三江源地区不同植被土壤固氮微生物的群落结构研究

The community and structure of nitrogen-fixing microorganism in Sanjiangyuan Natural Reserve

利用PCR_RFLP和测序分析法对位于青藏高原腹地三江源自然保护区的高寒草甸、高寒草原和高山森林等不同植被类型的土壤固氮微生物的群落组成进行了探讨。经过PCR_RFLP分析固氮基因nifH ,在3个样品中共得到2 33个克隆和99个可操作分类单元(OTUs) ,NQ_1样地具有最多的克隆数和OTUs,多样性为4 9 74 % ,在所有样品中分别具有1～2个明显的优势种群(占总克隆数>15 % ) ,并且具有4个共同的OTUs。选取了2 6个克隆进行基因测序分析,通过DNAMAN比较表明,这些序列间具有6 6 %～98%的相似性,并且在GenBank数据库中没有发现完全匹配的序列,因此这些序列可能代表着新的固氮生物株系。最后利用ClustalW与Mega软件构建了系统发育树,结果发现,这些序列被分为4个不同的簇,部分序列与属于蛋白细菌(Proteobacteria)的已知细菌具有近的亲缘关系,但是更多的序列与已知细菌具有较远的亲缘关系。

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 ofnifH 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 thenifH gene fragment using PCR by the primers ofnifH_34F 5′_AAAGG(C/T)GG(A/T) ATCGG(C/T)AA(A/G) TCCACCAC_3′ andnifH_491R 5′_TTGTT(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 andRsaI 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 26nifH clones that were sequenced from all samples. Sequence comparison showed that thenifH clones were 66% to 98% similar. The phylogenetic tree was constructed by the Clustal W and Mega softwares. 26 sequences could be subdivided into 4 clusters in the phylogenetic tree, and some of them had the closely similar toProteobacteria, 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.