基于高通量测序的黄河入海口细菌资源多样性及群落结构研究

Microorganisms are important part of biosphere on the earth, as well as play important roles in biogeochemical cycling, energy flowing and protection of ecosystem. The Yellow River estuary lies in the intersection of Yellow River, Bohai Sea and Laizhou Bay. Shengli Oilfield is located in the estuary, and the wetland ecosystem nearby the estuary is formed due to sinking of sands in water. Our preliminary researches have demonstrated that the community composition and diversity of bacteria in the Yellow River estuary are different from that only in earth, ocean or estuary because of its specific geographical position and habitat. This project will choose water column and sediments from different phases (flood season and non-flood season) and sites in Yellow River estuary as research samples, extract the total DNA and RNA from environmental samples, combining 454 pyrosequencing with bioinformatics methods, to investigate the total / active bacterial diversity and community composition in Yellow River estuary. The result will reveal the spatiotemporal distribution characters of bacterial community, and explore the bacterial community associated with the protection of estuary ecosystem. Moreover, combining the physical and chemical parameters, correlation analysis will be performed to reveal the relationship among bacterial commnity composition, diversity and environment factors. These results of this project will play important roles in the protection of ecosystem in Yellow River estuary, and it will also be benefit to the exploitation and application of microbiological resources in Yellow River estuary in future.

微生物是地球生物圈的重要组成部分，在生物地球化学循环、能量流动及生态系统维持等方面起着重要作用。黄河入海口地处黄河、渤海和莱州湾交汇处，是胜利油田的所在地，大量泥沙的沉积形成了入海口湿地生态系统。我们初步的研究证明黄河入海口独特的地理位置和生境使其细菌群落组成和多样性区别于单纯的陆地、海洋乃至河口环境。本项目以黄河入海口不同时期（汛期和非汛期）、不同地点的水体和沉积物为研究对象，分别提取环境样品的总DNA和总RNA，采用454焦磷酸测序的方法，结合生物信息学分析，全面解析黄河入海口总细菌类群和活性细菌类群的群落结构、多样性与时空分布特征；探讨与入海口生态系统维持紧密相关的细菌类群；结合各环境样品的理化指标，进行相关性分析，进而揭示各种环境因子与该地区细菌群落结构、多样性之间的关系。本项目的研究成果对黄河入海口生态系统的保护具有重要价值，也为黄河入海口微生物资源的开发与利用奠定基础。

入海口作为河海交汇、海陆交互的地带，盐度及物质浓度梯度变化剧烈，使其承载着巨大的环境生态压力。水体和沉积物作为构成入海口环境的两大主体，其中的微生物在缓解河口生态压力中扮演着核心角色。黄河入海口作为我国重要的入海口生态系统，目前还缺乏对该地区微生物生态的系统研究。本研究采用高通量测序及荧光定量PCR等技术对该地区水体及沉积物中微生物（细菌、古菌）丰度、群落结构和多样性进行解析。丰度结果表现为沉积物中微生物丰度显著高于水体，同类型样品中细菌丰度显著高于古菌，且细菌与古菌16S rDNA丰度比值与样品类型紧密相关。高通量测序表明水体优势细菌类群为α-变形菌纲、放线菌门、蓝细菌门和疣微菌门等，沉积物优势细菌类群为δ-变形菌纲、酸杆菌门、绿弯菌门、浮霉菌门、硝化螺旋菌门等。广古菌门和奇古菌门为黄河口的绝对优势古菌类群。黄河入海口优势微生物类群与碳（尤其是甲烷和有机质代谢）、氮（尤其是氨氧化）、硫（尤其是硫酸盐还原）等代谢密切相关，表明微生物在清除该地区污染物、维持生态平衡中发挥重要作用。细菌群落结构在水体和沉积物间差异显著，而古菌则不显著。多样性分析表明黄河口细菌多样性高于古菌，沉积物微生物多样性高于水体。环境因子与微生物群落的相关性分析表明，相比沉积物，水体微生物对环境因子响应更敏感。硝态氮、铵态氮等是影响黄河口微生物群落的主要环境因子，因此，有效控制氮输入应该是改善黄河口生态的有效措施。本研究证实，环境因子、扩散限制、微生物自身适应性及微生物间互作关系等因素共同塑造了黄河口独特的微生物生态特征。在上述结果指示下我们又对该地区氨氧化、反硝化及亚硝酸盐依赖的甲烷氧化等功能微生物类群进行了深入研究，也分离得到了一些功能菌株，初步实现了对黄河入海口地区从微生物生态到功能再到资源开发的研究目标。研究结果将对了解微生物在河口的分布规律、黄河口生态维持及微生物资源开发与利用有较高的理论和实际价值。