一株Ⅱ型甲烷氧化菌中甲烷单加氧酶基因和16S rDNA的分析

Molecular analysis of soluble methane monooxygenase and 16S rDNA from a type Ⅱ methanotroph

【目的】利用分子生物学方法对一株甲烷氧化菌Methylosinus trichosporium IMV3011中的16S rDNA和溶解性甲烷单加氧酶基因序列进行分析并探索其进化分类地位。【方法】利用基因数据库已有的基因序列信息,设计PCR扩增引物和基因测序引物,对溶解性甲烷单加氧酶基因和16S rDNA进行扩增和测序,并进行溶解性甲烷单加氧酶的6个基因和氨基酸序列与同类菌株的相应序列进行联配分析。【结果】获得了全长为5319bp甲烷单加氧酶基因序列和长度为1290bp的16S rDNA序列。该菌株与Methylosinus trichosporium OB3b中相对应基因的同一性是99.0%～82.7%,MMOX氨基酸序列的同一性为99.4%～81.8%,相似性为99.8%～89.2%。基于以上分析表明MMOX组分有很高的序列保守性,特别是在活性中心区域。【结论】菌株IMV3011属于甲基弯菌属,最近似的菌株是Methylosinus trichosporium OB3b。

The soluble methane monooxygenase （sMMO） from Methylosinus trichosporium IMV 3011 catalyzes the conversion of methane to methanol. [Objective] To identify the novel species Methylosinus trichosporium IMV 3011 and to describe its evolution status. [Methods] With the aid of the information from GenBank, we designed several sets of primers for PCR amplification and sequencing, the 16S rDNA and complete of genes sequence for soluble methane monooxygenases were gene sequenced and analyzed with biology software. [Results] We obtained a 5319 bp of full-length DNA of soluble methane mouooxygenases and a 1290 bp of 16S rDNA. Software analysis for six open reading frames and the deduced amino acid sequences of soluble methane monooxygenases has shown that 99.0 % to 82.7 % identity to the counterpart of Methylosinus trichosporium OB3b, 99.4 % to 81.8 % identity and 99.8 % to 89.2 % similarity tc the predicted amino acid of mrnoX genes in compared five strains. The multiple alignments of MMOX amino acid residues reveal that there is high conservation in MMOX, especially in two Fe binding regions. [Conclusion] These results indicated that strain IMV 3011 should be a true member of Methylosinus trichosporium, and it is closer to the species Methylosinus trichosporium OB3b.