荷斯坦奶牛瘤胃微生物元基因组BAC文库的构建与分析

采用未培养技术和脉冲场电泳技术直接从瘤胃微生物提取到大小在2Mb左右混合微生物DNA,经HindⅢ不完全酶切获得50～100kbDNA片段,将其连接在pCC1BAC载体上,转化E.coliEPI300,得到瘤胃微生物BAC文库,经对文库的鉴定分析,该文库的平均插入片段54.5kb,空载体率小于2%,库容837Mb,共保存15360个克隆。通过对该文库进行部分酶活性筛选,获得具有淀粉酶活性的克隆16个;纤维素酶活性的克隆26个,而且能降解纤维素的克隆中25个呈现多酶活性。这些结果表明该文库具有重要研究价值。     

显微观察中国荷斯坦牛瘤胃纤毛虫的物种多样性北大核心CSCD

瘤胃纤毛虫是反刍动物瘤胃微生物的重要组成部分,在维持瘤胃微生态和对宿主动物供能等方面起重要作用。本研究采集5头中国荷斯坦牛(Bos taurus)瘤胃液样品固定染色,而后通过光学显微镜计数瘤胃纤毛虫的密度和属的组成及组成比,并对其中常见属进行观察和特征描述,旨在了解中国荷斯坦牛瘤胃纤毛虫的物种多样性,为反刍动物的瘤胃内纤毛虫生物多样性研究提供资料。结果发现,中国荷斯坦牛瘤胃纤毛虫在瘤胃液中的平均密度为(3.2±3.4)×10^(8)个/L,共检出13属瘤胃纤毛虫,其中检出率最高的3个属依次为内毛属(Entodinium)、单甲属(Eremoplastron)和双毛属(Diplodinium),检出率最低的3个属依次为鞘甲属(Elytroplastron)、后毛属(Metadinium)和多甲属(Polyplastron)。内毛属是中国荷斯坦牛瘤胃液中最主要的属,其平均组成比为84.0%±10.2%,而其他属的组成比都低于5%。本研究表明,中国荷斯坦牛瘤胃液中瘤胃纤毛虫具有丰富的物种多样性。     

荷斯坦奶牛瘤胃微生物元基因组Fosmid文库的构建与分析

采用包埋法提取荷斯坦奶牛瘤胃微生物大片段总DNA,纯化后脉冲场电泳回收大小为36～48 kb,与pcc2FOS vector连接,转染至大肠埃希菌EPI 300宿主细胞,构建瘤胃微生物Fosmid基因组文库.对文库进行鉴定,该文库平均插入片段大小约35 kb,共保存30 000个克隆,空载体率小于2%,库容达1 050 Mb.     

广西水牛瘤胃中的细菌多样性

[目的]了解广西水牛瘤胃中细菌的组成及其可能的降解纤维素细菌的主要类群。[方法]提取水牛瘤胃内容物和高效降解滤纸的水牛瘤胃内容物的富集培养物的宏基因组DNA,以宏基因组DNA为模板,扩增16S rRNA基因序列,构建该两种样品的细菌的16S rRNA基因文库。通过对16S rRNA基因序列的分析,了解这两种样品的细菌群体种类及数量。 [结果] 水牛瘤胃内容物与其富集培养物中均主要含有LGCGPB (low G+C Gram-Positive Bacteria)、CFB (Cytophaga-Flexibacter-Bacteroides)两大类菌群和少数的螺旋体菌（Spirochaetes）,且LGCGPB所占的比例都是最高的,LGCGPB在水牛瘤胃内容物细菌中的比例为56.66%,而在富集培养物细菌中的比例升高为73.33%。在水牛瘤胃内容物中,丝状杆菌（Fibrobacteres）占3.33%,但在富集培养物中未被检测到。而在富集培养物中占13.33%的变形杆菌（Proteobacteria）,在水牛瘤胃内容物中未被检测到。本研究还发现了分类地位尚未明确的一菌群（R46）。[结论]细菌类群LGCGPB、Proteobacteria可能在水牛瘤胃中的纤维素降解过程中起重要作用。此外,水牛瘤胃中的细菌组成和牦牛、牛、羊瘤胃中的细菌组成较相似但比例有所不同。     

瘤胃微生物多样性及功能性研究方法概述

庞大的瘤胃微生物群系之间存在着共生关系,影响着宿主的代谢,是反刍动物营养学的研究热点之一.通过基于16S rRNA的分子生物学方法,如探针法、实时定量PCR法、DGGE/TGGE,RAPD和RFLP技术等研究瘤胃微生物多样性及组成结构,应用宏基因组学如建立YAC文库、BAC文库等研究方法对瘤胃微生物功能特征进行更深入的研究,实现改善反刍动物乳、肉产品品质的目的.     

新疆泥火山细菌遗传多样性

为了解新疆乌苏泥火山细菌多样性,从泥火山泥浆样品中直接提取总DNA,构建了含150个有效转化子的泥火山细菌16S rDNA基因文库,转化子经菌液PCR及HaeⅢ酶切后获得16个不同带型,克隆测序结果表明,其分属于16个不同的分类单元.一部分序列与已知细菌类群的16S rDNA序列相似性较高,归属变形菌门(Proteobacteria),厚壁菌门(Firmicutes),梭杆菌门(Fusobacteria),放线菌门(Actinobacteria);另外一部分序列与已知细菌类群的16S rDNA序列同源性较低,可能代表新的分类单位.研究结果显示,泥火山环境中微生物种群丰富,值得进一步研究.     

瘤胃细菌GH48家族糖苷水解酶基因多样性

【目的】了解瘤胃细菌第48家族糖苷水解酶基因(GH48)多样性,为木质纤维素高效降解提供新的基因资源。【方法】通过基因序列比对,设计gh48的简并引物;同时提取两个瘤胃样品的总DNA和总RNA,并将总RNA逆转录成cDNA。以总DNA和cDNA为模板,通过PCR扩增分别建立克隆文库并对克隆文库进行测序;对所得序列进行out种类划分和聚类分析。【结果】本研究共得到了455条编码GH48家族蛋白的基因序列,核苷酸序列之间的相似性为58.65%-100%。对序列的进一步分析表明,89%可以作为区分其种类的界定标准。以此为依据确定所得到的基因序列分别编码66种不同的GH48家族蛋白,分别聚为5个相对独立的类群,其中新类群C中OTU65所代表的序列是cDNA克隆文库中的优势序列,分别占两个cDNA克隆文库的36.4%和19.5%。我们的结果揭示瘤胃细菌gh48基因具有丰富的多样性,同时,其中也存在优势表达的GH48家族蛋白。     

第六次北极科学考察海洋沉积物可培养细菌的多样性分析

【目的】研究北极海洋沉积物可培养细菌的菌种资源多样性。【方法】采用海水Zobell2216E培养基和涂布平板法对第六次北极科学考察获得的海洋沉积物开展细菌分离培养,通过16S rRNA基因系统发育分析了解可培养细菌的多样性。【结果】根据菌落形态特征,从40个站位的北极海洋沉积物样品中共分离并获得16S rRNA基因有效序列的细菌达445株;基于16S rRNA基因的相似性分析与系统发育研究结果表明,分离获得的细菌分属于细菌域的4个门、6个纲、13个目、28个科、49个属、91个种,其中γ-Proteobacteria占大多数;有12株与模式菌株的16S rRNA基因序列相似性小于97%,可能代表了6个潜在的细菌新物种;此次获得的细菌种类组成与以往第五次北极科考获得的相比,在属水平上差异较大。【结论】北极海洋沉积物中存在着丰富的微生物菌种资源,具有很多新型微生物仍未被发现,是亟待开发的微生物资源宝库。     

美洲大蠊成虫肠道可培养细菌多样性研究

【目的】了解美洲大蠊成虫肠道可培养细菌的多样性。【方法】运用纯培养法、数值分类和16S rRNA基因序列的系统发育分析对样品中可培养细菌多样性进行研究。【结果】从NA培养基中分离得到54株细菌,根据形态观察和部分生理生化特性,选取32个代表性菌株进行16S rRNA基因序列的系统发育多样性分析。结果表明,数值分类中的代表菌株在82%相似水平上可分为12个表观群;这些分离菌株代表20个物种,属于4个大的系统发育类群(Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria)的10个科、15个属。多数菌株属于Proteobacteria门(15株,占46.9%)和Bacteroidetes门(10株,占31.3%)。【结论】美洲大蠊成虫肠道内存在较为丰富的细菌多样性。     

载锌凹凸棒石黏土对瘤胃体外发酵细菌多样性的影响

【目的】研究载锌凹凸棒石黏土对瘤胃体外发酵细菌多样性的影响。【方法】本试验采用离子交换法制备载锌凹凸棒石黏土,利用16S rDNA测序技术分析了载锌凹凸棒石黏土对奶牛瘤胃体外发酵细菌菌群和多样性的影响。【结果】研究共获得1490959个有效序列和87662个OTUs。测序结果表明,与对照组相比,试验Ⅳ组中的细菌多样性在体外发酵24 h时提高,试验Ⅳ组中的细菌丰度在体外发酵48 h时提高。细菌菌群组成分析表明,60个样本中的优势菌门主要是厚壁菌门、拟杆菌门、变形菌门和黏胶球形菌门。与对照组相比,各试验组的厚壁菌门在体外发酵24 h和48 h时均显著增加(P0.05),试验Ⅳ组中拟杆菌门在发酵48 h时显著降低(P0.05);60个样本中共获得124个细菌菌属,其中对照组与试验组中普氏菌属含量均没有显著变化(P0.05)。在体外发酵48 h时,试验Ⅳ组中密螺旋体属含量与对照组相比显著增加(P0.05),而食物谷菌属和假丁酸弧菌属含量与对照组相比均显著降低(P0.05)。【结论】载锌凹凸棒石黏土对奶牛瘤胃体外发酵中细菌多样性产生一定影响,其影响随发酵时间和添加剂量而不同。     

丛生盔形珊瑚共附生可培养真菌多样性分析

旨在研究珊瑚共附生可培养真菌多样性。运用稀释平板法和基于ITS-rDNA基因序列的系统发育分析对珊瑚共附生可培养真菌多样性进行研究, 将所得到的基因序列与NCBI数据库GenBank中的序列进行相似性比较并构建系统发育树。实验中从丛生盔形珊瑚表面和内部共分离得到19株菌落形态各异的真菌。ITS-rDNA序列分析及形态学鉴定表明, 丛生盔形珊瑚共附生真菌主要包括曲霉菌Aspergillus sp.、枝孢霉菌Cladosporium sp.、炭角菌Xylariales sp.、青霉菌Penicillium sp.、葡萄穗霉菌Stachybotrys sp.、赤霉菌Gibberella moniliformis、镰刀霉菌Fusarium sp.等。分离得到的菌株中, 4-13与GenBank中已报道的基因序列的相似性仅为89%。结果表明, 与丛生盔形珊瑚共附生的可培养真菌较为丰富, 是潜在的新的微生物菌种资源, 具有进一步研究的价值。     

微生物系统发育多样性及其保护生物学意义

近年来,分子系统发育分析方法,特别是rBNA基因同源性分析方法,在微生物多样性的研究中发挥着越来越重要的作用．它克服了传统的微生物分离培养方法的限制,极大地促进了人们对微生物多样性的理解．在遗传信息同源性分析基础上得出的微生物系统发育多样性为多样性的保护提供了新的视点．它不但可以作为微生物多样性评价的手段,而且为多样性保护中优先秩序的确定提供了依据．同时也为生物多样性保护确定了目标,即最大程度地保护系统发育关系中所包含的信息．本文对微生物系统发育多样性的特点及其保护生物学意义进行了简要评述．     

Phylogenetic and functional diversity of bacterioplankton during Alexandrium spp. blooms

The phylogenetic and functional diversity of the bacterioplankton assemblage associated with blooms of toxic Alexandrium spp. was studied in three harbours of the NW Mediterranean. Denaturing gradient gel electrophoresis and DNA sequence analysis revealed the presence of a bacterium within the Roseobacter clade related to the presence of Alexandrium cells. Phylogenetic diversity was affected by the presence of Alexandrium spp., geographic situation and seasonality. In contrast, functional diversity, assessed with Biolog plates, was clearly affected by seasonality, but not by the presence of Alexandrium, indicating that the presence of the bacterium associated with the blooms was not enough to modify the metabolic pattern of the bacterioplankton assemblage.     

Phylogenetic diversity of planktonic archaea in the estuarine region of East China Sea

To examine the diversity and structure of archaeal communities in the Yangtze River estuarine region of East China Sea (ECS), the 16S rRNA gene clone libraries of two typical sites were constructed with the archaea-specific primers. In total, 71 clones randomly selected were screened by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and 21 clones with unique RFLP pattern were sequenced. All the sequences are clustered into the two groups of Marine Group I (MGI) and Marine Group II (MGII) which are affiliated with the phyla Crenarchaeota and Euryarchaeota, respectively. MGI clones dominate both libraries with 20 MGI sequences obtained. The majority of sequences are closely related to uncultured marine archaea except for two sequences of which the nearest neighbor is a newly identified isolate of nitrifying marine archaeon Nitrosopumilus maritimus (98% identity). The results indicate that ECS coastal waters are inhabited by archaeal community with low dominance and high diversity corresponding to the complex estuarine environments, suggesting that archaea may perform an important role in the estuarine ecosystem.     

A re-appraisal of the diversity of the methanogens associated with the rumen ciliates

The diversity of methanogenic archaea associated with different species of ciliated protozoa in the rumen was analysed. Partial fragments of archaeal SSU rRNA genes were amplified from DNA isolated from single cells from the rumen protozoal species Metadinium medium, Entodinium furca, Ophryoscolex caudatus and Diplodinium dentatum. Sequence analysis of these fragments indicated that although all of the new isolates clustered with sequences previously described for methanogens, there was a difference in the relative distribution of sequences detected here as compared to that of previous work. In addition, many of the novel sequences, although clearly of archaeal origin have relatively low identity to the sequences in database which are most closely related to them.     

繁茂膜海绵细胞内微生物多样性的研究

采用海绵组织离散、细胞分离的方法,对繁茂膜海绵细胞进行纯化、胞内微生物DNA提取,构建了繁茂膜海绵细胞内微生物的16SrDNA克隆,对其遗传多样性进行了分析,发现海绵细胞内微生物16SrDNA序列主要归类于紫硫细菌门(Proteobacteria)中的α-亚门、γ-亚门和浮霉菌门(Planctomycetes)等类群。与研磨直接提取海绵组织DNA所得海绵组织中总微生物多样性相比,海绵细胞内存在丰富的浮霉菌(23%),说明浮霉菌主要存在于海绵细胞胞内。     

Phylogenetic diversity and ecological features in the Egyptian flora

Until fairly recently, regional-scale ecological and evolutionarypatterns have tended to be ignored as conservation efforts have been concernedwith species and their habitats. Here we compare frequencies in the Egyptianflora of particular rank sizes (order, family and genus) with patterns ofspecies abundance (classified as very rare, rare, common, or very common) and anarray of life-history attributes. The angiosperm flora of Egypt is representedby 2446 taxa (2088 species), including taxa in 10 subclasses, 51 orders, 120families, and 742 genera. A high degree of monotypism was observed: four ordersare monotypic (each existing as single species), and have very rare overallabundances; 30 families are monotypic (17 of which are very rare or rare); and 354genera are monotypic (over 70% of which are very rare or rare). Fourteenfamilies (in particular the Resedaceae and Zygophyllaceae) have at leastone-fifth of their global species represented in the Egyptian flora. Introducedspecies in general, and tree, aquatic herb and liana life forms all are especially well represented among monotypic genera. Native taxa are highlyrepresented among rare and very rare abundance classes, while introduced taxadid not differ significantly in their abundance patterns, compared to overallflora values. Few large genera (>20 spp.) occur in the flora, with mostspecies concentrated in genera containing 8–19 species per genus.Similarly, few families were highly speciose. Annual and herbaceous species weresignificantly over-represented, mainly among large, speciose genera andfamilies. However, perennials, trees, shrubs, aquatic herbs, lianas and parasiticspecies were found mainly in families and genera having very few taxa.Life-history attributes may have important implications to speciation rates.Taxonomically based results, involving abundances and life-history attributes,are discussed in the context of biodiversity and conservation.     

Ammonia Assimilation in Rumen Bacteria: A Review

In the rumen bacteria, ammonia as the end product of nitrogen is incorporated into carbon skeleton (α-ketoglutarate) to yield glutamine and glutamate which are important nitrogen donors in nitrogenous compounds metabolism in cells. The enzymes glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase are involved in these processes. Some experimental results have proven that the global nitrogen regulation system may participate in the regulation of assimilation of ammonia in rumen bacteria. This review offers a current perspective on the pathways and key enzymes of ammonia assimilation in rumen bacteria with the possible molecular regulation strategy, while points out the further research direction.