16S rRNA基因高通量测序分析牛粪发酵细菌多样性

将养殖粪便进行资源化处理,尤其是将粪便堆肥发酵后变为生物肥料还田,具有重要的经济、社会和生态效益。之前关于细菌在堆肥过程中的研究,大部分采用实验室培养、分离、鉴定的方法,由于受培养方式的限制,仅能分析粪肥中有限的细菌类别。16S r RNA基因作为生物物种的特征核酸序列,被认为是最适于细菌系统发育和分类鉴定研究的指标。本研究使用16S r RNA基因高通量测序技术,分析了牛粪自然发酵与添加益生菌剂发酵过程中细菌种群的多样性变化。结果表明,1)新鲜牛粪、自然发酵1个月、自然发酵6个月的牛粪中细菌种群并没有明显的变化规律,说明自然发酵过程主要依赖于新鲜牛粪中携带的细菌种群;2)添加益生菌发酵后,细菌种群明显不同于不自然发酵过程中的细菌种群,其中变形菌门(Proteobacteria)细菌显著增加,而厚壁菌门(Firmicutes)细菌显著减少,说明益生菌剂能够显著改变堆肥过程中的细菌种群。本研究对于理解牛粪堆肥过程、提高堆肥效果,以及新型堆肥益生菌剂的开发都具有重要意义。     

西藏牦牛mtDNA 16S rRNA基因的克隆及其遗传多样性分析

本研究首次通过克隆、测序获得了11个西藏牦牛类群共111头个体的mtDNA 16S rRNA基因全序列,并分析了西藏牦牛的遗传多样性、分类关系、起源和分化,为进一步保护和合理利用西藏牦牛遗传资源以及探讨牦牛类群的划分提供分子依据。结果表明：西藏牦牛16S rRNA基因全序列长度为1571 bp或1570 bp（LWQ4）;G＋C平均含量37.8%,具有明显的碱基偏倚性;平均核苷酸多样性（Pi）为0.00291,平均单倍型多样性（Hd）为0.8501±0.0009,111个样本共发现48种单倍型并聚为2簇,表明西藏牦牛具有较高的遗传多样性并存在2个母系起源;Kimura双参数遗传距离范围为0.00098-0.00694,11个西藏牦牛类群划分为2大类：嘉黎牦牛、巴青牦牛、丁青牦牛、工布江达牦牛、帕里牦牛、斯布牦牛、康布牦牛、桑桑牦牛、江达牦牛、桑日牦牛为一类,类乌齐牦牛单独为一类。本研究发现类乌齐牦牛具有较丰富的遗传多样性,并且发现了一个序列特异个体LWQ4,需要对其进行更深入的研究。牛种间的聚类结果显示,西藏牦牛与美洲野牛的亲缘关系最近,与普通牛、水牛的亲缘关系相对较远,本研究支持将牦牛从牛属（Bos taurus）中分离出来,作为一个独立的牦牛属（Poephagus）的观点。     

基于16SrRNA序列分析红耳龟肠道拟杆菌和厚壁杆菌菌群多样性

脊椎动物肠道内定植了大量的微生物群落,在正常肠道菌群中,拟杆菌菌群和厚壁杆菌菌群作为优势菌群存在,对宿主的生理健康起着重要的作用。为了更好地了解外来入侵物种红耳龟的肠道拟杆菌和厚壁杆菌菌群多样性组成,本研究提取3只成体红耳龟粪便的宏基因组DNA,利用最新的Roche454测序技术对样本菌群的16SrRNA基因V3～V5区域进行测序。测序结果显示拟杆菌菌群为2纲5科11属,其中拟杆菌纲（98．82±1．24）％是绝对优势菌群;厚壁杆菌菌群被鉴定为3纲11科38属,主要由梭菌纲（95．81±0．88）％构成。此外,测序还检测到大量未知的拟杆菌类群（4．38±1．64）％和厚壁杆菌类群（14．96±8．15）％。本研究表明成体红耳龟粪便中拟杆菌菌群和厚壁杆菌菌群具有多样性。基于16SrRNA基因的Roche454测序分析可以很好地揭示肠道菌群的组成结构。     

Tufa microbial diversity revealed by 16S rRNA cloning in Taroko National Park, Taiwan

Tufa is a carbonate sediment contains inorganic and organic substances such as algae, microorganism and invertebrate. Microbial diversity of tufa found in Taroko National Park was investigated using 16S rRNA cloning and fluorescent in situ hybridization (FISH). Eleven 16S rRNA phylotypes and 37 genus and group of bacteria were identified. Of total 381 clones isolated, proteobacteria occupied 25-30% whereas cyanobacteria dominated 16-28% in total microbial population in the three sites. Acidobacteria, agricultural soil bacterium, verrucomicrobia and firmicutes were, generally, distributed in the three sampling sites. Among the three sampling sites, Baiyang walkway is found to be the most diverse site in its tufa microbial composition, indicated by species richness plot and FISH.     

Vineyard soil bacterial diversity and composition revealed by 16S rRNA genes: Differentiation by geographic features

Here, we examine soil-borne microbial biogeography as a function of the features that define an American Viticultural Area (AVA), a geographically delimited American wine grape-growing region, defined for its distinguishing features of climate, geology, soils, physical features (topography and water), and elevation. In doing so, we lay a foundation upon which to link the terroir of wine back to the soil-borne microbial communities. The objective of this study is to elucidate the hierarchy of drivers of soil bacterial community structure in wine grape vineyards in Napa Valley, California. We measured differences in the soil bacterial and archaeal community composition and diversity by sequencing the fourth variable region of the small subunit ribosomal RNA gene (16S V4 rDNA). Soil bacterial communities were structured with respect to soil properties and AVA, demonstrating the complexity of soil microbial biogeography at the landscape scale and within the single land-use type. Location and edaphic variables that distinguish AVAs were the strongest explanatory factors for soil microbial community structure. Notably, the relationship with TC and TN of the <53 μm and 53–250 μm soil fractions offers support for the role of bacterial community structure rather than individual taxa on fine soil organic matter content. We reason that AVA, climate, and topography each affect soil microbial communities through their suite of impacts on soil properties. The identification of distinctive soil microbial communities associated with a given AVA lends support to the idea that soil microbial communities form a key in linking wine terroir back to the biotic components of the soil environment, suggesting that the relationship between soil microbial communities and wine terroir should be examined further.     

A phylogenetic microarray targeting 16S rRNA genes from the bacterial division Acidobacteria reveals a lineage-specific distribution in a soil clay fraction

We designed an oligonucleotide microarray using probe sequences based upon a phylogenetic analysis of 16S rRNA genes recovered from members of the bacterial division Acidobacteria. A total of 42,194 oligonucleotide probes targeting members of the Acidobacteria division at multiple phylogenetic levels were included on a high-density microarray. Positive control hybridizations revealed a linear relationship between hybridization signal and template concentration, and a substantial decrease in non-specific hybridization was achieved through the addition of 2.5 M betaine to the hybridization buffer. A mean hybridization signal value was calculated for each Acidobacteria lineage, with the resultant lineage-specific hybridization data revealing strong predictive value for the positive control hybridizations. The Acidobacteria phylochip was then used to evaluate Acidobacteria rRNA genes from a Wisconsin soil and within a soil clay fraction. The Acidobacteria hybridization profile revealed the predominance of Acidobacteria subdivisions four and six, and also suggested a decrease in the abundance of subdivision six relative to subdivision four in the soil clay fraction. The change in relative abundance of these subdivisions in a soil clay fraction was supported by data from quantitative PCR. These results support the utility of a phylogenetic microarray in revealing changes in microbial population-level distributions in a complex soil microbial assemblage.     

Intragenomic variation in the internal transcribed spacer regions between 16S–23S rRNA genes among the three copies of Sinorhizobium fredii strains

The soybean-nodulating Sinorhizobium fredii strain has been reported to possess three copies of rRNA gene operons. In the present study, we investigated the diversity of the 16S–23S rDNA internal transcribed spacer (ITS) regions of S. fredii strains. Based on the sequences of the ITS regions, we divided the sequences of the S.   fredii strains into two groups, type A and type B. A dot-matrix analysis indicated that the region flanked by tRNA-Ile and tRNA-Ala is longer in type A than in type B, whereas type B sequences possess longer regions upstream of tRNA-Ile and downstream of tRNA-Ala than those of the type A sequence. Restriction fragment length polymorphism of polymerase chain reaction product (PCR-RFLP) of the ITS region in the cloned plasmids as templates could reconstruct the PCR-RFLP pattern from the total DNA as a template. The results of Southern hybridization using the insert sequence between tRNA-Ile and tRNA-Ala in type A as a probe indicated differences in the copy numbers of the type A ITS regions among the strains tested. These results indicated that S. fredii strains possess the type A and type B sequences of the ITS regions at ratios of 3:0, 2:1, 1:2 or 0:3. These S. fredii strains may be useful biological materials for the study of intraspecific variations.     

Development of a 16S rRNA microarray approach for the monitoring of rhizosphere Pseudomonas populations associated with the decline of take-all disease of wheat

So far, the analysis of microbial populations associated with wheat monocropping-induced decline of take-all disease (Gaeumannomyces graminis var. tritici) has focused mainly on culturable biocontrol pseudomonads. The objective of this study was to develop a taxonomic rrs (16S rRNA gene) microarray to assess the changes in Pseudomonas populations taking place during take-all decline. The microarray contains 12 probes for five Pseudomonas phylogenetic clusters chosen because they include well-known plant-beneficial pseudomonads. Four of the clusters are within the ‘Pseudomonas fluorescens’ species complex. PCR primers were selected to target these five clusters, and they were validated using 53 pseudomonads belonging or not to these clusters. Microarray analysis of the pseudomonads enabled discrimination between strains from several Pseudomonas clusters. Rhizosphere samples were collected from field plots grown with wheat for 1 (low level of take-all disease), 5 (high level of disease) or 10 years (low level of disease, suppressiveness reached). Microarray data could distinguish Pseudomonas populations from some of the wheat plants grown in the same plot. When comparing treatments, there was a difference between years 1 and 10. Cloning–sequencing of rrs enabled to define more precisely this difference by identifying two major Pseudomonas populations, one associated with year 1 and the other with year 10 (disease suppressiveness), which represent new clades within the ‘P. fluorescens’ complex. These populations may be useful as soil quality indicators. In conclusion, the combination of microarray and cloning–sequencing approaches highlighted changes in the prevalence of two major Pseudomonas populations, giving new insights on the dynamics of root-associated pseudomonads during take-all decline.     

Genetic diversity of Rhizobium present in nodules of Phaseolus vulgaris L. cultivated in two soils of the central region in Chile

Although Phaseolus vulgaris L. is native from the Americas and is currently cultured in diverse areas, very little is known about the diversity of symbiotic nitrogen fixing Rhizobium (mycrosymbiont) in many of those cultures. Therefore, the aim of this study was to assess the genetic diversity of Rhizobium present in nodules of P. vulgaris in the central region of Chile. A method to extract DNA from surface-sterilized nodules was applied to two populations of the same seed variety grown in different fields. The 16S rRNA and nifH genes were amplified directly from the DNA extracted. DGGE analysis and clone libraries showed a restricted genetic diversity of the microsymbiotic populations that nodulate P. vulgaris. Both molecular markers revealed the presence of a microsymbiont closely related to Rhizobium etli in all the plants from the soils studied, indicating that the populations of Rhizobium sp. nodulating P. vulgaris in the central region of Chile displayed an extremely low genetic diversity. The level of genetic diversity in microsymbiont populations in plants grown in soils with different origin suggested that other factors rather than the indigenous soil rhizobial populations play a major role in the selection of the symbiotic partner in P. vulgaris.     

含油污泥的堆肥处理对微生物群落结构的影响

采用堆肥方法处理含油污泥,评价堆肥处理对含油污泥中石油烃的去除效果,并采用Biolog方法和构建16SrRNA基因克隆文库的方法对处理过程中微生物碳源利用特征和微生物群落结构进行了研究。结果表明,含油污泥经过90d的堆肥处理,石油烃降解率达53.3%±9.5%,显著高于对照处理。堆肥处理可以显著促进石油烃降解,是一种处理含油污泥的有效措施。Biolog分析结果表明,堆肥处理的孔的平均颜色变化率（AWCD）显著高于对照处理,堆肥处理提高了土壤微生物代谢活性。主成分分析结果表明,对照处理和堆肥处理的微生物碳源利用特征明显不同,堆肥处理改变了含油污泥中微生物的代谢功能特征。对照处理和堆肥处理的16SrRNA基因克隆文库之间存在显著差异,对照处理的优势类群是γ-Proteobacteria,堆肥处理的优势类群是Bacteroidetes,堆肥处理显著改变了含油污泥中的微生物群落结构。Marinobacter和Alcanivorax是对照处理中的优势菌,可能与石油烃的自然降解过程有关,而Pusillimonas和Agrobacterium可能对堆肥处理中石油烃的降解起一定作用。