Development and application of a PCR-denaturing gradient gel electrophoresis tool to study the diversity of Nitrobacter-like nxrA sequences in soil

A new PCR-denaturing gel gradient electrophoresis (DGGE) tool based on the functional gene nxrA encoding the catalytic subunit of the nitrite oxidoreductase in nitrite-oxidizing bacteria (NOB) has been developed. The first aim was to determine if the primers could target representatives of NOB genera: Nitrococcus and Nitrospira. The primers successfully amplified nxrA gene sequences from Nitrococcus mobilis, but not from Nitrospira marina. The second aim was to develop a PCR-DGGE tool to characterize NOB community structure on the basis of Nitrobacter-like partial nrxA gene sequences (Nb-nxrA). We tested (1) the ability of this tool to discriminate between Nitrobacter strains, and (2) its ability to reveal changes in the community structure of NOB harbouring Nb-nrxA sequences induced by light grazing or intensive grazing in grassland soils. The DGGE profiles clearly differed between the four Nitrobacter strains tested. Differences in the structure of NOB community were revealed between grazing regimes. Phylogenetic analysis of the sequences corresponding to different DGGE bands showed that Nb-nxrA sequences did not group in management-specific clusters. Most of the nxrA sequences obtained from soils differed from nxrA sequences of NOB strains. Along with existing tools for characterizing the community structure of nitrifiers, this new approach is a significant step forward to performing comprehensive studies on nitrification.     

Molecular and functional diversity in soil micro-organisms

Traditional approaches to the study of microbial diversity have relied on laboratory cultivation of isolates from natural environments and identification by classical techniques, including analysis of morphology, physiological characteristics and biochemical properties. These approaches provide information on fine-scale diversity but suffer from bias, resulting from the media and cultivation conditions employed, and from the inability to grow and isolate significant proportions of natural communities in the laboratory. An alternative approach is the amplification of ribosomal RNA and functional genes from nucleic acids extracted directly from environmental samples, with subsequent analysis by `fingerprinting' methods or by sequencing and phylogenetic analysis. This approach avoids the need for laboratory cultivation and has provided major insights into species and functional diversity of bacterial and archaeal populations. This article reviews molecular approaches to the characterisation of prokaryote diversity in natural environments, their more recent application to fungal diversity and the advantages and limitations of molecular analyses.     

黄顶菊入侵对土壤中主要功能细菌的影响

以我国北方大面积发生的入侵植物黄顶菊为研究对象,对黄顶菊根际土壤中可培养的主要功能细菌进行了分离筛选,通过rep-PCR聚类和多样性分析研究了其群落结构的变化,并利用16S rRNA序列比对,对主要优势菌群进行鉴定.结果表明: 相对于本地植物万寿菊和空白对照,黄顶菊显著增加了根际土壤中固氮菌、有机磷细菌、无机磷细菌和钾细菌的数量.rep-PCR分析显示,黄顶菊根际4种功能细菌的种群结构与本地植物和对照相比有显著差异,3种土壤中相同的聚类群极少.多样性分析表明,黄顶菊根际微生物物种多样性更加丰富,群落结构更加复杂,优势种群比较明显,生态多样性较高.对从3种土壤中分离得到的主要优势菌群的鉴定结果也进一步证明了这一结论.研究结果为阐明黄顶菊入侵的微生态机制提供了理论基础.     

基因打靶技术在植物基因功能研究中的应用

水稻、拟南芥等模式植物基因组测序计划的完成,验证预测基因的功能成为植物功能基因组学的重要内容,基因打靶(gene targeting)技术在哺乳动物中的成功应用为该技术在植物上展现了广阔的应用前景。现对植物基因打靶技术的影响因素、基因打靶在植物中的应用现状作一介绍。     

Characterization of monooxygenase gene diversity in benzene‐amended soils

Aim: To understand soil benzene monooxygenase gene diversity by clone library construction and microarray profiling. Methods and Results: A primer set was designed, and benzene monooxygenase gene diversity was characterized in two benzene‐amended soils. The dominant sequence types in the clone libraries were distinct between the two soils, and both sequences were assigned to novel clusters. Monooxygenase gene richness and diversity increased after benzene degradation. Oligonucleotide probes for microarray analysis were designed to detect a number of sequenced clones and reported monooxygenase genes. The microarray detected several genes that were not detected in the clone libraries of the same samples. Six probes were detected in more than one soil. Conclusions: The primer set designed in this study successfully detected diverse benzene monooxygenase genes. The level of diversity may have increased because the degradation of benzene differed from soil to soil. Microarrays have great potential in the comprehensive detection of gene richness as well as the elucidation of key genes for degradation. Significance and Impact of the Study: This study introduces a new primer set that may be used to identify diverse benzene monooxygenase genes in the environment; moreover, it demonstrates the potential of microarray technology in the profiling of environmental samples.     

不同林型土壤微生物有机碳降解基因的多样性

应用寡聚核苷酸基因芯片,分析了米亚罗林区冷杉原始林（M—Y）和20世纪60年代云杉人工林（M-60）土壤微生物的功能基因多样性。该功能基因芯片含有与有机碳降解、碳固定、氮、磷、硫循环和金属抗性相关的1961个基因探针。在M—Y和M-60样地中分别检测到39和62个具有较强杂交信号（SNR≥2）的功能基因,其基因多样性水平指数分别为3．59和4．04,杂交信号强度总值分别为480280和630560。M—Y和M-60样地中分别检测到32个和37个有机碳降解基因,占总基因的82％和60％,这些基因分属于22个不同的基因类群,分别参与木质素、木聚糖、几丁质等有机碳的降解过程。有机碳降解基因在两个样地中存在较大的多样性和丰度差异。这些结果说明了大多数的土壤微生物直接参与了土壤有机碳的降解,同时,林型不同显著影响了土壤微生物群落结构和有机碳降解微生物的多样性。     

Effects of a surface wildfire on soil nutrient and microbial functional diversity in a shrubbery

The aim of the study was to determine effects of a wildfire on soil nutrients and soil microbial functional diversity in short-term time scales. Burned and unburned control soil samples were collected 1 day, and 2, 4, 8, 10, 12 and 15 months after a shrubbery fire in Yumin county of Xinjiang, Northwest China. Nutrients of soil in each sampling time were detected and soil microbial functional diversity was measured by Biolog Eco plates. Results of the study showed that soil nutrients were significantly affected by fire. Soil pH increased immediately after the wildfire and was higher than that of unburned soil during 15 months post fire. Soil organic matter and total N significantly decreased immediately after the fire and was even lower than control soil at the 15th month post fire. Soil available P level increased sharply during the 4th month after the fire, and later reached to the maximum value with eight times higher than that of unburned soil. Soil available N and available K were more than the control site in 2 months after the fire, then decreased, but available N began to increase, when vegetations restored 1 year after the fire. Soil microbial activity and functional diversity recovered gradually after fire. The average well color development (AWCD) and functional diversity indices (Shannon index, Simpson index, and McIntosh index) decreased significantly 1 day after the fire, but then increased and were similar to that of undisturbed soil 15 months after the fire, when plant started to regenerate in burned area. The changes in soil nutrients after the fire affected soil microbial activity and functional diversity. Correlation analysis revealed that AWCD was negatively correlated with soil pH and positively correlated with soil total N and available N, Shannon and Simpson index had positive significantly correlation with soil total N and McIntosh index had positive significantly correlation with available N. Result of principal component analysis based on the data of carbons metabolism showed that microbial catabolic profiles of burned soils of each sampling time after the wildfire were different and all were distinct from those of unburned soils, which might suggest that microbial community structure of fire-impacted area changed dynamically on monthly scale and was distinct from that of the control site in 15 months after fire, although microbial activity or richness showed similar to pre-fire level at the 15th month post-fire.     

土壤中甲烷厌氧氧化菌多样性的分子检测

甲烷厌氧氧化作用是减少海洋底泥甲烷释放的重要生物地球化学过程,然而在陆地生态系统中甲烷厌氧氧化作用及其功能菌群的生态功能仍然不确定。对甲烷厌氧氧化菌多样性的研究可为减少甲烷排放提供重要科学依据。与传统的分离培养方法比较,分子检测方法是一种更为快速和高效的研究手段,可直接和全面的反映参与甲烷厌氧氧化作用的功能微生物。以DNA分子标记物为研究对象,重点探讨三类主要的分子标记基因,即16S rRNA,mcr A和pmo A,所采用的相关探针和引物信息,同时从定性和定量两个角度综述土壤甲烷厌氧氧化菌的多样性研究的主要进展,最后提出厌氧甲烷氧化菌多样性研究中存在的一些问题和相应的解决思路。     

Characterization of rhizosphere prokaryotic diversity in a horizontal subsurface flow constructed wetland using a PCR cloning-sequencing based approach

Performance of biological wastewater treatment systems may be related to the composition and activity of microbial populations they contain. However, little information is known regarding microbial community inhabiting these ecosystems. The purpose of this study was to investigate archaeal and bacterial diversity, using cultivation-independent molecular techniques, in a constructed wetland receiving domestic wastewater. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by PCR using primers specific for archaeal and bacterial domains. A high microbial diversity was detected. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 31.3 % of the OTUs, followed by the Bacteroidetes (14.8 %), Planctomycetales (13.8 %), Actinobacteria (12 %), and Chloroflexi (8.2 %). Sequences affiliated with minor phylogenetic divisions such as the TM7, Nitrospira, OP10, and BRC1 are represented by <6 % of total OTUs. The Archaea domain was represented by the Thaumarchaeota phylum dominated by the Candidatus Nitrososphaera genus.     

亚热带两种森林土壤担子菌漆酶基因多样性比较

漆酶是降解森林凋落物中木质素的关键酶之一,直接影响着森林生态系统碳循环过程.运用TA克隆、测序技术,研究了两种亚热带森林(原生常绿落叶阔叶混交林和人工马尾松林)凋落物层(O层)和土壤表层(A层,0～20 cm)降解木质素的担子菌漆酶基因多样性.结果表明:同一土壤层位,原生林土壤中担子菌漆酶基因多样性和种群丰富度高于马尾松林;同一森林生态系统,原生林土壤O层中担子菌漆酶基因多样性和种群丰富度略高于土壤A层,而马尾松林则O层明显低于A层;两森林土壤具有相同含漆酶基因的担子菌优势种群,且大部分优势种群与伞菌目小菇属或侧耳属有较高的氨基酸相似性;与原生林土壤A层和马尾松林土壤O层相比,原生林土壤O层和马尾松林土壤A层中含漆酶基因的担子菌种群分布相对均匀;马尾松林O层与A层之间漆酶基因核苷酸序列的相似性较原生林土壤O层与A层之间的高.表明植被和土壤层位显著影响漆酶基因多样性和群落结构,而植被和土壤层位引起的担子菌可利用底物和土壤pH值的差异可能直接驱动这种影响.     

青藏高原高寒草甸土壤环境对线虫功能多样性的影响

土壤线虫是地下食物网的重要组成部分, 在生态系统能量流动和物质循环中起着至关重要的作用。大量研究报道了肥力等土壤环境对土壤线虫物种多样性及各功能群多度的影响, 而我们对土壤线虫功能多样性如何响应土壤环境变化依然知之甚少。本研究以群落水平个体大小和个体大小多样性表征土壤线虫功能多样性。在青藏高原高寒草甸选择3个研究点, 调查和分析了不同生境(沟底平地、阴坡、阳坡和山顶)土壤线虫物种多样性、各功能群多度和功能多样性及其与土壤理化因子和植物多样性的关系。结果表明: (1)土壤线虫个体多度和物种多样性在阳坡最高, 随土壤pH值和土壤总磷增加而升高; 而基于个体大小的土壤线虫功能多样性主要受土壤养分影响, 随土壤总氮和有机质增加而增加, 随土壤总磷含量增加而减少; (2)食细菌和食真菌线虫多度在沟底最高, 植食与捕食杂食线虫多度在山顶最低; 除捕食杂食线虫外, 各功能群多度也主要随土壤磷增加而升高; 除食真菌线虫外, 各功能群多度随植物物种丰富度的增加而减少。本研究强调了土壤线虫物种和功能多样性受不同土壤环境因子的影响, 丰富了土壤线虫多样性研究的内容, 为理解高寒草甸土壤动物多样性形成、维持和变化提供了更广阔的 视角。     

Functional gene diversity of soil microbial communities from five oil-contaminated fields in China

To compare microbial functional diversity in different oil-contaminated fields and to know the effects of oil contaminant and environmental factors, soil samples were taken from typical oil-contaminated fields located in five geographic regions of China. GeoChip, a high-throughput functional gene array, was used to evaluate the microbial functional genes involved in contaminant degradation and in other major biogeochemical/metabolic processes. Our results indicated that the overall microbial community structures were distinct in each oil-contaminated field, and samples were clustered by geographic locations. The organic contaminant degradation genes were most abundant in all samples and presented a similar pattern under oil contaminant stress among the five fields. In addition, alkane and aromatic hydrocarbon degradation genes such as monooxygenase and dioxygenase were detected in high abundance in the oil-contaminated fields. Canonical correspondence analysis indicated that the microbial functional patterns were highly correlated to the local environmental variables, such as oil contaminant concentration, nitrogen and phosphorus contents, salt and pH. Finally, a total of 59% of microbial community variation from GeoChip data can be explained by oil contamination, geographic location and soil geochemical parameters. This study provided insights into the in situ microbial functional structures in oil-contaminated fields and discerned the linkages between microbial communities and environmental variables, which is important to the application of bioremediation in oil-contaminated sites.     

天坑森林植物群落叶功能性状、物种多样性和功能多样性特征

以天坑内部-边缘-外部森林植物群落为研究对象,通过调查植物的群落结构、叶功能性状,探究天坑内外森林植物群落叶功能性状、物种多样性和功能多样性变化特征及其内在关联,为深入了解负地形森林生态系统的功能和恢复退化喀斯特地区的植被提供一定参考。研究结果如下：（1）比叶面积（SLA: 198.75 cm2/g)）、叶面积（LA: 42.70 cm2）、叶磷含量（LPC: 1.70 g/kg）和叶钾含量（LKC: 10.27 g/kg）在天坑内部最高,叶组织密度（LTD: 0.32 g/cm3）和叶干物质含量（LDMC: 0.41 g/g）在天坑外部最高,天坑内外森林均易受到磷限制,表明随天坑内部-边缘-外部生境变化,植物对环境的适应机制和生存策略发生了部分调整,物种的防御策略增强,生长投入策略减弱。（2）Shannon-Wiener指数（2.82）、Simpson指数（0.92）和Pielou’s均匀度指数（0.87）均以天坑外部最高,功能丰富度（1.05）、功能离散度（1.88）和Rao’s二次熵（4.52）以天坑内部最高,表明随天坑内部-边缘-外部生境的变化,植物功能性状的差异减少,物种分布及其功能性状分布总体上更为均匀、物种数量增多。（3）物种多样性指数之间、功能多样性指数之间存在较强的相关性,表明物种多样性指数之间、功能多样性指数之间存在不同的制约关系。（4）叶功能性状与物种多样性、功能多样性的相关性强,物种多样性和功能多样性之间相关性较弱,表明叶性状对生态学过程的变化较为敏感,叶功能性状与物种多样性之间存在较强的耦合关系。     

恩诺沙星对土壤微生物群落功能多样性的影响

为了评价恩诺沙星(enrofloxacin)对土壤微生物群落的影响,借助BIOLOG检测法比较了不同浓度恩诺沙星影响下的土壤微生物的群落特征。结果表明,各添加药物组与空白对照组的土壤微生物群落代谢多样性差异显著(P<0.05),空白对照组土壤微生物在BIOLOG微平板上的平均每孔颜色变化率(AWCD)和微生物代谢多样性指数(丰富度和多样性)显著高于添加药物组;药物显著影响微生物群落对BIOLOG微平板中碳源的利用能力,较低浓度的恩诺沙星(0.01μg/g)就显著影响了土壤微生物群落对α-Ketobutyric Acid、L-Phenylalanine、1-Erythritol、D-Xylose等碳源的利用代谢能力(P<0.05),且土壤微生物群落利用各类碳源的能力随药物浓度加大而降低。由此可见,恩诺沙星在土壤中残留时对土壤微生物的影响不容忽视。     

泛基因组研究在遗传多样性和功能基因组学中的应用

相对于单个参考基因组仅聚焦于个体遗传信息的挖掘,泛基因组研究则能够反映整个物种或类群全部的遗传信息。随着基因组测序和分析技术的不断发展,泛基因组学逐渐成为新的研究热点,并已在植物、动物和微生物多个物种中获得了广泛应用,为全面解析物种或类群水平的遗传变异和多样性、功能基因组和系统进化重建等研究提供了强有力的工具,取得了很多显著的研究成果。尽管如此,由于泛基因组学研究尚处于发展阶段,测序费用和分析成本仍然较高,难以广泛普及; 且存在分析标准不一、数据挖掘不够全面深入、理论难以应用于生产实际等尚待解决的问题,仍有较大的发展空间。该文系统总结了泛基因组在生物遗传多样性挖掘和功能基因组学中的研究进展,主要包括其在泛基因组图谱的构建、基因组变异和有利基因的发掘、功能基因的多态性、群体遗传多样性和系统进化等多个领域中的应用和研究,探讨了其在不同领域的应用潜力。同时,讨论了目前泛基因组研究中存在的局限性和可能的解决方法,并对其将来的发展前景进行了展望。     

Evolutionary genomics analysis reveals gene expansion and functional diversity of arylalkylamine N-acetyltransferases in the Culicinae subfamily of mosquitoes

Arylalkylamine N-acetyltransferase (aaNAT), considered a potential new insecticide target, catalyzes the acetylation of arylalkylamine substrates such as serotonin and dopamine and, hence, mediates diverse functions in insects. However, the origin of insect aaNATs (iaaNATs) and the evolutionary process that generates multiple aaNATs in mosquitoes remain largely unknown. Here, we have analyzed the genomes of 33 species to explore and expand our understanding of the molecular evolution of this gene family in detail. We show that aaNAT orthologs are present in Bacteria, Cephalochordata, Chondrichthyes, Cnidaria, Crustacea, Mammalia, Placozoa, and Teleoste, as well as those from a number of insects, but are absent in some species of Annelida, Echinozoa, and Mollusca as well as Arachnida. Particularly, more than 10 aaNATs were detected in the Culicinae subfamily of mosquitoes. Molecular evolutionary analysis of aaNAT/aaNAT-like genes in mosquitoes reveals that tandem duplication events led to gene expansion in the Culicinae subfamily of mosquitoes more than 190 million years ago. Further selection analysis demonstrates that mosquito aaNATs evolved under strongly positive pressures that generated functional diversity following gene duplication events. Overall, this study may provide novel insights into the molecular evolution of the aaNAT family in mosquitoes.     

长期施肥对稻田土壤细菌、古菌多样性和群落结构的影响

稻田土壤是“迷失碳”的重要吸纳场所之一,也是温室气体(CH4和N2O等)的重要排放源.大气温室气体的动态变化与土壤碳氮转化的微生物过程紧密相关.以湖南桃江国家级稻田肥力变化长期定位试验点为平台,采用PCR-克隆测序和实时荧光定量PCR技术,研究不施肥(CK)、施氮磷钾肥(NPK)和氮磷钾肥+秸秆还田(NPKS)3种长期施肥制度(＞25 a)对稻田土壤细菌和古菌群落结构及数量的影响.细菌和古菌16S rRNA基因文库分析结果表明:稻田土壤细菌主要类群为变形菌、酸杆菌、绿弯菌,而古菌主要为泉古菌和广古菌.长期施肥导致土壤细菌和古菌种群结构产生明显差异,与CK相比,NPK和NPKS处理稻田土壤的变形菌、酸杆菌和泉古菌相对丰度增加.LIBSHUFF软件分析结果也表明,16S rRNA基因文库在CK、NPK及NPKS处理间存在显著差异.3种施肥处理的稻田土壤细菌16S rRNA基因拷贝数为每克干土0.58× 1010～1.06×1010个,古菌为每克干土1.16×106 ～ 1.72×106个.施肥(NPK和NPKS)后,细菌和古菌的多样性和数量增加,且NPKS＞NPK.说明长期施肥显著影响土壤细菌和古菌群落结构、多样性及数量.     

Microbial community composition and functional diversity in the rhizosphere of maize

This study investigates the small-scale stratification of bacterial community composition and functional diversity in the rhizosphere of maize. Maize seedlings were grown in a microcosm with a horizontal mesh (53 M) creating a planar root mat and rhizosphere soil. An unplanted microcosm served as control. Thin slices of soil were cut at different distances from the mesh surface (0.2–5.0 mm) and analysed for bacterial community composition by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) of 16S rDNA and tested for activities of different enzymes involved in C, N, P and S cycling. Bacterial community composition and microbial functional diversity were affected by the presence of the maize roots. The bacterial composition showed a clear gradient up to 2.2 mm from the root surface, while no such gradient was observed in the unplanted pot. Invertase and phosphatase activities were higher in the close vicinity of maize roots (0.2–0.8 mm), whereas xylanase activity was unaffected. This study shows that the changes in bacterial community composition and functional diversity induced by roots may extend several millimetres into the soil.     

Microbial functional diversity covaries with permafrost thaw‐induced environmental heterogeneity in tundra soil

Permafrost soil in high latitude tundra is one of the largest terrestrial carbon (C) stocks and is highly sensitive to climate warming. Understanding microbial responses to warming‐induced environmental changes is critical to evaluating their influences on soil biogeochemical cycles. In this study, a functional gene array (i.e., geochip 4.2) was used to analyze the functional capacities of soil microbial communities collected from a naturally degrading permafrost region in Central Alaska. Varied thaw history was reported to be the main driver of soil and plant differences across a gradient of minimally, moderately, and extensively thawed sites. Compared with the minimally thawed site, the number of detected functional gene probes across the 15–65 cm depth profile at the moderately and extensively thawed sites decreased by 25% and 5%, while the community functional gene β‐diversity increased by 34% and 45%, respectively, revealing decreased functional gene richness but increased community heterogeneity along the thaw progression. Particularly, the moderately thawed site contained microbial communities with the highest abundances of many genes involved in prokaryotic C degradation, ammonification, and nitrification processes, but lower abundances of fungal C decomposition and anaerobic‐related genes. Significant correlations were observed between functional gene abundance and vascular plant primary productivity, suggesting that plant growth and species composition could be co‐evolving traits together with microbial community composition. Altogether, this study reveals the complex responses of microbial functional potentials to thaw‐related soil and plant changes and provides information on potential microbially mediated biogeochemical cycles in tundra ecosystems.