转mapk双链RNA干扰表达载体黄瓜对根际土壤细菌多样性的影响

随着RNA干扰技术的发展,通过植物表达病原物特异的dsRNA来防治植物病害的转基因作物越来越多.转根结线虫mapk双链RNA表达载体的黄瓜能够通过RNA干扰作用沉默线虫的mapk基因,对根结线虫具有良好的防治效果.为了评价该转基因黄瓜的安全性,明确mapk双链RNA干扰表达载体转基因黄瓜植株对根际土壤细菌多样性的影响,采用16S rRNA基因克隆文库方法对非转基因黄瓜和转基因黄瓜土壤细菌群落多样性进行分析.结果表明,非转基因黄瓜土壤细菌文库包含124个OTU(可操作分类单元),转基因黄瓜土壤细菌文库包含122个OTU.2个文库共同拥有的OTU为115个.2个文库都包含1 3个类群细菌,Acidobacteria、Actinobacteria、Armatimonadetes、Bacteroidetes、candidate division BRC1、Chloroflexi、Firmicutes、Gemmatimonadetes、Nitrospira、Planctomycetes、Proteobacteria、Verrucomicrobia、unclassified_Bacteria. 其中 Proteobacteria、Bacteroidetes 、Chloroflexi和Acidobacteria是优势菌群.其他细菌类群数量相对较少.在纲分类水平上,两个文库包含的细菌类群一致,且各类群细菌比例差异不大.在Acidobacteria门中,Acidobacteria_Gp6为优势菌群.在Bacteroidetes门中,Sphingobacteria纲细菌数量最多.在Chloroflexi门中,unclassified Chloroflexi细菌最多.在Proteobacteria门中,其中Betaproteobacteria纲的细菌数量最多.从多样性指数角度分析,两种土壤细菌群落的Shannon、Simpson和Chao值差异不大.总体来看,两种土壤细菌类群差异不显著,转基因黄瓜未对根际土壤细菌群落产生明显影响.     

16S rDNA克隆文库法探索转基因香石竹对土壤细菌群落的影响

【目的】通过研究转基因香石竹对土壤细菌群落的影响,为转基因香石竹的环境安全性评价提供依据。【方法】通过构建16S rDNA克隆文库,分析种植转基因和非转基因香石竹的土壤中细菌的群落结构组成。【结果】转基因和非转基因香石竹土壤中,共有的菌群有变形菌门(Proteobacteria)、浮霉菌门(Planctomycetes)、酸杆菌门(Acidobacteria),其中α-变形菌门、β-变形菌门、浮霉菌门为优势菌群;而在放线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia)及未培养菌(Uncultured bacterium clone)等菌群存在部分差异。【结论】通过16S rDNA克隆文库方法揭示了转基因香石竹的土壤中细菌多样性十分丰富,其栽培对土壤细菌群落结构影响有限。     

转β-1,3-葡聚糖酶基因和几丁质酶基因棉花

为了提高棉花的抗枯、黄萎病的能力,用花粉管通道法,将烟草β-1,3-葡聚糖酶基因和菜豆几丁质酶基因导入棉花,获得了抗卡那霉素的转化植株。经PCR、PCR－Southern Blot检测,证明两种基因已插入到棉花基因组中。     

应用16SrDNA-RFLP方法分析宁夏地区稻田土壤细菌的多样性

水稻是宁夏地区主要粮食作物, 水稻种植也具有维持生态系统平衡, 防止土地荒漠化等重要的生态功能。而稻田土壤细菌是维持土壤生态功能的基础。但长期以来缺乏对干旱地区稻田土壤细菌多样性的认识。本研究采用非培养技术提取稻田土壤样品总DNA, 构建其16S rDNA克隆文库, 用PCR-RFLP分析进一步测序后聚类分析细菌群落的多样性。从稻田土样中分离获得了大于23 kb的DNA片段。PCR-RFLP共得到74种酶切带型, 序列分析发现77.3%的克隆序列与环境中未培养细菌的16S rDNA序列有较高的相似性, 仅有22.7%的克隆序列与数据库中可培养细菌有较高的相似性, 表明宁夏稻区土壤中的多数细菌尚未培养。系统发育研究发现74个序列分属于12个类群, 其中变形细菌所占比例最大(37.8%), 依次为酸杆菌(16.2%)、放线菌(12.2%)、拟杆菌(10.8%)、绿屈挠菌(10.8%)、浮游霉菌(8.1%), 另外有少量厚壁菌门、芽单胞菌门和疣微菌门细菌克隆。在变形细菌的序列中包括、、γ和δ 4个类型, 比例分别为13.5%、5.4%、12.2%和6.8%。表明宁夏稻区土壤中优势细菌类群为变形杆菌和酸杆菌, 且土壤细菌类群具有丰富的多样性。     

锦鸡儿植物对盐碱地土壤理化性质和细菌群落的影响

锦鸡儿属的多种植物在干旱/半干旱区域水土保持等方面具有显著的作用效果,该研究以柠条锦鸡儿、中间锦鸡儿和小叶锦鸡儿为材料,选择宁夏银北西大滩核心试验站的盐渍化荒地为研究对象,分析了3种锦鸡儿建植1年对盐碱地土壤理化性质及土壤细菌群落的影响,以了解该类植物在盐碱地改良中的作用及生态效应。结果显示:(1)种植锦鸡儿1年能够显著降低盐碱地土壤全盐含量和pH,提高土壤全氮和全磷含量。(2)种植锦鸡儿不影响其非根际土壤细菌群落的α多样性,但小叶锦鸡儿非根际土壤细菌群落的香浓指数最低。(3)不同种锦鸡儿对盐碱地细菌群落结构的影响有所不同,小叶锦鸡儿根际/非根际细菌群落的结构分别与对照土壤以及柠条、中间锦鸡儿根际土壤存在明显差异;在属水平,包括假黄色单胞菌属、新鞘氨醇菌属及根瘤菌属在内的12个优势细菌属的相对丰度在小叶锦鸡儿非根际土壤中显著增加。(4)db-RDA分析表明,盐碱地土壤全盐、全氮及全磷含量分别解释了锦鸡儿非根际细菌群落结构变异信息的23.5%、25.4%和22.2%,3种因子累计共解释48.5%的细菌群落变异(P0.05),但这些因子对锦鸡儿根际细菌群落的变化无显著影响。研究表明,短期种植锦鸡儿属植物对盐碱地土壤理化性质具有一定改良效果,且不影响非根际土壤细菌群落的α多样性,但不同种锦鸡儿对土壤细菌群落结构的影响存在差异。     

结合宏基因组末端随机测序和16S rDNA技术分析温室黄瓜根围土壤细菌多样性

土壤细菌在温室土壤环境中具有十分重要的生态功能,与温室作物以及微生物内部存在互作关系。研究土壤细菌的群落结构组成,有助于了解土地利用变化与生态环境效应之间的关系。结合16S rRNA基因克隆文库和宏基因组末端测序对温室黄瓜根围土壤细菌的多样性进行了分析。在16S文库中,根据97%的序列相似性水平划分OTU,共有35个OTU,其中优势菌群是γ-Proteobacteria,其次为Firmicutes,Bacillus为优势细菌。在纲分类水平上,16S文库和宏基因组末端测序结果均包含γ-Proteobacteria、α-Proteobacteria、δ-Proteobacteria、β-Proteobacteria、Actinomycetales和Firmicutes,各纲比例有差别;在优势种群属水平上,末端测序的结果包含的属多于16S文库(4035);在优势细菌种类上,两者反映的结果一致,均为Bacillus。但是,宏基因组末端测序包含了大多数的弱势种群,更能反映细菌多样性的真实水平。与露地土壤细菌16S文库相比较,土壤细菌多样性降低,这可能与温室多年连作,种植蔬菜种类单一直接相关。     

中国典型冻土区土壤可培养细菌多样性

[目的]对比分析中国典型高纬度冻土区和高海拔冻土区土壤可培养细菌的多样性.[方法]采用NM、TSA 、R2A 3种培养基分离培养不同冻土区土壤可培养细菌,用通用引物扩增分离的细菌16S rRNA基因,根据系统发育分析进行鉴定.[结果]从6个样品中得到冻土土壤可培养细菌的菌落数量为4.70×103 -2.57×105 cfu/g(土壤干重),根据不同的菌落形态分离出144株可培养细菌.纯培养物的16S rRNA基因部分序列分析表明:我国高纬度冻土区土壤样品中的细菌分别属于Firmicutes分支(59.52％)、Gammaproteobacteria 分支(38.10％)、Betaproteobacteria分支(2.38％),其中假单胞菌属(Pseudomonas)、芽胞杆菌属(Bacillus)、类芽胞杆菌属(Paenibacillus)的菌株为该区域的三大优势菌群.我国高海拔冻土区土壤样品中分离细菌属于Gammaproteobacteria分支(89.22％)、Firmicutes分支(8.82％)和Bacteroidetes分支(1.96％)o优势菌群为假单胞菌属( Pseudomonas).[结论]我国高纬度冻土区和高海拔冻土区土壤具有较高的可培养细菌多样性;不同类型冻土区土壤可培养细菌群落组成不同.本文研究结果将为我国冻土区土壤细菌资源研究与利用提供理论依据.     

土壤类型对花生根际土壤细菌群落多样性和产量的影响

为了解不同土壤类型花生根际土壤细菌菌群多样性差异与产量的关系,明确不同土壤类型花生生产特性的区域优势,以6个花生主产区代表性土壤为研究对象,采用盆栽实验利用高通量测序技术研究不同土类花生根际土与非根际土细菌群落结构和变化特征。对6个土壤样品细菌菌群丰富度和多样性分析显示,湖南邵阳红壤细菌丰富度和多样性均较低,河北滦县褐土细菌丰富度和多样性均较高。放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、绿弯菌门(Chloroflexi)、酸杆菌门(Acidobacteria)及芽单胞菌门(Gemmatimonadetes)等为共有优势菌门,但在不同土壤类型样本中的菌群丰度存在明显差异,其中湖南邵阳红壤中绿弯菌门优势最明显,其他土壤类型放线菌门和变形菌门优势较明显。根际土与非根际土细菌门丰度间有差异,湖南邵阳红壤根际土与非根际土各细菌门(包括放线菌门、变形菌门、绿弯菌门、酸杆菌门、芽单胞菌门以及厚壁菌门)丰度变化程度较其它地区大。冗余分析表明,土壤酸碱度(pH)、有机质(ORM)、碱解氮(N),速效磷(P)和速效钾(K)等土壤理化因子对细菌菌群组成和物种丰度均有...     

宁夏地区春季奶牛场牛舍土壤细菌群落特征

【背景】现代规模化生产模式下,牛舍环境管理是影响奶牛高效健康生产的重要因素。【目的】探讨牛场不同牛舍土壤细菌群落特征,为奶牛健康生产提供理论依据。【方法】采集宁夏某规模化奶牛场的哺乳犊牛岛、断奶犊牛舍、育成牛舍、低产泌乳牛舍、高产头胎泌乳牛舍、高产经产泌乳牛舍、干奶牛舍和病牛舍这8个不同牛舍的土样,每个牛舍6个重复,共48份土样。利用16S rRNA基因扩增子测序分析细菌群落结构与多样性,并对细菌群落的功能进行预测。【结果】不同牛舍土样细菌群落组成存在差异,并且8个牛舍中高产头胎泌乳牛舍土样的细菌群落多样性最高。哺乳犊牛岛土壤与其他牛舍土壤细菌群落在门水平上差异较大;泌乳期牛舍土样之间的细菌群落结构相似度较高。在门的水平上,拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和厚壁菌门(Firmicutes)是这8个牛舍土样共有的优势菌门。在属的水平上,嗜盐碱的盐单胞菌属(Halomonas)、具有潜在降解特性的Fermentimonas和栖海面菌属(Aequorivita)及致病菌的鸟杆菌属(Ornithobacterium)是犊牛期牛舍土样的优势菌属;嗜盐碱的Truepera是育成牛舍土样的优势菌属;致病菌的不动杆菌属(Acinetobacter)和Parapedobacter、耐药菌的Pedobacter是泌乳期牛舍土样的优势菌属。【结论】致病菌和参与硝酸盐呼吸的细菌主要分布在哺乳犊牛岛,嗜盐碱菌主要分布在断奶犊牛舍和育成牛舍,产甲烷的细菌主要分布在高产头胎泌乳牛舍。本研究分析了不同牛舍土壤细菌群落多样性,为奶牛健康生产提供理论依据。     

Molecular bacterial diversity of a forest soil under residue management regimes in subtropical Australia

A major operational change in exotic pine plantations of subtropical Australia has been the decision to retain postharvest residues on site. A long-term field experiment was established in February 1996 to examine the impacts of residue management regimes [i.e. the postharvest residues removed (G0R), natural amount of residues retained (G1R) and residue quantity doubled and retained (G2R)] on tree growth (F1 hybrid pine) and sustainable soil management. Twelve soil samples, which included the above three residue regimes with four replicates, were collected at plantation age 6.4 years. A 16S rRNA gene clone library was established following soil community DNA extraction, polymerase chain reaction amplification and cloning. A total of 324 clones, including 27 from each sample, were randomly selected and sequenced to represent the bacterial composition and diversity of the clone library and thus the soil bacterial community under the residue management regimes. Phylogenetic analyses indicated that Acidobacteria (37.6%) and Proteobacteria (35.6%) were the dominant components of the soil bacterial community, followed by Actinobacteria (14.7%), Chlamydiae/Verrucomicrobia (7.3%), Unclassified Bacteria (3.8%) and Gemmatimonadetes (1.0%). Analysis of molecular variance revealed that there was no significant difference in bacterial composition and diversity among the residue management regimes or their replicated samples.     

Molecular analysis of the bacterial community in a continental high-temperature and water-flooded petroleum reservoir

Water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China was analysed for its bacterial community and diversity. The bacteria were characterized by their 16S rRNA genes. A 16S rRNA gene clone library was constructed from the community DNA, and using restriction fragment length polymorphism analysis, 337 randomly selected clones were clustered with 74 operational taxonomic units. Sequencing and phylogenetic analyses showed that the screened clones were affiliated with Gammaproteobacteria (85.7%), Thermotogales (6.8%), Epsilonproteobacteria (2.4%), low-G+C Gram-positive (2.1%), high-G+C Gram-positive, Betaproteobacteria and Nitrospira (each <1.0%). Thermopilic bacteria were found in the high-temperature water from the flooded petroleum reservoir, as well as mesophilic bacteria such as Pseudomonas-like clones. The mesophilic bacteria were probably introduced into the reservoir as it was being exploited. This work provides significant information on the structure of bacterial communities in high-temperature, long-term water-flooded petroleum reservoirs.     

Analysis of bacterial community structure in the natural circulation system wastewater bioreactor by using a 16S rRNA gene clone library

A variety of physical and chemical parameters are routinely monitored during operation of the Natural Circulation System, a wastewater purification bioreactor in which only natural materials and no synthetic chemicals are used. However, the microbial community structures existing in the Natural Circulation System have not been well characterized. Thus, bacterial community structure and composition in this system were studied using clone library analysis of 16S ribosomal RNA genes amplified using PCR with universal bacterial primer sets. The PCR products were then subcloned into the pGEM-T vector. Each unique restriction fragment length polymorphism pattern, created by using two pairs of restriction endonucleases, was designated as an operational taxonomic unit (OTU). The Natural Circulation System comprises five tanks, the second and third of which play a major role in the bioreactor. Clone library pro-files and principal component analysis revealed differences in the bacterial community structures of the second (anaerobic chamber) and the third (aerobic chamber) tanks. However, the beta-proteobacteria, Bacteroidetes/ Chlorobi and gamma-proteobacteria groups were dominant in both tanks. Bacterial composition was more complex in the second tank (107 OTUs) than in the third tank (68 OTUs). Of a total of 154 OTUs in the clone libraries, only 21 were common to the two tanks. The results obtained in this study should provide important information for future research into and management of the Natural Circulation System wastewater bioreactor.     

盐碱旱地转基因抗虫棉田昆虫群落多样性

棉花是耐干旱和耐盐碱的经济作物。随着土壤的盐碱化和干旱化, 在人口数量和植棉成本剧增的背景下, 我国黄河流域和长江流域棉花种植面积锐减, 棉花种植被迫向滨海盐碱地和内陆及西北干旱地区转移。本文于2013年和2014年在山东东营滨海盐碱地和河北枣强半干旱轻度盐碱地以非转基因棉(‘中棉所49’)为对照, 以转Bt基因棉(‘中棉所79’)为试验材料, 分别作施农药和不施农药处理, 于每年5月初到9月中旬, 调查取样点棉株及地面上害虫及其天敌的种类和数量, 并分析不同施药处理下转基因和非转基因棉田昆虫群落的生物多样性参数差异。结果表明, 施药和不施药转Bt基因棉田昆虫群落和害虫亚群落昆虫的个体总数均低于非转基因棉田, 其中昆虫群落和害虫亚群落个体数在二者之间差异显著; 转Bt基因棉田昆虫群落和害虫亚群落昆虫的多样性指数和均匀度指数均高于非转基因棉田, 而优势集中性指数均低于非转基因棉田, 但差异均不显著。施药条件下两种棉田的昆虫群落和害虫亚群落昆虫个体总数、多样性指数和均匀度指数均低于不施药棉田, 优势集中性指数均高于不施药棉田, 但转基因棉田和非转基因棉田之间无显著差异。表明转基因抗虫棉在盐碱旱地对棉田靶标害虫具有较好的控制作用, 棉田昆虫群落稳定性较高, 昆虫群落对外界的入侵和干扰缓冲能力强, 而化学农药的使用对昆虫群落杀伤力较大, 容易导致某种昆虫的抗性产生和昆虫群落的不稳定, 但比非盐碱旱地棉田昆虫群落生物多样性低, 棉田生态系统更简单。     

Molecular phylogenetic diversity of the microbial community associated with a high-temperature petroleum reservoir at an offshore oilfield

The microbial community and its diversity in production water from a high-temperature, water-flooded petroleum reservoir of an offshore oilfield in China were characterized by 16S rRNA gene sequence analysis. The bacterial and archaeal 16S rRNA gene clone libraries were constructed from the community DNA and, using sequence analysis, 388 bacterial and 220 archaeal randomly selected clones were clustered with 60 and 28 phylotypes, respectively. The results showed that the 16S rRNA genes of bacterial clones belonged to the divisions Firmicutes, Thermotogae, Nitrospirae and Proteobacteria, whereas the archaeal library was dominated by methanogen-like rRNA genes (Methanothermobacter, Methanobacter, Methanobrevibacter and Methanococcus), with a lower percentage of clones belonging to Thermoprotei. Thermophilic microorganisms were found in the production water, as well as mesophilic microorganisms such as Pseudomonas and Acinetobacter-like clones. The thermophilic microorganisms may be common inhabitants of geothermally heated specialized subsurface environments, which have been isolated previously from a number of high-temperature petroleum reservoirs worldwide. The mesophilic microorganisms were probably introduced into the reservoir as it was being exploited. The results of this work provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs at offshore oilfields.     

Microbial community succession and bacterial diversity in soils during 77,000 years of ecosystem development

The origins of the biological complexity and the factors that regulate the development of community composition, diversity and richness in soil remain largely unknown. To gain a better understanding of how bacterial communities change during soil ecosystem development, their composition and diversity in soils that developed over c. 77 000 years of intermittent aeolian deposition were studied. 16S rRNA gene clone libraries and fatty acid methyl ester (FAME) analyses were used to assess the diversity and composition of the communities. The bacterial community composition changed with soil age, and the overall diversity, richness and evenness of the communities increased as the soil habitat matured. When analysed using a multivariate Bray-Curtis ordination technique, the distribution of ribotypes showed an orderly pattern of bacterial community development that was clearly associated with soil and ecosystem development. Similarly, changes in the composition of the FAMEs across the chronosequence were associated with biomarkers for fungi, actinomycetes and Gram-positive bacteria. The development of the soil ecosystem promoted the development of distinctive microbial communities that were reminiscent of successional processes often evoked to describe change during the development of plant communities in terrestrial ecosystems.