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类型 基础研究 预答辩日期 2018-01-09
开始(开题)日期 2013-11-27 论文结束日期 2017-11-28
地点 邵逸夫科技馆3楼会议室 论文选题来源 973、863项目     论文字数 11 (万字)
题目 太湖蓝藻水华的宏基因组学研究
主题词 太湖,蓝藻水华,微生物,基因组,宏基因组
摘要 蓝藻水华是湖泊富营养化的一个主要问题,水华及其释放的毒素会对水体生态系统、人和畜禽等造成严重危害。尽管很多学者已经从水文气象、营养盐、食物链以及基因组等角度,开展了大量研究来探索蓝藻水华暴发机制,但是至今仍未完全阐明。细菌在水体循环、能量转化和信息传递等方面均发挥着重要作用。因此,探索细菌和蓝藻水华的关系,对于揭示蓝藻水华的暴发机制具有重要意义。本研究利用高通量测序技术,基于宏基因组学的方法,从微生物的结构和功能角度,结合水文、气象和营养盐等环境因子,探索太湖蓝藻水华的暴发机制。主要内容包括以下几个方面: (1) 建立了基于16S rRNA的太湖细菌多样性研究方法体系。通过对比Cetyl Trim -ethyl Ammonium Bromide (CTAB)、Xanthogenate-SDS(XS)、Mobio DNA solation和Omega DNA solation 试剂盒等4种常用Meta DNA (Metagenomic DNA, Meta DNA)提取方法对蓝藻群体颗粒、原水和过滤水样的提取和分析结果,发现不同方法对多样性结果具有显著影响。XS适合于蓝藻群体颗粒样本,而Omega 试剂盒则合适原水和过滤样本的DNA提取。通过SILVA RefNR 123数据库模拟和实验验证,从细菌的覆盖率(Coverage)、专一性(Specificity)、覆盖范围(Spectrum)和注释率 (POAOs) 等方面对16Sr RNA的V区选择进行了评估和论证。结果表明, V4区表现最好,并推荐S-D-Bact-0564-a-S-15|S-D-Bact-0785-b-A-18作为该区的扩增引物。此外,通过对比SILVA、Greengenes、RDP和FW数据库,建议在以太湖蓝藻水华为主题的细菌多样性研究中,以Greengenes数据库对16S rRNA扩增子测序获得的OTU进行注释。 (2) 明确了太湖水体细菌的多样性和演替规律。太湖水体细菌主要以Cyanobacte -ria、Proteobacteria、Verrucomicrobia、Actinobacteria和Planctomycetes类群为主。群落结构存在明显的季节演替规律,主要以Microcystis、ACK-M1、C111、Synechococcus、Actinomycetales、Pirellulaceae和Sphingobacteriaceae 等类群为主线进行演替。两季演替间存在显著或极显著差异的物种主要为,冬春:Candidatus_Xiphinematobacter、Synechococcus、Flavobacterium;春夏:Microcystis、Synechococcus、Planctomyces;夏秋:Microcystis、Planctomyces、Opitutus;秋冬:Microcystis、Methylotenerar、Candidatus_Xiphinematobacte。此外,浮游植物、水温和N是驱动太湖细菌群落演替的主要因子。 (3) 探明了太湖蓝藻群体颗粒附生细菌(Bacteria Associated with Cyanobacterial Aggregates, BACA)群落的多样性。水温是驱动BACA演替的主要因子。BACA不但存在季节上的演替,同时也存在季节相同而生命周期不同的演替。因此,附生细菌积极参与了蓝藻水华的暴发过程。此外,上浮和下沉蓝藻群体颗粒样本间BACA差异不显著,然而惠氏微囊藻、片状微囊藻、铜绿微囊藻和水华微囊藻的BACA存在一定的宿主特异性。这些特异性的附生细菌不但参与了群体形态的形成,更为重要的是可能参与了微囊藻毒素的降解,以及氮、磷和碳等物质循环,从而在功能上“影响”和“塑造”了相应的宿主。因此,从微囊藻附生细菌(Bacteria Associated with Microcystis Aggregates, BAMA) 的角度探索微囊藻的分类,也许是微囊藻分类值得探索的一个重要方向。 (4) 构建了Microcystis panniformis FACHB 1757基因组完成图。2012年在太湖发 现了中国微囊藻属的一个新记录种M. panniformis FACHB 1757。利用Pacific Bio RSII平台,对M. panniformis FACHB 1757进行了全基因组测序,共得到48倍覆盖度的原始数据。经过组装,得到一个完整的5.69 Mb基因组序列和一个39 Kb的质粒,分别注释出6,519和49个蛋白编码序列。通过与微囊藻属模式物种铜绿微囊藻M. aeruginosa NIES 843和其他一些可形成蓝藻水华的物种基因组比较研究,发现片状微囊藻在基因组水平上发生了大规模的结构重排,基因组大小也有较大变化。此外,在全基因组水平还发现了36个基因岛候选位点。因此,微囊藻在基因组水平上展现了较强的可塑性和多变性,是一个开放型基因组,这也是其在水体中能够大量繁殖和全球分布的一个重要原因。 (5) 表征了蓝藻群体颗粒微生物(Meta-microbes of Cyanobacterial Aggregates, MMCA) 的生理生态学特征。不同蓝藻水华暴发阶段的MBCA和代谢通路均存在显著性差异。蓝藻水华暴发前(1-3月),早(4-6月)和中(7-10月)期分别可以分别用低丰度的Rheinheimera、Pseudomonas、Phormidium,以及高丰度的Rheinheimera、Pseudomonas、Paucibacter和Microcystis、 Bdellovibrio、Bryobacter、Rickettsia来进行表征。N代谢通路主要有DNRA (Dissimilatory nitrate reduction to ammonium, DNRA)、ANRA (Assimilatory nitrate reduction to ammonium, ANRA) 和反硝化过程,存在一定的固氮过程,但是没有发生硝态氮的硝化过程和厌氧氨氧化过程。其中DNRA和ANRA在三个阶段均有发生,而为主要代谢通路,而在暴发早期和中期,MMCA没有发现反硝化作用。值得注意的是,MMCA的P代谢通路在三个阶段不存在显著性差异。 (6) 揭示了太湖微囊藻和长孢藻(Dolichospermum)的演替机理。微囊藻和长孢藻 的附生细菌存在明显的宿主特异性。以微囊藻和长孢藻为主的两个水华阶段可以分别用Gemmatimonas和Sediminibacterium、Saprospira进行表征。同时,两个水华阶段在N的代谢上存在显著差异,其中DNRA、ANRA和固氮作用在两个阶段均有存在,而反硝化作用在微囊藻水华阶段缺失。尽管微囊藻本身不固氮,但是其与附生微生物所组建的MMCA,作为一个整体可以进行固氮,这是其作为非固氮种类在N相对缺乏状态下获得竞争优势的重要原因。此外,太湖N负荷持续下降被认为是长孢藻水华在冬春季出现的主要原因,N和水温是微囊藻和长孢藻在冬春季节演替的主要原因。
英文题目 METAGENOMIC STUDIES ON CYANOBACTERIAL BLOOMS IN LAKE TAIHU
英文主题词 Lake Taihu, cyanobacterial blooms, microbes, genome, metagenome
英文摘要 The massive development of bloom-forming cyanobacteria is causing serious problems in eutrophic water bodies worldwide. Many cyanobacterial species have been reported to be able to produce toxins, which threaten many aquatic ecosystems and cause serious and occasionally fatal human liver, digestive, neurological, and skin diseases. Despite the extensive researches in the areas of hydrometeorology, food chains, and genomics, the mechanism underlying the cyanobacterial bloom formation largely remained elusive. Microbial interaction with each other and their environment is known to play a significant role in maintaining the various components of the biological ecosystems at equilibrium. In cyanobacterial blooms, various microorganisms have been reported to interact with bloom-forming cyanobacterial species. To get insights into the mechanisms of cyanobacterial bloom formation, exploration of interaction among the bloom-forming cyanobacterial species and other co-existing species is indispensable. In this study, metagenomics, nutrient hydrology, meteorology and environment factor datasets based integrated approaches were utilized to reveal the mechanism of cyanobacterial bloom formation. Main findings of the present study are summarized below. (1) An experimental platform method based on 16S rRNA amplicon for bacterial diversity survey of Taihu Lake was established. Extraction of the DNA is a key step involved in identifying the bacterial diversity and community composition. To find the reliable DNA extraction method, four widely used DNA extraction methods, including CTAB, XS, commercial Mobio DNA solation kit and Omega DNA solation kit were evaluated using three different kinds of samples collected from Lake Taihu. To perform comprehensive assessments different factors including yield, purity, and integrity of Meta DNA were also considered along with “spectrum”. The results showed that XS is the most suitable for cyanobacterial aggregates (Attached bacteria), and Omega kit for origin water (Total bacteria) and filtered water (Bacterioplankton). Furthermore, to evaluate the performances of the V regions metrics including “coverage”, “specificity”, “spectrum”, and “POAOs” were considered and V4 was found as most prominent V region for achieving good domain specificity, higher coverage and a broader spectrum in the bacterial domain. S-D-Bact-0564-a-S-15/S-D-Bact-0785-b-A-18 was found as a promising primer set for surveying bacterial diversity in eutrophic lakes. Moreover, Greengenes was observed as a robust database for aligning sequences reads recovered from Lake Taihu, as compared to other available reference databases including SILVA and RDP. (2) The bacterial succession pattern and dynamics in community structures, in Lake Taihu, were also observed. Overall, Cyanobacteria, Proteobacteria, Verrucomicrobia, Actinobacteria and Planctomycetes were the dominant bacterial groups cross the four seasons in Lake Taihu. Significant differences in community structure among the four seasons and an obvious seasonal succession pattern of bacteria were observed therein. The differences between two seasons succession were significantly observed, where main succession groups were Microcystis, ACK-M1, C111, Synechococcus, Actinomycetales, Pirellulaceae and Sphingobacteriaceae. Candida -tus_Xiphinematobacter, Synechococcus, Flavobacterium were involved in winter-spring; Microcystis, Synechococcus, Planctomyces for spring-summer; Microcystis, Planctomyces, Opitutus for summer-autumn; Microcystis, Methylotenerar, Candidatus_Xiphinematobacte for autumn-winter. Moreover, phytoplankton, water temperature, and N were found as main driving factors for a succession of bacterial community. (3) The community diversity of BACA was ascertained and temperature found playing key roles in structuring BACA. Bacterial community succession was observed on long-term (Seasonal) and even short-term basis (Weekly) in Lake Taihu. Therefore, BACA might have an active role in the outbreak of cyanobacterial blooms and no significant difference between the floating and sinking BACA samples was observed. BACA of M. wesenbergii, M. panniformis, M. aeruginosa, and M. flos-aquea demonstrated the host specificity among the Microcystis. Importantly, BACA was found involved in carbon cycle and degradation of microcystin, nitrogen, and phosphorus, which affected their host from a functional perspective. Therefore, the classification of Microcystis, combined with morphology and its BACA could serve to differentiate the Microcystis spp. in their natural population. (4) Complete genome sequence and genomic characterization of M. panniformis FACHB 1757. A strain of Microcystis, M. panniformis FACHB1757, was isolated from Meiliang Bay of Lake Taihu in August 2011. The whole genome was sequenced using PacBio RS II sequencer with 48-fold coverage. The complete genome sequence with no gaps contained a 5,686,839 bp chromosome and a 38,683 bp plasmid, which coded for 6,519 and 49 proteins, respectively. Comparison with strains of M. aeruginosa and some other water bloom-forming cyanobacterial species revealed large-scale structure rearrangement and length variation at the genome level along with 36 genomic islands annotated genome-wide, which demonstrates high plasticity of the M. panniformis FACHB1757 genome and reveals that Microcystis has a flexible genome evolution. Therefore, high plasticity and flexibility in genome evolution might be the reason behind providing superiority to Microcystis in ecological competition and global distribution. (5) Microbial profiles of MMCA in physiological and ecological functions. There were Signifi -cant differences among the different phases of cyanobacterial blooms for the MBCA diversity and metabolic pathways were observed. Early phase of cyano-blooms (Jan. to Mar.) was characterized by low abundance of Rheinheimera, Pseudomonas, Phormidium, and exponential phase of cyanobacterial blooms (Apr. to Jun) was characterized by high abundance of Rheinheimera, Pseudomonas, Paucibacter, whereas, stationary phase of cyanobacterial blooms (Jul. to Oct.) was characterized by high abundance of Microcystis, Bdellovibrio, Bryobacter, and Rickettsia, respectively. DNRA, ANRA, and denitrification were the main processes occurring on MMCA, and nitrogen fixation proportion was not that high, and nitrification process was not found. DNRA and ANRA were observed in all the three phases and were particularly found dominate in N metabolic pathway, whereas no denitrification during the exponential and stationary phase of cyanobacterial blooms was observed. Notably, no significant difference in P metabolic pathway among the three phases of cyanobacterial blooms process was observed. (6) The mechanism of the succession of Microcystis and Dolichospermum is revealed in Lake Taihu. Host specificity was observed between Microcystis and Dolichospermum, which can be characterized by Gemmatimonas and Sediminibacterium, Saprospira. Meanwhile, DNRA, ANRA, and nitrogen fixation were observed in both Microcystis and Dolichospermum dominated blooms, whereas denitrification was absent in Microcystis blooms. Microcystis is non-N2 fixing cyanobacteria; however, MMCA can be a functional unit to fix nitrogen. This might be the possible explanation that non-N2 fixing genus (Microcystis) can maintain dominance in Lake Taihu. Additionally, the continuous decline of N load in Taihu Lake is considered to be the main reason for the occurrence of Dolichospermum blooms during winter and spring. It was further inferred from the analysis that N and water temperature are the main reasons for the succession of Microcystis and Dolichospermum during winter and spring in Lake Taihu.
学术讨论
主办单位时间地点报告人报告主题
中国海洋湖沼学会藻类学分会 2017年6月29-30日 无锡 张军毅 太湖蓝藻水华及其水质:十年回顾与展望
中国海洋湖沼学会藻类学分会 2016年6月25日 温州 张军毅 太湖微囊藻和鱼腥藻属演替规律探讨
中国海洋湖沼学会藻类学分会 2015年11月27-30日 厦门 张军毅 太湖蓝藻水华监测预警及其研究
Indiana University School of Medicine November 13-15, 2015 Indianapolis, IN, USA Chris Sander Systems Biology in Action: Prediction of Large Protein 3D Structures and
中国海洋湖沼学会藻类学分会 2014年11月15-16日 上海 王全喜 中国硅藻分类及其研究
中国环境科学学会 2014年8月22-23日 成都 张军毅 太湖蓝藻“水华”和“菹草”的监测及其研究
南京师范大学 2014年6月24日 南京 张军毅 五里湖藻类及其演替
     
学术会议
会议名称时间地点本人报告本人报告题目
中国海洋湖沼学会藻类学分会 2017年6月29-30日 无锡 太湖蓝藻水华及其水质:十年回顾与展望
中国环境科学学会 2014年8月22-23日 成都 太湖蓝藻“水华”和“菹草”的监测及其研究
     
代表作
论文名称
Microbial profiles of a drinking water resource based on different 16S rRNA V regions during a heavy
Complete genome sequence and genomic characterization of Microcystis panniformis FACHB 1757 by third
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
李仁辉 正高 研究员 博导 中国科学院水生生物研究所
李涛 正高 研究员 博导 中国科学院水生生物研究所
李建宏 正高 教授 博导 南京师范大学
高光 正高 研究员 博导 中国科学院南京地理与湖泊研究所
卢晓林 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
葛芹玉 副高 副教授 东南大学