分子生物学方法在环境微生物生态学中的应用研究进展

随着分子生物学方法的不断发展和改进,微生物在生态系统中的作用被更好的挖掘出来。目前快速发展的先进的分子生物学技术,已经开始应用于分析环境微生物的多样性、微生物的生物地理学及微生物对气候变化的响应等。一般环境微生物的研究目标主要有3个,即确定微生物的种类和多样性、微生物的功能或潜在作用及在特定时间点活跃的微生物等。然而,现有微生物的研究方法复杂多样,容易给研究者在方法的选择上带来困惑。将从微生物的多样性和功能研究两个方面介绍和分析相应的分子生物学方法,尤其是近年来快速发展的高通量测序、宏组学和单细胞水平研究方法(如纳米二次离子质谱与荧光原位杂交相结合的方法)等新技术及其应用情况,以期为研究者选择合适的研究方法进行环境微生物的研究提供依据。     

功能代谢组学技术在微生物研究中的应用

功能代谢组学是以代谢组学技术发现关键代谢物为基础,结合体内体外实验和分子生物学等技术手段,研究差异代谢物及相关蛋白、酶和基因的功能,从而揭示生物体内在的分子调控机制。功能代谢组学技术具有精准识别关键调控代谢物及其相关基因或酶的特性,近年来在微生物相关疾病的防控和工业化生产等方面受到了广泛的关注。本文介绍了功能代谢组学技术的分析流程、相关研究方法与平台及其在微生物研究方面的应用,其中重点阐述了真核、原核以及病毒微生物的代谢特性、调控靶点及相关防控策略等。最后,提出功能代谢组学研究在未来面临的问题与挑战,为后续功能代谢组学的研究与发展提供新的思路。     

功能代谢组学技术在微生物研究中的应用

功能代谢组学是以代谢组学技术发现关键代谢物为基础,结合体内体外实验和分子生物学等技术手段,研究差异代谢物及相关蛋白、酶和基因的功能,从而揭示生物体内在的分子调控机制。功能代谢组学技术具有精准识别关键调控代谢物及其相关基因或酶的特性,近年来在微生物相关疾病的防控和工业化生产等方面受到了广泛的关注。本文介绍了功能代谢组学技术的分析流程、相关研究方法与平台及其在微生物研究方面的应用,其中重点阐述了真核、原核以及病毒微生物的代谢特性、调控靶点及相关防控策略等。最后,提出功能代谢组学研究在未来面临的问题与挑战,为后续功能代谢组学的研究与发展提供新的思路。     

稳定性同位素探测技术在微生物生态学研究中的应用

稳定性同位素标记技术同分子生物学技术相结合而发展起来的稳定性同位素探测技术（stableisotope probing,SIP）,在对各种环境中微生物群落组成进行遗传分类学鉴定的同时,可确定其在环境过程中的功能,提供复杂群落中微生物相互作用及其代谢功能的大量信息,具有广阔的应用前景.其基本原理是：将原位或微宇宙（microcosm）的环境样品暴露于稳定性同位素富集的基质中,这些样品中存在的某些微生物能够以基质中的稳定（性同位素为碳源或氮源进行物质代谢并满足其自身生长需要,基质中的稳定性同位素被吸收同化进入微生物体内,参与各类物质如核酸（DNA和RNA）及磷脂脂肪酸（PLFA）等的生物合成,通过提取、分离、纯化、分析这些微生物体内稳定性同位素标记的生物标志物,从而将微生物的组成与其功能联系起来.在介绍稳定性同位素培养基质的选择及标记方法、合适的生物标志物的选择及提取分离方法的基础上,举例阐述了此项技术在甲基营养菌、有机污染物降解菌、根际微生物生态、互营微生物、宏基因组学等方面的应用.     

分子生物学方法在微生物多样性研究中的应用

微生物多样性是生物多样性的重要组成部分。由于微生物和大生物（动、植物）相比,存在着多种显著差异,因此其多样性,保护及利用也有所不同,尤其是研究方法亟待完善,提高。近年来,分子生物学方法广泛用于微生物多样性的研究并取得了一系列研究成果。本文从四个方面加以介绍：１）微生物总ＤＮＡ制备及其遗传多样性检测方法;２）１６ＳｒＲＮＡ基因序列研究;３）核酸杂交分析技术;４）ＤＮＡ动力学的研究。今后的发展趋势是加     

分子生物学方法在食品微生物检测中的应用

近年来,食品安全受到广泛关注。及时、准确地检测出食品中的病原微生物是食品安全检测的重要内容,食品病原微生物的分子生物学检测技术因其特异性和灵敏性而备受瞩目。我们主要介绍了基因探针检测法、PCR检测法和基因芯片检测法的原理、开发及其在食品微生物检测中的应用。     

荧光原位杂交技术在医学微生物学中的应用

以16S rRNA 为靶序列的寡核苷酸探针荧光原位杂交技术已广泛应用于分析复杂环境中的微生物群落构成,包括监测和鉴定病原微生物以及未被培养微生物．通过对临床样品中微生物细胞的检测能提供微生物在人体中的种类、数量和空间分布等信息．其结果快速准确,较之传统的病原微生物诊断方法具有明显的优越性,在临床应用中有广泛的前景．     

碳同位素示踪技术及其在陆地生态系统碳循环研究中的应用与展望

碳同位素示踪技术具有高度的专一性和灵敏度, 经过几十年的发展, 形成了一系列成熟的标记方法, 在陆地生态系统碳循环过程的研究中已得到广泛应用。目前, 自然丰度法、与¹³C贫化示踪技术结合的自由空气中气体浓度增加(FACE)实验、脉冲与连续标记法以及碳同位素高丰度底物富集标记法是研究陆地生态系统碳循环过程常用的碳同位素示踪方法; 通过将长期定位实验和室内模拟实验结合, 量化光合碳在植物-土壤系统的传输与分配特征, 明确植物光合碳对土壤有机质的来源、稳定化过程的影响及其微生物驱动机制; 阐明土壤碳动态变化(迁移与转化)和新碳与老碳对土壤碳库储量的相对贡献, 评估有机碳输入、转化与稳定的生物与非生物微观界面过程机制。然而, 生态系统碳循环受气候、植被、人为活动等多因素影响, 碳同位素技术需要结合质谱、光谱技术实现原位示踪, 结合分子生物学技术阐明其微生物驱动机制, 从而构建灵敏、准确、多尺度、多方位的同位素示踪技术体系。因此, 该文以稳定碳同位素为主, 综述了碳同位素示踪技术的原理、分析方法和在陆地生态系统碳循环过程中的应用进展, 归纳总结了碳同位素示踪技术结合原位检测技术和分子生物学技术的研究进展和应用前景, 并对碳同位素示踪技术存在的问题进行了分析和展望。     

分子生物学在石油微生物多样性研究中的应用

该文较系统地概述了目前以16SrRNA为基础的核酸探针检测技术、利用引物的PCR技术、DNA序列分析技术和电泳分离及显示技术在油藏微生物群落多样性和石油环境污染研究中的研究进展,并探讨了未来分子生物学技术在石油微生物研究中的发展方向。     

氮稳定同位素技术在陆地生态系统氮循环研究中的应用

在过去几十年中, 氮(N)稳定同位素技术的发展提高了人们对于陆地生态系统氮循环的认识。该文回顾了氮稳定同位素技术在研究生态系统氮循环中的历史, 综述了最近十多年来氮稳定同位素技术在陆地生态系统氮循环研究中的典型案例, 包括利用氮同位素自然丰度鉴定植物氮来源、指示生态系统氮状态和量化过程速率, 利用¹⁵N标记技术示踪氮的去向和再分布等。该文同时指出这些应用中存在的问题, 以及在陆地生态系统上氮稳定同位素技术今后研究的重点发展方向。     

Humboldt's spa: microbial diversity is controlled by temperature in geothermal environments

Over 200 years ago Alexander von Humboldt (1808) observed that plant and animal diversity peaks at tropical latitudes and decreases toward the poles, a trend he attributed to more favorable temperatures in the tropics. Studies to date suggest that this temperature–diversity gradient is weak or nonexistent for Bacteria and Archaea. To test the impacts of temperature as well as pH on bacterial and archaeal diversity, we performed pyrotag sequencing of 16S rRNA genes retrieved from 165 soil, sediment and biomat samples of 36 geothermal areas in Canada and New Zealand, covering a temperature range of 7.5–99 °C and a pH range of 1.8–9.0. This represents the widest ranges of temperature and pH yet examined in a single microbial diversity study. Species richness and diversity indices were strongly correlated to temperature, with R² values up to 0.62 for neutral–alkaline springs. The distributions were unimodal, with peak diversity at 24 °C and decreasing diversity at higher and lower temperature extremes. There was also a significant pH effect on diversity; however, in contrast to previous studies of soil microbial diversity, pH explained less of the variability (13–20%) than temperature in the geothermal samples. No correlation was observed between diversity values and latitude from the equator, and we therefore infer a direct temperature effect in our data set. These results demonstrate that temperature exerts a strong control on microbial diversity when considered over most of the temperature range within which life is possible.     

俄罗斯堪察加地区热泉及其周边生境的泉古菌多样性

【目的】泉古菌为陆地热泉系统的主要古菌类群,可能在自然界生源元素的地球化学循环中发挥着重要作用。本研究旨在揭示俄罗斯堪察加地区热泉以及热泉周边区域的泉古菌多样性,同时基于之前已获得的我国云南地区热泉数据,比较两地区泉古菌群落差异。【方法】通过构建16S rRNA基因片段克隆文库获得序列信息和丰度,随后进行物种多样性、系统发育和群落结构差异分析。【结果】高温热泉Burlyashi Liza(BSL,89℃)中的泉古菌全部属于热变形菌纲(Thermoprotei)内的物种。中温热泉TF Vent 2(TFV,49℃)的群落结构主要由不确定的热变形菌纲类群、不确定的泉古菌类群、高温水环境泉古菌类群Ⅱ(HWCG-Ⅱ)和奇古菌下的Group1.1b类群组成。热泉周边常温环境的主要物种与热泉环境的代表性克隆pJP41一起聚成一个较大的遗传分枝。Jackknife聚类树和主坐标分析(Principal coordinates analysis,PCoA)的结果显示:温度相似的样点,其泉古菌群落结构相对来说更为相似。【结论】俄罗斯堪察加地区与我国云南地区热泉中的泉古菌存在着一定程度上的不同。陆地热泉系统影响着其周边环境的泉古菌类群。热泉中泉古菌群落结构受温度的明显影响。     

极地陆域微生物多样性研究进展

极地是指高纬度、高海拔地区,包括南极(60°S以南)、北极(60°N以北)和被称为“第三极”的青藏高原地区(平均海拔4,500 m).这些地区气温极低、养分极度贫乏,生态系统非常脆弱,对全球气候变化极为敏感,该地区生态系统一旦破坏将很难恢复.尽管极地地区自然条件恶劣,但在这些极端环境中栖息着大量微生物,是元素生物地球化学循环的主要驱动者,对极地生态系统的构建和维持具有非常重要的作用.本文综述了极地土壤、湖泊和冰川等陆域环境微生物研究进展.在这些极地环境中,目前已发现了Acidobacteria,Actinobacteria,Bacteroidetes,Cyanobacteria和Firmicutes等类群,这些微生物具有嗜盐/耐盐及耐低温等特征.我国在极地微生物生态学研究方面落后于发达国家,建议优先发展较易到达的青藏高原地区微生物生态学长期定位观测,这将有助于较快提升我国极地微生物多样性研究水平,深入了解极端生命过程及其生态学效应.     

冰川生态系统固碳微生物研究进展

[目的] 海南海口含有丰富的温泉资源,对温泉微生物多样性进行研究,有助于进一步开发和利用海南温泉微生物资源。[方法] 本文采用Illumina HiSeq高通量测序技术对海口3个温泉[海甸岛荣域温泉（S1）、火山口开心农场温泉（S2）和西海岸海长流温泉（S3）] 水样中微生物ITS序列和16S rRNA基因V3-V4区进行测序及生物信息学分析,探究海口市3个不同区域的温泉真菌多样性与细菌多样性。[结果] （1）α多样性分析表明,真菌群落中,S3 > S1 > S2,而在细菌群落中,S2 > S1 > S3。β多样性分析表明,3个温泉真菌群落和细菌群落组成差异皆显著。（2）分类分析表明,温泉真菌群落优势菌门为子囊菌门（Ascomycota）和担子菌门（Basidiomycota）,细菌群落优势菌门为变形菌门（Proteobacteria）、拟杆菌门（Bacteroidetes）、Thermi、硝化螺旋菌门（Nitrospirae）、绿菌门（Chlorobi）、厚壁菌门（Firmicutes）、绿弯菌门（Chloroflexi）、放线菌门（Actinobacteria）。（3）CCA（Canonical correspondence analysis）分析表明,3个温泉的真菌群落主要影响因子是温度,细菌群落主要影响因子是总磷。[结论] 海南省海口市温泉中含有丰富的微生物资源,其微生物群落组成受多种环境因子影响,且影响真菌和细菌的主要环境因子不同。     

Life in Hot Spring Microbial Mats Located in the Trans-Mexican Volcanic Belt: A 16S/18S rRNA Gene and Metagenomic Analysis

The geothermal system of the Araró region, located in the Trans-Mexican Volcanic Belt of México, hosts various hot springs with unique physicochemical characteristics, including temperatures ranging from 45°C to 78°C. The microbial diversity in these hot springs has been explored only by culture-dependent surveys. In this study, we performed metagenomic Illumina MiSeq, and 16S and 18S rRNA pyrosequencing analysis of the microbial life are residing in the microbial mats of the springs called “Tina–Bonita”. Our results show the presence of 186 operational taxonomic units, 99.7% of which belong to bacteria, 0.27% to eukaryotes, and 0.03% to archaea. The most abundant bacterial divisions are the Proteobacteria, Chloroflexi, and Cyanobacteria, which include 105 genera. The ecological indexes indicate that the microbial mats have moderate microbial diversity. An abundant group of genes that participate in photosynthesis, including photosynthetic electron transport, as well as photosystems I and II, were detected. Another cluster of genes was found that participates in sulfur, nitrogen, and methane metabolism. Finally, this phylogenetic and metagenomic analysis revealed an unexpected taxonomic and genetic diversity, expanding our knowledge of microbial life under specific extreme conditions.     

Metagenomics of microbial and viral life in terrestrial geothermal environments

Geothermally heated regions of Earth, such as terrestrial volcanic areas (fumaroles, hot springs, and geysers) and deep-sea hydrothermal vents, represent a variety of different environments populated by extremophilic archaeal and bacterial microorganisms. Since most of these microbes thriving in such harsh biotopes, they are often recalcitrant to cultivation; therefore, ecological, physiological and phylogenetic studies of these microbial populations have been hampered for a long time. More recently, culture-independent methodologies coupled with the fast development of next generation sequencing technologies as well as with the continuous advances in computational biology, have allowed the production of large amounts of metagenomic data. Specifically, these approaches have assessed the phylogenetic composition and functional potential of microbial consortia thriving within these habitats, shedding light on how extreme physico-chemical conditions and biological interactions have shaped such microbial communities. Metagenomics allowed to better understand that the exposure to an extreme range of selective pressures in such severe environments, accounts for genomic flexibility and metabolic versatility of microbial and viral communities, and makes extreme- and hyper-thermophiles suitable for bioprospecting purposes, representing an interesting source for novel thermostable proteins that can be potentially used in several industrial processes.     

Validation of astrobiology technologies and instrument operations in terrestrial analogue environments

Terrestrial analogue environments are places on Earth that present geological or environmental conditions that are similar to those found on an extraterrestrial body. Analogue environments serve four functions: (1) learn about planetary processes on Earth and elsewhere; (2) test technologies, methodologies, and protocols; (3) train highly-qualified personnel, as well as science and operations teams; (4) engage the public, space agencies, media, and educators. Analogue studies also enable the development and validation of biosignatures and detection techniques. Analogue programs include the Canadian Space Agency's Canadian Analogue Research Network, NASA's Astrobiology Science and Technology for Exploring Planets, and NASA's Analog Missions. Examples of technology and instrument testing and validation in analogue environments include the Haughton-Mars Project Research Station, the Arctic Mars Analog Svalbard Expedition (AMASE), the Rio Tinto basin, and NASA's Field Integrated Design and Operations (FIDO).     

A Window to the Subsurface: Microbial Diversity in Hot Springs of a Sulfidic Cave (Kaklik,Turkey)

Caves are windows to the extreme habitats of deep subsurface, and provide answers of unknowns about the underground life. Furthermore, sulfidic caves are important analogues for the early Earth environments, since some environmental conditions are common, such as high sulfur concentration, high temperature and oxygen-poor conditions. Kaklik Cave (Denizli, Turkey) with its travertine formation, carbonate- and sulfur-rich thermal springs, exhibits a unique ecosystem as a sulfidic cave. This study represents the first molecular survey of the microbial community in the Kaklik Cave, Turkey using high-throughput 16S rRNA gene amplicon sequencing analysis. An average of 859–2,416 operational taxonomic units per sample were observed including 25 bacterial phyla and 3 archaeal phyla. The bacterial diversity profiles were generally dominated by Epsilonproteobacteria and Gammaproteobacteria. At the carbonate-rich hot spring, that formed travertine structure, 9.7% of sequence reads affiliated with Thiofaba spp. In contrast, 38.74% of the total sequence reads at the sulfidic hot spring samples associated with the genus Sulfurimonas and Sulfurovum. In the archaeal community composition, Thermoplasmata was the most abundant group in all sampling areas. The 454-pyrotag results provide leads about ammonia-, nitrite- and sulfur-oxidation as well as sulfur-reduction, carbon dioxide fixation, and nitrogen fixation.     

高通量测序分析云南腾冲热海热泉真菌多样性

【目的】本研究揭示云南腾冲热海热泉真菌多样性及群落分布格局,探讨其理化因子对真菌群落结构的影响。【方法】利用Illumina Hi Seq2500高通量测序平台对腾冲热海热泉沉积物宏基因组ITS基因进行测序,并进行生物信息分析。【结果】从5个热泉样品中共检测到343484条有效序列,包括5个真菌门,20个纲,66个目。右姐妹泉(JMQR)、左姐妹泉(JMQL)、蛤蟆嘴泉(M)、桥泉(QQ)及鼓明泉(GMQP)分别以Agaricales、Eurotiales、Capnodiales和Hypocreales等为优势目。在属水平上,共获得365个属,从JMQR中检测到212个属,以裂褶菌属(Schizophyllum)为最优势;从JMQL中挖掘到197个属,以青霉属(Penicillium)为最优势类群;从M和QQ中分别获得222个和270个属,均以枝孢属(Cladosporium)为最优势;从GMQP中发现179个属,以侧齿霉属(Engyodontium)丰度最高。NH4+含量、温度及pH影响不同优势真菌的分布,其中以pH与优势类群(OTU1%)结构变化显著性最高(P=0.05)。【结论】云南腾冲热海高温热泉蕴藏着极其丰富的真菌物种,其不同样品真菌分布具有差异性,pH可能是影响热泉真菌群落分布的重要因素之一。     

Molecular marker technologies for plant improvement

The exploitation of DNA polymorphisms by an ever-increasing number of molecular marker technologies has begun to have an impact on plant genome research and breeding. Restriction fragment length polymorphisms, micro- and mini-satellites and PCR-based approaches are used to determine inter- and intra-specific genetic diversity and construct molecular maps of crops using specially designed mapping populations. Resistance genes and other agronomically important loci are tagged with tightly linked DNA markers and the genes isolated by magabase DNA technology and cloning into yeast artificial chromosomes (YAC). This review discusses some recent developments and results in this field.The authors are with Plant Molecular Biology; Biozentrum, Frankfurt University, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany