黄土高原土壤中细菌群落结构多样性的PCR-DGGE分析

利用细菌通用引物,对从黄土高原5个地区土壤样品以及西峰剖面26个土壤样品中提取的总DNA进行聚合酶链式反应(PCR)扩增,采用变性梯度凝胶电泳(DGGE)技术对PCR产物进行分析,以揭示黄土高原土壤中细菌群落结构的多样性.针对黄土高原土壤微生物特点进行DGGE试验条件优化,获得最佳变性梯度为40%～70%,最佳电泳时间为17 h(100 V).对不同深度土壤细菌DGGE图谱的聚类分析表明,不同深度土壤细菌呈现2种不同的群落结构,推测其成因可能与季风性气候引起的温湿环境变化及冰期有关.在夏季风加强而冬季风减弱的时期,气候温暖潮湿,风化成壤作用强烈,在DGGE图谱中表现为土壤样品条带多而密;反之,风化成壤作用较弱,在DGGE图谱中表现为条带少而疏.对洛川、西峰等5个地区土壤细菌Shannon-Weiner指数的测定结果表明,Shannon-Weiner指数受条带亮度及迁移率的影响.     

转Bt-cry1Ac基因棉花对意大利蜜蜂肠道细菌群落的影响*

采用基于16 SrDNA的变性梯度凝胶电泳(DGGE)的方法,研究了转Btcry1Ac基因棉花SGK321对益虫——意大利蜜蜂(Apis mellifera ligustica)肠道细菌群落的影响.分别用转基因/非转基因棉花花粉和高浓度外源DNA饲喂蜜蜂7d,通过DGGE分析经不同处理的蜜蜂肠道细菌群落组成的差异,同时用PCR检测是否发生外源基因的横向转移.结果显示,各个处理组DGGE图谱中的条带数目在11.3～15.0之间,Shannon’s指数在0.87～1.05之间,相似性系数介于68.3%～84.7%,不同处理组肠道细菌组成差异不明显,用PCR的方法也没有检测到外源基因的横向转移.因此初步判定转基因棉花SGK321短期内对蜜蜂细菌群落尚无不利影响.图2表3参22     

北运河水体浮游细菌群落的空间分布特征及其与水质的关系

污染源汇入和闸坝拦截等因素能够影响城市河流水生生态环境质量,主要表现在群落结构和功能的改变。分别于春季、夏季和冬季3个季节对北运河干流的10个点进行水样采集,通过16S rDNA末端限制性片段长度多态性(T-RFLP)分析手段研究了北运河河道水体浮游细菌的群落结构,以揭示流域微生物多样性以及浮游细菌群落与水质因子的响应关系。共得到47种不同的片段,其中218bp片段是优势菌。T-RFs片段计算微生物多样性指数和均匀度指数结果表明,目前北运河水体生态结构已经较为脆弱。冬季与其他季节的群落组成有明显差别,夏季细菌丰度高于冬季。通过CANOCO软件分析浮游细菌群落结构与水质指标的空间特点,发现水体流动性降低、支流汇入和污染源的汇入都会引起微生物群落结构在空间上的改变;环境因子与微生物群落组成的相关性研究表明,总磷、溶解性有机碳(DOC)和温度对北运河微生物群落结构影响较大。     

云南玉溪烟区轮作与连作土壤细菌群落多样性比较研究

为评价云南玉溪烟区轮作和连作对土壤细菌群落多样性影响,从土壤中直接提取土壤细菌总DNA,用细菌16S rDNA特异性引物进行PCR扩增和变性梯度凝胶电泳(DGGE),每个处理样品3次重复,在DGGE图谱中相似性较高,基本聚在一起,从整体证明了试验操作方法较为精确.从泳道分析图可知,轮作土壤细菌群落各泳道条带数量13～29条不等,平均为21条.连作土壤细菌群落各泳道条带数量从4～20条不等,平均为12条.从每条泳道的条带数方面和光密度值分别进行细菌群落多样性各指标的比较,结果表明,轮作处理细菌群落丰富度指数均大于连作处理,轮作香农-威纳和辛普森指数(1/D)均大于连作,表明轮作微生物多样性较连作高,轮作方式可以提高植烟土壤细菌群落的多样性.     

基于浮游植物群落的纵向连通性初步研究

目前鲜有从生物群落角度定量分析连通性。作者以建有多级节制闸的沙颍河干流为对象,研究河流纵向连通性对沙颍河干流浮游植物群落的影响,并尝试基于梯度分析方法从浮游植物群落角度定量评估河流连通性。调查结果显示,沙颍河干流浮游植物339种,分为8个门类,以硅藻门浮游植物种类(170种)最多,其次是绿藻门(93种)和蓝藻门(37种)。从季节来看,夏秋季浮游植物种类显著多于春季;从浮游植物密度和生物量来看,春秋季以隐藻门浮游植物为主,而在夏季蓝藻门浮游植物密度最高。优势度排前10的种类蓝藻门有4种,隐藻门3种,硅藻门2种,绿藻门1种。通过比较不同季节浮游植物群落组成可知,闸坝阻隔对浮游植物群落结构具有较大影响,群落结构组成从常见的以硅藻为主的河流群落特征转变为以蓝藻、隐藻为主的湖泊群落特征。根据DCA(Detrended Correspondence Analysis)第1轴长,均采用PCA(Principal Component Analysis)排序法对不同季节浮游植物密度和生物量进行分析,并绘制排序图,结果表明,不同河段浮游植物群落特征可在排序图中区分开来,表明不同河段之间的浮游植物群落结构存在差异;不同河段之间浮游植物群落特征在排序图中的相对位置及河段的实际相对位置,可以在群落空间中明确表征河流连通性的潜在生态梯度。从水生生物群落角度定量表征连通度的大小,将有助于建立基于生物群落计算河流纵向连通程度的方法。     

辽东湾大凌河口湿地土壤微生物群落分析

土壤微生物对湿地生态系统的物质转化和能量流动具有重要作用,目前对天然湿地的微生物资源及群落结构缺少研究。应用PCR-DGGE技术研究辽东湾大凌河口湿地土壤中的微生物群落结构,并对其群落组成进行分析,结合冗余分析（RDA）方法考察环境因子对微生物群落结构的影响。共设定7个站位采集土壤样品,直接提取样品中微生物的总DNA,以巢式PCR扩增细菌16S rDNA-V3区片段,应用变性梯度凝胶电泳（DGGE）技术分离片段,研究河口区湿地土壤细菌的群落结构。结果表明：辽东湾大凌河口区土壤具有较为丰富的细菌种群,主要包含Proteobacteria、Actinobacteria和Bacteroidetes几大类门,且同一季节采集的样品在聚类分析图上聚类成簇。RDA分析表明,土壤的温度与盐度2个变量分别解释了6.5%、2.3%的土壤细菌群落结构变化,表明大凌河口区土壤的群落结构变化是多种环境因子共同作用的结果,其中温度的影响略大于盐度。     

基于16S rDNA-DGGE和FDC技术对富营养化湖泊不同生态修复工程区细菌群落结构的研究

于2005年10月在太湖梅梁湾水体生态修复工程区中采集不同生物净化区水样,直接提取水样中的总DNA,以细菌16SrDNA通用引物进行V3区PCR扩增,PCR产物经DGGE(变性凝胶梯度电泳)分离后,获得水体细菌群落的16S rDNA指纹图谱;并运用FDC(表面荧光直接计数)方法对不同生物净化区的细菌丰度进行了测定.结果表明,随着净化处理程度的增加,细菌群落多样性呈现上升趋势,DGGE条带从未实施生态修复的梅梁湾中的13条上升到沉水植物恢复区的25条;细菌数量的变化则呈现显著下降趋势,由工程区外围(重富营养区)的5.36×106cells/mL下降到生态修复区的2.06×106cells/mL.生态修复工程的实施改善了水体的生境条件,提高了水体的自净能力,促进了细菌多样性的恢复.图6表1参40     

酸性矿山排水影响下水库及其入库流域水体细菌多样性与环境因子的关系

为了解酸性矿山废水排水处沿流域至水库底层水体细菌群落结构的沿程变化,探索其环境影响因素.采用高通量测序技术并结合主成分分析（principal component analysis,PCA）方法,对2019年9月采集受酸性矿山废水（acid mine drainage,AMD）影响的12个水样进行研究.结果表明,入库流域水样中细菌群落多样性变化趋势不明显,从表层至底层细菌多样性不断升高.在门水平上受AMD影响的细菌主要由变形菌门（Proteobacteria）、拟杆菌门（Bacteroidetes,0.21%—12.96%）、厚壁菌门（Firmicutes,0.15%—14.4 8%）和放线菌门（Actinobacteria,0.06%—4.64%）构成.其中,变形菌门中的α-变形菌纲（Alphaproteobacteria,2.42%—61.99%）、β-变形菌纲（B etaproteobacteria,1.29%—63.53%）和γ-变形菌纲（Gammaproteobacteria,6.41%—63.09%）为优势菌类.从不同空间位置浮游细菌群落组成来看,不动杆菌属（Acineto...     

府河水体及沉积物细菌群落结构分布特征及其影响因素

城市河流承接不同类型的废水导致水质恶化,影响河流生态系统.通过分析河流细菌群落结构变化,不仅可以判断河流污染特征,还将有助于河流污染治理与生态修复.笔者研究了府河夏季上下游水体和沉积物理化性质,并采用高通量测序法分析了细菌群落结构特征.结果 表明,府河上游水质化学需氧量(COD)、氨氮(NH3-N)和总磷(TP)浓度显著高于下游(P＜0.05),沉积物中重金属镉、锌和铅含量较高,均值分别为(0.44±0.03)、(182.17 ±0.34)和(35.76±0.20) mg· kg-1.府河沉积物细菌多样性及丰富度明显高于相应上覆水体,变形菌门(Proteobacteria)是水体和沉积物中的第一优势菌,水体中参与氮循环的蓝藻菌门(Cyanobacteria)的高丰度说明府河存在一定的富营养化,此外,可以分解有机物的放线菌门(Actinobacteria)在下游丰度高于上游;沉积物中具有致病作用的拟杆菌门(Bacteroidetes)在上游城市河段丰度较高.细菌群落空间异质性结果表明下游硝化细菌(nitrifying bacteria)和反硝化细菌(denitrifying bacteria)的丰度(分别为62.25％和31.29％)明显比上游高.水体细菌多样性和温度、pH、总有机碳有显著相关性(P＜0.05),冗余分析显示NH3-N、总氮(TN)、TP和pH对细菌群落结构影响较大;而pH、TN和重金属镉是影响下游沉积物细菌群落结构的主要环境因子.     

烟区轮作与连作土壤细菌群落多样性比较

采用从土壤中直接提取土壤细菌总DNA,并用细菌16SrDNA特异性引物进行PCR扩增和变性梯度凝胶电泳（DGGE）的方法,对比研究了贵州福泉烟区轮作和连作对土壤细菌群落多样性的影响,每个处理样品3次重复在DGGE图谱中相似性较高,基本聚在一起,从整体证明了试验操作方法较为精确。由DGGE图谱可知,轮作土壤微生物条带数量15-31条不等,平均为23条。连作土壤微生物条带数量从7-25条不等,平均为18条。分别从每条泳道的条带数和光密度值两方面,对细菌群落多样性指标进行了比较。结果表明,轮作处理细菌群落丰富度指数均大于连作处理,轮作Shannon-wiener和辛普森指数（1/D）均大于连作,表明轮作微生物多样性较连作高,轮作方式可以提高植烟土壤细菌群落的多样性。     

基于高通量定量PCR研究城市化小流域微生物污染特征

水体微生物污染(包括致病菌、病毒、寄生虫)会引起多种传染病和寄生虫病,对生产生活用水安全和人体健康造成重要威胁。本研究应用基于Taq Man探针的高通量荧光定量PCR技术对厦门市后溪流域冬季微生物污染进行检测,包含了5种粪便污染源(人源、反刍动物源、猪源、家禽源、狗源)微生物源示踪分子标记物与12种病原微生物。结果表明,该流域在上游及水库5个位点没有粪便污染,仅在其中一个水库位点检测出棘阿米巴,微生物污染极小;中下游检测出人类、反刍动物、猪、家禽、狗粪便污染,并且检测出产气荚膜梭菌、肠聚集性大肠杆菌、肠毒素型大肠杆菌、幽门螺杆菌、霍乱弧菌、副溶血弧菌、棘阿米巴、克雷伯氏肺炎杆菌等病原菌,其中流经旧城区居民生活生产区水样微生物污染严重,下游新城区微生物污染较小。这些结果暗示着城市人类活动是流域微生物污染主要来源,应从污染源头加强微生物污染控制。     

Monitoring microbial diversity and natural product profiles of the sponge<Emphasis Type="Italic"> Aplysina cavernicola</Emphasis> following transplantation

In order to assess the stability of the microbial community of the sponge Aplysina cavernicola under in situ conditions, sponges were transplanted from their original location (>40 m depth) to shallower, more light-exposed sites (7-15 m depth). Transmission electron microscopy revealed that the microbial community remained visually unchanged and free of cyanobacteria over the experimental time period of 3 months. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA gene sequences allowed a distinction between the variable and permanent fraction of the bacterial community. Comparative sequence analysis of four variable DGGE bands revealed high sequence similarity to representatives of the Alpha- and Gammaproteobacteria and the phylum Bacteroidetes, which have been recovered previously from Mediterranean seawater as clone sequences or by cultivation. Seven (out of 12) permanent DGGE bands showed high sequence similarity to a sponge-specific, monophyletic 16S rRNA gene sequence cluster within the Acidobacteria division, and to a sequence cluster of uncertain affiliation. These sequence clusters represent members of a common microbial community that is shared among distantly related sponges from different, non-overlapping geographic regions. Four additional permanent DGGE bands showed high sequence similarity to a Betaproteobacterium, Burkholderia cepacia, which is not typically known as a marine bacterium. High-performance liquid chromatography analyses of sponge tissues revealed no changes in metabolite pattern, indicating that these compounds are expressed constitutively irrespective of the variations resulting from the transplantation experiment.     

Biogeography of bacterioplankton in lakes and streams of an Arctic tundra catchment

Bacterioplankton community composition was compared across 10 lakes and 14 streams within the catchment of Toolik Lake, a tundra lake in Arctic Alaska, during seven surveys conducted over three years using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified rDNA. Bacterioplankton communities in streams draining tundra were very different than those in streams draining lakes. Communities in streams draining lakes were similar to communities in lakes. In a connected series of lakes and streams, the stream communities changed with distance from the upstream lake and with changes in water chemistry, suggesting inoculation and dilution with bacteria from soil waters or hyporheic zones. In the same system, lakes shared similar bacterioplankton communities (78% similar) that shifted gradually down the catchment. In contrast, unconnected lakes contained somewhat different communities (67% similar). We found evidence that dispersal influences bacterioplankton communities via advection and dilution (mass effects) in streams, and via inoculation and subsequent growth in lakes. The spatial pattern of bacterioplankton community composition was strongly influenced by interactions among soil water, stream, and lake environments. Our results reveal large differences in lake-specific and stream-specific bacterial community composition over restricted spatial scales (<10 km) and suggest that geographic distance and connectivity influence the distribution of bacterioplankton communities across a landscape.     

亚麻温水脱胶液中细菌菌群结构分析

为了探讨实验室条件下模拟的工厂亚麻温水脱胶液中细菌菌群结构,采用纯培养技术和PCR-DGGE技术(Denaturing gradient gel electrophoresis)对细菌菌群结构进行了研究.用纯培养方法分离获得9类菌落,其中假单胞菌属(Pseudomonas)在有氧培养条件下总是处于优势.梭菌属(Clostridium)在厌氧培养过程中总是处于优势.微球菌属(Micrococcus)和葡萄球菌属(Staphylococcus)只有亚麻脱胶初期才有发现.PCR-DGGE指纹图谱显示,亚麻温水脱胶过程中条带数量较少且没有明显种群群落结构演替过程.通过对不同时期沤麻液中16S rDNAV3片段PCR产物d、e两个DGGE条带进行分子克隆、序列测定和Blas份析,发现e条带包含的16S rDNAV3片段除e 35外均属于假单胞菌属.d条带包含着较多不同的16S rDNAV3片段,其中有传统方法没有分离到的泛菌属(Pantoea)细菌、一些NCBI未收录的序列及一些非可培养微生物序列.纯培养技术和PCR-DGGE技术的共同使用,可以更全面准确地提供细菌多样性方面的信息.图4参15     

太湖中微囊藻群落的季节变化分析

采用显微观察和PCR-DGGE 2种方法分析太湖(河口、梅梁湾湾心、湖湾交汇处和湖心)4个代表性区域微囊藻群落的季节变化,研究太湖中微囊藻群落的季节变化规律.显微形态分析未发现微囊藻属的主要优势种铜绿微囊藻(Microcystis aeruginosa)、惠氏微囊藻(Microcystis wesenbergii)和水华微囊藻(Microcystis flos-aquae)存在明显的季节变化规律(P>0.05).DGGE条带测序结果的亲缘分析表明,太湖夏季和冬季样品中的微囊藻彼此分离成2大特征类群,而春季和秋季样品中的微囊藻彼此混杂.遗传距离分析结果显示,同一季节内微囊藻的遗传距离小于不同季节间的遗传距离(P<0.01),太湖夏季水华主要由亲缘关系很近的微囊藻类群构成.     

Spatial characteristics analysis by PCR-DGGE for bacterial communities in surface sediments of Tangxi River,Hefei City北大核心CSCD

Microbes play an important role in material circulation and pollutant release in urban sediments, and its community structure can provide an important basis for evaluating the pollution load and ecosystem health of an urban river. In this study, bacterial communities in sediments from different locations of Tangxi River in Anhui Province were analyzed and compared, with an objective of evaluating the effects of aquatic environment on spatial characteristics of bacterial communities and the feasibility of using bacterial community composition as an indicator of urban river health. PCR-DGGE was applied to analyze the bacterial communities; fifteen major bands of 16S rDNA genes fragments from DGGE profiles of sediment samples were further eluted from gel, reamplified and sequenced. The sequences of these fragments were compared with the database in GenBank (NCBI). The collected samples were clustered based on UPGM Aanalysis. In addition, relationships between bacterial communities and environmental factors were analyzed by Monte Carlo test and redundancy analysis (RDA). The DGGE profiles indicated that upstream surface sediments had about 30 bands, but the differences in brightness were not significant. Although midstream surface sediments had only 18 bands on average, the brightness of some bands was relatively high, indicating that some dominant species of bacteria existed in these sediments. The amounts of bands in downstream sediments were between those of upstream and midstream, with some bands of high brightness. The NCBI comparison results showed that ten sequences shared 98-100% homology with known sequences, one with 97%, and the other four with uncultured bacteria. Shannon index (H) of bacterial diversity from upstream surface sediments was 3.31 on average, which was significantly (P < 0.05) higher than that of midstream surface sediments, and slightly higher than that of downstream surface sediments. UPGMA results showed both the distribution of bacteria communities and the diversity were strongly related with the sampling locations (Eigenvalue = 0.188, P = 0.134). Monte Carlo test and RDA analysis showed that the ion exchangeable form of nitrogen (IEF-N) was the main factor influencing the spatial characteristics of bacterial communities in the sediments. The aquatic plants had a significant effect on the richness of bacterial communities in the sediments, and the effectiveness of plant species was more notable than that of the vegetation coverage. The above results indicated that bacterial community structure and diversity in the surface sediments can well reflect the degree of urbanization development and effectiveness of ecological restoration in Tangxi River region.     

Denaturing gradient gel electrophoresis profiling of bacterial communities composition in Arabian Sea

Denaturing gradient gel electrophoresis (DGGE) was used to elucidate spatial and temporal variations in bacterial community composition (BCC) from four locations along the central west coast of India. DNA extracts from 36 water samples collected from surface, mid-depth (-10 m) and dose to bottom (-20 m) during premonsoon, postmonsoon, monsoon were analyzed by PCRfor amplifying variable region of 16S rRNAgene and subsequently through DGGE. Prominent bands were excised, cloned and sequenced indicated the preponderance of gammaproteobacteria, bacteroidetes and cyanobacteria. Non-metric dimensional scaling of the DGGE gels indicated that the spatial variations in BCC were prominent among the sampling locations. Temporal variations in the BCC appear to be influenced by monsoonal processes. The canonical correspondence analyses suggest that the concentration of chlorophyll a and nitrate are two important environmental factors for both spatial and temporal variations in BCC. Chlorophyll a seems to be impart a top-down control of BCC while nitrate, the bottom-up control. Our results also suggest that BCC can vary over a small geographic distance in highly dynamic, seasonally predisposed tropical coastal waters.     

厌氧发酵过程pH对微生物多样性和产物分布的影响

采用PCR-DGGE技术,研究了不同pH条件下蔬菜类有机垃圾厌氧发酵过程中的微生物多样性,探讨了微生物群落结构与发酵产物分布的关系.Shannon指数分析表明,pH=7和pH=8时的微生物多样性较高,随时间变化规律相似,而pH=5时的微生物多样性较低.UPGMA聚类分析和PCA分析结果也表明,pH=7和pH=8时的微生物群落结构相似,pH=5与之显著不同.在不同pH条件下的优势菌属都是乳酸细菌和梭菌。微生物多样性的变化与发酵产物分布具有一定相关性,pH通过影响微生物群落结构的变化最终影响发酵产物的分布.图4表2参27     

Climatic/edaphic controls on soil carbon/nitrogen response to shrub encroachment in desert grassland.

The proliferation of woody plants in grasslands over the past 100+ years can alter carbon, nitrogen, and water cycles and influence land surface-atmosphere interactions. Although the majority of organic carbon in these ecosystems resides belowground, there is no consensus on how this change in land cover has affected soil organic carbon (SOC) and total nitrogen (TN) pools. The degree to which duration of woody plant occupation, climate, and edaphic conditions have mediated SOC and TN responses to changes in life-form composition are poorly understood. We addressed these issues at a desert grassland site in Arizona, USA, where the leguminous shrub velvet mesquite (Prosopis velutina) has proliferated along an elevation/precipitation/temperature gradient and on contrasting soil morphologic surfaces. On sandy loam complexes of mid-Holocene origin, mean SOC and TN of soils in the grassland matrix increased approximately 68% and approximately 45%, respectively, with increasing elevation. Soil organic carbon pools were comparable and TN pools were approximately 23% higher in Pleistocene-aged clay loam complexes co-occurring with Holocene-aged soils at the upper elevation/climatic zone. Across the site, belowground resources associated with large Prosopis plants were 21-154% (SOC) and 18-127% (TN) higher than those in the grassy matrix. The variance in SOC and TN pools accounted for by Prosopis stem size (a rough surrogate for time of site occupation) was highest at the low- and mid-elevation sites (69-74%) and lowest at the upper elevation site (32-38%). Soil delta15N values ranged from 5.5 per thousand to 6.7 per thousand across the soil/elevation zones but were comparable in herbaceous and shrub-impacted soils and exhibited a weak relationship with Prosopis basal stem diameter (r2 < 0.1) and TN (r2 < 0.08). The SOC delta13C values decreased linearly with increasing Prosopis basal diameter, suggesting that size and isotopic composition of the SOC pool is a function of time of Prosopis site occupation. Isotopic mixture models indicate that encroachment of C3 woody plants has also promoted SOC additions from C4 plant sources, indicative of long-term herbaceous facilitation. Grassy sites in contrasting soil/elevation combinations, initially highly distinctive in their SOC pool size and delta13C, appear to be converging on similar values following approximately 100 years of woody plant proliferation.