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倭蜂猴粪便微生物苯酚羟化酶和邻苯二酚1,2-双加氧酶基因多样性研究
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国家自然科学基金项目(No. 30960165,31160229,31360268,31560305)


Analysis gene diversity of phenol hydroxylase and catechol 1,2-dioxygenase from fecal microbiome of Nycticebus pygmaeus
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    摘要:

    【目的】分析倭蜂猴粪便微生物中苯酚羟化酶(Phenol hydroxylase,PH)和邻苯二酚1,2-双加氧酶(Catechol 1,2-dioxygenase,C12O)的基因多样性。【方法】利用简并引物,以倭蜂猴粪便微生物宏基因组DNA为模板,通过PCR扩增,分别构建PH和C12O基因克隆文库,并对克隆进行测序分析。【结果】倭蜂猴粪便微生物来源的PH和C12O基因序列经BLAST比对分析,与GenBank中相应酶的序列一致性分别介于92%?100%和87%?100%。系统进化树分析表明PH基因序列与Neisseria、Burkholderia、Alcaligenes、Acinetobacter 4个属来源的PH序列相关;C12O基因序列全部与Acinetobacter来源的C12O序列相关。序列比对结果表明PH序列具有LmPH (Largest subunit of multicomponent PH)中高保守的两个DEXRH结构域;C12O序列具有能被Ag+和Hg2+抑制的位点(半胱氨酸)。【结论】倭蜂猴粪便微生物来源的PH为多组分PH,其降解苯酚的中间产物邻苯二酚可以被C12O通过邻位开环途径裂解。

    Abstract:

    [Objective] The gene diversity of phenol hydroxylase and catechol 1,2-dioxygenase were investigated from fecal microbiome of Nycticebus pygmaeus. [Methods] Degenerate primers were used to amplify phenol hydroxylase and catechol 1,2-dioxygenase gene fragments from metagenomic DNA. Phenol hydroxylase and catechol 1,2-dioxygenase gene clone libraries were constructed, and some of clones were sequenced separately. [Results] The BLAST analysis of phenol hydroxylase and catechol 1,2-dioxygenase sequences showed 92%?100% and 87%?100% identities to the known phenol hydroxylase and catechol 1,2-dioxygenase sequences in GenBank. Phylogenetic analysis showed that phenol hydroxylase sequences in gene clone libraries had high similarity with phenol hydroxylase sequences from Neisseria, Burkholderia, Alcaligenes, Acinetobacter. And catechol 1,2-dioxygenase sequences in gene clone libraries had high similarity with catechol 1,2-dioxygenase sequences from Acinetobacter. Sequence alignment showed two DEXRH motifs of LmPH sequences were detected in phenol hydroxylase sequences, and the conserved cysteine was detected in catechol 1,2-dioxygenase sequences which was inhibited by Ag+ and Hg2+. [Conclusion] The phenol hydroxylase from fecal microbiome of Nycticebus pygmaeus was multicomponent phenol hydroxylase, and catechol that middle production of phenol degradation can be cleaved by catechol 1,2-dioxygenase through ortho-pathway.

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熊彩云,许波,戴利铭,李俊俊,唐湘华,慕跃林,杨云娟,周峻沛,丁俊美,黄遵锡. 倭蜂猴粪便微生物苯酚羟化酶和邻苯二酚1,2-双加氧酶基因多样性研究[J]. 微生物学通报, 2015, 42(11): 2189-2197

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  • 在线发布日期: 2015-11-10
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