【作者】 付媛媛；

【导师】 邵宗泽；

【作者基本信息】 国家海洋局第三海洋研究所 ， 微生物学， 2011， 硕士

【摘要】 当前,海洋的石油污染正日趋加剧,海上溢油事故的频繁发生对海洋环境和生态系统造成了长期和恶劣的影响。生物修复已经成为治理海洋石油污染的重要方向。因此,开展烷烃降解菌的研究对于认识和利用海洋微生物在油污清除过程中的作用具有非常重要的意义。本论文研究了北部湾2008年受海底溢油污染的13个站位样品(其中一个样品为水样,其他均为沉积物)的石油降解菌多样性。经过富集筛选,共得到80株不同的细菌,分属于31个属。16S rRNA基因序列系统进化分析表明,它们主要属于变形菌纲变形菌门、放线菌类群放线菌门、拟杆菌门以及厚壁菌门的细菌,其中尤以变形菌纲α和γ亚群以及放线菌类群居多。这些菌株中包括17株潜在的新种,并对其中一株细菌F44-8进行系统的分类鉴定,结果显示菌株F44-8为黄杆菌属的一个新种,命名为北部湾黄杆菌(Flavobacterium beibuense)。本文还对分离自不同海域的12株咸水球形菌属(Salinisphaera)细菌和该属三株模式菌株,通过16S rRNA基因、gyrB基因和BOX-PCR聚类分析的方法,进行了系统的比较分析,发现这15株菌总体进化关系较一致,与其它属种相比较独立成一分支。通过16S rRNA基因分析,这些菌株在属内被区分为4个小分支;gyrB基因将它们分成6个小分支;BOX-PCR分析将它们分成8个小分支。进一步,对这些菌株的烷烃羟化酶(alkB)基因进行了克隆与系统进化分析,发现该属的烷烃降解基因(alkB)具有丰富多样的特征。此外,生理生化特性分析表明,这15株细菌的生理生化特性各异,突出特点是都具有烷烃降解能力,降解范围从C5-C38,大多数菌株对中长链烷烃的降解率达到50%以上。在系统进化分析基础上,选取降解效果较好的菌株Salinisphaera sp. C84B14作代表进行了全基因组测序,获得了框架图。结果显示,该菌基因组大小为3.85M,G+C含量为63.35 mol%,拼接成61个Scaffolds,预测有3872个开放阅读框。初步分析发现,该菌共有5个潜在的烷烃单加氧酶基因。RT-PCR分析表明其中的2个功能基因即一个烷烃单加氧酶(Alkane 1-monooxygenase)基因和一个依赖FMNH2的单加氧酶(FMNH2-dependent monooxygenase)基因在中长链烷烃的诱导下有上调表达的现象,表达量比非诱导对照提高2-3倍。此外,对这两个单加氧酶基因的排布进行了分析,发现基因上下游含有许多与烷烃降解相关的醇脱氢酶基因和醛脱氢酶基因。综上,本论文一方面对北部湾13个站位的石油降解微生物进行了多样性的分析,获得了大量的石油降解细菌资源并鉴定了一株石油降解细菌新种。此外,初步研究了15株Salinisphaera属细菌的系统进化关系和烷烃羟化酶基因的多样性;测定了其中一株菌的全基因组框架图,并对其2个烷烃单加氧酶基因的诱导表达水平进行了RT-PCR分析。这些结果对于认识海洋降解微生物多样性及其环境作用具有参考价值。更多还原

【Abstract】 Nowadays, intensified marine oil pollution and the frequent occurrence of oil spill accidents lead to persistent and serious pollution on marine environments and ecosystems. Bioremediation has been used widely to administer upon marine oil pollution. Additionally, one of the most important environmental bioremediation technologies is making the use of microbial degradation. Thus, the isolation and identification of oil-degrading bacteria and study of their degradation genes (alkane hydroxylase) are very important and necessary, which has very important significance in today’s removing marine oil pollution process.Analysis of oil degrading bacteria in 13 samples (one is sea water sample, the other are all sediment samples) of Beibu Gulf oil spill pollution area was studied. After enrichment and screening, 80 different strains belonging to 31 genera were isolated. They belonged to Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, among which theα-, g-Proteobacteria and Actinobacteria consisted of the majority. Additionally, there were 17 potential novel strains. Results of systematic classification and identification of strain F44-8 showed that it represents a novel species in the genus Flavobacterium, of which the name Flavobacterium beibuense sp. nov. is proposed.Also analysis of phylogenetic evolution of 12 strains (salinisphaera) isolated from different marine area and three type strains based on 16S rRNA gene, gyrB gene and BOX-PCR clustering showed that they had consistent evolution and formed an independent branch from other genera. However, these strains were divided in genus by 16S rRNA gene, gyrB genes and BOX-PCR, which divided the 15 strains into 4, 6 and 8 small branches respectively. Phylogenetic analysis based on alkB genes from 15 strains of the genus salinisphaera showed that the genus had a great diversity of alkB genes. In addition, these strains had different physiological and biochemical characteristics and had wide range of alkane degradation (C5-C38). The degradation ability of most strains have got to more than 50%.The whole genome sequencing of strain salinisphaera sp. C84B14 with excellent degradation effect was carried out. The genome is about 3.85M, consisted of 61 Scaffolds and containing about 3872 ORFs. The content of G+C mol% is 63.35 mol%. After preliminary analysis of alkane-degradation genes, 5 potential functional genes for alkane-degradation were found. Then the experiments of induction and expression with n-alkanes were conducted using RT-PCR, and preliminary analysis suggested there were two functional genes, named Alkane 1-monooxygenase gene and FMNH2-dependent monooxygenase which have the phenomenon of regulated expression during the induction and expression with n-alkanes and the quantity is twice or three times of the control. Finally, gene cluster arrangement of the two functional genes indicated that there were a lot of alcohol dehydrogenase genes and aldehyde dehydrogenase genes ralated with alkane degradation in the upstream and downstream of the two functional genes.In sum, research on the diversity of oil-degrading microorganism of 13 samples from oil pollution area in Beibu gulf was carried out and a large number of oil-degrading bacteria were gained and a novel strain was identified; a lot of degrading gene resources were gained in preliminary studies on systematical evolution and alkane-degradation functional genes of these 12 strains of genus salinisphaera and three type strains. These results have reference value in understanding of marine oil-degrading microorganism and environmental function.更多还原