[The community and structure of nitrogen-fixing microorganism in Sanjiangyuan natural reserve]

Research on the diversity of microorganism community in natural environment has been concerned hot spot using the newly molecular biotechnology in the world now. To understand the composition and structure of nitrogen-fixing bacteria communities in the Qingzang plateau, the molecular diversity and phylogenetic of nifH genes of Sangjiangyuan natural reserve were examined by using the PCR-RFLP based cloning approach. The 3 samples were come from different sites and different plant types, and their biogeochemical parameters were diverse. DNA was directly extracted from the soil microorganism and amplified the nifH gene fragment using PCR by the primers of nifH-34F 5'-AAAGG(C/T)GG(A/T) ATCGG(C/T)AA(A/G) TCCACCAC-3' and nifH-491R 5'-TFGTT(G/C)GC(G/C)GC(A/G)TACAT(G/C)GCCATCAT-3'. For the nifH gene segment, diverse PCR products were characterized by cloning, restriction fragment length polymorphism (RFLP) analysis and sequencing. A total of 233 clones and 99 operational taxonomic units (OTUs) which were digested the clones by the restriction enzymes MspI and RsaI were obtained from all samples. YS-1 had 63 clones and 24 OTUs, ZD-1 had 75 clones and 28 OTUs, and NQ-1 had 95 clones and 47 OTUs, respectively. They were found 1-2 significant domain groups of clones and shared 4 OTUs in all samples. A wide range of sequence divergence was observed in the 26 nifH clones that were sequenced from all samples. Sequence comparison showed that the nifH clones were 66% to 98% similar. The phylogenetic tree was constructed by the Clustal W and Mega software. 26 sequences could be subdivided into 4 clusters in the phylogenetic tree, and some of them had the closely similar to Proteobacteria, but The majority of the clones were not closely related to any known cultivated nitrogen-fixing bacteria, Therefore, most of them are unique and may represent novel sequences of nitrogen-fixing bacteria.