高通量测序技术在微生物分子生态学研究中的应用

微生物在众多的自然和人工生态系统中发挥着核心的作用,但能够被培养分离的微生物在大部分生态系统中只占极少一部分,极大地限制了人们对微生物组成、功能及其潜在应用的认识。分子生物学方法,尤其是高通量测序技术应用到微生物生态学研究中,为认识微生物多样性、群落结构组成及其生态功能提供了有利手段。高通量测序作为一种新兴的免培养分子生物学技术,具备检测快速、准确、信息全面丰富等特点。随着高通量测序技术的不断升级换代,测序通量、读长和准确度的不断提升以及成本的大幅下降,该技术在过去十几年间被迅速应用于土壤、水体和肠道等微生物区系的研究中。本文简述了基于高通量测序技术的PCR产物测序技术和宏基因组学测序技术的原理、发展历程、数据分析方法与应用,以及宏基因组学测序技术在病毒学领域的应用,以期为微生物分子生态学研究提供参考。     

环境微生物基因组学的生态学管窥

自然界约99%的微生物不能通过传统的分离筛选途径进行培养(即未培养微生物),为获得新的基因资源,更全面地认识微生物多样性和微生物在自然环境和生物圈中的重要作用,近年来随着分子生物学的快速发展及其在微生物研究中的广泛运用,以环境中未培养微生物为研究对象的新兴学科--环境微生物基因组学成为国内外学者的研究热点.本文介绍了环境微生物基因组学的分析研究策略及生态作用:从环境样品中直接提取所有微生物的总DNA,采用适宜的载体克隆到宿主细胞中构建宏基因组文库,再筛选新的活性物质或基因;从生态学角度分析微生物不可培养的原因,可利用环境微生物基因组技术进行土壤污染修复、畜禽养殖除臭、鉴定新物种以及确定特定生态环境体系中未培养微生物种群与群落的结构组成及物种的进化模式.     

城乡复合生态系统土壤微生物群落特征及功能差异

土壤微生物在陆地生态系统多个过程中发挥着重要作用,而城市化过程使得城市及其周边地区土地利用发生剧烈变化,形成了异质性环境梯度,直接或间接地影响了土壤微生物群落的组成和功能,进而影响了其承载的生态系统服务。本文综述了城乡复合生态系统不同景观单元土壤微生物群落的组成特征、主要影响因素及其功能差异,发现城市化对土地利用的改变驱动了土壤微生物群落的组成、结构和功能差异,土地利用、土壤污染物、植被覆盖、土壤性质等因素共同影响土壤微生物群落,并且在不同景观中影响土壤微生物的主导因素有所不同。进一步探讨了土壤微生物的生态服务功能,并分析了不同景观中土壤微生物功能存在的差异性。今后需进一步解析社会—经济—自然复合生态系统格局特征对土壤微生物的影响,揭示城乡复合生态系统不同功能区土壤微生物对土壤生态服务的产生和维持机制,明确变化环境下土壤微生物对土壤安全和人类健康的维持机制,以提升土壤生态服务功能、维护城乡土壤安全和人居环境健康。     

土壤微生物分离新技术的研究进展

土壤是环境微生物学研究中最具挑战性的环境,也是生物分子、遗传资源开发的热点区域。在"宏-组学"方法快速发展的今天,土壤微生物分离培养技术在微生物环境功能研究、代谢途径的阐明、特定功能的验证及基础实验和生产实践的应用等方面仍然发挥着重要作用。本文首先分析了大多数环境微生物不能被分离培养的几个原因。然后重点综述了近年来在土壤微生物分离培养技术方面的探索,主要包括生长培养基的优化和重新设计如培养基稀释分离、添加生长限制因子、更换凝固剂等,在延长培养时间、改善含氧量、降低培养温度等方面对生长条件进行改进,采用"扩散盒"、"原位培养陷阱"等技术对微生物进行天然环境原位培养,对环境微生物群体培养和共培养,利用"宏基因组学""宏蛋白质组学"等非培养技术辅助分离等。最后,作者对未来土壤微生物分离研究的方向提出了自己的建议。     

红壤微生物群落结构及其演变影响因素的研究进展

南方红壤丘陵区面临土壤肥力和生物功能退化问题,研究红壤微生物资源分布及其演变规律是培育红壤生物肥力的理论基础。本文综述了环境和人为因素对红壤微生物群落组成及其演变的影响,提出了红壤微生物群落组成及其功能调控研究的重点。土壤微生物群落组成受历史因素(地理距离、土壤类型)和现代因素(气候和土壤条件的变化)的共同影响,但不同因素间的相对贡献仍不清楚。土地利用方式和耕作施肥的改变均影响了红壤微生物群落的结构特征,但微生物结构和功能在耕作施肥过程中的长期变化规律仍需进一步研究。未来需要加强土壤?根系?微生物系统中生物交互作用及其对养分协同代谢和转化的影响,建立最佳管理措施修复退化红壤的微生物功能。     

江西典型红壤区土壤细菌基因组文库构建及功能初步分析

地球上的微生物,仅原核生物细胞总数就大约在4×1030～6×1030,包含的独立基因型在106～108种之间[1].因此,微生物所具备的分类、功能、遗传和系统发育等多样性在维持生物圈生态平衡和为人类提供资源方面起着重要的作用[2].然而长期以来由于受到研究方法和手段的限制,土壤中绝大多数微生物的功能还不清楚.环境样品的宏基因组学(metagenomics)是对环境样品中微生物群体基因组进行的分析,该方法在众多用于获得未培养微生物的生理和遗传特性的方法中,逐渐显示出强大的优势[3,4].国外已有从不同环境样品的宏基因文库中,通过功能性筛选获得新的抗生素、脂肪酶、丁质酶、膜蛋白、4-羟基丁酸脱氢酶等合成基因或相关基因,以及对群落结构组成和功能进行分析的报道[5～9].国内也逐步开始有相关研究[10,11].     

植物根际微生物组的研究进展

根际微生物组 (rhizosphere microbiome),是植物从其种子库土壤微生物组中有选择性地招募在根际聚集的动态微生物集群。随着近年来高通量测序技术、宏基因组学等的飞速发展,根际微生物组与植物宿主及土壤微生物组间的紧密联系引起了全球关注和研究热潮。根际微生物组被视作植物第二基因组,其与植物间的互作极为复杂,有正相也有负相。植物通过从土壤微生物组中招募到根际的某些组分获得积极反馈。正确管理植物根际微生物组不仅能促进宿主营养吸收、抵抗病虫害及适应环境胁迫,还可能促进健康土壤的形成,增强土壤生态系统的服务功能。对根际微生物组的定义、驱动因素、研究方法及其与农业生产的关系4个方面进行综述,并重点关注了根际微生物组与植物宿主间的互作过程,以期为更好的开发利用这类生物资源提供新思路。     

河套灌区盐渍土壤原核生物群落特征及其潜在功能研究

辨明盐渍土原核生物群落特征及潜在功能对盐渍化程度和土地利用类型的响应，对理解盐渍土壤元素循环与植物互馈效应、构建良性循环农田生态系统具有重要意义。以河套灌区不同盐渍化程度的农田和荒地为研究对象，结合土壤基本理化性质分析与原核生物高通量测序方法，探究盐渍化土壤中原核生物的群落组成特征、环境驱动要素及其潜在功能。结果表明，农田土壤盐渍化程度显著低于荒地，其原核生物多样性更高，特别是富集了大量农田特有的ASV（扩增子序列变体，amplicon sequence variant）；原核生物的群落组成在农田和荒地间差异最大，并主要受到土壤电导率（EC）、pH和有机质（SOM）等环境因子的驱动。基于群落组成和功能预测的差异分析结果表明，盐渍化农田中具有较高丰度的氮循环相关微生物以及潜在的植物促生菌，如亚硝化球菌（Nitrososphaeraceae）、亚硝化单胞菌（Nitrososmonadaceae）、诺卡氏菌（Nocardioidaceae）和鞘氨醇单胞菌（Sphingomonadaceae）等；而盐渍化荒地富集了以盐杆菌（Halobacterota）为代表的古菌和具有烃类化合物分解功能的原核生物类群。本研究对于明晰北方灌区盐渍土原核生物群落特征与土壤微环境的互馈关系、揭示土壤养分周转对提升土壤-植物-微生物跨域有益协同具有指导意义。     

土壤微生物多样性的科学内涵及其生态服务功能

土壤微生物多样性是指土壤生态系统中所有的微生物种类、它们拥有的基因以及这些微生物与环境之间相互作用的多样化程度,当前研究主要集中在物种多样性、遗传多样性、结构多样性及功能多样性等4个方面。土壤微生物多样性作为全球性研究迄今仅有10余年时间,但已呈现对象广、内容多、水平宽、方法新等特点,特别是分子生物学技术在很大程度上决定并体现了其研究水平和发展进程。然而,如何进一步改进土壤微生物的培养技能、加强土壤宏基因组学分析与应用、耦合土壤微生物多样性与生态功能、揭示土壤微域结构的影响机制是土壤微生物多样性研究的4个关键科学问题。当然,土壤微生物多样性的生态系统服务功能是其根本价值所在,主要包括有机物分解、物质循环和生态安全调控等3个方面。今后,土壤微生物多样性研究应紧紧围绕其与土壤生物过程、生态服务功能三者之间的联系,着重建立土壤微生物多样性的研究指标和方法体系,进而阐明人类生产活动影响土壤微生物多样性及其生态服务功能的土壤生物过程。     

氮磷富集对森林土壤碳截存的影响研究进展

大气氮磷沉降增加森林土壤养分的可利用性,改变底物的化学质量、土壤微生物组成和功能,进而影响土壤有机质的储量与稳定性。然而,现有研究主要集中在氮素富集对自然森林生态系统碳截存的影响,有关磷富集以及氮磷交互对人工林土壤有机碳(SOC)截存的影响及其微生物学机制尚不清楚。本文综述了氮磷富集对森林土壤碳转化和净交换通量、土壤有机质(SOM)的激发效应、SOM组成与稳定性以及介导碳转化功能微生物群落的影响,并指出各个研究环节的不足,包括:(1)森林土壤碳通量及其组分对氮磷富集的非线性响应方程及临界阈值尚未确定;(2)氮磷富集对森林SOM激发效应的影响程度与潜在机制知之甚少;(3)SOM的物理-化学协同稳定机制研究不够深入;(4)土壤活性微生物群落组成、SOM化学结构与SOC累积之间的耦联关系尚不清晰。据此,指出未来研究重点与研究思路:基于多水平氮磷添加控制试验和~(13)C标记培养实验,利用原位监测、土壤化学(~(13)C-NMR和Py-GC/MS)、宏基因组测序的分子生物学方法,重点研究氮磷添加及其交互作用对人工林土壤碳排放与流失通量、微生物激发效应、SOM组成与化学稳定性以及功能微生物群落组成的影响,确定土壤碳输出通量对氮磷添加的非线性响应方程与氮沉降临界负荷,阐明分解微生物群落组成与土壤碳转化及稳定性的耦联关系,揭示氮磷交互影响人工林土壤碳积累与损耗的微生物学机制。研究结果有助于控制森林尤其是人工林土壤碳损失,有效降低陆地"氮促碳汇"评估的不确定性,并可为森林生态系统应对全球变化提供科学依据。     

磷脂脂肪酸分析方法及其在土壤微生物多样性研究中的应用

磷脂脂肪酸（PLFA）是活体微生物细胞膜的重要组分，不同类群的微生物可通过不同的生化途径合成不同的PLFA。一些PLFA可作为分析微生物量和微生物群落结构变化的“生物标记”。在土壤微生物分析中，越来越多地采用了PLFA方法。本文介绍了表征微生物的一些PLFA、从土壤中提取PLFA的方法以及用GC-MS分析PLFA的原理。本文还将常用的研究微生物多样性的几种方法进行了比较；传统的分析土壤微生物群落的方法依赖于培养技术，只能培养和分离出一小部分微生物群落；Biolog方法可用于研究土壤微生物群落生理多样性，即可反映微生物群落如何利用各种碳源底物，但对快速生长和适合在Biolog实验条件下生长的小部分群落成员有强烈的选择性；核酸分析方法的主要缺点是不能对土壤微生物进行定量分析；而PLFA方法相对于上述几种方法有诸多优势。本文通过一些实例证明PLFA方法的特色或独到之处，也分析了其缺点。采用PLFA方法并结合其他方法有助于获取土壤微生物群落多样性的更多和更全面而完整的信息。     

Size Does Matter: Application-driven Approaches for Soil Metagenomics

Metagenomic analyses can provide extensive information on the structure, composition, and predicted gene functions of diverse environmental microbial assemblages. Each environment presents its own unique challenges to metagenomic investigation and requires a specifically designed approach to accommodate physicochemical and biotic factors unique to each environment that can pose technical hurdles and/or bias the metagenomic analyses. In particular, soils harbor an exceptional diversity of prokaryotes that are largely undescribed beyond the level of ribotype and are a potentially vast resource for natural product discovery. The successful application of a soil metagenomic approach depends on selecting the appropriate DNA extraction, purification, and if necessary, cloning methods for the intended downstream analyses. The most important technical considerations in a metagenomic study include obtaining a sufficient yield of high-purity DNA representing the targeted microorganisms within an environmental sample or enrichment and (if required) constructing a metagenomic library in a suitable vector and host. Size does matter in the context of the average insert size within a clone library or the sequence read length for a high-throughput sequencing approach. It is also imperative to select the appropriate metagenomic screening strategy to address the specific question(s) of interest, which should drive the selection of methods used in the earlier stages of a metagenomic project (e.g., DNA size, to clone or not to clone). Here, we present both the promising and problematic nature of soil metagenomics and discuss the factors that should be considered when selecting soil sampling, DNA extraction, purification, and cloning methods to implement based on the ultimate study objectives.     

Culture-independent molecular techniques for soil microbial ecology

The advent of nucleic acid-based molecular methods, in particular the polymerase chain reaction (PCR), has revolutionised the study of soil microbial ecology, previously constrained by an inability to culture the majority of cells detected by direct microscopic observation. Extraction of DNA and RNA directly from cells in soil circumvents the requirement to grow microorganisms in laboratory culture, avoiding problems associated with the differential growth rates of the estimated 1% that can be grown routinely. However, not all cells that contain DNA are capable of growth, and in some conditions such as air-dried soil, DNA can be extracted from non-viable microorganisms after 140 years of storage. To investigate the active microbial community, RNA can also be isolated directly from soil. Analysis of ribosomal RNA (rRNA) indicates the dominant active population in any particular set of conditions and the large, constantly increasing electronic database of gene sequences for the small subunit of rRNA (16S for prokaryotes, 18S for eukaryotes) provides identification of many soil bacteria, archaea and fungi with varying degrees of certainty to the genus, species or sub-species level. More precise information on which functional genes are active can be obtained from messenger RNA (mRNA). Newer methods including high-throughput (massively parallel) sequencing and microarrays offer further advances. We describe a range of molecular techniques used to investigate soil microbial communities, discuss how they relate to other methods for investigating bacterial and fungal activity, and explore their drawbacks and limitations.     

土壤原生动物——研究方法及其在土传病害防控中的作用

原生动物是原生生物的一种,是土壤食物网中的消费者,能捕食细菌和真菌等其他微生物。除了对土壤微生物群落和物质循环产生重要影响外,根际原生动物与细菌、真菌等土壤微生物共同组成生物网络屏障,并在抵御土传病原菌入侵作物根系的过程中发挥着重要作用。然而,相对于根际有益细菌和有益真菌,国内外关于原生动物防控土传病害的效果及作用机制的研究非常有限。本文梳理了土壤原生动物在土传病害防控中的效果、作用机制及其相关研究方法,并对未来原生动物的研究和应用作出了展望。呼吁更多学者关注土壤原生动物及其在生态系统中的功能,挖掘其在土壤健康和农业可持续发展中的重要价值。     

Surveying soil faunal communities using a direct molecular approach

Soil faunal communities are often phylogenetically diverse and the accurate assessment of the taxonomic structure of these communities is both time-consuming and requires a high level of taxonomic expertise. Here we describe a DNA sequence-based methodology for characterizing soil micro- and mesofaunal communities that is similar to the molecular approaches commonly used to survey soil microbial diversity. The technique involves the direct extraction of faunal DNA from soil, PCR amplification of the extracted DNA with metazoan-specific primers, followed by the construction of clone libraries and direct sequencing of individual PCR products. We used this technique to characterize micro- and mesofaunal community composition from six individual soils representing two land-use types. The technique captured the more abundant faunal groups in the soils (nematodes, Collembola, Acari, tardigrades, enchytraeids) and provided sufficient taxonomic resolution to describe the overall structure of the communities. We compared the results obtained using this molecular approach to results obtained using a traditional, microscopy-based approach and found that the results were broadly similar. However, since biases are inherent in both methods it remains unclear which method provides a more accurate assessment of soil faunal community composition. Although this molecular approach has some distinct disadvantages over the more widely-used direct extraction methods, one advantage is that the taxonomic identification it can provide will be more accurate and consistent across research groups, facilitating effective comparisons of mesofaunal surveys.     

Potential applications of soil microbial ecology and next-generation sequencing in criminal investigations

The complex relationships between the changes in microbial community profiles and postmortem interval (PMI) estimates have recently been discussed in the forensic literature. Edaphic, necrobiomic microorganisms at the cadaver-soil interface construct multi-species communities that change in richness and activity when the host body dies and begins to decompose. Characterization of these dynamic changes has been made possible by current advances in high throughput, next-generation platforms. The effectiveness of these metagenomic technologies is that they pride the foundations of a framework for identification of grave sites and the determination of postmortem timelines, or “microbial clocks.” The proposed clocks may help substantiate the estimation of PMI. Studies have demonstrated the differences between soils collected at grave sites and control soils which may be useful in identifying clandestine grave sites. In this review is the discussion of the recent and formative findings involving sequencing applications of soil microbial communities relating the differences in taxon richness and abundance patterns as molecular tools with broad and important applications in forensics.     

转Bt基因棉花根际细菌与古菌群落结构分析

在一年内棉花的四个生长时期（苗期,蕾期,花铃期,吐絮期）分别采集转Bt基因抗虫棉GK12和非转基因亲本棉花泗棉3号根际土壤,以及未种植棉花的背景土壤,利用末端标记限制性片段长度多态性（T-RFLP）分析技术,分析三种土壤中细菌和古菌的16S rRNA基因片段多态性,结合克隆文库建立和测序,研究了土壤中细菌和古菌群落结构的变化.结果表明：在棉花生长的各个时期,背景土壤中细菌群落结构发生了明显的变化,生物多样性指数明显降低,古菌群落结构也有一定的变化,说明季节性变化对土壤中微生物群落产生了明显的影响.与背景土壤相比,棉花种植后根际土壤中细菌和古菌群落发生显著的变化.转基因棉花与非转基因棉花相比,根际土壤细菌和古菌的种类和种群大小的分布也发生了明显的改变.克隆文库和测序结果表明土壤中主体微生物为目前未培养的、功能特性未知的细菌和古菌,转基因棉花种植对这些细菌和古菌影响的原因、环境危害和生态风险目前尚不清楚.与古菌群落相比,棉花种植对细菌群落结构的影响较小.     

Salt effects on the soil microbial decomposer community and their role in organic carbon cycling: A review

Salinization of soil is recognised as one of the most pressing environmental challenges to resolve for the next century. We here conduct a synoptic review of the available research on how salt affects decomposer microbial communities and carbon (C) cycling in soil. After summarizing known physiological responses of microorganisms to salinity, we provide a brief overview and qualification of a selection of widely applied methods to assess microorganisms in soil to date. The dominant approaches to characterise microbial responses to salt exposure have so far been microbial biomass and respiration measurements. We compile datasets from a selection of studies and find that (1) microbial biomass-carbon (C) per C held in soil organic matter shows no consistent pattern with long-term (field gradients) or short-term (laboratory additions) soil salinity level, and (2) respiration per soil organic C is substantially inhibited by higher salt concentrations in soil, and consistently so for both short-term and long-term salinity levels. Patterns that emerge from extra-cellular enzyme assessments are more difficult to generalize, and appear to vary with the enzyme studied, and its context. Growth based assessments of microbial responses to salinization are largely lacking. Relating the established responses of microbial respiration to that of growth could provide an estimate for how the microbial C-use efficiency would be affected by salt exposure. This would be a valuable predictor for changes in soil C sequestration. A few studies have investigated the connection between microbial tolerance to salt and the soil salinity levels, but so far results have not been conclusive. We predict that more systematic inquiries including comprehensive ranges of soil salinities will substantiate a connection between soil salinity and microbial tolerance to salt. This would confirm that salinity has a direct effect on the composition of microbial communities. While salt has been identified as one of the most powerful environmental factors to structure microbial communities in aquatic environments, no up-to-date sequence based assessments currently exist from soil. Filling this gap should be a research priority. Moreover, linking sequencing based assessments of microbial communities to their tolerance to salt would have the potential to yield biomarker sets of microbial sequences. This could provide predictive power for, e.g., the sensitivity of agricultural soils to salt exposure, and, as such, a useful tool for soil resource management. We conclude that salt exposure has a powerful influence on soil microbial communities and processes. In addition to being one of the most pressing agricultural problems to solve, this influence could also be used as an experimental probe to better understand how microorganisms control the biogeochemistry in soil.     

Microbial community development and unseen diversity recovery in inoculated sterile soil

Soil is considered as one of the most biodiverse environments on Earth; yet, the taxonomy, occurrence, and role of its different microbial populations are largely unknown. Here, two sterilized soils (from England and Italy) were inoculated with a subsample of their initial microbial communities and/or those from the other soil to study their microbial community evolution. This approach compared two driving factors (original community and soil physico-chemical characteristics) for microbial community definition. After 2 months of incubation and based on metagenomic datasets, the two inoculated communities (from an English grassland and an Italian forest) possessed similar functional and taxonomical structures when inoculated in the same sterile soil. For example, the newly colonized Italian soil was dominated by Actinobacteria related organisms (>66 % of the detected community) with a functional distribution independent of the inoculated soil origin. In addition, some of the organisms that dominated the different inoculated communities after 2 months were similar for a given sterile soil whether they came from the English grassland or the Italian forest, and they had not been detected in the original microbial community from either soil. Thus, similar microorganisms with low representation from the two distinct communities emerged in each sterilized soil, thus increasing the microbial diversity recovered from the microbial community of the donor soil. So far, these observations support the idea that different temperate soil microbial communities have different evenness due to environmental physico-chemical variations, yet have similar community composition (richness), and thus develop similarly when colonizing the same habitat.