The diversity of denitrifying bacteria in the alpine meadow soil of Sanjiangyuan natural reserve in Tibet Plateau

This is the first time to describe the diversity of denitrifying bacteria in Sanjiangyuan natural reserve in Tibet Plateau by investigating the molecular diversity and phylogenetic analysis of nirK and nosZ genes using PCR-RFLP and sequencing. Four soil samples were collected from alpine meadow communities from over an altitude of 4600 m which had different physicochemical properties by principal component analysis (PCA). For the genes fragment of nirK and nosZ, the diverse PCR products were characterized by cloning, restriction fragment length polymorphism (RFLP) analysis and sequenced. A total of 253 nirK clones and 283 nosZ clones were received in four samples, and 78 operational taxonomic units (OTUs) of nirK and 120 OTUs of nosZ by the restriction enzymes MspI and RsaI digested. The analysis of environmental factors showed that altitude and C/N ratio in soil may be the key factors to the denitrifying bacteria community. 36 nirK clones and 17 nosZ clones were sequenced, and their levels of nucleotide identity were from 69% to 98% and 57% to 97%, respectively. The phylogenetic tree was constructed using the Clustal W and Mega softwares, and all the sequenced clones could be subdivided into 4 groups. Some of clone sequences were related to the nirK and nosZ genes belonging to three phylogenetic subdivisions (α-, β- and γ subclasses of the Proteobacteria), while most of the clones were closely related to the genes of the uncultured bacteria. The sequence distributions were not clear relating to the sample sites in the tree.