Microbial Diversity Of Alpine Meadow Soil From Mila Mountain In Tibetan Plateau

Qinghai-Tibetan Plateau is the highest and largest plateau on the Earth, which is frequently referred to as an extreme environment for special microbial resources. But most of them could not be utilized for their unculturable property. And togather with the global climate change and human impact, the microbial ecosystem of the Plateau is being destroyed. Extraction of total microbial DNA with cultured-in-dependent method and construction of metagenomic library is an alternative method to exploit uncultured microorganisms. To exploiting new microbial resources and investigating ecological changes on the Plateau, meadow soil from Tibetan Mila mountain was collected as alpine soil type. And this dissertation first systematically studied the microobial diversity of the alpine meadow soil in Tibetan Plateau with metagenomic method.A pre-experiment indicated that the alpine meadow soil has high organic matter and humic acid content, which resulted in a great difficulty in DNA extraction. We compared different extraction and purification methods, the result showed that the DNA obtained by directly extraction from the soil was in small DNA fragments with high contamination. For its easy operating and high efficiency, it is suitable for mircrobial diversity research after column purification step. Comparison with direct extraction, the DNA extracted from cells separated from combing low speed centrifugation and Nycodenz gradient centrifugation has larger molecular weigh and higher purity with comparative diversity.Metagenomic libraries were constructed by pUC19 and pCC2FOS? vector respectively. The average insert fragments of the two constructed pUC19 libraries (ZMp-1 and ZMp-2) were 1 kb and 4 kb individually, and the Fomisd library (ZMf-vsmfba) contained 30 624 clones of 35 kb average insert fragments, which made it has a total genomic storage capacity over 1 Gb. Bioassay demonstrated that both of the constructed libraries have large capability of insert DNA with unkown species. The method for constructing library provides a powerful tool for soil samples from extreme environment and the constructed library provides resources for finding and utilizing new functional genes from Tibetan altiplano.As 16S rRNA and its gene has proven to be useful and powerful markers for the presence of microorganisms in environmental samples, we determined the diversity of 16S rRNA gene of Archaea and Bacteria with library method. The result showed that the microorganisms, especially Archaea, have special characteristics. Taxonomic analysis recovered only Unclassified_Thermoprotei of Crenarchaeota and Unclassified_Archaea, no Euryarchaeota clone was found. Comparing to the Archaea, the bacterial group covered most of the basic soil bacteria types. But for the climate change and human effective, the microbial structure of degrading alpine meadow soil sample was changed.And with the meadows'retrogressing, the carbon/nitrogen are releasing and affecting environment severely. Much study has been focused on the process of biogeochemical cycle, whereas none was done with the ammonia-oxidizing microorganisms (AOM), whose diversity and factors influencing them are keys to understanding the movement of nitrogen. We estimated the amoA (ammonia monooxygenase subunits A) gene diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in two soil samples with different human and climate impact. The result suggested that AOA might play more important role in the degenerated grassland than AOB, and the opposite was true in undegraded sample. Besides, the amoA sequences from degenerated sample were closest to protected lands'sequences, which indicated that the model of nitrification in degenerated grassland of the alpine meadow soil in Tibetan Plateau is likely to that of protected lands in greenhouse.Furthermore, for exploring new protease, which was considered to be important nematode biocontrol reagent, we designed PCR amplification primers according to the serine proteases isolated from nematophagous fungi, which could decompose nematode body wall and egg cuticle. The amplified result with metagenomic DNA of alpine meadow soil showed that no achieved sequences were close to target genes, but the high diversity implicated rich available resources. Moreover, partial bidirectional-terminal sequencing of pUC19 plasimid library (ZMp-2) clones resulted into three protease-like genes with double-ends and four with single-end liking protease genes, and functional screening of the Fosmid library resulted into two new genes similar to protease. But the retrieved nine protease-like genes from two metagenomic libraries had very low similarity to reported sequences in GenBank (30~62%), we deduced they are brand-new protease genes.To sum up, there are much undiscovered special microbes and their gene information in the alpine meadow soil of the Tibetan Plateau. But with the environmental and anthropic impacts, the alpine meadows were degenerating and the microbial communities were adapting by changing their activity, biomass and structure. Therefore, the alpine meadows require our protection and recover for further utilizing and environmental protection purpose.