可降解TCE的甲烷氧化菌16S rDNA与pmoCAB基因簇序列分析

Sequence analysis of 16S rDNA and pmoCAB gene cluster of trichloroethylene-degrading methanotroph

摘要:

甲烷氧化菌可高效催化甲烷和多种氯代烃降解，开展甲烷单加氧酶的基因簇序列分析研究有助于深入了解催化机理，并提升其在污染物生物降解领域中的应用价值。以甲烷为唯一碳源富集分离甲烷氧化菌，取5种氯代烃作为共代谢基质考察其降解情况，利用MEGE5.05软件构建基于16S rDNA的系统发育树对所选菌株进行初步鉴定，用半巢式PCR法分段扩增菌株的颗粒性甲烷单加氧酶 (pMMO) 基因簇并进行T-A克隆测序，通过ExPASy计算pMMO三个亚基的理论分子量。筛选到了一株甲基孢囊菌Methylocystis sp. JTC3，在三氯乙烯 (TCE) 初始浓度为15.64 μmol/L条件下，反应5 d后降解率可达93.79%。经扩增、测序、拼接得到了3 227 bp的pmoCAB基因簇序列，包括771 bp的pmoC基因、759 bp的pmoA基因、1 260 bp的pmoB基因和2个非编码中间序列，所对应γ、β、α亚基理论分子量分别为29.1 kDa、28.6 kDa和45.6 kDa。通过Blast比对发现Methylocystis sp. JTC3的pmoCAB基因簇序列与Methylocystis sp. strain M的pmoCAB一致性较高，其中pmoA的序列相对保守。JTC3菌株可高效降解TCE，对pmoCAB基因簇序列的详细分析可为pMMO活性中心特征、氯代烃类底物选择性等方面的深入研究提供信息数据基础。

Abstract:

Methanotrophs could degrade methane and various chlorinated hydrocarbons. The analysis on methane monooxygenase gene cluster sequence would help to understand its catalytic mechanism and enhance the application in pollutants biodegradation. The methanotrophs was enriched and isolated with methane as the sole carbon source in the nitrate mineral salt medium. Then, five chlorinated hydrocarbons were selected as cometabolic substrates to study the biodegradation. The phylogenetic tree of 16S rDNA using MEGE5.05 software was constructed to identify the methanotroph strain. The pmoCAB gene cluster encoding particulate methane monooxygenase (pMMO) was amplified by semi-nested PCR in segments. ExPASy was performed to analyze theoretical molecular weight of the three pMMO subunits. As a result, a strain of methanotroph was isolated. The phylogenetic analysis indicated that the strain belongs to a species of Methylocystis, and it was named as Methylocystis sp. JTC3. The degradation rate of trichloroethylene (TCE) reached 93.79% when its initial concentration was 15.64 μmol/L after 5 days. We obtained the pmoCAB gene cluster of 3 227 bp including pmoC gene of 771 bp, pmoA gene of 759 bp, pmoB gene of 1 260 bp and two noncoding sequences in the middle by semi-nested PCR, T-A cloning and sequencing. The theoretical molecular weight of their corresponding gamma, beta and alpha subunit were 29.1 kDa, 28.6 kDa and 45.6 kDa respectively analyzed using ExPASy tool. The pmoCAB gene cluster of JTC3 was highly identical with that of Methylocystis sp. strain M analyzed by Blast, and pmoA sequences is more conservative than pmoC and pmoB. Finally, Methylocystis sp. JTC3 could degrade TCE efficiently. And the detailed analysis of pmoCAB from Methylocystis sp. JTC3 laid a solid foundation to further study its active sites features and its selectivity to chlorinated hydrocarbon.