新疆艾比湖湿地博乐河入口处土壤细菌多样性分析

【目的】了解新疆艾比湖湿地国家级自然保护区非培养土壤细菌群落组成及多样性。【方法】采用非培养法直接从湿地土壤提取总DNA进行16S r RNA基因扩增,构建细菌16S r RNA基因克隆文库。使用MspⅠ和AfaⅠ限制性内切酶对阳性克隆进行16S r RNA基因扩增片段的限制性酶切分析(Amplified r DNA restriction analysis,ARDRA),挑取具有不同双酶切图谱的克隆进行测序,序列比对并构建16S r RNA基因系统发育树。【结果】从土壤细菌的16S r RNA基因文库中随机挑取75个不同谱型的克隆子,共得到58个OTUs,系统发育归类为8个细菌类群:绿弯菌门(Chloroflexi)、蓝藻门(Cyanobacteria)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrob)和芽单胞菌门(Gemmatimonadetes)。其中,变形菌门为第一优势菌群,拟杆菌门为第二优势菌群,两者约占总克隆的65%。【结论】艾比湖湿地博乐河入口处土壤细菌多样性丰富,且存在一定数量的潜在微生物新种。     

弗氏柠檬酸菌1,3-丙二醇合成的代谢工程研究

【目的】研究弗氏柠檬酸菌(Citrobacter freundii) 1,3-丙二醇合成的代谢过程。【方法】构建甘油脱氢酶基因GSR-lacZ、1,3-丙二醇氧化还原酶基因PDO-lacZ和甘油脱水酶基因GL-lacZ等报告基因。在此基础上,构建3个相应的转座子突变文库。【结果】筛选到6株突变子,其相应关键酶表达水平提高1?11倍,1,3-丙二醇产量提高幅度为3%?50%。对转座子插入位点分析显示,5株突变子插入位点均为β-内酰胺酶(CKO_02592)编码基因,1株突变子插入位点为二氢硫辛酰胺基转移酶(CKO_02433)编码基因。进一步分析发现,β-内酰胺酶基因突变显著提高甘油脱水酶和甘油脱氢酶的表达水平,而1,3-丙二醇氧化还原酶表达水平没有变化;二氢硫辛酰胺基转移酶基因突变显著提高1,3-丙二醇氧化还原酶表达水平,其他两种关键酶基因表达水平不变。【结论】β-内酰胺酶和二氢硫辛酰胺基转移酶基因能够分别影响1,3-丙二醇合成代谢途径关键酶的表达,为构建工程菌株打下基础。     

Construction of a bacterial artificial chromosome (BAC) library of Lycopersicon esculentum cv. Stevens and its application to physically map the Sw-5 locus

The Sw-5 gene is a dominantly inherited resistance gene in tomato and functional against a number of tospovirus species. The gene has been mapped on chromosome 9, tightly linked to RFLP markers CT220 and SCAR421. To analyse the Sw-5 locus, a BAC genomic library was constructed of tomato cv. Stevens, homozygous for the Sw-5 gene. The library comprised 18 816 clones with an average insert size of 100 kb, corresponding to two genome equivalents. The library was screened by PCR using primers designed for the CT220 and SCAR421 sequences, resulting in a 250 kb contig of known orientation on the long arm of chromosome 9. Using degenerate primers based on homologous sequences in the nucleotide binding site of resistance gene sequences, three discrete PCR fragments obtained from this contig were cloned and sequenced. Analysis of these fragments revealed a high similarity with numerous resistance genes or resistance gene like sequences. The present data indicate that at least three different resistance gene candidate (RGC) sequences are present in the vicinity of marker CT220, supporting the view that a resistance gene family may be responsible for the unusually broad resistance to tospoviruses conferred by the Sw-5 locus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Construction and Characterization of the Microclone Library from Chromosome 1R in Rye

Two and five 1R chromosomes were microdissected from the metaphase spreads of rye ( Secale cereale L. ) root-rip cells with the aids of glass needles. The dissected chromosomes were amplified in vitro by the Sau3A linker adaptor mediated PCR technique, by which 0.3 to 2.5 kb smear DNA fragments were obtained. After hybridized with DIG labeled probes, it was confirmed that the PCR products of the microdissected chromosomes were homologous with the rye genomic DNA, and derived from the 1R chromosome as well. Then, the second round PCR products from five chromosomes of 1R were microcloned to construct the plasmid library, including 220 000 clones. 172 randomly selected clones were evaluated ranged in size from 300 to 1 800 bp. Furthermore, the genomic dot hybridization results indicated that the library contained nearly 42% medium/high repetitive sequences and 58% low/single copy sequences, and its redundancy was very low. In this research, many aspects of the 1R chromosome microclone library exceeded or approached those of the previous reports in the literatures. Those are potential for construction of a high density genetic map of chromosome IR, from which some important genes can be tagged and isolated.     

Genomic structure and localization of the human protein phosphatase 2A BRgamma regulatory subunit.

In the course of our analysis of genomic sequence from the human chromosome 4p16.1 region harboring both the Wolfram and Ellis van Creveld syndrome genes we have identified a sequence with high homology (98% at the amino acid level) to the rat cDNA coding for the protein phosphatase 2A BRgamma (PP2ABRgamma) regulatory subunit. Although the human cDNAs for both the BRalpha and BRbeta isoforms have been described previously, the BRgamma subunit has not yet been identified in humans. Here we describe the precise genomic organization and genetic localization of the human PP2ABRgamma gene.     

Sequence-ready 1-Mb YAC, BAC and cosmid contigs covering the distal imprinted region of mouse chromosome 7.

We have constructed approximately 1-Mb contigs of yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones covering the imprinted region in mouse chromosome band 7F4/F5. This region is syntenic to human chromosome 11p15.5, which is associated with Beckwith-Wiedemann syndrome (BWS) and certain childhood and adult tumors. These contigs provide the basis for genomic sequencing, identification of genes and their regulatory elements, and functional studies in transgenic and knockout mice, which should be of help to understand not only the mechanisms of imprinting but also the molecular events involved in the genesis of BWS and tumors.     

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。     

三氯乙烷污染地地下水中Dehalobacter属细菌定量与多样性分析

[目的]对某公司6个以1,1,1-三氯乙烷(1,1,1-Trichloroethane,1,1,1-TCA)为主要污染物的地下水样品中的降解微生物Dehalobacter spp.(Dhb)进行相对定量和多样性分析.[方法]采用气相色谱法测定6个样品中1,1,1-TCA、1,1-二氯乙烷(1,1-DCA)和氯乙烷(CA)的浓度;通过定量PCR法分别测定6个样品中Dhb占总菌的百分比;以16S rRNA基因通用引物和Dhb特异性引物扩增获得的PCR产物构建了6个样品的Dhb特异性克隆文库,所得序列与GenBank中的最相似序列构建系统发育树.[结果]6个样品中均有1,1-DCA和(或)CA的检出,推测此6处地下水中1,1,1-TCA可能存在生物降解.定量PCR结果表明,6个样品中Dhb丰度差异较大.6个Dhb特异性克隆文库获得41条序列,序列比对结果表明,与它们最相似的已知分类地位的序列全部属于Dhb属.这些序列按99％的相似性被划分成7个可操作性分类单元(OTU).其中24条序列属于OTU1,该OTU的序列与已知能阵解1,1,1-TCA的Dehalobacter sp.str.TCA1的16S rRNA基因序列相似性达98％;文库中的3个OTU与GenBank中16S rRNA基因序列同源性最高仅为95％-96％.[结论]该污染场地地下水中存在多样性较丰富的降解微生物Dehalobacter属细菌,它们可能与现场的1,1,1-TCA生物降解有关.     

敦煌莫高窟第98窟壁画表面菌斑的群落结构分析

【目的】确定第98窟壁画表面白色污染物内微生物微观特征, 分析其群落组成、结构特点及诱发壁画病害微生物产生的因素, 为石窟寺保护和旅游管理提供建议。【方法】利用无菌手术刀收集壁画表面白色污染物样品; 利用扫描电子显微镜(SEM)分析样品中微生物体微观形貌; 通过提取样品总DNA、扩增细菌16S rDNA、构建克隆文库、测序和系统发生关系分析等技术研究壁画微生物群落组成与结构特点。【结果】壁画白色污染物中存在大量具有微生物特征的结构体, 形态多呈短杆状和卵圆形, 大小在 (3.0?5.5)?μm×(1.5?2.5) μm之间。共得到克隆文库序列111条, 主要为变形菌门γ-变形菌亚门肠杆菌科(Enterobacteriaceae)与假单孢菌科(Pseudomonadaceae)成员。群落组成和结构分析表明所得序列主要隶属于肠杆菌属(Enterobacter)、埃希菌属(Escherichia)、固氮菌属(Azotobacter)、沙雷氏菌属(Serratia)和克雷伯菌属(Klebsiella); 埃希菌属和肠杆菌属为优势属, 分别占克隆文库中总序列的46.8%和35.1%, 二者在自然界分布广泛, 大多属于人类致病菌。【结论】莫高窟第98窟壁画表面白色污染物主要为病害细菌生长所形成的菌斑群落集成。变形菌门在壁画细菌克隆文库中占绝对优势, 壁画病害微生物的出现和蔓延可能与该洞窟之前长期旅游开放存在一定关联。