【作者】 刘欣；

【导师】 肖天；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生态学， 2010， 博士

【摘要】 本文采用了变性梯度凝胶电泳(DGGE)和16S rRNA基因文库分析相结合的方法,对胶州湾海域沉积物样品细菌多样性及细菌群落的组成、空间分布和季节演替规律进行了研究,结果表明胶州湾沉积物细菌具有高度多样性。系统进化分析表明,共包含17个门类,其中较优势的菌群包括变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria)、拟杆菌门(Bacteroidetes)、浮霉菌门(Planctomycetes)、放线菌门(Actinobacteria)(高G+C革兰氏阳性菌)、疣微菌门(Verrucomicrobia)、绿弯菌门(Chloroflexi)和硝化螺旋菌门(Nitrospirae),同时还包括少量厚壁菌门(Firmicutes)、梭杆菌门(Fusobacteria)、蓝细菌门(Cyanobacteria)、芽单胞菌门(Gemmatimonadetes)、螺旋体门(Spirochaetes)、Elusimicrobia、脱铁杆菌门(Deferribacteres)、黏胶球形菌门(Lentisphaerae)、异常球菌-栖热菌门(Deinococcus-Thermus)等类群的存在,以及0.9%的尚不确定分类地位的类群(unclassified),胶州湾沉积物中蕴藏着巨大的微生物资源。从胶州湾10个不同站位采集4个季度的沉积物样品,直接提取样品细菌总基因组DNA,利用细菌16Sr DNA V3区特异引物扩增后进行变性梯度凝胶电泳(DGGE),研究胶州湾沉积物细菌群落结构及时空演替规律。发现细菌群落结构存在较明显时空变化,对于整个胶州湾范围而言,菌群结构在不同季节的变化比空间的不同所形成的差异更为显著。根据分析结果可将10个不同站位分为3种类型,A3、A5和B2三个站位归为一种类型;C3, D3, D6和D7站位代表第二种类型;而C1, C4和D1站位代表第三类特殊的类型,这类站位沉积物菌群结构在各季节都较为特殊,且彼此互不相同。尤其是D1站位,其菌群时空变化规律在10个站位中最为特殊,几乎不受季节影响。将DGGE结果与沉积物中总碳、总氮等理化因子相结合进行多元统计分析,结果再一次显示对于整个胶州湾而言,沉积物菌群结构受季节变化影响较大,同时,沉积物理化因子的变化对菌群结构也有一定影响,尤其是靠近广阔外海站位的沉积物,其菌群结构更易受某些沉积物理化因子影响。根据DGGE结果与环境条件相结合,应用PCR-RFLP构建细菌16S rDNA克隆文库方法,系统全面的比较研究四个季度中四个典型站位细菌多样性及群落结构演替。建立四个站位、四个季节共16个细菌16S rDNA全长克隆文库。2560个阳性克隆共得到5120个RFLP结果,包含1231个不同的RFLP分型,根据97%的相似度标准归类后,获得746种操作分类单元(OTUs)。文库分析结果也表明四个典型站位的细菌群落结构时空差异明显。UniFrac分析表明由空间上不同区域所形成的菌群结构的差异大于季节变化的影响,其结果也反映了环境相似站位菌群结构相似性高,同时证明了根据DGGE结果选择四个站位的合理性;而不同季节对四个站位的菌群结构影响并无明显规律,这也从侧面证明了胶州湾复杂的空间环境、季节、沉积学等因素的差异共同造成沉积物菌群复杂的分布规律特征。多样性指数(H′)和物种丰富度指数(Schaol)等结果也表明,胶州湾沉积物细菌多样性非常高。此外,对数据库中与本研究所获得序列具最近亲缘关系序列的来源环境进行分析表明,胶州湾中细菌群落受航运活动、水产养殖、重金属污染等人类活动的明显影响,同时这些活动表现出一定的空间特异性。更多还原

【Abstract】 The composition, distribution and the dynamics of bacterial community in the sediment of Jiaozhou Bay China) were examined using both the 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) and the 16S rRNA gene clone library analyses. The benthic bacterial communities in the sediment of Jiaozhou Bay displayed a highly diversified composition including members from 17 bacterial phylum and some unclassified sequences (0.9%). Proteobacteria, Acidobacteria, Bacteroidetes, Planctomycetes, Actinobacteria, Verrucomicrobia, Chloroflexi and Nitrospirae were the rather dominant groups and members of Firmicutes, Fusobacteria, Cyanobacteri, Gemmatimonadete, Spirochaetes, Elusimicrobia, Deferribacteres, Lentisphaerae and Deinococcus-Thermus were also found in sediment of Jiaozhou Bay.Sediment samples were collected at 10 stations in four seasons from Jiaozhou Bay. Total genomic DNA was extracted from each sample and used as template to amplify 16S rRNA V3 region gene with universal V3 specific primers. PCR-denaturing gradient gel electrophoresis (DGGE) was then performed to study the spatial and temporal variations of bacteria communities. Considering all the ten stations of Jiaozhou Bay as a whole, variations in bacterial community composition were obvious and particularly evident temporally. Less pronounced changes were also detected spatially. The analysis of bacterial communities was used to divide the 10 sampling stations into three types: stations A3, A5, and B2 were relatively in similar situations and were classified as one type; stations C3, D3, D6, and D7 represented another type. Stations C1, C4, and D1 formed the third peculiar type and they were different each other. The dynamics of bacterial communities at station D1 were the most irregular and were not temporally driven at all. Multivariate statistical analysis was used combing the DGGE bands with the sediment physico-chemical properties such as total nitrogen and total carbon content. It revealed again the strong and obvious variation of bacterial communities in temporal. Besides, changes in bacterial communities driven by environmental factors also occurred, especially in some outer bay stations.Four stations were selected according to DGGE result and the geographical factors. The construction of 16S rDNA clone libraries and Restriction Fragment Length Polymorphism (RFLP) analysis were applied not only to give a comprehensive view of the diversity and composition of bacterial communities in these stations but also to investigate a detail spatial-temporal variability of bacterial communities. For each of the 16 samples collected from the four stations and four seasons, comprehensive full-length 16S rDNA clone libraries were built. In total, 2560 positive clones were retained, and 1231 different RFLP patterns occurred in all the 5120 RFLP patterns. The number of distinct operational taxonomic units (OTUs) was 746 based on a taxa cutoff set at 97% similarity.It also revealed a high diversity and temporal-spatial difference in bacterial communities of each sample by the construction of clone libraries. UniFrac analyses showed that bacterial community shifted more obviously in spatial than in temporal when in this relatively small scale. It also indicated that similar environmental features selecting for similar bacterial communities, and the validity of the station selection by DGGE result was tested and proved here. The influence of seasons on bacterial communities in the four stations was quite irregular and this result reflects from another perspective that the bacterial community variations in Jiaozhou Bay result from a combination of many factors such as spatial, temporal and sedimentological factors. The high diversity Shannon-Wiener index and species richness (Schaol) estimates indicated that our sampling stations might have a higher bacterial diversity.Moreover, comparative phylogenetic analysis of the closest relatives of the sequences obtained in Jiaozhou Bay implied the presence of influences of human activities such as the influence of harbor, aquaculture and heavy metal contamination, which exhibited spatial and temporal specificities.更多还原