一株不动杆菌降解石油烃的特性及关键烷烃降解基因分析

Characterization of key alkane degradation genes from a petroleum hydrocarbon degradation Acinetobacter sp. Tust-DM21

摘要:

【背景】Acinetobacter sp. Tust-DM21 (GenBank登录号KX390866)是本实验室前期从渤海湾海洋石油勘探船废油收集区采集的水油混合样中分离出的一株高效石油降解菌，其对短、中、长链烷烃均表现出很强的降解能力，有较好的应用前景。【目的】从应用层面探究其最佳降解条件，同时从生物信息层面探究其降解基因的作用。【方法】将其在不同温度、pH下培养144 h，通过GC-MS内标法测定石油烃各组分的变化情况，计算出其最佳降解条件；同时，通过生物信息学手段确定基因组中的降解基因，每个基因分别选择7个同源基因，对它们的蛋白序列进行比较；最后对2个降解基因在0?144 h的表达情况进行了Real-time PCR分析。【结果】Acinetobacter sp. Tust-DM21最佳降解条件为35 °C、pH 8.5，该条件下对石油降解率可达97.5%，其中，对长链烷烃降解率达98.5%，对环烃为81%，对芳香烃为87%；同时，研究发现基因组中含有常见烷烃降解基因alkB (Genbank登录号MH368539)和长链烷烃降解基因almA (Genbank登录号MH357335)，2个降解基因的蛋白经比较均与其同源蛋白表现出一定的相似性，同属菌的相似性最高；通过Real-time PCR发现这2个基因在0?144 h的相对表达量随时间逐步提高。【结论】Acinetobacter sp. Tust-DM21在最佳降解条件下对石油各组分都显示出了优良的降解能力，特别对长链烷烃的降解能力尤为突出；将2个降解基因的相对表达量结合该时间段的生长趋势，证明了菌株Acinetobacter sp. Tust-DM21的生长和降解与alkB和almA基因的上调表达存在关联。

Abstract:

[Background] Acinetobacter sp. Tust-DM21 (GenBank: KX390866) is a highly efficient petroleum degrading bacteria isolated early from the water-oil mixed sample collected from the waste oil area of the Bohai Bay offshore oil exploration vessel in this laboratory. It exhibits strong degradability to short, medium and long chain alkanes. [Objective] To investigate the optimal conditions of the strain for degradation at the application level and to investigate the role of degradation genes at the bioinformatics level. [Methods] The strain was incubated at different temperatures and pH for 144 h. Changes in petroleum hydrocarbon components were determined by GC-MS internal standard methods, and the best degradation condition was calculated. Meanwhile, the degrading genes were identified in its genome by bioinformatics analysis, and seven homologous genes were selected for each gene and their protein sequences were compared. Real-time PCR analysis of these two genes at 0?144 h were performed for their transcription expression research. [Results] The optimal degradation conditions were 35 °C and pH 8.5. The relative degradation level of petroleum hydrocarbon under this optimal condition can reach up to 97.5%. The degradation rate was 98.5% for long-chain alkanes, 81% for cycloalkanes and 87% for aromatics. Meanwhile, the common alkane degradation gene alkB (Genbank: MH368539) and the long-chain alkane degradation gene almA (Genbank: MH357335) were found in its genome, the proteins of the two degradation genes are similar to their homologous proteins, and the similarity of the same genus is the highest. Real-time PCR showed that gene relative expression increased gradually over time. [Conclusion] It exhibits strong degradability to short, medium and long chain alkanes under the optimal degradation conditions, especially for long-chain alkanes. Combining with the growth trend in this time period, it was demonstrated that the strain growth and degradation were associated with up-regulated expression of the alkB and almA genes.