三江源高寒草甸土固氮基因(nifH)的多样性和系统发育研究

Molecular diversity and phylogenetic analysis of nitrogen-fixing (nifH) genes in alp prairie soil of Sanjiangyuan natural reserve

首次利用PCR_RFLP和测序分析对位于青藏高原腹地的青海三江源保护区高寒草甸土壤微生物固氮基因(nifH)的多样性和系统发育进行了探讨。在 2个样地中 ,共得到 14 3个阳性克隆 ,用限制性内切酶MspⅠ和RsaⅠ进行RFLP分析后得到 35个不同的RFLP谱带型 ,多样性为 2 4 . 5 % ,其中ZD样品中获得 82个阳性克隆和 2 1个不同的RFLP谱带型 ,多样性为 2 5 6 % ,而YS样品中获得 6 1个阳性克隆和 19个不同的RFLP谱带型 ,多样性为 31 1% ,2个样地中有 5个相同的RFLP谱带型。在各样地都发现一个明显的优势种群 ,ZD样地的明显优势种群占克隆数的2 9 3% ,YS的优势种群占克隆数的 32 .8%。对 2 1个克隆进行了部分序列的测定 ,序列的相似性在 71%～ 98%之间 ,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软件和已有序列构建了系统发育树 ,结果发现 ,2 1个序列分为 4个不同的簇 ,大部分的克隆与Proteobac teria的 3个系统发育亚簇 (α、β和γ)具有较高的相似性 ,其中主要的序列都落在第一和第二簇内。YS样地中的优势种群与α_Proteobacteria中的Rhodobactersphaeroides具有较高的相似性 ,而ZD样地中的优势种群则与 β_Proteobac

Research on the diversity of microorganism community in natural environment has been concerned hot spot using the newly molecular biotechnology in the world now. This was the first description of the molecular diversity and phylogenetic analysis of nitrogen-fixing (nifH) genes in alp prairie soil of Sanjiangyuan natural reserve. 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′-TTGTT(G/C)GC(G/ C)GC(A/G)TACAT(G/C)G CCATCAT-3′. For the gene fragment, diverse PCR products were characterized by cloning, restriction fragment length polymorphism (RFLP) analysis and sequencing. 143 clones and 35 different RFLP patterns were received in two samples by the restriction enzymes MspI and RsaI digested. ZD sample had 82 clones and 21 different RFLP patterns, and YS sample had 61 clones and 19 different RFLP patterns. There were shared 5 RFLP patterns in two samples. The analysis result found a significant dominant group of clones occurring in both samples which account for 29.3 and 32.8, respectively, and several minor groups were also detected. 21 clones were sequenced, and their levels of nucleotide identity were from 71 to 98. None of the sequenced nifH gene was completely identical to any deposited in the data banks, and therefore each of them belong to a noncharacterized bacterium. Finally, the phylogenetic tree was constructed by the Clustal W and Mega softwares. 21 sequences can be subdivided into 4 clusters in the phylogenetic tree, and most of them had the closely similar toα-,β-, and γ-Proteobacteria. The significant dominant group in YS sample and ZD sample had the closely related with Rhodobacter sphaeroides and Delftia tsuruhatensis, respectively. The YS-nifH-11 was the only sequence which had highly similar to Cyanobacteria.