应用DGGE技术分析青藏铁路沿线的土壤细菌种群多样性

选择青藏铁路沿线不同海拔高度的10个地点采集土壤样品,直接提取样品中的总DNA,以巢式PCR扩增细菌16S rDNA片段,应用变性梯度凝胶电泳(DGGE)技术分离PCR扩增的16S rDNA片段,研究土壤细菌的种群多样性.结果表明,青藏铁路沿线高海拔地区具有较为丰富的细菌种群多样性,植被类型是影响青藏铁路沿线土壤细菌种群多样性的重要因素,也是影响土壤细菌种群结构相似性的重要因素,而海拔高度等是次要的影响因素;具有相似植被类型的土壤样品,其细菌种群多样性随海拔的升高而下降.     

RISA、DGGE和2D-PAGE技术对大豆根际土壤细菌多样性分析的比较

采用基于PCR扩增的核糖体间隔区分析(RISA)、变性梯度凝胶电泳(DGGE)和双向电泳(2D-PAGE)3种分子生态学技术对大豆根际土壤细菌多样性比对分析.结果表明:2D-PAGE技术得到的土壤细菌多样性(基因点)最丰富,其次为DGGE技术(基因片段),RISA技术(基因片段)最低.RISA技术得到的条带数最少,但结...     

应用DGGE法对青海相邻两盐湖中细菌多样性的快速检测

分别对青海相邻两盐湖柯柯盐湖、茶卡盐湖土样、泥样进行富集培养后,从中提取的DNA用两套不同的细菌通用引物进行扩增,分别得到包含V8和V9高变区的16SrDNA片断。经变性梯度凝胶电泳(DGGE)分析,结果显示这两个盐湖富集样品中细菌多样性具有较大的差异,而且同一盐湖不同性质样品中的细菌多样性差异也较大,两湖的泥样富集样品中均表现出了稍丰富的多样性。     

利用PCR-DGGE技术分析内蒙古西部地区土壤细菌的多样性

为明确内蒙古西部地区土壤细菌的多样性,利用聚合酶链反应-变性梯度凝胶电泳PCR-DGGE(polymerase chain reaction-denaturing gradient gel electrophoresis)技术对170份土壤样品中的细菌丰度和群落结构组成进行了分析,并研究了土壤细菌多样性与肥力参数的关系。结果表明:内蒙古西部地区土壤细菌的多样性是比较丰富的,丰富度指数处于4到60之间,香浓指数处于1.38到4.09之间。不同土壤类型,其细菌的多样性有明显差异。其中,新积土、棕钙土、栗钙土和灰钙土中的细菌多样性指数均高于其他类型土壤,而灰漠土的多样性指数最低。且土壤的不同利用方式也会对土壤细菌多样性有所影响,其中耕地土壤细菌多样性指数最高,而未利用土壤的多样性指数最低。细菌多样性与土壤肥力参数的相关性分析结果显示二者之间并无显著的相关性。内蒙古西部地区土壤中的优势种群包括Proteobacteria(变形菌门)、Bacteroidetes(拟杆菌门)、Firmicutes(厚壁菌门)、Actinobacteria(放线菌门)、Acidobacteria(酸杆菌门)、Gemmatimonadetes(芽单胞菌门)、Nitrospira(硝化螺旋菌门)。可为该地区的土壤生态系统治理与恢复及未来该地区特有微生物资源的开发利用、农业生产指导等提供一定的科学依据。     

滦河干流中上游浮游细菌群落多样性及其影响因素

浮游细菌是河流生态系统中营养元素的转运者和能量流动的主要贡献者,其群落组成和多样性会受沿途河岸带环境的影响,但目前对开放流动水体中浮游细菌群落的研究还不足。研究使用高通量测序方法,获取了滦河中上游21个样点的浮游微生物群落数据,并结合遥感数据和水体理化指标进行分析,探讨浮游细菌群落结构与水环境因子的关系。结果表明：(1)变形菌门和放线菌门是滦河干流中占据优势地位的门类。(2)群落Chao1丰富度指数与Shannon′s多样性指数均与采样点距河流源头距离存在显著负相关(P<0.05),其中Chao1丰富度指数在河流中段的反常升高暗示着外源性偶见种在该河段的大量涌入。(3)采样点距源头距离与细菌群落组成相关性最高;以距源头距离作为控制变量,只有总氮、硝酸盐氮、氨氮与细菌群落组成相关(P<0.05),表明距源头距离所代表的滞水时间和氮营养盐对细菌群落组成存在显著影响。(4)在4种土地利用因子中,无论是否以距源头距离作为控制变量,只有建设用地占比与细菌群落组成相关(P<0.05);若以硝酸盐氮、电导率作为控制变量,则建设用地占比与细菌群落组成在α=0.05的显著性水平下不再显...     

玉米秸秆青贮过程中优势细菌多样性分析

通过从发酵0、1、3、5、15、30和60d自然发酵的青贮玉米(CK)和采用菌剂处理的青贮玉米(处理1)上取样,分别进行pH值测定和菌群总DNA的提取。总DNA经纯化后采用引物341F-GC和518R进行细菌16S rDNA V3区PCR扩增,扩增产物进行变性梯度凝胶电泳(DGGE)分离并回收优势条带和变化明显的条带进行测序分析,了解秸秆饲料发酵过程中的优势细菌多样性及动态变化规律,从而为现有青贮接种菌的改良奠定理论基础。研究结果表明:采用菌剂处理的青贮玉米pH值下降更快,其优势细菌种类更丰富,且各优势菌的丰度比对照组中相应菌的丰度更强。     

利用DGGE评价不同培养基回收番茄根际细菌类群的能力

用营养肉汤、YG、根系分泌物、土壤浸渍液4种培养基从番茄根际分离培养细菌,并结合变性梯度凝胶电泳(DGGE)技术,对4种培养基回收番茄根际细菌种群的能力进行了比较研究。结果表明,不同培养基和培养温度,回收到的细菌种群有一定差异;低营养浓度的YG培养基在较低的培养温度20℃下进行较长时间的培养,比高营养浓度营养肉汤培养基产生更多、更具代表性的细菌;以根系分泌物为基础的培养基从番茄根际回收到的优势菌群最多。该研究初步建立了用DGGE技术对不同培养基回收分离细菌种群能力进行评价的方法。     

陕北地区苹果树根内细菌多样性分析

以延安市4个不同县区苹果树根为研究对象，分析苹果树根内生细菌的群落组成和多样性，揭示内生细菌的群落结构及多样性差异。采用高通量测序技术分析4个不同县区苹果树根内生细菌16S rRNA基因V3~V4 变异区序列，使用生物信息学方法分析其内生细菌的群落结构及多样性。结果表明，测序共获得994 571条有效序列，在97%一致性下共产生1 559个有效的操作分类单元(operational taxonomic uunits，OTUs)。在门水平上，4个样地苹果树根内生细菌群落组成中共有的优势类群分别为放线菌门(Actinobacteria)和变形菌门(Proteobacteria)，其相对丰富度分别为33.1%~60.7%和30.9%~61.6%；属水平上的优势菌属为假单胞菌属(Pseudomonas)、节杆菌属(Arthrobacter)、链霉菌属(Streptomyces)、诺卡氏菌属(Nocardioides)、叶杆菌属(Phyllobacterium)、黄杆菌属(Flavobacterium)、农杆菌属(Agrobacterium)和贪噬菌属(Variovorax)。Alpha多样性和Beta多样性分析表明，宜川县(YC)的Chao1指数(433.54)和Shannon指数(6.45)最大，基于韦恩图分析4个样地苹果树根内生细菌群落存在354个共有OTUs，延长县(YS)、宝塔区(BT)、宜川县(YC)、洛川县(LC)独有苹果树根内生细菌群落的OTUs分别为378、430、486和477。典范对应分析(Canonical Correlation Analysis, CCA)结果表明，土壤pH是影响4个样地苹果树根内生细菌的群落多样性变化的主要因素。4个样地苹果树根内生细菌种类丰富，但群落结构组成和丰富度的差异不明显，功能预测分析表明无论是Level 1还是Level 2，均未发现显著性差异的代谢通路，此结果表明苹果树根系招募了特异型有益的内生细菌菌群。本研究可为苹果树根内生细菌菌种资源的开发和应用提供参考。     

乌梁素海湖滨湿地细菌群落结构多样性

【目的】了解乌梁素海湖滨湿地水陆过渡带细菌群落结构及多样性变化,探讨富营养化湖泊湿地基质条件对细菌群落结构的影响。【方法】应用变性梯度凝胶电泳(PCR-DGGE)技术,分析和比较了依陆向分布的4个水陆过渡带样点的湿地细菌群落结构多样性,采用典型对应分析(CCA)探讨了湿地基质因子对细菌多样性的影响。【结果】DGGE图谱显示依湖泊水体沉积物(S-1)→湖滨芦苇沼泽沉积物(S-2)→湖滨碱蓬盐化草甸土壤(S-3)→岸上白刺荒漠土壤(S-4),4个样点的条带数依次减少,对应菌群结构及多样性变化显著;多样性指数分析结果显示,Shannon-Wiener指数(H)、均匀度(E)、丰富度(S)以及Simpson指数(DS)均显示依陆向分布逐步下降的规律,即:S-1S-2S-3S-4。序列比对结果显示,沉积物及土壤细菌分属于变形菌门(78.6%)、酸杆菌门(7.1%)、拟杆菌门(14.3%)这3个细菌类群,优势菌门为变形菌门,而变形菌门又分为5个亚群,其中ε变形菌纲为优势亚群;CCA结果表明,图中各条带对应物种的分布受铵态氮、总氮、有机碳、水溶盐总量、氯离子以及钾离子影响最大。【结论】乌梁素海富营养化湖泊的水陆过渡带湿地细菌群落结构存在较大差异,富营养化相关基质因子对细菌多样性影响较大。这为研究富营养化湖泊湿地水陆过渡带的细菌结构多样性及空间异质性提供了科学依据。     

中国白酒大曲细菌群落结构的未培养方法分析

用于白酒酿造的酒曲被西方学者誉为中国的第五大发明[1],是白酒生产中微生物及酶的主要来源,不同的酒曲在微生物群落结构上存在差异,并直接影响酒的出酒率及酒质。因而,研究和鉴定白酒生产中与特征风味物质相关的关键微生物种类,对生产工艺改进具有重要意义。     

Change of bacterial communities in sediments along Songhua River in Northeastern China after a nitrobenzene pollution event

More than 100 tons of nitrobenzene and related compounds were released into Songhua River due to the explosion of an aniline production factory in November, 2005. Sediment samples were taken from the heavily polluted drainage canal, one upstream and three downstream river sites. The change of bacterial community structures along the river was studied by denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing of 16S rRNA genes with five clone libraries constructed and 101 sequences acquired representing 172 clones. Both DGGE profiles and sequences of 16S rRNA genes from clone libraries demonstrated that the contaminated drainage canal and three downstream river sites were similar in that all had Betaproteobacteria , mainly grouped into Comamonadaceae , as the dominant group of bacteria, and all had Firmicutes , primarily as Clostridium spp. These results suggest that these latter two groups of bacteria may play potential roles in degradation and detoxification of nitrobenzene in the present contaminated river environments.     

新疆阿克苏地区盐碱地细菌类群多样性及优势菌群分析

【目的】研究新疆阿克苏地区盐碱土样中细菌类群多样性和优势种群,及其与环境因子的相关性。【方法】采用基于16S rDNA的变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)、克隆测序和典型相关性分析(CCA)的方法。【结果】优势菌群序列分析表明有29个序列属于未培养的微生物,其他43个序列分属细菌的9个目:粘球菌目(Myxococcales)、假单胞菌目(Pseudomonadales)、根瘤菌目(Rhizobiales)、芽孢杆菌目(Bacillales)、伯克氏菌目(Burkholderiales)、放线菌目(Actinomycetales)、海洋螺菌目(Oceanospirillales)、黄杆菌目(Flavobacteriales)、交替单胞菌目(Alteromonadales),21个属。【结论】新疆阿克苏地区土壤中的微生物丰富度非常高,存在大量的细菌类群,优势菌群不尽相同,盐碱土样中微生物群落结构与环境因子是密切相关的。     

应用rpoB和16S rDNA基因的变性梯度凝胶电泳技术对山羊瘤胃细菌多样性的研究

采用免培养的rpoB和16S rDNA基因的变性梯度凝胶电泳技术(DGGE)对3种山羊(波尔山羊,内蒙古绒山羊,四川南江黄羊)瘤胃细菌优势菌群结构进行了比较分析。研究结果显示rpoBDGGE图谱中条带数目少于16S rDNA图谱,并且条带分离效果明显,更有利于分析瘤胃细菌群落组成。从两种DGGE图谱中均可以发现3种山羊瘤胃细菌具有一定的相似性,种内个体间相似性明显高于种间相似性,这说明寄主品种是影响瘤胃细菌种群构成的一个重要因素。同时进行了部分优势细菌16S rDNA基因V6-V8区序列的系统发育分析。基因序列分析表明,DGGE图谱中优势条带的16S rDNA基因序列中有4条克隆的序列与基因库最相似菌的相似性大于97%,余下的克隆序列相似性在89%～96%之间,其中13条序列的与之相似性最高的序列均来自于未被鉴定的瘤胃细菌。     

基于DGGE技术的茯砖茶发花过程细菌群变化分析

变性梯度胶电泳是当前微生物生态学研究重要技术之一。为研究茯砖茶发花过程中细菌群落结构和种类,对发花过程中不同时段细菌16S rDNA的V3可变区扩增,经变性梯度胶电泳(DGGE)后、对细菌DGGE条带进行克隆、测序和比对。结果表明,在发花过程的第0—4天、6—8天、10—14天茯砖茶发花存在3个差异较大的细菌优势种群结构的演变;从16SrDNA的V3可变区比对结果证明黑毛茶发花过程中有短波单胞菌属、诺卡氏菌属、新鞘脂菌属、突那梭菌属、韦龙氏假单胞菌属、乳杆菌属、克雷伯氏菌属以及不可培养ε-变形菌、腐败螺旋菌属、粘球菌属、根瘤菌属和6种未知分类地位的不可培养细菌,说明采用DGGE指纹图谱能更系统、更真实地反映茯砖茶发花发酵过程中细菌群落结构和多样性变化。     

基于高通量测序技术对山羊盲肠细菌多样性的分析

【背景】由于反刍动物特殊的生理结构,以往研究者主要集中对其瘤胃微生物的结构与组成进行了大量研究,严重忽略了盲肠微生物在营养物质消化和肠道健康方面发挥的重要作用。【目的】采用高通量测序技术分析山羊盲肠细菌的多样性及菌群结构。【方法】选用12只10月龄健康母羊,其平均体重为20.70±1.60kg,饲喂20d后,采集每只山羊的盲肠内容物,提取微生物总DNA,用细菌通用引物对细菌16S rRNA基因的高可变区进行PCR扩增,利用Illumina MiSeq平台对扩增子进行高通量测序,并用QIIME等软件对测序序列进行生物信息学分析。【结果】山羊盲肠微生物测序共获得813 496条有效序列与6 883个OTU,并且稀释曲线和Coverage指数反映此次测序结果比较全面的覆盖了山羊盲肠微生物群落。α多样性和β多样性分析表明,山羊个体之间盲肠微生物的多样性存在差异。在门水平,各样品的优势菌门均为厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes);属水平,核心菌群由梭菌属(Clostridium)、瘤胃球菌属(Ruminococcus)和6个未分类的细菌组成。PICRUSt基因预测表明,山羊盲肠微生物以代谢功能为主,主要包括:碳水化合物代谢、氨基酸代谢、能量代谢和脂质代谢等。【结论】山羊盲肠与瘤胃细菌的多样性存在显著差异,与粪便微生物组成相似;与单胃动物相比,两者盲肠微生物的组成既有共性,也存在差异。     

Temporal and Spatial Variation of Methylmercury in Sediments in the Second Songhua River,China

The methylmercury (MeHg) content in the Second Songhua River was investigated in this study. Compared with the former data, the following trends in temporal variation were observed. The MeHg content decreased in relation to the distance from the pollution source in 1977; it showed a decline in 1983 after the pollution source had been shut off for one year and continued to decline from 1983 to 1991 when the Second Songhua River was in a cleansing period. The MeHg content in most segments investigated in this study was higher than in 1991. Along the river, sediment samples were collected from the Baishan Segment to the Sifangtai Segment, and from the surface to the underlying layer to check the vertical trend. The MeHg content was high in the segments upstream due to the gold mines existing, with highest content in the Toudaogou Segment (10.0 ng/g). The MeHg content declined from the Jiapigou Segment (6.2 ng/g) to the Hongshi Segment (0.69 ng/g), and it gradually increased from the Hongshi Segment (0.69 ng/g) to the Hadawan Segment (1.8 ng/g). It was still lower than upstream. The MeHg content gradually decreased from the Hadawan Segment to the Zhaoyuan Segment. However, there was no clear trend from the Zhaoyuan Segment to the Sifangtai Segment, where the order of the MeHg content was the Sanzhan Segment > the Sifangtai Segment > the Zhaoyuan Segment > the Laozhou Segment. The vertical variation in sediments showed that the MeHg content in the surface layer was higher than in the underlying layer in all segments with the exception of the Hongshi Segment and the Zhaoyuan Segment. The pollution index of MeHg content in the Second Songhua River was also discussed.     

Mercury Contamination and Dynamics in the Sediment of the Second Songhua River,China

The Second Songhua River (SSR) was subjected to a large amount of mercury discharge from petrochemical industries in Jilin City from the 1960s to 1980s. The objectives of this study were to investigate the spatial and temporal change of mercury concentration in the sediments of the river and to assess Hg pollution in sediment employing enrichment ratio. Bottom sediments sampled in 2005 were digested with various acids followed by analysis by atomic fluorescence spectrometry for Hg, ICP-MS for Cd, Pb and Sc, and ICP-OES for Co, Cr, Cu, Zn, Mn, Ni, P, Pb, Sb, Ti, V, Al, Fe, Mg, Ca, Na, and K, in order to measure the total concentrations of these elements in the sediments. Results indicated that mercury concentrations in the sediments were strongly related with distance from the historic industrial point source, decreasing at an exponential rate from 1.27 mg kg^?1 at Jilin City to 0.01 mg kg^?1 at downstream Haerbin City. In addition, mercury concentration decreased from 16.8 mg kg^{? 1} y^?1 in 1974 to 0.09 mg kg^?1 y^?1 in 2005 in the sediments at effluent discharge site, and from 0.006 mg kg^?1 y^?1in 1974 to 0.004 mg kg^?1 y^?1 in 2005 at Songyuan City 257 km downstream. In the sedimentary sections of the river, deeper sediments contained higher concentrations of mercury as compared to the surface sediments, suggesting discharges of higher levels of mercury in the past and its subsequent burial over the years by less polluted sediments. Background concentrations of mercury in the surface sediments, reconstructed by tracer Sc, were 0.011 to 0.018 mg kg^?1. Enrichment ratios of Hg in the sediments of SSR was 5 to 75, indicating moderate to extreme pollution, while the sediment of Songhua River is less contaminated, with enrichment ratios of 0.9 to 1.5. At present, the previously accumulated and buried mercury in sediments may not significantly affect water quality of the SSR, but might pose a potential ecological risk to aquatic and amphibian animals. Natural attenuation seems to be an economic remedial choice for these sediments.     

Molecular assessment on impact of uranium ore contamination in soil bacterial diversity

Impact of uranium (U) ore and soluble uranium (at pH 4.0) contamination on agricultural soil bacterial diversity was assessed by using laboratory microcosms for one year. Diversity and abundance of metabolically active bacterial populations in periodically collected microcosm’s samples were analyzed by extracting total RNA and preparation of cDNA followed by analysis of 16S rRNA gene by DGGE and real time PCR. DGGE analysis revealed prominent shift of soil bacterial population due to uranium ore contamination within 12 months while uranium ore along with soluble U completely destroyed the soil bacterial diversity within first six months. Real time PCR based analysis indicated 100–200 folds increase in 16S rRNA gene copies of total as well as individual bacterial taxa in both U ore amended and unamended soils in first six months while increase in incubation period upto 12 months showed reduction of the same only in U ore amended soil. Antagonistic effect of U ore contamination on soil bacterial diversity indicated the severe impact of U mining likely to have on nearby ecosystems. Role of U at acidic pH in destroying the diversity completely is noteworthy as it corroborated the disastrous consequence of acid mine drainage generated from U mine sites.     

Bacterial community diversity assessment in municipal solid waste compost amended soil using DGGE and ARISA fingerprinting methods

Bacterial community structure and diversity of Tunisian agricultural soil treated with different amounts of municipal solid waste compost (MSWC) and other fertilizers were studied using DGGE and ARISA fingerprinting methods. Sequence analysis of dominant DGGE bands revealed the presence of three major clusters, Cytophaga/Flexibacter/Bacteroides (CFB) group, Proteobacteria and Acidobacteria group. Using ARISA profiles, dominant populations were assigned to low and high GC Gram positive bacteria, Cyanobacteria, Spirochetes and Cytophagales. The two methods revealed the absence of significant bacterial community shifts related to the different MSWC applications. Moreover, indigenous bacterial population of the used loam-clayey soil was observed to limit proliferation and survival of Proteobacteria, initially dominant in MSWC and farmyard manure. Effectiveness of the two methods for soil bacterial community studying was shown. While DGGE was more accurate for bacterial identification, ARISA was more practical for handling and rapid estimation of dominant bacteria.     

Characterization of bacterial diversity in two aerated lagoons of a wastewater treatment plant using PCR-DGGE analysis

Aerated lagoons are commonly used for domestic and industrial wastewater treatment due to their low cost and minimal need of operational requirements. However, little information is known regarding microbial communities that inhabit these ecosystems. In this study, a 16S-DGGE approach was used to estimate bacterial diversity and to monitor community changes in two aerated lagoons from a wastewater treatment plant receiving urban and industrial effluents. Pronounced shifts between bacterial communities collected in winter–spring and summer–autumn months were detected. Temperature, dissolved oxygen (DO) and pH were the variables that most influenced the bacterial communities. Phylogenetic affiliation of predominant members was assessed by the determination of the 16S rDNA sequence of correspondent bands. Affiliations to Cytophaga–Flexibacter–Bacteroides (CFB) group, Firmicutes, and β- and ε-proteobacteria were found.