甘肃甘南传统发酵牦牛乳中细菌菌群的多样性研究

为了研究甘肃甘南传统发酵牦牛乳中细菌菌群的多样性,本试验以甘肃甘南地区传统发酵牦牛乳为研究对象,基于Illumina MiSeq高通量测序技术,对13份发酵牦牛乳的16S rRNA中V3-V4区进行测序,分析甘肃甘南地区传统发酵牦牛乳中细菌菌群的多样性.结果表明,最终获得720 221条高质量16S rRNA基因序列,...     

饲喂纳豆枯草芽胞杆菌对荷斯坦犊牛瘤胃细菌区系的影响

本研究选取8头(60日龄)犊牛随机分为对照组和处理组,对照组犊牛饲喂开食料,处理组在开食料中添加纳豆枯草芽胞杆菌(Bacillus subtilis)(1×10~6 CFU/g日粮)菌液,于断奶后2个月进行屠宰,采集瘤胃食糜构建16S rDNA克隆文库,随机挑取克隆进行测序,对照组16S rDNA克隆文库共有111个克隆,可分为88个操作分类单元;处理组16S rDNA克隆文库中有142个克隆,可分为131个操作分类单元.序列分析和多样性指数分析表明,两组犊牛瘤胃细菌区系多样性存在显著差异.拟杆菌门(Bacteroidetes)和壁厚菌门(Firmicutes)是两克隆文库中主要的代表菌群.对照组和处理组中的拟杆菌门分别占文库中总克隆数的38%和25%,而壁厚菌门分别占47%和57%.与瘤胃球菌相关的克隆在对照组克隆文库中分别占5%,而在处理组文库中分别占10%.RT-PCR结果显示,处理组中白色瘤胃球菌(R.albus)(log_(10)7.7/mL)和黄色瘤胃球菌(R.flavefaciens)(log_(10)8.1/mL)的数量比对照组(分别为log_(10)7.2/mL和log_(10)7.7/mL)分别增加了3倍和2.4倍.研究结果提示,饲喂纳豆枯草芽胞杆菌有助于促进断奶后犊牛瘤胃中细菌区系的建立,促进纤维分解菌群的定植和生长.     

小叶满江红内生细菌DGGE及EM多样性分析

摘要：用16S rDNA-PCR-DGGE技术、电子显微镜和平板培养的方法系统地揭示小叶满江红（Azolla microphylla）中内生细菌的遗传多样性和表型多样性,根据16S rDNA-PCR-DGGE的指纹图谱,作者认为在小叶满江红－蓝藻共生体内存在一个以蜡质芽孢杆菌及其变种为优势种群的复杂、多样的微生物区系,内生细菌在体内呈现的各异的超微结构特征和体外培养生成的不同大小、形态和色素的菌落便是证明。文中也讨论了应用DGGE研究遗传多样性的优点、不足以及内生细菌在满江红－蓝藻共生体的系统发育和协同进化中的可能作用。     

五氯酚厌氧生物降解体系中细菌的多样性分析

为了解五氯酚(PCP)降解过程中参与PCP降解的微生物多样性,本文应用16SrRNA基因克隆文库方法对PCP厌氧生物降解体系中细菌群落的组成和相对丰度进行了研究。结果表明,TM7类群的微生物在整个细菌群落中占有最大丰度(48.6%),检测到的序列与在三氯乙烯污染的地下水中检测的克隆子有一定的序列相似性(93.6%)。丰度位居第二的微生物类群为β-变形菌纲(Betaproteobacteria)细菌,其中的一些克隆子(10.8%)与脱氯微生物Dechlorosoma suillum具有极高的序列同缘性(99.7%)。此外,也检测到少数Clostridium属[厚壁菌门(Firmicutes)类群]的微生物。克隆文库中发现许多序列(占整个克隆文库的51.3%)与Gen Bank中已报道的序列具有较远的同源性(小于93.4%),它们可能代表新的微生物。本研究进一步拓宽了对PCP降解微生物多样性的认识。     

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。     

PCR-RFLP技术分析沼气池厌氧活性污泥细菌的多样性

应用PCR-RFLP和rRNA分析法研究了户用沼气池厌氧活性污泥细菌的多样性。采用直接提取法提取了户用沼气池微生物宏基因组DNA,构建了细菌的16S rDNA克隆文库。随机挑取了144个准确含有16S rDNA的阳性克隆进行PCR-RFLP分析,聚类得到46个OTUs（operational taxonomic units）,其中3个OTUs是优势类群,分别占14％,10％和9％,21个OTUs只含有单个克隆。随机挑取了26个克隆进行测序,并构建了系统发育进化树。结果表明：农村户用沼气池中细菌种类较为丰富,占优势的类群分别为Firmicutes（28％）、Delta-proteobacteria（18％）和Bacteroidetes（17％）,大多数16S rDNA序列与GenBank数据库中未培养细菌相似性最高（91％～99％）,为进一步研究、利用沼气池能源提供了基础资料。     

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。     

连作对再植枸杞根际细菌群落多样性和群落结构的影响研究

受枸杞道地产区土地资源等因素限制,连作障碍已成为影响枸杞产业发展的重要原因之一,导致严重的经济损失.研究连作条件下枸杞农田土壤生态系统微生物群落的演替规律对枸杞产业的可持续发展具有重要的理论意义.以宁夏银川市南梁农场连作多年的枸杞地为研究对象,利用Illumina MiSeq测序技术分析了连作对再植枸杞根际/非根际细菌群落的影响.结果表明,连作地显著抑制再植枸杞苗地径的增加,且其土壤pH较对照样地显著降低(p<0.05).测序结果证实,与对照样地相比,连作地再植枸杞根际土壤细菌物种数显著降低(p<0.05),细菌群落α多样性下降(p>0.05).主坐标分析表明,连作和对照样地间枸杞非根际细菌群落结构无明显差异,但连作显著改变再植枸杞根际细菌的群落结构.对细菌群落丰度的统计分析发现,连作地枸杞根际浮霉菌门、非根际假单胞菌门的相对丰度较对照样地显著降低(p<0.05).此外,冗余分析结果表明:枸杞园土壤pH和有效磷含量是影响枸杞非根际土壤细菌群落结构变化的主要因素,分别解释了41.8%和35.4%的群落结构变化(p<0.05),其他土壤因子无统计学意义,但土壤理化因子对再植枸杞根际细菌群落结构变化的影响均未达显著水平.这些结果证实连作能够显著抑制再植枸杞生长、影响再植枸杞根际细菌群落结构和多样性,干扰枸杞与土壤细菌群落间的互作关系.这些研究结果将为解析枸杞连作障碍机制提供理论基础.     

毛竹竹鞭内生细菌的特征及16S rDNA多样性分析

本研究利用传统的细菌分离方法,结合16S rDNA序列分析对毛竹竹鞭内生细菌的特征和多样性进行了分析。从福建省武夷山、将乐、长汀毛竹竹鞭中分离到34株内生细菌,初步归属于14属,20种。来源于不同地区的毛竹竹鞭内生细菌组成存在较大差异,其优势菌群为产碱杆菌属（Alcaligenes）和葡萄球菌属（Staphylococcus）。     

不同施肥水平下旱地土壤细菌群落多样性的RFLP分析

采用直接提取土壤微生物总DNA的方法,通过对不施肥(CK)、低量施肥(F1,N∶P2O5∶K2O为0·1gkg-1∶0·075gkg-1∶0·15gkg-1)、适量施肥(F2,N∶P2O5∶K2O为0·2gkg-1∶0·15gkg-1∶0·3gkg-1)和过量施肥(F3,N∶P2O5∶K2O为1·0gkg-1∶0·75gkg-1∶1·5gkg-1)等4种不同施肥水平土样DNA提取,扩增细菌16SrDNA基因片段,建立克隆文库。用限制性内切酶HhaI和RsaI进行PCR-RFLP分析,分别得到146、133、187、170个酶切类型。采用α多样性的测度对试验结果进行分析统计表明,不同处理间土壤细菌的多样性和物种丰富度均为F2>F3>CK>F1,表明合理施肥有利于土壤细菌的多样性;λ、R2、dMa和E指数在不同施肥处理间的变异系数达到12·86%～118·9%,尤其是Simpson指数λ变化最为敏感,处理间的差异最大。序列分析和系统发育树结果表明,不同施肥处理下供试土壤优势细菌的分布发生了改变。     

球等鞭金藻三级培养过程中细菌群落多样性分析

球等鞭金藻三级培养过程中生长差异明显,在完全灭菌的一级培养中生长最佳,在未灭菌的三级培养中生长最差。为揭示培养过程中藻际细菌群落多样性与其生长差异的相关性,以球等鞭金藻(Isochrysis galbana)为研究对象,利用Illumina HiSeq平台通过高通量测序的方法对球等鞭金藻三级培养过程中细菌群落多样性进行研究。结果表明,一、二、三级培养组之间的藻际细菌群落组成差异明显。Shannon和Simpson指数分析说明,一级培养组中细菌群落多样性显著低于二级和三级培养组。MetaStat分析发现,一级和三级培养组之间存在两株丰度显著差异的细菌,其中麦氏交替单胞菌(Alteromonas macleodii)在三级培养下丰度显著高于一级,而红球菌(Rhodococcus erythropolis)相反。进一步通过2216E平板涂布法分离并鉴定了麦氏交替单胞菌等17株球等鞭金藻藻际环境细菌;系统进化树构建结果显示,整个进化树分成13个分支,分别对应13个属,包括交替单胞属(Alteromonas)、假交替单胞属(Pseudoalteromonas)、海杆菌属(Marinobacter...     

Changes of Bacterial Community Structure in Copper Mine Tailings After Colonization of Reed (Phragmites communis)

Soil samples were collected from both bare and vegetated mine tailings to study the changes in bacterial communities and soil chemical properties of copper mine tailings due to reed (Phragmites communis) colonization. The structures of bacterial communities were investigated using culture-independent 16S rRNA gene sequencing method. The bacterial diversity in the bare mine tailing was lower than that of the vegetated mine tailing. The former was dominated by sulfur metabolizing bacteria, whereas the latter was by nitrogen fixing bacteria. The bare mine tailing was acidic (pH = 3.78), whereas the vegetated mine tailing was near neutral (pH = 7.28). The contents of organic matter, total nitrogen, and ammonium acetate-extractable potassium in vegetated mine tailings were significantly higher than those in the bare mine tailings (P< 0.01), whereas available phosphorus and electrical conductivity were significantly lower than those in the bare mine tailings (P< 0.01). The results demonstrated that 16S rRNA gene sequencing could be successfully used to study the bacterial diversity in mine tailings. The colonization of the mine tailings by reed significantly changed the bacterial community and the chemical properties of tailings. The complex interactions between bacteria and plants deserve further investigation.     

番石榴结合态多酚对小鼠肠道菌群结构及多样性的影响研究

为探究番石榴结合态多酚对小鼠肠道菌群的影响,本试验将CD-1小鼠分为空白组、高浓度组(500 mg·kg^-1)和低浓度组(100 mg·kg^-1)3组,在灌胃5周后,收集小鼠的无菌粪便,扩增肠道菌群16S rRNA基因高变域V3-V4,通过16S rRNA基因扩增子测序技术检测小鼠肠道菌群的α多样性、β多样性,以及门、属水平的物种组成。结果表明,与空白组相比,低浓度组中菌群的均匀性和丰富度显著提高(P<0.05);基于门水平分析,厚壁菌门、拟杆菌门和疣微菌门3个菌门的相对丰度值较大;在属水平中,高低浓度组均能降低拟杆菌属相对丰度,低浓度组能提高乳杆菌相对丰度,并且高浓度组中艾克曼菌属的相对丰度大幅提高。综上所述,番石榴结合态多酚能提高小鼠肠道菌群的均匀性和丰富度,且对菌群结构具有调节作用,既能提高益生菌的相对丰度,也能降低有害菌的相对丰度。本研究结果可为番石榴结合态多酚的活性研究和实际应用提供理论参考。     

Leaf litter is the main driver for changes in bacterial community structures in the rhizosphere of ash and beech

The rhizosphere and the surrounding soil harbor an enormous microbial diversity and a specific community structure, generated by the interaction between plant roots and soil bacteria. The aim of this study was to address the influences of tree species, tree species diversity and leaf litter on soil bacterial diversity and community composition. Therefore, mesocosm experiments using beech, ash, lime, maple and hornbeam were established in 2006, and sampled in October 2008 and June 2009. Mesocosms were planted with one, three or five different tree species and treated with or without litter overlay.Cluster analysis of DGGE-derived patterns revealed a clustering of 2008 sampled litter treatments in two separated clusters. The corresponding treatments sampled in 2009 showed separation in one cluster. PCA analysis based on the relative abundance of active proteobacterial classes and other phyla in beech and ash single-tree species mesocosm indicated an effect of sampling time and leaf litter on active bacterial community composition. The abundance of next-generation sequencing-derived sequences assigned to the Betaproteobacteria was higher in the litter treatments, indicating a higher activity, under these conditions. The Deltaproteobacteria, Nitrospira and Gemmatimonadetes showed an opposite trend and were more active in the mesocosms without litter. The abundance of alphaproteobacterial sequences was higher in mesocosms sampled in 2009 (P = 0.014), whereas the Acidobacteria were more active in 2008 (P = 0.014). At the family level, we found significant differences of the litter vs. non-litter treated group. Additionally, an impact of beech and ash as tree species on soil bacterial diversity was confirmed by the Shannon and Simpson indices. Our results suggest that leaf litter decomposition in pH-stable soils affect the soil bacterial composition, while tree species influence the soil bacterial diversity.     

芦苇定居后铜尾矿细菌群落结构的变化

Soil samples were collected from both bare and vegetated mine tailings to study the changes in bacterial communities and soil chemical properties of copper mine tailings due to reed (Phragmites communis) colonization. The structures of bacterial communities were investigated using culture-independent 16S rRNA gene sequencing method. The bacterial diversity in the bare mine tailing was lower than that of the vegetated mine tailing. The former was dominated by sulfur metabolizing bacteria, whereas the latter was by nitrogen fixing bacteria. The bare mine tailing was acidic (pH = 3.78), whereas the vegetated mine tailing was near neutral (pH = 7.28). The contents of organic matter, total nitrogen, and ammonium acetate-extractable potassium in vegetated mine tailings were significantly higher than those in the bare mine tailings (P < 0.01), whereas available phosphorus and electrical conductivity were significantly lower than those in the bare mine tailings (P < 0.01). The results demonstrated that 16S rRNA gene sequencing could be successfully used to study the bacterial diversity in mine tailings. The colonization of the mine tailings by reed significantly changed the bacterial community and the chemical properties of tailings. The complex interactions between bacteria and plants deserve further investigation.     

Community structure of bacteria on different types of mineral particles in a sandy soil

Various types of mineral particles in a soil probably provide different microenvironments for microorganisms. The purpose of this study is to investigate whether different types of mineral in a soil harbor different bacterial populations. DNA was extracted from five types (quartz, feldspar, pyroxene, magnetite, iron-coated reddish brown particles) of sand-size mineral particles separated from a sandy soil, and was amplified for partial 16 S rRNA gene by polymerase chain reaction (PCR). Twenty-nine to 69 amplicons per each type of mineral were cloned and sequenced, followed by phylogenetic affiliation of the sequences. As a result, some types of bacteria were detected on all of the types of mineral including the orders Rhizobiales, Bacillales, and Acidobacteriales. In the case of Acidobacteriales, higher percentages were found on magnetite and quartz. Some taxa were restricted to specific types of mineral; the class Actinobacteria was found on pyroxene but not on quartz, and rarely on magnetite and feldspar. Bacterial diversity at the order level estimated by Chao1 value was higher in feldspar and pyroxene than the other three types of mineral. The UniFrac Significance test indicated that the differences in bacterial communitiy structures among the particles were suggestive except that between feldspar and pyroxene. These results support the idea that different communities of bacteria were associated with each of the mineral types.