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.     

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。     

厦门后溪流域沿城乡梯度浮游细菌多样性及其与环境因子的关系

为研究城市化对河流浮游细菌的影响,选取厦门后溪流域沿城乡梯度采集样品,并利用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)和多元统计方法对浮游细菌群落空间分布格局进行分析.结果表明,Shannon-Weiner多样性指数和DGGE条带数随着采样站点和城市中心距离的减小呈现先升高后下降的趋势.聚类分析(Cluster)和多维尺度分析(Multidimensional scaling,MDS)将12个站点的浮游细菌群落分为4组,测序结果显示β-变形菌(Betaproteobacteria)是优势类群,占40.0%.统计分析表明,pH和总氮(Total nitrogen,TN)是影响后溪浮游细菌群落组成最主要的环境因子,他们共同解释了47.6%的群落组成变化.总之,沿城乡梯度随着水体理化指标的变化,浮游细菌群落也发生明显转变,表明后溪浮游细菌受到城市化的强烈影响.此外,结果也提示DGGE可以作为监测与评价河流生态系统健康的重要工具.图6表2参33     

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     

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。     

转Bt基因玉米种植对土壤细菌数量及多样性的影响

利用三室根箱装置获得玉米生长室土壤(S I)、根际土(SⅡ)、非根际土(SⅢ)3个不同根区土壤,采用传统平板计数培养与变性梯度凝胶电泳(DGGE)技术相结合的方法,研究了转基因玉米Btll及其非转基因亲本在播种后40、50、60 d各根区土壤细菌数量及多样性的变化.结果表明,转Bt基因玉米播种后50、60 d S I根区土壤可培养细菌数量显著低于非转基因亲本,播种后40、50 d SⅡ根区土壤可培养细菌数量较亲本玉米显著增加,而在其他时期和根区与亲本玉米之间均无显著差异.DGGE图谱显示,3个采样时期各根区DGGE图谱条带数、土壤细菌多样性指数和均匀度指数均无显著差异(P＞0.05).     

富集培养下的若尔盖高原湿地低温纤维素降解细菌群落结构

若尔盖高原湿地常年低温,泥炭沼泽土分布广泛,为了解该特殊生境中的低温纤维素降解菌群落结构,将采自3个乡(阿西茸、黑河、达扎寺)的泥炭沼泽土样品于10℃条件下富集培养于羧甲基纤维素钠(CMC-Na)液体培养基中,用3,5-二硝基水杨酸(DNS)法测定纤维素酶活性,并利用变性梯度凝胶电泳技术(Denaturing gradient gel electrophoresis,DGGE)分析原始土壤样品和富集培养系中的细菌群结构.酶活测定结果表明,低温富集传代过程中,3个地区样品的相对酶活均随着培养代数的增加而增加,第3代的相对酶活最高,为25.5 U.DGGE分析表明:相对于原始土壤样品,富集系中的微生物菌群变简单;不同富集代数间,阿西茸乡样品细菌群落结构随传代次数增加而增加,而黑河乡和达扎寺乡样品表现为减少.对DGGE图谱条带序列的系统发育分析表明,富集培养系中的细菌属于α-变形菌纲(α-proteobacteria)、β-变形菌纲(β-proteobacteria)、γ-变形菌纲(γ-proteobacteria)、酸杆菌门(Acidobacteria)、疣微菌门(Verrucomicrobia)等类群.本研究结果揭示若尔盖高原湿地泥炭沼泽土低温纤维素降解菌种群丰富,具有潜在工业开发应用前景.(图3表2参35)     

转双价抗真菌病害基因大豆对根际土壤微生物群落结构的影响

采用传统的平板计数法和变性梯度凝胶电泳技术,对转chi+rip(几丁质酶和核糖体失活蛋白)双价抗真菌基因大豆转基因G0431以及其亲本非转基因大豆黑农35种植后的根部土壤可培养细菌(含芽胞杆菌、产荧光假单胞菌)、真菌、放线菌进行平板计数及细菌和真菌的群落结构分析.结果表明,二者根部土壤可培养细菌(含芽胞杆菌和产荧光假单胞)、真菌和放线菌的数量并无显著差异;此外,变性梯度凝胶电泳图谱的聚类分析结果显示,时间变化是造成根部土壤细菌和真菌群落结构变化的主要因素,而同一时间内转基因G0431和黑农35根部土壤的细菌和真菌群落结构之间并无显著不同.在大豆的整个生长期内,根部土壤细菌群落多样性指数为5.32～5.37,群落分布均匀度指数为0.91~0.95;根部土壤真菌群落多样性指数为4.78～4.91,群落分布均匀度指数为0.81～0.89.以上结果说明,几丁质酶和核糖体失活蛋白双价基因的导入并没有对大豆根际可培养细菌(含芽胞杆菌和产荧光假单胞)、真菌和放线菌的数量以及细菌和真菌的群落结构产生显著影响.图4表2参22     

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     

化学-生物絮凝污水处理工艺中微生物群落结构变化分析

利用分子生物学技术，直接从化学-生物絮凝工艺的活性污泥样品中提取DNA，对16S rDNA V3区进行PCR扩增，结合DGGE（变性浓度梯度凝胶电泳），分析了活性污泥中微生物群落结构，并对Shannon多样性指数进行分析讨论，通过研究指出系统中细菌数量的增加或减少．测定了活性污泥中部分菌种的16S rDNA V3区片段序列，通过NCBI（美国国立生物技术信息中心）基因库比对，初步确定细菌的属．结果表明，PCR—DGGE结合测序技术是一种完全可行的快速进行环境样品微生物研究的分析方法．图3表4参13     

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.     

基于高通量测序的工业园区地下水和土壤细菌群落结构比较研究

为探究工业园区地下水和土壤细菌群落结构、多样性变化特征,采用高通量测序技术对地下水和土壤细菌16S r RNA基因高变区域进行序列测定。通过对Alpha多样性、物种组成、丰度和群落结构的分析,比较地下水和土壤细菌群落结构的异同。Alpha多样性的比较结果表明,土壤细菌群落多样性和丰富度明显高于地下水,地下水细菌群落多样性指数反映出地下水已受到周边污染源的影响。物种注释结果表明,地下水样品共检出48个细菌门,土壤样品共检出50个细菌门。变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)是地下水细菌群落的优势类群,共占93.54%,且该工业园区地下水细菌群落呈现出典型的淡水种群特征;土壤中优势细菌门为Proteobacteria、放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)、Firmicutes和芽单胞菌门(Gemmatimonadetes),共占85.21%。由于地下水和土壤两者的生态系统和理化环境的差异,致使Actinobacteria、Acidobacteria、绿弯菌门(Chloroflexi)、硝化螺旋菌门(Nitrospirae)、α-变形菌纲(Alphaproteobacteria)、δ-变形菌纲(Deltaproteobacteria)和Gemmatimonadetes占比在地下水和土壤细菌群落间差异显著,同时使地下水和土壤细菌群落各含有一些特有的优势细菌属(地下水2个,土壤4个)。基于高通量测序技术对工业园区样品的测序结果可以为地下水和土壤环境的生态评价提供方法依据。     

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     

不同植被下红壤性质对细菌碳源利用的影响

选取中国科学院红壤生态实验站的8种植被,用BIOLOG法检测了土壤细菌群落的碳源利用,并分析了16种土壤性质,利用主成分分析与回归分析方法研究了土壤性质对细菌群落碳源利用的影响.结果表明,7项土壤性质解释了细菌碳源利用总变异的54.9%,其中,土壤溶解性有机质碳氮比解释了14.3%,砂粘比、微生物碳氮比和溶解性有机碳解释了22.6%.水解氮和微生物氮解释了12.3%,速效钾解释了5.7%,表明红壤不同植被下细菌群落碳源利用受土壤性质的影响很大.     

A comparative study of microbial communities in soils amended by manures from pigs fed with organic versus synthetic feeds

We applied pyrosequencing to determine bacterial communities in soil samples taken from test fields amended by manures of pigs fed with green feeds (GPMS), synthetic feeds (APMS), and the reference field without swine manure amendment (NPMS), respectively. For each sample, 7050 effective sequences were selected and utilized to do the bacterial diversity and abundance analysis, respectively. In total, 2986, 2873, and 1602 operational taxonomic units were obtained at a 3% distance cut-off in GPMS, APMS, and NPMS, respectively. Bacterial phylotype richness in GPMS was higher than the other samples. NPMS had the least richness. The most dominant class in both GPMS and APMS is Betaproteobacteria, whereas Alphaproteobacteria is dominant in NPMS. Circa 7.5% sequences in GPMS were found to be affiliated with Burkholderiales order. Bacterial diversity and abundance in the soil varied with swine manure amendment, due to nutrient elements as well as toxic metals in swine manures. Compared with the soil amendment by manures from pigs bred with synthetic feeds, more bacterial diversity but less potentially pathogenic bacterial genera in the soil amended by manures of pigs fed with green feeds were found, which indicated that these manures were better land-applied organic fertilizers.     

菲和铬(Ⅵ)单一及复合暴露对土壤微生物多样性的影响

有机物和重金属已成为我国土壤环境中常见的2类污染物质,二者间复合污染引起的土壤生态环境风险不容忽视。本研究以多环芳烃模式物菲和典型重金属铬(VI)作为受试物质,采用PCR-DGGE分子指纹图谱技术,探讨这2种污染物单一及复合暴露对土壤微生物群落多样性的影响,并选用主成分分析、聚类分析和戴斯系数3种算法对微生物群落相似性进行了比较。结果表明,在暴露实验第1天,菲单一暴露低浓度组中微生物群落相似性产生了极为明显的变化,而到第7天时,菲和铬(VI)单一暴露高浓度组均对微生物群落结构相似性产生最大程度的影响;采用香农指数法评价微生物群落的多样性,发现在暴露实验第7天,菲和铬(VI)单一暴露高浓度组对微生物群落多样性的影响比复合暴露高浓度组更强,二者复合暴露的相互作用方式表现为拮抗效应。本研究证明低浓度菲短期暴露的效应高于高浓度暴露结果,因而多环芳烃菲自身及其在复合暴露中所扮演的角色尤其值得关注。     

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.