西藏米拉山土壤古菌16S rRNA及amoA基因多样性?分析

摘要：【目的】硝化作用在全球土壤氮循环中具有重要的作用,虽然细菌一度被认为单独负责催化这个过程的限速步骤,但是最近一些研究结果表明泉古菌具有氨氧化的能力。本文通过构建古菌16S rRNA 基因克隆文库和氨氧化古菌amoA基因文库,分析西藏米拉山高寒草甸土壤中古菌及氨氧化古菌群落结构组成情况,为揭示青藏高原高寒草甸土壤古菌的多样性提供理论基础。【方法】采用未培养技术直接从土壤中提取微生物总DNA,分别利用通用引物构建古菌16S rRNA 基因和氨氧化古菌amoA基因克隆文库。【结果】通过构建系统发育树,表明古菌16S rRNA 基因克隆文库包括泉古菌门和未分类的古菌两大类,并且所有泉古菌均属于热变形菌纲。氨氧化古菌amoA基因克隆文库中序列均为泉古菌。通过DOTUR软件分析,古菌16S rRNA基因和古菌amoA基因克隆文库分别包括64个OTUs和 75个OTUs。【结论】西藏米拉山高寒草甸土壤中古菌多样性比较丰富,表明古菌在高寒草甸土壤的氮循环中可能具有重要的作用。所获得的一些序列与已知环境中土壤、淡水及海洋沉积物中获得的一些序列具有很高的相似性,其古菌及氨氧化古菌来自不同环境的可能性比较大,可能与青藏高原的地质历史变迁过程有关。米拉山古菌及氨氧化古菌与陆地设施土壤中相似性最高,说明与西藏米拉山高寒草甸土壤的退化有关。     

黄河三角洲盐碱地花生根层土壤菌群结构多样性

花生属豆科固氮作物,具较强的抗旱耐盐性,土壤微生物在盐碱土生态系统中具有重要的生态功能。以花生平作、花生/棉花间作为对象,通过16S rRNA基因克隆文库技术分析了黄河三角洲滨海盐碱地花生旺盛生长期不同含盐量盐碱地和非盐碱地0—40cm根层非培养土壤微生物群落组成及其多样性,分析了盐碱地花生根层土壤细菌群落与非盐碱地花生根层土壤细菌群落的差异,为揭示盐碱地花生根层土壤微生物的多样性以及土地利用变化与生态环境效应间的关系奠定基础。利用免培养技术直接从土壤样品提取总DNA,针对细菌基因组16S rRNA基因的V3高变区进行PCR扩增;利用焦磷酸测序的方法对V3高变区PCR产物进行高通量测序,并对测序数据进行生物信息学分析。结果表明,(1)黄河三角洲滨海盐碱土较高含盐量土壤中根层土壤微生物种类、优势种群数量和群落功能多样性较非盐碱土壤较为丰富。(2)盐碱土花生平作或花生//棉花间作两种种植方式基本不影响二者0—40cm根层土壤微生物优势类群;不同土壤类型和种植模式下,花生和棉花根层土壤中优势菌群均为变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、绿弯菌门(Chloroflexi)和酸杆菌门(Acidobacteria) 4种菌群,其总丰度为80%—90%。非盐碱土壤中花生根层的酸杆菌门(Acidobacteria)丰度是盐碱土壤中的3倍以上,嗜热油菌纲(Thermoleophilia)和放线菌纲(Actinomycetales)丰度远高于各种盐碱土壤花生平作和花生//棉花间作两种植模式下的花生根层土壤;非盐碱土平作花生0—40cm土层中Rubellimicrobium、Pontibacter和Lamia细菌则显著缺失。(3)土壤类型对土壤微生物菌群类型影响较大,聚类分析表明,10个土壤样本依据土壤含盐量高低和根系分布深度聚为3类,即非盐碱土壤归为1类,盐碱土壤根系密集分布层0—20cm、20—40cm各归为1类。     

新疆两典型微咸水湖水体免培养古菌多样性

微咸水湖是湖泊演化过程中的一个重要中间状态,以新疆两典型微咸水湖-赛里木湖和柴窝堡湖水为研究对象,采用微孔滤膜收集菌体,SDS-酚-氯仿抽提法直接提取湖水总DNA,利用古菌16S rRNA基因通用引物进行PCR扩增,分别构建两湖古菌16S rRNA基因克隆文库。用限制性内切酶Hae Ⅲ对随机挑选的阳性克隆子进行酶切分型,分别得到7个和8个可操作分类单元(Operational Taxonomic Units, OTUs),两文库覆盖率均大于98%。BLAST比对和系统发育分析表明赛里木湖全部克隆子归属于泉古菌门(Crenarchaeota),97%的克隆子与不同环境免培养氨氧化泉古菌有较高的序列相似性(>97%)。柴窝堡湖水古菌归为3个门:Thaumarchaeota (81.2%)、广古菌门(Euryarchaeota)(13%)和泉古菌门(Crenarchaeota) (5.8%),81.2%的克隆子与具有氮代谢功能的氨氧化古菌纯培养物具有较高的序列相似性(97%-98%),13%的克隆子与已分离到的产甲烷古菌序列同源性大于97%。研究发现新疆微咸水湖可能存有大量新划分的古菌Thaumarchaeota门类群、可培养氨氧化及产甲烷古菌类群,两典型微咸水湖泊中古菌类群多样性较低且群落组成差异大。     

引物应用策略：基于2对古菌16S rRNA基因通用引物对环境样品古菌多样性分析

【目的】找到适宜的16S rRNA基因通用引物应用策略,应对复杂环境微生物多样性调查,尤其目前高速发展的高通量测序技术带来的巨大挑战。【方法】用Oligocheck软件分别将两对应试的古菌16S rRNA基因通用引物与RDP(Ribosomal database project)数据库中古菌16S rRNA基因序列进行匹配比对。用两对应试引物分别构建海洋沉积物样品的古菌16S rRNA基因文库。【结果】软件匹配结果显示引物f109/r958与目的基因的匹配程度高于引物f21/r958。该结果与古菌16S rRNA基因文库RFLP分析、古菌多样性指数分析结果相吻合。数据还表明,2对引物的综合文库能更好满足该沉积物样品的古菌多样性分析。【结论】选用与数据库中目的基因匹配性高的通用引物和多个引物的联合使用,可以有效提高环境样品微生物多样性调查的分辨率。     

新疆艾比湖和伊吾湖可培养嗜盐古菌多样性

新疆地区盐湖密布,蕴藏着丰富的微生物资源。为保护和利用微生物物种与基因资源,作者从新疆准噶尔盆地的艾比湖和天山山间盆地的伊吾湖分离纯化嗜盐微生物。采用PCR方法扩增其中65株嗜盐古菌16SrRNA基因序列。序列分析表明,分离的嗜盐古菌分属6个属,艾比湖以Haloterrigena和Natrinema属的菌株为主,伊吾湖由Haloarcula和Halorubrum两个属的菌株构成。通过多样性指数、丰富度指数、均匀度指数和物种相对多度模型对分离的菌株进行多样性分析和比较,结果表明,盐湖嗜盐古菌的多样性指数、丰富度指数和均匀度指数具有一定相关性,艾比湖可培养嗜盐古菌的多样性高于伊吾湖。研究发现了一些新的物种资源,表明新疆盐湖中孕育的特色微生物资源亟待保护与利用。     

海洋古菌多样性研究进展

海洋古菌是海洋微生物中的一个大的类群,然而绝大多数的古菌不能分离培养.近年来分子生物学的方法广泛地应用于微生物多样性的研究中,研究发现,海洋古菌广泛地生活在各类海域环境中,而不仅仅是生活在极端的环境中.海洋古菌为海洋生态系统中主要的原核细胞成分,在海洋生态系统中的物质与能量循环中扮演着重要角色.主要阐述了生活在海洋不同环境中海洋古菌的多样性,有海洋浮游古菌的多样性、海底环境及海洋沉积物中古菌的多样性、附着或寄共生古菌多样性等的研究状况,以及研究海洋古菌多样性的分子生物学的主要方法.     

不同土壤采样设计下土壤表层微生物α多样性的差异分析

【背景】土壤采样是土壤研究的基础,采样方案的不同可能会对土壤微生物多样性的研究结果产生一定影响。【目的】研究不同的土壤采样设计方案对土壤样品16S rRNA基因高通量测序结果的影响。【方法】对2个不同生境样地的土壤进行网格化采样,对采集的18个土壤样品进行16S rRNA基因测序分析,通过模拟5种常见土壤采样方法,对比不同采样方式所获得的测序结果。【结果】不同采样方式会产生不同的测序结果。在测序深度有效的情况下,细菌总物种数随着采样数的增加而逐渐增长,增长速度在采样数大于5以后趋于平缓;样品中的优势物种(序列数200以上)只需很少的采样数(1-3)即可观察到全部物种;Shannon-Wiener指数与Simpson指数的变化较相似,当采样数由1到3时两指数均有较大增长,之后变化放缓。【结论】土壤细菌微生物测序研究中,土壤样地采样数量低于3个会影响测序结果的可靠性,采样方案选择梅花形采样法或蛇形采样法较为适宜。     

转mapk双链RNA干扰表达载体黄瓜对根际土壤细菌多样性的影响

随着RNA干扰技术的发展,通过植物表达病原物特异的dsRNA来防治植物病害的转基因作物越来越多.转根结线虫mapk双链RNA表达载体的黄瓜能够通过RNA干扰作用沉默线虫的mapk基因,对根结线虫具有良好的防治效果.为了评价该转基因黄瓜的安全性,明确mapk双链RNA干扰表达载体转基因黄瓜植株对根际土壤细菌多样性的影响,采用16S rRNA基因克隆文库方法对非转基因黄瓜和转基因黄瓜土壤细菌群落多样性进行分析.结果表明,非转基因黄瓜土壤细菌文库包含124个OTU(可操作分类单元),转基因黄瓜土壤细菌文库包含122个OTU.2个文库共同拥有的OTU为115个.2个文库都包含1 3个类群细菌,Acidobacteria、Actinobacteria、Armatimonadetes、Bacteroidetes、candidate division BRC1、Chloroflexi、Firmicutes、Gemmatimonadetes、Nitrospira、Planctomycetes、Proteobacteria、Verrucomicrobia、unclassified_Bacteria. 其中 Proteobacteria、Bacteroidetes 、Chloroflexi和Acidobacteria是优势菌群.其他细菌类群数量相对较少.在纲分类水平上,两个文库包含的细菌类群一致,且各类群细菌比例差异不大.在Acidobacteria门中,Acidobacteria_Gp6为优势菌群.在Bacteroidetes门中,Sphingobacteria纲细菌数量最多.在Chloroflexi门中,unclassified Chloroflexi细菌最多.在Proteobacteria门中,其中Betaproteobacteria纲的细菌数量最多.从多样性指数角度分析,两种土壤细菌群落的Shannon、Simpson和Chao值差异不大.总体来看,两种土壤细菌类群差异不显著,转基因黄瓜未对根际土壤细菌群落产生明显影响.     

结合宏基因组末端随机测序和16S rDNA技术分析温室黄瓜根围土壤细菌多样性

土壤细菌在温室土壤环境中具有十分重要的生态功能,与温室作物以及微生物内部存在互作关系。研究土壤细菌的群落结构组成,有助于了解土地利用变化与生态环境效应之间的关系。结合16S rRNA基因克隆文库和宏基因组末端测序对温室黄瓜根围土壤细菌的多样性进行了分析。在16S文库中,根据97%的序列相似性水平划分OTU,共有35个OTU,其中优势菌群是γ-Proteobacteria,其次为Firmicutes,Bacillus为优势细菌。在纲分类水平上,16S文库和宏基因组末端测序结果均包含γ-Proteobacteria、α-Proteobacteria、δ-Proteobacteria、β-Proteobacteria、Actinomycetales和Firmicutes,各纲比例有差别;在优势种群属水平上,末端测序的结果包含的属多于16S文库(4035);在优势细菌种类上,两者反映的结果一致,均为Bacillus。但是,宏基因组末端测序包含了大多数的弱势种群,更能反映细菌多样性的真实水平。与露地土壤细菌16S文库相比较,土壤细菌多样性降低,这可能与温室多年连作,种植蔬菜种类单一直接相关。     

Analyses of soil fungal communities in adjacent natural forest and hoop pine plantation ecosystems of subtropical Australia using molecular approaches based on 18S rRNA genes

Soil fungal communities were studied using 18S rDNA-based molecular techniques. Soil DNA was analyzed using temperature gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP), cloning and sequencing methods, following community DNA extraction and polymerase chain reaction (PCR). The extracted community DNA was successfully amplified using the primer pair of EF4f-Fung5r which produced ca. 550bp 18S rDNA fragments. TGGE screening of the PCR products showed some differences in band position and intensity between two soil samples in adjacent natural forest (YNF) and hoop pine plantation (YHP) ecosystems at Yarraman in subtropical Australia. TGGE and SSCP could be used for screening PCR products. However, care must be exercised when interpreting the TGGE and SSCP results with respect to microbial diversity, because one band may not necessarily represent one species. It is recommended that the PCR products should be purified before TGGE or SSCP screening. SSCP screening of the clone sequences revealed differences among the clones. Sequence and phylogenetic analyses revealed that all obtained clones were affiliated to the kingdom Fungi, including three phyla, i.e., Zygomycota, Ascomycota and Basidiomycota. Our results suggested that community DNA extraction, PCR, cloning, SSCP screening of clones, sequencing of selected clones and phylogentic analyses could be a good strategy in investigation of soil fungal community and diversity.     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

Mixed plantations of Metasequoia glyptostroboides and Bischofia polycarpa change soil fungal and archaeal communities and enhance soil phosphorus availability in Shanghai,China

Soil degradation has been found in urban forests in Shanghai, especially in the pure plantations. Mixed plantations are considered to improve soil quality because they can stimulate organic matter cycling and increase soil carbon and nutrient content. Although soil microbes play crucial roles in regulating soil biogeochemical processes, little is known about how mixed plantations affect soil microbial communities, including bacteria, archaea, and fungi. Here, we evaluated soil chemical properties, abundances and compositions of soil bacterial, archaeal, and fungal communities, and enzyme activities in pure and mixed Metasequoia glyptostroboides and Bischofia polycarpa plantations, located in Shanghai, China. The results showed that soil available phosphorus content in the mixed plantation of M. glyptostroboides and B. polycarpa was significantly higher than that in pure plantations, while no significant difference was observed in the content of soil organic carbon, total and available nitrogen, total and available potassium among the three studied plantations. We found higher fungal abundance in the mixed plantation, when compared to both pure plantations. Moreover, fungal abundance was positively correlated with the content of soil available phosphorus. No significant difference was found in the abundance and diversity of bacterial and archaeal community among the three studied plantations. A similarity analysis (ANOSIM) showed that mixed plantation significantly altered the community composition of archaea and fungi, accompanied with an increase of alkaline phosphatase activity. However, ANOSIM analysis of bacterial communities showed that there was no significant group separation among different plantations. Overall, results from this study indicated that fungal and archaeal communities were more sensitive to aboveground tree species than bacterial community. Moreover, mixed plantations significantly increased the activity of alkaline phosphatase and the content of soil available phosphorus, suggesting that afforestation with M. glyptostroboides and B. polycarpa is an effective way to alleviate phosphorus deficiency in urban forests in Shanghai, China.     

16S rRNA基因在微生物生态学中的应用

16S rRNA(Small subunit ribosomal RNA)基因是对原核微生物进行系统进化分类研究时最常用的分子标志物(Biomarker),广泛应用于微生物生态学研究中。近些年来随着高通量测序技术及数据分析方法等的不断进步,大量基于16S rRNA基因的研究使得微生物生态学得到了快速发展,然而使用16S rRNA基因作为分子标志物时也存在诸多问题,比如水平基因转移、多拷贝的异质性、基因扩增效率的差异、数据分析方法的选择等,这些问题影响了微生物群落组成和多样性分析时的准确性。对当前使用16S rRNA基因分析微生物群落组成和多样性的进展情况做一总结,重点讨论当前存在的主要问题以及各种分析方法的发展,尤其是与高通量测序技术有关的实验和数据处理问题。     

Shifts of tundra bacterial and archaeal communities along a permafrost thaw gradient in Alaska

Understanding the response of permafrost microbial communities to climate warming is crucial for evaluating ecosystem feedbacks to global change. This study investigated soil bacterial and archaeal communities by Illumina MiSeq sequencing of 16S rRNA gene amplicons across a permafrost thaw gradient at different depths in Alaska with thaw progression for over three decades. Over 4.6 million passing 16S rRNA gene sequences were obtained from a total of 97 samples, corresponding to 61 known classes and 470 genera. Soil depth and the associated soil physical–chemical properties had predominant impacts on the diversity and composition of the microbial communities. Both richness and evenness of the microbial communities decreased with soil depth. Acidobacteria, Verrucomicrobia, Alpha‐ and Gamma‐Proteobacteria dominated the microbial communities in the upper horizon, whereas abundances of Bacteroidetes, Delta‐Proteobacteria and Firmicutes increased towards deeper soils. Effects of thaw progression were absent in microbial communities in the near‐surface organic soil, probably due to greater temperature variation. Thaw progression decreased the abundances of the majority of the associated taxa in the lower organic soil, but increased the abundances of those in the mineral soil, including groups potentially involved in recalcitrant C degradation (Actinomycetales, Chitinophaga, etc.). The changes in microbial communities may be related to altered soil C sources by thaw progression. Collectively, this study revealed different impacts of thaw in the organic and mineral horizons and suggests the importance of studying both the upper and deeper soils while evaluating microbial responses to permafrost thaw.     

Cultivation-independent analysis reveals a shift in ciliate 18S rRNA gene diversity in a polycyclic aromatic hydrocarbon-polluted soil

Using cultivation-independent methods the ciliate communities of a clay-rich soil with a 90-year record of pollution by polycyclic aromatic hydrocarbons (PAH) (4.5 g kg(-1) PAH) were compared with that of a nonpolluted soil collected in its vicinity and with similar properties. A ciliate-specific set of 18S rRNA gene targeting primers was designed and used to amplify DNA extracted from both soils (surface and 20 cm depth). Four clone libraries were generated with PCR products that covered an 18S rRNA gene fragment of up to 670 bp. Comparative sequence analysis of representative clones proved that the primer set was highly specific for ciliates. Calculation of similarity indices based on operational taxonomic units after amplified ribosomal DNA restriction analysis of the clones showed that the community from the nonpolluted surface soil was highly dissimilar to the other communities. The presence of several taxa, namely sequences affiliated to the orders Phyllopharyngia, Haptoria, Nassophorea, Peniculida and Scuticociliatia in samples from nonpolluted soil, points to the existence of various trophic functional groups. In contrast, the 18S rRNA gene diversity was much lower in the clone libraries from the polluted soil. More than 90% of these sequences belonged to the class Colpodea, a well-known clade of mainly bacterivorous and r-selected species, thus potentially also indicating a lower functional diversity.     

宁夏地区春季奶牛场牛舍土壤细菌群落特征

【背景】现代规模化生产模式下,牛舍环境管理是影响奶牛高效健康生产的重要因素。【目的】探讨牛场不同牛舍土壤细菌群落特征,为奶牛健康生产提供理论依据。【方法】采集宁夏某规模化奶牛场的哺乳犊牛岛、断奶犊牛舍、育成牛舍、低产泌乳牛舍、高产头胎泌乳牛舍、高产经产泌乳牛舍、干奶牛舍和病牛舍这8个不同牛舍的土样,每个牛舍6个重复,共48份土样。利用16S rRNA基因扩增子测序分析细菌群落结构与多样性,并对细菌群落的功能进行预测。【结果】不同牛舍土样细菌群落组成存在差异,并且8个牛舍中高产头胎泌乳牛舍土样的细菌群落多样性最高。哺乳犊牛岛土壤与其他牛舍土壤细菌群落在门水平上差异较大;泌乳期牛舍土样之间的细菌群落结构相似度较高。在门的水平上,拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和厚壁菌门(Firmicutes)是这8个牛舍土样共有的优势菌门。在属的水平上,嗜盐碱的盐单胞菌属(Halomonas)、具有潜在降解特性的Fermentimonas和栖海面菌属(Aequorivita)及致病菌的鸟杆菌属(Ornithobacterium)是犊牛期牛舍土样的优势菌属;嗜盐碱的Truepera是育成牛舍土样的优势菌属;致病菌的不动杆菌属(Acinetobacter)和Parapedobacter、耐药菌的Pedobacter是泌乳期牛舍土样的优势菌属。【结论】致病菌和参与硝酸盐呼吸的细菌主要分布在哺乳犊牛岛,嗜盐碱菌主要分布在断奶犊牛舍和育成牛舍,产甲烷的细菌主要分布在高产头胎泌乳牛舍。本研究分析了不同牛舍土壤细菌群落多样性,为奶牛健康生产提供理论依据。