【作者】 邢孟欣；

【导师】 刘斌；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2013， 博士

【摘要】 本课题是利用分子手段和传统培养手段对养殖大菱鲆肠道菌群结构及功能特征进行研究。首先，利用宏基因组测序技术及16S rRNA分析技术对大菱鲆肠道菌群结构进行研究，共得到33,998条拼接序列（contigs）和95条操作分类单元序列（OTUs）。研究结果显示在大菱鲆肠道中变形菌门与厚壁菌门所占的比例最高（95%），且存在于肠道粘膜和内含物中。进一步将大菱鲆肠道菌群按照属来划分可知，弧菌属所占的比例最大，且存在于大菱鲆肠道的各个部分中，尤其在直肠内含物中所占比例最大。弧菌属中的四个主要类群，包括哈氏弧菌、霍乱弧菌、创伤弧菌以及副溶血弧菌，均被认为是海水鱼类的潜在致病菌。与其他物种肠道菌群结构相比，大菱鲆肠道中含有较高比例的-变形菌纲和柔膜菌纲，弧菌属和支原体属分别在这两个纲中占优势地位。16S rRNA基因序列分析结果显示，大菱鲆肠道各部分菌群的多样性是逐渐降低的（从胃到直肠），且大菱鲆肠道中的某些菌群可能来源于周围海水环境。其次，为了能够提高培养效率，本课题选择了18种固体培养基对大菱鲆肠道菌群进行培养，包括8种基础培养基，8种1%肠道上清培养基以及2%、4%肠道上清培养基。结果共得到1,711个菌落和24个OTUs，其中Zobell2216E/Zobell2216E+固体培养基中分离的菌落数和菌群多样性最高，而MRS/MRS+固体培养基中的培养结果最不理想。含有肠道上清的培养基（1%、2%、4%）中的菌群结构与基础培养基中的菌群结构存在差异，且肠道上清中可能存在某些物质能够提高大菱鲆肠道菌群的培养效率。对培养得到的菌落进行分类分析可知，-变形菌门所占的比例最大（82%），其次是厚壁菌门和放线菌门（分别为15.6%和2.4%）。进一步按照属来划分可知，弧菌属所占比例最大（49.4%）。此外，还检测到许多潜在的致病菌和益生菌。致病菌包括假单胞菌属、发光菌属以及肠杆菌属。益生菌包括芽孢杆菌属、类芽孢杆菌属、假单胞菌属以及希瓦氏菌属。另外，培养实验所得到的大多数OTUs均是首次在大菱鲆肠道中报道，这些细菌在大菱鲆肠道中的作用还需进一步研究。最后，本课题对大菱鲆肠道宏基因组拼接数据进行功能分析。结果显示，Clustering-based subsystems所占比例最大，约占大菱鲆肠道宏基因组拼接数据的15.9%，此外，与代谢相关的功能也占有较高比例。利用比较宏基因组学方法分析不同物种肠道宏基因组可知，在大菱鲆肠道宏基因组数据中与群体感应及生物膜形成系统相关的基因所占的比例较大，且这些基因均来源于弧菌属，包括创伤弧菌、霍乱弧菌以及副溶血弧菌。在养殖鱼类肠道宏基因组中，与压力应答以及蛋白折叠系统相关的基因所占比例较高，此外还检测到一些抗生素抗性以及重金属抗性基因。这些数据证明大菱鲆肠道微生物可能受到了养殖过程中某些人为因素的影响，从而产生相应的特殊功能。另外，与淡水鱼（杂交条纹鲈鱼）肠道宏基因组相比，大菱鲆肠道宏基因组中铁元素吸收及代谢系统所占的比例较大，这证明在海洋动物中可能存在着特有的代谢功能。更多还原

【Abstract】 Culture-independent methods and culture-dependent technologies were appliedto unveil the taxonomic composition and functional diversity of the farmed turbot(Scophthalmus maximus) gastrointestinal (GI) microbiome. First, metagenomicscombined with16S rRNA sequence analysis was used to analyze the taxonomicdistribution and a total of33,998contigs and95operational taxonomic units (OTUs)were identified, respectively. Proteobacteria and Firmicutes which existed in both GIcontent and mucus were dominated in the turbot GI microbiome. At the genus level,Vibrio was observed in all parts of turbot GI tract, especially in rectum content.Within Vibrio, the four main species, V. vulnificus, V. cholerae, V. parahaemolyticusand V. harveyi were recognized as potential pathogens which were responsible formany fish diseases. As compared taxonomic distribution between hosts,Gammaproteobacteria and Mollicutes were overabundant in turbot GI microbiomes.Among them, members of Vibrio and Mycoplasma were dominant.16S rRNAsequence analysis also indicated that the bacterial community diversity decreasedalong the turbot GI tract (from stomach to rectum) and the turbot GI tract may harborsome bacteria which originate from associated seawater.Second, to enhance the cultivation efficiency, eighteen agar media were tested forcultivating fish-gut-associated bacteria in farmed adult turbot, including agar mediawith or without1%gastrointestinal (GI) supernatant, and2%and4%GI supernatantagar media. A total of1,711colonies were analyzed, and24operational taxonomicunits (OTUs) were identified. Most colonies and OTUs were obtained from Zobell2216E agar media, while a low diversity was identified from MRS/MRS+agar media.Agar media with GI supernatant (1%,2%, and4%) yielded different profiles of OTUsfrom those of the corresponding original media and provided some substances thatenhanced the cultivation efficiency of bacteria in turbot GI tract.Gammaproteobacteria represented the large majority of the colonies (82%). Firmicutes and Actinobacteria represented15.6%and2.4%colonies, respectively. Atthe genus level,49.4%of all colonies belonged to Vibrio. Other potential pathogensincluding Pseudomonas, Photobacterium, and Enterobacter and potential probioticsincluding Bacillus, Paenibacillus, Pseudomonas and Shewanella were also obtainedfrom agar media. Additionally, most of OTUs identified in this study had highhomology with the species that were first described in turbot GI tract. The impact ofthese species on the turbot physiology and health should be further investigated.Last, functional analyses indicated that the clustering-based subsystem and manymetabolic subsystems were dominated in the turbot GI metagenome, accounting for15.9%of turbot GI metagenome. Compared to other gut metagenomes, quorumsensing and biofilm formation was overabundant in the turbot GI metagenomes.Genes associated with quorum sensing and biofilm formation was found in specieswithin Vibrio, including V. vulnificus, V. cholerae and V. parahaemolyticus. In farmedfish gut metagenomes, the stress response and protein folding subsystems wereover-represented and several genes regarding antibiotic and heavy metal resistancewere also detected. These data suggested that the turbot GI microbiome may beaffected by human factors in aquaculture. Additionally, iron acquisition andmetabolism subsystem was more abundant in the turbot GI metagenome thanfreshwater fish (hybrid striped bass) gut metagenome suggesting that uniquemetabolic potential may be observed in marine animals.更多还原