【作者】 杨大群；

【导师】 安黎哲；

【作者基本信息】 兰州大学 ， 植物学， 2008， 博士

【摘要】 低温环境是一个独特的生态系统,它被认为是天然的“菌种保藏中心”。近年来冷环境下微生物的研究取得了长足的发展,尤其是微生物生态学的研究。天山地区具有寒冷、中纬度和高海拔等特点,是研究低温微生物的理想生境。本文选取了天山2米深的雪坑、1.6米深的活动层冻土和3.0米深的永冻土作为材料,应用PCR-DGGE技术研究了其中微生物的群落结构和分布,分析和讨论了它们之间的关系及其与温室气体（CH₄和N₂O）排放的关系。得出了以下主要结果:1、从天山永冻土中检测出44条细菌序列,在系统进化树上聚类为7大类群:酸杆菌门（Acidobacteria）、放线菌门（Actinobacteria）、芽单胞菌门（Gemmatimonadetes）、绿弯菌门（Chloroflexi）、厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）和拟杆菌门（Bacteroidetes）。共包含Gemmatimonas、Carnobacterium、Bacillus、Acidobacterium、Arthrobacter、Pseudomonas、Rhodoplanes、Nordella、Herminiimonas、Denitratisoma、Ramlibacter、Flavobacterium、Thermoleiphilum和一些未确定种属的细菌。其中变形菌门包含α、β、γ和ε等4个亚门,是天山永冻土中的优势菌群。在所有检测到的细菌中与冷环境相关的细菌占22.7%（10/44）。此项研究表明天山永冻土特殊的生境孕育了丰富多样的细菌资源。2、从天山永冻土中检测出28条古细菌序列,在系统进化树上聚类为2大类群:广古菌门（Euryarchaeota）和泉古菌门（Crenarchaeota）。广古菌分为两大支,一是与盐细菌有相关性的广古菌Ⅰ,二是与甲烷菌有相关性的广古菌Ⅱ。泉古菌根据K（o|¨）nneke等（2005）的分类,归为“低温泉古菌”,只在较深的2.5米和3.0米的冻土中检测到。其中广古菌为永冻土中的优势菌群。在所有检测到的古细菌中与冷环境相关的古细菌占67.9%（19/28）。此项研究开启了高山寒区低温古菌的搜寻,为阐明生命低温起源和进化机制提供了更丰富的研究素材。3、从天山活动层冻土中检测出34条细菌序列,在系统进化树上聚类为4大类群:变形菌门（Proteobacteria）、厚壁菌门（Firmicutes）、放线菌门（Actinobacteria）和拟杆菌门（Bacteroidetes）。共包含Burkholderia、Xanthomonas、Sphingomonas、Pararubellimicrobium、Rudanella、Pedobacter、Dyadobacter、Nocardioides和一些未确定种属的细菌。其中变形菌门细菌为活动层冻土中的优势菌群。在所有检测到的细菌中与冷环境相关的细菌占61.8%（21/34）,说明了在活动层冻土存在反复性冻融的情况下冷适应细菌能够更好的被保存下来。4、从天山雪样中检测出40条细菌序列,在系统进化树上聚类为3大类群:变形菌门（Proteobecteria）、放线菌门（Actinobacteria）和拟杆菌门（Bacteroidetes）。共包含Janthinobacterium、Polaromonas、Acinetobacter、Pseudomonas、Rhodobacter、Nocardioides、Flectobacillus、Hymenobacter和一些未确定种属的细菌。其中变形菌门细菌为雪样中的优势菌群。在所有检测到的细菌中与冷环境相关的细菌占47.5%（19/40）。DGGE图谱表明雪样中的细菌种类与大气中的尘埃有着密切的关系,说明了大气的生物输送对雪样细菌的分布起着重要作用,可能成为一种反映气候环境变化的指标。5、比较雪样、活动层和永冻层中微生物的群落结构特征,发现它们互相之间几乎没有生物交换。微生物来源方式不同和样品年代的不同可能是造成这一结果的主要原因。6、在永冻层的上限（1.5米）处,发现存在着许多反硝化细菌（假单胞菌）和产甲烷菌,并且氨态氮和硝态氮的比值（NH₄-N:NO₃/NO₂-N）最大,表明浅层冻土中的微生物具有释放温室气体CH₄和N₂O的潜在能力,全球气候变暖已经开始影响到永冻土中的微生物,微生物也即将对全球气候变暖产生重要的反馈作用。7、冻土和积雪中存在着一些病原菌。如肉杆菌（Carnobacterium）、伯克氏菌（Burkholderia）和黄单胞菌（Xanthomonas）,随着冻土和积雪的融化,这些病原菌将会被释放。以上研究表明天山地区冷环境中存在着丰富的低温微生物资源,天山低温微生物多样性的研究拓展了低温生物学领域的研究范围,揭示了原核生物在高山环境生物地球化学作用中的分布与功能,为进一步的应用研究提供了理论依据和科学支撑。更多还原

【Abstract】 Low temperature environment is a special complex ecosytem and it is considered as a center for culture collection.Recently,much progress has been made in terms of microbial research in low temperature environment,especially in cold-adapted microbial ecology.Tianshan area represents a unique environment,low temperature, middle latitude and high altitude,is deemed to a perfect habitat for cold-adapted microorganisms.In this study,snow and frozen soils samples were collected from Tianshan,northwestern China.We investigated the distribution and diversity of the uncultured microorganisms using PCR-DGGE technique,analysed the relationship between microbial communities in different types of samples and discussed the capacity of these organisms to release the greenhouse gases N₂O and CH₄.The major results were obtained as follows:1.Forty-one representative bacterial bands were selected for sequencing and phylogenetic analysis from permafrost.The phylogenetic trees placed these clones into 7 major groups:Acidobacteria,Actinobacteria,Gemmatimonadetes,Chloroflexi, Firmicutes,Proteobacteria and Bacteroidetes,including genera Gemmatimonas, Carnobacterium,Bacillus,Acidobacterium,Arthrobacter,Pseudomonas,Rhodoplanes, Nordella,Herminiimonas,Denitratisoma,Ramlibacter,Flavobacterium, Thermoleiphilum and unidentified bacteria.The Proteobacteria,consisting of theα,β,γ, andεsubdivision,was a clearly dominant group at all depths studied.Of all bacteria, there were 22.7%（9/44）with highest sequence similarity to their closest relatives recovered from other low temperature environments.From this study,it was proposed that permafrost sediments provide a specific ecological niche for diverse microbial lineages.2.Twenty-eight representative archaeal bands were selected for sequencing and phylogenetic analysis from permafrost.The phylogenetic trees placed these clones into three phylogenetic clusters within the two kingdoms,Euryarchaeota and Crenarchaeota. Within the Euryarchaeota,methanogen-related groupⅡwas most abundant at shallow depth,whereas halobacterium-related groupⅠdominated at greater depths.A Low-Temperature Crenarchaeota group was only detected at 2.5 and 3.0 m.Of all archaea,there were 67.9%（19/28）with highest sequence similarity to their closest relatives recovered from others low temperature environment.This study help to the exploration of cold-adapted archaea in the cold alpine environment,and provided abundant opportunities to clarify cold origin of life and the mechanisms of evolution.3.Thirty-four representative bacterial bands were selected for sequencing and phylogenetic analysis from active layers.The phylogenetic trees placed these clones into 4 major groups:Actinobacteria,Firmicutes,Proteobacteria and Bacteroidetes, including genera Burkholderia,Xanthomonas,Sphingomonas,Pararubellimicrobium, Rudanella,Pedobacter,Dyadobacter,Nocardioides and unidentified bacteria.The most abundant and diverse bacteria were members of Proteobacteria.Of all bacteria,there were 61.8%（21/34）with highest sequence similarity to their closest relatives recovered from others low temperature environment.From this study,it was proposed that cold adaptation bacteria can be preservated on repeated freezing and thawing in active layers.4.Forty representative bacterial bands were selected for sequencing and phylogenetic analysis from snow samples.The phylogenetic trees placed these clones into 3 major groups:Actinobacteria,Proteobacteria and Bacteroidetes,including genera Janthinobacterium,Polaromonas,Acinetobacter,Pseudomonas,Rhodobacter, Nocardioides,Flectobacillus,Hymenobacter and unidentified bacteria.The most abundant and diverse bacteria were members of Proteobacteria.Of all bacteria,there were 47.5%（19/40）with highest sequence similarity to their closest relatives recovered from others low temperature environment.The DGGE pattern showed there were some positive relationship between bacterial species and micro-particles deposited,it suggested the main influence of atmospheric transportation on the microbial distribution in snow and microbial analysis of snow may provide proxy formation for past climates and environments.5.Compare with the bacterial diversity and abundance in snow,active layer and permafrost.We found the bacteria almost wouldn’t exchange from each niche.The main reasons caused were the different bacterial sources and different samples formation time.6.Specific-depth distribution of methanogen-related Euryarchaeota groupⅡand denitrifying bacteria of the genus Pseudomonas dominated at 1.5 m depth,accompanied with a distinct peak of NH₄-N:NO₃/NO₂-N ratio,implying the potential capacity of these organisms in near-surface permafrost to release the greenhouse gases N₂O and CH₄.7.Some pathogenic bacteria were found in permafrost and snow.Such as Carnobacterium,Burkholderia and Xanthomonas.They will be released accompanied with the permafrost degradation and snow thawing.The data obtained in this study on genetic diversity of uncultured bacteria from the frozen soils and snows in the Tianshan Mountains expand our knowledge on the extent of bacterial diversity in the cold realm and improve our understanding of the biogeographic distribution and function of prokaryotes in alpine environments.更多还原