分子生态学方法对油藏细菌群落结构分析的比较

目前对油藏细菌群落结构分析方法繁多,为得到准确全面的群落分析结果,必须选取合适的方法。群落分析方法的优劣进行比较分析,选取从新疆陆梁油田注水井筛出的11株单菌,测定其16srDNA序列,制成包含7个菌属、9个菌种的混合菌液。应用基于PCR扩增的三种分子生态技术DGGE、T-RFLP、建立16SrDNA文库比较和分析了混合菌液细菌多样性。使用PCR-DGGE方法时发现,DGGE方法可灵敏地检测到序列1 bp的变化,能将不同菌株分开,但该方法经过切胶测序后的的目标序列较短,信息量不大且有时有一定误差,较适合用于定性对比;而用于菌群结构的分析时应结合其他方法。T-RFLP法可区分大部分菌属,但数据库不够完整,不能确定细菌群落组成;因此也不适合单独用在细菌群落检测中,可用作多个样品种群多样性的对比或结合其他方法对样品进行系统透彻解析。建立16srDNA克隆文库法成功鉴定到7个菌属8个菌种16SrDNA序列,更适用于油藏细菌群落结构的分析。     

Protection of repetitive DNA borders from self-induced meiotic instability

DNA double strand breaks (DSBs) in repetitive sequences are a potent source of genomic instability, owing to the possibility of non-allelic homologous recombination (NAHR). Repetitive sequences are especially at risk during meiosis, when numerous programmed DSBs are introduced into the genome to initiate meiotic recombination. In the repetitive ribosomal DNA (rDNA) array of the budding yeast Saccharomyces cerevisiae, meiotic DSB formation is prevented in part through Sir2-dependent heterochromatin formation. Here we show that the edges of the rDNA array are exceptionally susceptible to meiotic DSBs, revealing an inherent heterogeneity in the rDNA array. We find that this localized DSB susceptibility necessitates a border-specific protection system consisting of the meiotic ATPase Pch2 and the origin recognition complex subunit Orc1. Upon disruption of these factors, DSB formation and recombination increased specifically in the outermost rDNA repeats, leading to NAHR and rDNA instability. Notably, the Sir2-dependent heterochromatin of the rDNA itself was responsible for the induction of DSBs at the rDNA borders in pch2Δ cells. Thus, although the activity of Sir2 globally prevents meiotic DSBs in the rDNA, it creates a highly permissive environment for DSB formation at the junctions between heterochromatin and euchromatin. Heterochromatinized repetitive DNA arrays are abundant in most eukaryotic genomes. Our data define the borders of such chromatin domains as distinct high-risk regions for meiotic NAHR, the protection of which may be a universal requirement to prevent meiotic genome rearrangements that are associated with genomic diseases and birth defects.     

不同海拔区域内牦牛肠道菌群结构组成多样性研究

探索不同海拔牦牛肠道菌群结构组成,为不同海拔区域间牦牛的饲养交流提供帮助. 本研究随机采集海拔2 897~4 717 m的12头牦牛的新鲜粪便样本,用16s rDNA技术对样本宏基因组测定,比较分析高低海拔牦牛肠道菌群的OTU丰度差异,研究高低海拔区域的牦牛肠道微生物菌群结构组成. 结果显示,高海拔与低海拔地区牦牛肠道菌群在门、纲、目、科、属、种层面菌群结构组成不同. 高低海拔区域的牦牛肠道菌群有8个菌在属水平差异显著（P＜005）. 结果表明,高海拔与低海拔牦牛肠道菌群结构组成存在一定差异,低海拔地区牦牛肠道菌群OTU丰度比高海拔的更为丰富.     

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.     

Characteristics of rDNA intergenic spacer region from a waterbloom cyanobacteria species Oscillatoria sp.

Sequence of rDNA intergenic spacer region (ISR) from a waterbloom cyanobacterial species Oscilla-toria sp. was determined and analyzed. The results of sequence comparison showed that the spacer had a high level sequence divergence, suggesting the sequence may be a target sequence for developing cyanobacteria genus- and species-specific oligonucleotide probes. In addition, a 20bp sequence of rDNA ISR was found highly conserved in all species of cyanobacteria, which was not found in other eubacteria. This conserved sequence within a variable region indicates that it might be a functional oligonucleotide in the processing of the rRNA precursor.     

Distribution of highly repeated DNA sequences in Haynaldia villosa and its application in the identification of alien chromatin

Haynaldia villosa (L.) is a wild relative species of common wheat that possesses many beneficial genes that can be used for wheat improvement. The accurate detection of H. villosa chromosomes in the genetic background of wheat is critical for transferring its beneficial genes to common wheat by chromosome engineering. The aim of the present study was to investigate the distribution patterns of two repeated DNA sequences, pSc119.2 and pAs1, as well as two rDNA multigene family sequences, 45S rDNA and 5S rDNA, in the individual chromosomes of H. villosa for the future precise identification of alien chromatin in germplasm development and breeding programs. A set of common wheat-H. villosa disomic addition 1V-7V lines was used to determine these specific signals on individual chromosomes of H. villosa. The results showed that two rDNA probes, pTa71 (45S rDNA) and pTa794 (5S rDNA), were located on 1VS and 5VS, respectively, and the signal could be discriminated exclusively in the common wheat background as effective markers of 1VS and 5VS. Furthermore, all seven chromosomes of H. villosa could be distinguished clearly by fluorescence in situ hybridization using pSc119.2 and pAs1 as probes in combination. The utilization of these cytogenetic markers of repetitive sequences, combined with other molecular markers sometimes, will make it possible for a precise identification of alien chromosomes with high efficiency.     

甲醇酵母表达系统pMIRH型载体研究

在为甲醇酵母（Ｈａｎｓｅｎｕｌａｐｏｌｙｍｏｒｐｈａ）表达系统成功构建高拷贝整合型表达载体ｐＭＩＲＨ－１的基础上,构建了更能满足外源基因表达需要的、大小仅为７３００ｂｐ的高拷贝整合型表达载体ｐＭＩＲＨ－２．有关转化、筛选及传代稳定性试验结果表明,ｐＭＩＲＨ－２亦具有与ｐＭＩＲＨ－１相似的转化和筛选特性及高拷贝整合能力,且二者均可在非选择条件下稳定传代６０代以上．     

灰黄霉素高产变株与出发菌株18S rRNA基因序列的比较分析

根据真菌18S rDNA的保守序列设计引物,对灰黄霉素高产突变菌Penicillium griseofulvum F208与出发菌株Penicillium griseofulvum 3.5190的18S rRNA进行克隆测序及对比分析,并登录GenBank(序列号为EF608151和EF607282).依据18S rDNA建立的系统进化树表明突变株F208与出发菌株3.5190的遗传距离较远,而与Penicillium urticae亲缘关系最近.出发菌株3.5190与Penicillium commune亲缘关系最近.18SrDNA作为分子标记可有效地鉴别灰黄霉素产生菌(Penicillium griseofulvum)高产与低产菌株.     

两株产人参皂苷糖苷酶酵母菌株的18S rDNA和ITS序列分析

对两株产人参皂苷糖苷酶的酵母菌株进行核糖体18S rDNA和ITS序列克隆测定,获得了长度分别为1 477和1 478 bp的18S rDNA序列和长度分别为791和727 bp的ITS序列,对获得的基因序列进行比对及同源性分析,结果显示,两株酵母菌的18S rDNA序列的相似性达100%,而ITS序列的相似性则为66%,均与NCBI数据库中登录的啤酒酵母的相应序列同源性最高.从GenBank中选取部分不同种属的酵母菌ITS序列,以ITS为对象构建系统发育树,从分子生物学角度确定了两株酵母菌为酵母菌属的不同种类.     

Organization of genetic variation in individuals of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi (Glomeromycota) are thought to be the oldest group of asexual multicellular organisms. They colonize the roots of most land plants, where they facilitate mineral uptake from the soil in exchange for plant-assimilated carbon. Cells of AM fungi contain hundreds of nuclei. Unusual polymorphism of ribosomal DNA observed in individual spores of AM fungi inspired a hypothesis that heterokaryosis--that is, the coexistence of many dissimilar nuclei in cells--occurs throughout the AM fungal life history. Here we report a genetic approach to test the hypothesis of heterokaryosis in AM fungi. Our study of the transmission of polymorphic genetic markers in natural isolates of Glomus etunicatum, coupled with direct amplification of rDNA from microdissected nuclei by polymerase chain reaction, supports the alternative hypothesis of homokaryosis, in which nuclei populating AM fungal individuals are genetically uniform. Intrasporal rDNA polymorphism contained in each nucleus signals a relaxation of concerted evolution, a recombination-driven process that is responsible for homogenizing rDNA repeats. Polyploid organization of glomeromycotan genomes could accommodate intranuclear rDNA polymorphism and buffer these apparently asexual organisms against the effects of accumulating mutations.     

细菌种特异性的16SrDNA寡核苷酸探针数据库的初步构建

核酸二级数据库是生物信息学研究的重要领域 ,对生命科学的研究和发展起重要作用 .目前 ,国际核酸序列公共数据库中存在大量的细菌 16SrDNA序列 ,本文利用这些已知细菌的 16SrDNA序列 ,设计了细菌种特异性的寡核苷酸探针 ,将其结果存入二级数据库 ,以计算机网络为载体 ,开发了界面友好的通过WWW浏览器实现对数据库查询的系统 ,其查询结果形象直观 ,为设计细菌的种特异性寡核苷酸探针提供了参考和帮助 ,从而可加速对细菌分类及鉴定的进程 .     

反硝化菌株GW1的筛选及特性研究

研究利用反硝化培养基,从实验室厌氧反硝化颗粒污泥中分离、筛选出1株反硝化优势菌株GW1,通过16SrDNA序列分析对其初步鉴定,并研究了温度、pH值、碳源、碳氮比和硝酸盐氮质量浓度对菌株GW1反硝化特性的影响。研究结果表明,菌株GW1的16SrDNA基因序列与Paracoccus versutus有最大相似性,达到99.9%,Genebank登录序列号为GU111570;分离菌株呈革兰氏阳性;最佳反硝化条件:丁二酸钠为碳源,温度为35～40℃,pH值为7.0～8.0,建议工程应用碳氮比为3∶1(质量比)。该菌株特性的研究为解决反硝化速率过慢问题提供了技术支持。     

甘肃张掖地区葡萄天然酵母菌的筛选及其系统发育分析

目的：通过对张掖地区酵母菌的筛选,了解张掖地区酵母菌的生物多样性。方法：采用YPD培养基分离纯化菌株,通过26SrDNA基因D1／D2区序列分析确定菌种的系统进化地位。结果：分离筛选出6个属天然酵母菌。分别为：假丝酵母属Candida、Meyerozyma属、红酵母属Rhodotorula、孢汉逊属Hanseniaspora、酿酒酵母Saccharomycescerevisiae和毕赤酵母属Pichia,共10个种,其中包括6个疑似种。     

Genetic engineering of factor VIII

Genetic manipulation of mammalian cells has provided a means of producing unlimited quantities of a high purity, virus-free preparation of factor VIII--the most complex protein manufactured through rDNA technology to date.     

褶累枝虫16SrRNA基因分析

以缘毛目褶累枝虫为研究材料,探索和建立了适用于较难培养的单细胞原生动物的分子生物学研究方法．并测定了褶累枝虫１６ＳｒＲＮＡ基因３′端１１１５个核苷酸．通过比较分析,从分子水平探讨了累枝虫属与缘毛目其它属之间的亲缘关系,为进一步重构原生动物的系统图提供最基本的资料．     

核糖体RNA基因在丝核菌分子系统学研究中的应用

随着分子标记技术的应用与发展,核糖体RNA基因(rDNA)因其特殊的组成结构和演化方式被广泛应用于真菌分子系统学研究中,系统介绍了真核生物核糖体RNA基因的组成特点、特定区域的通用性扩增引物,及近年来在丝核菌不同融合群的鉴定、系统进化关系、遗传分化等研究中的应用.     

螺旋状专性化能自养铁氧化细菌ML-04菌株的分子鉴定

对1株螺旋状专性化能自养铁氧化细菌ML-04的16S rDNA基因PCR扩增产物进行了序列分析，并以16Sr DNA序列同源性为基础构建系统发育树，结果表明，ML-04菌株与氧化亚铁钩端螺旋菌(Leptospirillum ferrooxidans)具有较大的差异性，而与嗜铁钩端螺旋菌(Leptospirillum ferriphilum)的相似度达99．7％以上，属于同一类群．     

基于18S rDNA序列研究腺介虫亚目系统5发育关系

基于1720 bp 的18S rRNA 基因序列以 Darwinula sp．和 Cytherelloidea．munechikai 为外群,采用最大简约法(MP 法)和邻接法(NJ 法)对腺介虫亚目(Cypridocopina)3总科5科9种动物的系统发育关系进行了分析．结果表明：1)现生腺介虫亚目动物为单系群；2)两种营海水生活的介形类—海介虫总科(Pontocypridoidea)和大介虫总科(Macrocypridoidea)间的亲缘关系较近；3)现生腺介虫总科(Cypridoidea)也为单系群,但该总科内各科间关系较复杂,主要分为两支,其中腺介虫科的 Dolerocypris sp．与萤光介虫科的 Candona holzkanpfi 聚成一支；而萤光介虫科的 Cypria crenulata 和泥介虫科的 Ilyocypris．angulata 同时与腺介虫科其它动物聚为一支．     

基于18S rDNA序列研究腺介虫亚目系统发育关系

基于1 720 bp的18S rRNA基因序列以Darwinula sp.和Cytherelloidea. munechikai为外群,采用最大简约法（MP法）和邻接法（NJ法）对腺介虫亚目（Cypridocopina）3总科5科9种动物的系统发育关系进行了分析.结果表明：1） 现生腺介虫亚目动物为单系群;2） 两种营海水生活的介形类—海介虫总科（Pontocypridoidea）和大介虫总科（Macrocypridoidea）间的亲缘关系较近;3） 现生腺介虫总科（Cypridoidea）也为单系群,但该总科内各科间关系较复杂,主要分为两支,其中腺介虫科的Dolerocypris sp.与萤光介虫科的Candona holzkanpfi聚成一支;而萤光介虫科的Cypria crenulata和泥介虫科的Ilyocypris. angulata同时与腺介虫科其它动物聚为一支.