this study,27subalpine meadow soil samples were collected in Kaka ditch Songpan, and90bacterial strains were isolated. Using BOXAIR-PCR,16S rDNA PCR-RFLP and16S rRNA gene sequence analysis, the genetic diversity and phylogeny of these bacteria were determined. Then, PCR-DGGE technique was used to analyze the soil bacterium diversity. The results showed as follows:1. A total of90bacterial strains were isolated and purified. Gram staining and microscopic observation revealed that66strains of them were Gram-positive (G+), the left24strains were Gram-negative (G), and bacteria strains belonged to Bacillus were the predominant community in the tested soils.2. Genetic diversity analysis showed that there were very diverse among90bacteria of subalpine meadow soil collected from Kaka ditch in Songpan. BOXAIR-PCR indicated31,29and30strains isolated from soil taken in January, March and May were clustered into5,4,5groups at the similarity of85%,81%and82%, respectively.16S rDNA PCR-RFLP analysis clustered these strains into6,4and4genotypes at the similarity of77%,75%and75%, respectively. The result of BOXAIR-PCR was good consistent with that of16S rDNA PCR-RFLP.3. Forty-five stains were selected for16S rRNA gene sequences analysis, and the phylogenetic trees were constructed using MEGA4.1software, the results showed that these strains belonged to genus Bacillus, Pseudomonas, Arthrobacter, Paenibacillus, Staphylococcus, Flavobacterium, and Janthinobacterium, and strains belonged to genus of Bacillus was the most predominant bacterial community.4. Using PCR-DGGE technique, the genetic diverisity of uncuturable bacteria in subalpine meadow soil was performed, the results suggested that the DGGE bands were very enrich, and the diversity was existed among the different treatment soils. Of these DGGE bands, some were similar types among the different soil samples, which showed that these bands represented the predominant bacteria in the treated soils; and the some distinctive bands suggested there were specific bacteria strains existed. Then,12repreaentive bands were coined and sequenced, and the phylogenic tree was constructed by MEGA4.1program. The results suggested that most bacteria belonged to uncultured bacteria, and these unculturable strains were predominant bacterial communities; while, only strains belonged to genus Pseudomonas, Janthinobacterium were amplified by PCR-DGGE.The results also suggested that soil bacterial flora revealed by the cultured method and the uncultured independent method (PCR-DGGE) was quite different. Hence, it is necessary to combine both culture and unculturable independent technique study the genetic diversity of soil bacteria.