Multi-scale variability analysis reveals the importance of spatial distance in shaping Arctic soil microbial functional communities

Understanding biological diversity and distribution patterns at multiple spatial scales is a central issue in ecology. Here, we investigated the biogeographical patterns of microbial functional genes in 24 heath soils from across the Arctic using GeoChip-based metagenomics and principal coordinates of neighbour matrices (PCNM)-based analysis. Functional gene richness varied considerably among sites, while the proportions of each major functional gene category were evenly distributed. Functional gene composition varied significantly at most medium to large spatial scales, and the PCNM analyses indicated that 14–20% of the variation in total and major functional gene categories could be attributed primarily to relatively large-scale spatial effects that were consistent with broad-scale variation in soil pH and total nitrogen. The combination of variance partitioning and multi-scales analysis indicated that spatial distance effects accounted for 12% of the total variation in functional gene composition, whereas environmental factors accounted for only 3%. This small but significant influence of spatial variation in determining functional gene distributions contrasts sharply with typical microbial phylotype/species-based biogeographical patterns (including these same Arctic soil samples), which are primarily determined by contemporary environmental heterogeneities. Therefore, our results suggest that historical contingencies such as disturbance events, physical heterogeneities, community interactions or dispersal barriers that occurred in the past, have some significant influence on soil functional gene distribution patterns.     

Functional diversity of soil organisms — a review of recent research activities in Germany

The aim of this review is to provide an overview of recent investigations on the functional diversity of soil organisms and to elucidate whether a combination of different phenotypic and genotypic assessment methods can give new insights into the relation of structural (phylogenetic) and functional diversity of soil microbial and faunal communities. The knowledge of functional gene sequences for the major microbial transformations enables studies of their presence and diversity in soils. The concomitant evaluation of phylogenetic identification and functional activity of even individual microbial cells in situ is now possible using such as fluorescence in situ hybridization and microautoradiography. Studies about microbial‐faunal interactions clarifies the importance of soil organisms for soil processes.     

谷子硫解酶基因家族鉴定及表达模式分析

硫解酶(thiolase)是脂肪酸代谢的关键酶,在植物次生代谢合成、生长发育以及抵抗非生物胁迫中均发挥重要作用.为探究谷子硫解酶基因家族的基本特征,本研究利用生物信息学方法,对谷子硫解酶基因家族成员进行了鉴定,对蛋白理化性质及系统进化、基因结构、染色体位置、启动子、基因进化进行了分析;采用转录组测序进行了根、茎、叶、穗...     

基于知识图谱分析的土壤氮循环功能基因研究进展

土壤氮循环功能基因广泛地参与包括固氮、氨化以及硝化和反硝化作用等一系列生态过程,是氮素生物地球化学循环的关键组成部分,在很大程度上影响土壤生产力、全球环境变化以及碳中和可持续发展。近几十年来,分子生物学和微生态学技术的快速发展极大地促进了与土壤氮循环密切相关的功能基因以及其功能微生物群落特征等方面的研究。本研究利用检索自Web of Science数据库中2001—2020年期间土壤氮循环功能基因相关文献,结合R语言科学知识图谱分析方法,从发文量、高被引论文、高频关键词及历史直接引文等方面对土壤氮循环功能基因研究现状进行了系统分析,并总结了土壤氮循环功能基因领域的研究动态、热点和发展趋势。结果表明：1）应用分子生物学技术挖掘土壤氮循环相关微生物功能基因及群落结构探索土壤氮循环的微生物学机制,是当前土壤氮循环研究领域的热点及切入点。2）土壤氮循环功能基因研究主要集中于3个方面：(1)利用宏基因组学等技术对土壤氮循环相关的功能基因进行筛选、识别和注释,从而发现新的微生物功能基因序列、更新引物数据库等;(2)环境因子及管理措施对土壤氮循环相关微生物指标的影响;(3)利用功能基因丰度表征土壤氮...     

参与土壤氮素循环的微生物功能基因多样性研究进展

土壤氮素循环是生物地球化学循环的重要组成部分, 不但影响着土壤生产力和可持续发展, 还影响着全球环境变化.土壤微生物在土壤氮循环中发挥着不可替代的作用, 参与了包括固氮作用、氨化作用、硝化作用和反硝化作用等重要生态过程.近十年中, 分子生物学技术的发展为从功能基因角度研究与土壤氮循环密切相关的微生物功能群结构、组成和丰度的变化提供了新的契机.本文综述了参与土壤氮循环的微生物功能基因多样性研究进展, 并展望了未来发展方向.     

Efficient cell reprogramming as a target for functional‐marker strategies? Towards new perspectives in applied plant‐nutrition research

The review aims at visualizing and strengthening approximation of current strategies in plant breeding, plant nutrition, and molecular biology. Innovations in new breeding strategies on quantitative traits are based on the development of functional DNA markers. This requires knowledge on robust physiological key reactions or parameters in view of the desired agronomic trait. To understand the significance of adaptive molecular‐physiological factors for the expression of agronomic traits in quantitative terms, systems analyses have to demonstrate the phenotypic effect of differential gene activities. The logistic to advance in applied systems biology is currently being strongly discussed. In the present contribution, identification of target cells, which are important for agronomic traits, is stressed as a key for future modeling and virtual experimentation. Integration of target cells in systems analysis should allow to link top‐down approaches, that start at the whole‐plant level, with bottom‐up approaches, that come from the molecular level. To illustrate the importance of adaptive cell reprogramming for agronomic traits, reprogramming of rhizodermic cells to trichoblasts is pointed out in its role for nutrient efficiency (NE). The nature of molecular factors, which may serve as functional markers in breeding, is discussed in view of future marker developments.     

Extraction of DNA from soil

There is an increased interest in the extraction of nucleic acids from various environmental samples, since molecular techniques allow less biased access to a greater portion of uncultivable microorganisms. Two strategies have been developed to improve DNA recovery in terms of yield, purity and unbiased representation of the microbial diversity. The first approach consists of the direct extraction of nucleic acids from soil through in situ cell lysis followed by DNA purification. The alternative approach is based on the separation of bacteria from the soil particles followed by cell lysis and then DNA purification. Several published methods describe the recovery of highly purified nucleic acids that are well-suited for molecular purposes even though a new challenge concerns the recovery of large bacterial DNAs essential for functional investigation of gene clusters and biosynthetic pathways. This review presents an overview of the available methods to achieve this challenging objective.     

TAIL-PCR和 Tn5 转座子质粒拯救法快速分离水稻条斑病菌致病相关基因

构建植物病原菌突变体库是进行新基因发掘和功能基因组学研究的有效途径之一。为了快速准确地确定Tn5的插入位点和相应的功能基因,本研究采用TAIL-PCR和Tn5转座子质粒拯救两种技术对本实验室在筛选Tn5转座子插入水稻条斑病菌的突变体库过程中获得的8个在水稻上毒性减弱的突变体进行了鉴别。结果显示,TAIL-PCR和Tn5质粒拯救相互结合使用,可有效鉴别Tn5的插入位点和相应的功能基因。TAIL-PCR明确的5株突变体是Tn5分别插入在gspF、cAMP-regulatory protein、Integral membrane protease subunit、S-adenosylmethionine decarboxylase proenzyme和gpsJ 基因上从而导致水稻条斑病菌的毒性发生了改变;Tn5质粒拯救法明确的3株突变体是Tn5分别插入在pathogencity protein、conserved hypothetical protein和flagellum-specific ATP synthase 基因上。同源分析发现,8株突变体Tn5插入的基因与已基因组测序的BLS256菌株中的基因同源性达100%。这表明,TAIL-PCR和Tn5质粒拯救技术可有效应用于植物病原菌Tn5突变体库的鉴别和目标基因的分离。     

玉米脱镁叶绿酸氧化酶基因ZmPAO表达与叶绿素含量动态变化的关联分析

【目的】分析玉米脱镁叶绿酸氧化酶基因ZmPAO序列多态性,并挖掘该基因与玉米成熟后期穗位叶叶绿素组分含量相关的功能位点,为基于ZmPAO开发功能标记提供结构信息,有助于对玉米成熟后期叶绿素代谢遗传机制的理解。【方法】以141份具有广泛遗传变异的玉米自交系为试验材料组成关联群体,以2个环境7个时间点的叶绿素组分含量为表型数据,利用Tassel 5.0通过混合线性模型 (MLM,mixed linear model) 开展玉米脱镁叶绿酸氧化酶基因 (ZmPAO) 与成熟后不同时期叶绿素组分变化的相关变异位点的关联分析,并对性状的有效关联位点进行单倍型分析。【结果】在玉米生长后期大部分取样时间点的叶绿素组分含量变异较大,叶绿素a普遍低于叶绿素b的含量,最终总叶绿素 (叶绿素a与叶绿素b的和) 有下降趋势。结果共鉴定ZmPAO中19个有效功能位点,其中4个处于外显子区,1个位于UTR区域,其他均位于内含子区域;功能位点对叶绿素组分含量变异的表型解释率在3.89%～16.57%,总表型效应在5.24%～41.78%。来自第6个内含子的位点S3235对于Yang-chlb6有高达16.57%的表型解释率;第7外显子S3675分别解释了Yang-chla1和Yang-chlb1表型变异的12.16%和14.14%。性状显著单倍型中有利位点和关联分析的变异位点偏好相似。【结论】有效功能位点挖掘和性状单倍型分析表明,ZmPAO外显子发生了2个氨基酸变异,均由疏水氨基酸转化为亲水氨基酸,说明该基因可能通过蛋白结构的变异进行调控,但较多关联位点处于非编码区,说明该基因也受转录水平的调控。转录水平受环境影响较大,故导致该基因出现不同地点因播期和生育期的不同找到的关联位点并不一致,但有效变异位点的存在具有普遍性。     

Structural and functional diversity of soil bacterial and fungal communities following woody plant encroachment in the southern Great Plains

In the southern Great Plains (USA), encroachment of grassland ecosystems by Prosopis glandulosa (honey mesquite) is widespread. Mesquite encroachment alters net primary productivity, enhances stores of C and N in plants and soil, and leads to increased levels of soil microbial biomass and activity. While mesquite’s impact on the biogeochemistry of the region is well established, it effects on soil microbial diversity and function are unknown. In this study, soils associated with four plant types (C₃ perennial grasses, C₄ midgrasses, C₄ shortgrasses, and mesquite) from a mesquite-encroached mixed grass prairie were surveyed to in an attempt to characterize the structure, diversity, and functional capacity of their soil microbial communities. rRNA gene cloning and sequencing were used in conjunction with the GeoChip functional gene array to evaluate these potential differences. Mesquite soil supported increased bacterial and fungal diversity and harbored a distinct fungal community relative to other plant types. Despite differences in composition and diversity, few significant differences were detected with respect to the potential functional capacity of the soil microbial communities. These results may suggest that a high level of functional redundancy exists within the bacterial portion of the soil communities; however, given the bias of the GeoChip toward bacterial functional genes, potential functional differences among soil fungi could not be addressed. The results of this study illustrate the linkages shared between above- and belowground communities and demonstrate that soil microbial communities, and in particular soil fungi, may be altered by the process of woody plant encroachment.     

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。     

茶树黄酮醇合成酶基因的克隆与原核表达

本研究采用EST测序技术和RT—PCR技术,获得了一个茶树茶多酚代谢中的重要基因——黄酮醇合成酶（FLS）基因,在GenBank登录（GenBank accession No．EF205150）,其序列全长1317bp,其中开放阅读框长996bp,编码331个氨基酸,3]端有一个明显的多聚腺苷酸加尾信号,推测的蛋白分子量约为37．5kD,理论等电点为5．80。序列分析表明它与葡萄FLS基因序列的亲缘关系比较近。将该基因重组到表达载体pET-32a（＋）中进行原核表达,经IPTG诱导、SDS-PAGE检测,结果表明茶树黄酮醇合成酶基因能在大肠杆菌BL21中表达,电泳检测到一条大约61kD的外源蛋白,与预测的融合蛋白分子量相符。用Ni-NTA亲和层析柱对融合蛋白进行纯化,得到了纯度在90%以上的纯化蛋白,为进一步研究PET-FLS融合蛋白的活性及功能奠定了基础。     

一个基因序列及其注释数据库的管理系统

以微软公司的数据处理软件M icrosoft Exce l 2003为平台,利用其VBA语言,编写了能够从大量的基因及其注释结果中自动大规模地提取并处理关键信息项的宏程序系统——基因序列及其功能注释数据管理系统M DSA(m anagem en t system for database of sequences and functiona l annotation of genes)。由于具有多种文件输出格式,M DSA可与许多数据处理的操作系统兼容,如M icroG ridTA S(B ioR obotics L td,UK)微阵列制作仪中的基因信息上传软件系统。该管理系统是M icrosoft Exce l软件在专业数据库管理中二次开发的成功尝试,在前期的玉米EST和cDNA数据库管理工作中发挥了重要作用。     

FUM13 encodes a short chain dehydrogenase/reductase required for C-3 carbonyl reduction during fumonisin biosynthesis in Gibberella moniliformis

Fumonisins are polyketide-derived mycotoxins produced by the filamentous fungus Gibberella moniliformis (anamorph Fusarium verticillioides). Wild-type strains of the fungus produce predominantly four B-series fumonisins, designated FB(1), FB(2), FB(3), and FB(4). Recently, a cluster of 15 putative fumonisin biosynthetic genes (FUM) was described in G. moniliformis. We have now conducted a functional analysis of FUM13, a gene in the cluster that is predicted by amino acid sequence similarity to encode a short chain dehydrogenase/reductase (SDR). Mass spectrometric analysis of metabolites from FUM13 deletion mutants revealed that they produce approximately 10% of wild-type levels of B-series fumonisins as well as two previously uncharacterized compounds. NMR analysis revealed that the new compounds are similar in structure to FB(3) and FB(4) but that they have a carbonyl function rather than a hydroxyl function at carbon atom 3 (C-3). These results indicate that the FUM13 protein catalyzes the reduction of the C-3 carbonyl to a hydroxyl group and are the first biochemical evidence directly linking a FUM gene to a specific reaction during fumonisin biosynthesis. The production of low levels of FB(1), FB(2), FB(3), and FB(4), which have a C-3 hydroxyl, by the FUM13 mutants suggests that G. moniliformis has an additional C-3 carbonyl reductase activity but that this enzyme functions less efficiently than the FUM13 protein.     

基于CiteSpace的功能性有机肥研究动态与热点分析

功能性有机肥是推动废弃物资源化利用、化肥减量行动的重要路径,本文采用文献计量学方法系统梳理当前该领域的研究热点及趋势。本文从CNKI、Web of Science核心数据集中检索获取文献,从发文量、作者、机构、关键词、热点等方面进行统计分析。分析结果表明：(1)功能性有机肥研究正处于热点状态,单年发文量国内学者已超过国外学者且影响力优于国外学者。(2)国内沈其荣、刘强等学者团队是研究重要力量,机构以南京农业大学、中国农业大学、中国农科院等农业类的科研机构、高校为主。(3)国内外研究关键词均主要围绕功能性有机肥的研发及施用后功能效益分析方面,同时产生了功能基因、碳、多样性等新的热点词。(4)时间线图谱分析发现国内外均形成了以有机肥、多样性、吸附等主要聚类类型,有机肥、生物炭、腐殖物质、细菌群落、多样性是当前热点。研究认为国内外学者应加大制备材料的功能性、有效性、稳定性研究和施用后的环境效益分析,包括人体健康、环境残留、养分缓释、固碳等方面以推动绿色发展。     

粘细菌纤维堆囊菌的基因组学研究现状

纤维堆囊菌（S.cellulosum）属于粘细菌中的堆囊菌属,它们能产生丰富的次级代谢产物,而且是目前基因组最大的原核生物,现已测序的S.cellulosum So ce56和So 0157-2分别拥有13.03 Mb和14.78 Mb的环状染色体。本文阐述了这两株纤维堆囊菌在序列的组装验证和基因组的分析注释等方面的完成情况,以及人们在测序的基础上,对其展开的结构基因组学、比较基因组学和功能基因组学的研究。通过介绍这两株已测序纤维堆囊菌的基因组学研究现状,我们总结了从纤维堆囊菌的全基因组序列中挖掘遗传信息的方法,为发现纤维堆囊菌中新的功能基因提供一些参考。     

毛白杨叶绿体基因启动子和终止子的克隆及其功能鉴定

摘 要：从毛白杨(Populus tomentosa)无性系植株组培苗中提取基因组总DNA，通过合成3对特异性引物，用PCR的方法扩增了包含psbA基因启动子、终止子和16S rRNA基因启动子的叶绿体DNA序列。用BLAST及DNAMAN对克隆序列进行了分析，并结合前人的研究工作，确定了两个启动子Prrn、PpsbA的-35区、-10区以及翻译调控序列等重要调控序列元件和终止子的“TAA”序列。并在序列分析的基础上合成新的引物，准确克隆了毛白杨的叶绿体16S rRNA基因启动子Prrn、psbA基因启动子PpsbA及其终止子TpsbA。利用叶绿体基因启动子的原核表达特性，分别用启动子Prrn（其后添加rbcL基因的RBS序列）和PpsbA及终止子TpsbA分别构建了GFP基因的原核表达载体pSGmT、pPGmT，并以T7启动子为对照构建了GFP基因的原核表达载体pETGm，进行了初步的原核表达实验，检测结果表明：克隆到的叶绿体基因启动子和终止子是有生物学功能的，在大肠杆菌BL21中呈组成型表达，同时还证明了RBS序列在基因表达中的重要性。     

丹参海藻糖-6-磷酸合成酶基因（SmTPS）的克隆及其表达分析

对丹参EST序列进行Blast分析,得到一条海藻糖-6-磷酸合成酶（TPS）基因,将其命名为SmTPS,GenBank注册号为JF937196。该基因cDNA全长2836 bp,包含一个长为2574 bp的ORF,编码857个氨基酸。生物信息学分析表明,分子量为97.04 kD,等电点为5.76,含α-螺旋、β转角、延伸连和无规则卷曲。该蛋白同时具有海藻糖-6-磷酸合成酶（TPS）和海藻糖-6-磷酸磷酸酯酶（TPP）的功能结构域。系统进化树分析表明丹参TPS功能域和拟南芥AtTPS6属于一类,而TPP功能域与低等生物酵母ScTPS2属于一类。实时定量PCR结果分析表明,SmTPS基因在丹参不同组织器官中差异表达,其茎中表达量最高。在干旱和低温处理下,其表达量与对照相比均可上调约2倍,表明SmTPS基因在丹参抵抗干旱和低温胁迫中发挥一定的作用。     

Soil aggregate size mediates the impacts of cropping regimes on soil carbon and microbial communities

Understanding the influence of long-term crop management practices on the soil microbial community is critical for linking soil microbial flora with ecosystem processes such as those involved in soil carbon cycling. In this study, pyrosequencing and a functional gene array (GeoChip 4.0) were used to investigate the shifts in microbial composition and functional gene structure in a medium clay soil subjected to various cropping regimes. Pyrosequencing analysis showed that the community structure (β-diversity) for bacteria and fungi was significantly impacted among different cropping treatments. Functional gene array-based analysis revealed that crop rotation practices changed the structure and abundance of genes involved in C degradation. Significant correlations were observed between the activities of four enzymes involved in soil C degradation and the abundance of genes responsible for the production of respective enzymes, suggesting that a shift in the microbial community may influence soil C dynamics. We further integrated physical, chemical, and molecular techniques (qPCR) to assess relationships between soil C, microbial derived enzymes and soil bacterial community structure at the soil micro-environmental scale (e.g. within different aggregate-size fractions). We observed a dominance of different bacterial phyla within soil microenvironments which was correlated with the amount of C in the soil aggregates suggesting that each aggregate represents a different ecological niche for microbial colonization. Significant effects of aggregate size were found for the activity of enzymes involved in C degradation suggesting that aggregate size distribution influenced C availability. The influence of cropping regimes on microbial and soil C responses declined with decreasing size of soil aggregates and especially with silt and clay micro-aggregates. Our results suggest that long term crop management practices influence the structural and functional potential of soil microbial communities and the impact of crop rotations on soil C turnover varies between different sized soil aggregates. These findings provide a strong framework to determine the impact of management practices on soil C and soil health.