冲绳海槽热液区环境微生物及共生微生物研究

IOCAS-IR > 实验海洋生物学重点实验室

	冲绳海槽热液区环境微生物及共生微生物研究
	孙庆磊
学位类型	博士
导师	孙黎
	2016-05-19
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	冲绳海槽热液区沉积物阿尔文虾微生物群落新菌
摘要	微生物多样性是微生物生态学研究的重点，了解一个地区的微生物多样性及其群落结构，有助于我们了解微生物在此地区发挥的功能作用。深海热液被认为是地球上环境最为恶劣的地区之一，但却存在着丰富多样的微生物，它们都是什么，又发挥着什么样的作用？这些都令科学家们心驰神往。本文利用2014年 “科学号”冲绳海槽热液航次获得的沉积物及大型生物样品，分别采用培养方法和非培养方法对其中的微生物多样性进行了研究。在此基础上，我们对沉积物中的可培养微生物生物活性进行了检测，并对发现的两株潜在新菌进行了多相分类学研究。除此之外，借助于高通量测序技术和克隆文库技术，探究了深海热液大型生物共生微生物的构成及碳代谢机制。采用多种培养方式，我们从冲绳海槽两个热液区沉积物中分离细菌213株，其中128株来自伊平屋脊热液区，85株来自伊平屋北热液区。通过16S rDNA鉴定，这些菌分布在4个门, 5个纲，12个目，22个科，33个属，96个种。γ-变形菌纲在两个热液区均占主导地位，共有146株，其次是芽孢杆菌纲，有53株；其它的包括α-变形菌纲9株和黄杆菌纲3株；放线菌纲有2株，只在伊平屋脊热液区发现。我们分析这些分离株的蛋白酶、脂肪酶及抗菌活性，发现44株至少表现出一种活性/抗性。其中来自于9个属的25株菌表现出胞外蛋白酶活性，来自11个属的21株菌表现出胞外脂肪酶活性，来自8个属的11株菌具有抗菌活性。这些结果表明热液细菌具有潜在的工农业和医药应用价值。我们自环境微生物中发现了两株潜在新菌，并对其进行了多相分类学研究。其中一个菌株S6为革兰氏阳性杆菌，属于Domibacillus属，与之16S rDNA序列相似度最高的模式菌株是D. robiginosus（98.6 %），其次是D. indicus（97.6 %）, D. enclensis（97.6 %）和D. tundrae （97.6 %）。DNA-DNA杂交分析表明，菌株S6与相近的模式菌株的DNA-DNA杂交值均小于70%。进一步研究发现菌株S6与相似模式菌株在细菌形态、生理生化等表型特征上存在一定差别。这些结果表明菌株S6属于Domibacillus属的一个新种，命名为Domibacillus iocasae sp. nov, 该种的模式菌株为S6（= DSM 29979 = CCTCC AB 2015183）。我们发现的另一潜在新种为菌株S61，是革兰氏阴性菌。基于16S rDNA的系统发育分析表明菌株S61属于Algoriphagus属，与之序列相似度最高的模式菌株为A. lutimaris（98.12 %），A. halophila（98.12 %），A. chungangensis（97.95 %）和A. aestuarii （97.35 %）。菌株S61与相近的模式菌株的DNA-DNA杂交值均小于70 %，并且其与相似模式菌株在细菌形态、生理生化等表型特征上不同。这些结果表明菌株S61属于Algoriphagus属的一个新种，命名为Algoriphagus iocasae sp. nov, 该种的模式菌株为S61。热液共生微生物是热液微生物的重要组成部分。阿尔文虾是冲绳海槽热液区常见的一种虾类。目前对阿尔文虾共生微生物的群落结构及功能报道还非常有限。本文采用高通量测序技术和克隆文库技术首次对热液阿尔文虾鳃和肠道共生微生物的群落结构及碳固定途径进行了比较研究。我们发现尽管两个组织内的群落组成相似，但某些细菌组成丰度存在很大差异。ε-变形菌纲在鳃中的丰度要高于肠道，而γ-变形菌、梭状芽孢杆菌纲（Clostridia）正好相反。我们进一步分析了鳃和肠道中的微生物的碳固定途径，发现在两个组织内均存在卡尔文本森循环和还原性三羧酸循环途径中重要酶的编码基因，其丰度在鳃中明显高于肠道内。这些结果表明鳃和肠道至少同时存在两条固碳途径，并且鳃内微生物可能有更高的自养生产力。
其他摘要	Microbial diversity is an important research part in microbial ecology. The study for microbial diversity and structure in one certain region contributed to our understanding the function of microbes inside it. The deep-sea hydrothermal vents were considered as one area of the most hostile environtment on earth. However, there are a large amount of microorganisms with versatile metabolic pathways living in the hydrothermal ecosystems, which have drawn a great deal of attention of microbiologists all over the world. In 2014, we obtained sediments and some shrimps sampled from the Okinawa Trough hydrothermal field during the “Okinawa Trough hydrothermal fields” cruise of the vessel KEXUE. Microbial diveristiy in sediments and shrimps were analyzed via culture-dependent and culture-independent methods, respectively. Further, we detected some bioactivity characteristics of the culturable bacteria; performed polyphasic taxonomy studies of two potential novel bacterial species; explored the carbon metabolic pathway of the shrimp-associated microbial community. A total of 213 strains were isolated from two hydrothermal fields (HV1 and HV2) of Okinawa Trough. Of these isolates, 128 were from HV1 and 85 were from HV2. Based on near full-length 16S rRNA gene sequence analysis, the 213 isolates obtained from HV1 and HV2 belonged to 4 phyla, 5 classes, 12 orders, 22 families, 33 genera, and 96 species. Gammaproteobacteria were the most abundant, followed by bacilli, and Alphapreobacteria and Flavobacteria were minor groups in two hydrothermal fields; Actinobacteria was occurred only in HV1. Bioactivity analysis revealed that 25 isolates belonging to 9 different genera exhibited extracellular protease activities, 21 isolates from 11 genera exhibited extracellular lipase activities, and 13 isolates of 8 genera displayed antimicrobial activities. This is the first observation of a large population of bacteria with extracellular bioactivities existing in deep-sea hydrothermal vents. A novel Gram-positive bacterium, designated as strain S6^T, isolated from the deep sea sediment in Okinawa Trough. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain S6^T belonged to the genus Domibacillus. The closest phylogenetic neighbors of strain S6^T were D robiginosus (98.6%), D. indicus (97.6%), D. enclensis (97.6%), and D. tundrae (97.6%). The identities between strain S6^Tand other close members were below 96%. The values of DNA–DNA relatedness between strain S6^T and its closest relatives in genus Domibacillus were well below 70%. Phylogenetic, physiological, biochemical, and morphological analyses suggest that this strain represents a novel species of genus Domibacillus, and the name Domibacillus iocasae sp. nov. is proposed with the type species S6^T(= DSM 29979^T= CCTCC AB 2015183^T). A novel Gram-negative bacterium, designated as strain S61^T, isolated from the deep sea sediment in Okinawa Trough. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain S61^T belonged to the genus Algoriphagus. The closest phylogenetic neighbors of strain S6^T were A. lutimaris (98.12%), A. halophila (98.12%), A. chungangensis (97.95%) and A.aestuarii (97.35%). The identities between strain S6^Tand other close members were below 97%. The values of DNA–DNA relatedness between strain S6^T and its closest relatives in genus Algoriphagus were well below 70 %. Phylogenetic, physiological, biochemical, and morphological analyses suggest that this strain represents a novel species of genus Algoriphagus, and the name Algoriphagus iocasae sp. nov. is proposed with the type species S61^T. Alvinocaris longirostris is a species of shrimp existing in the hydrothermal fields of Okinawa Trough. To date the structure and function of the microbial community associated with A. longirostris are essentially unknown. In this study, by employment of the techniques of high through-put sequencing and clone library construction and analysis, we compared for the first time the community structures and metabolic profiles of microbes associated with the gill and gut of A. longirostris in a hydrothermal field of Okinawa Trough. Fourteen phyla were detected in the gill and gut communities, of which 11 phyla were shared by both tissues. Proteobacteria made up a substantial proportion in both tissues, while Firmicutes was abundant only in gut. Although gill and gut communities were similar in bacterial diversities, the bacterial community structures in these two tissues were significantly different. Further, we discovered for the first time the existence in the gill and gut communities of the genes encoding the key enzymes of Calvin-Benson-Bassham (CBB) cycle and the reductive tricarboxylic acid (rTCA) cycle, and that both CBB- and rTCA-associated genes were significantly more abundant in gill than in gut. Taken together, these results provide the first evidence that at least two carbon fixation pathways are present in both the gill and gut communities of A. longirostris, and that the communities in different tissues likely differ in autotrophic productivity.
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/112548
专题	实验海洋生物学重点实验室
作者单位	中科院海洋所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	孙庆磊. 冲绳海槽热液区环境微生物及共生微生物研究[D]. 北京. 中国科学院大学,2016.

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