小麦种子中几丁质酶基因的扩增及序列分析

根据http://www.tigr.org中与小麦几丁质酶基因相关的序列TC187877,设计引物,分别从小麦品种Gamenya和苏麦3号中扩增到大小约为1 000bp的片段。经序列测定和软件分析比较,发现这些片段所编码的蛋白质氨基酸序列,都有CHITINASE-19.1和CHITINASE-19.2的基序,为第II类几丁质酶基因。扩增的核酸序列在GenBank上发表,登录号分别为AY973229和AY973230。     

Molecular cloning and primary sequence analysis of a gene encoding a putative chitinase gene in Brassica oleracea var.capitata

INTRODUCTIONPlantshavedevelopedseveralbi0chemicaldefensemechanismsinresp0nsetopath0gensandabioticstress.Fo1l0wingpathogenattack,plantsynthesizephenyl-propaniodpr0ductssuchaslignin,l0wm0l.wt.antimicrobia1comp0undsknownasphyt0alexins,andseveraldefense-relatedproteins.Amongthesepr0teinsare"pathogenesis-relatedproteins"includingthefungalcellwalldegradingenzymeschitinaseandP-1,3-glucanase[1].Endochitinasefromhigherplantscatalyzethehydr0lysis0fchitin,aP-1,4-linkedhomop0lymerofN-acetyl-D-glucos…     

一株异养硝化细菌的分离及系统发育分析

从大棚土壤中分离到一株异养型硝化细菌,命名为菌株HN,分离菌株为革兰氏染色阳性,球状或杆状。菌落颜色为橙红色。该菌株能以乙酰胺为唯一碳源和氮源进行氨化作用和硝化作用并产生亚硝酸。以硝酸钠为氮源时能进行反硝化作用。部分长度的16S rDNA 序列分析表明,分离菌株HN与Rhodococcus ruber 具有99％相似性。并用PHYLIPS程序将该菌株与报道的相关硝化细菌进行系统发育进化分析。本文首次报道Rhodococcus sp.HN为异养型亚硝化细菌。     

西藏根瘤菌新表观群的DNA同源性及16S rDNA全序列分析

在前期数值分类工作的基础上,对7株与Rhizobium关系较密切的分离自西藏部分地区豆科植物Trigonellaspp.和Astragalusspp.的根瘤菌所形成的独立表观群,通过DNA同源性测定及16S rDNA全序列分析进行了分类地位的进一步确定。结果表明:该独立表观群菌株的(G C)mol%为59.5%~63.3%,群内菌株间DNA同源性在74.3%~92.3%之间,中心菌株XZ2-3与相关Rhizobium种之间的DNA同源性在0%~47.4%之间,是不同于Rhizobium内各种的新DNA同源群。另外,16S rDNA全序列分析结果也表明,中心菌株XZ2-3占居Rhizobium系统发育分支中的一个独立亚分支,其与临近R.leguminosarumUSDA2370T和R.etliCFN42T之间的序列相似性分别为96.55%和96.62%。根据国际系统细菌学委员会提出的细菌种属分类标准,该独立表观群构成了一个不同于Rhizobium内各种的新种群。该研究结果丰富了现有根瘤菌分类系统,将为国际上现有Rhizobium的14个种中再增添一个新的分类单元。     

Sporichthya polymorpha represents a novel line of descent within the order Actinomycetales

Abstract The 16S rDNA of Sporichthya polymorpha was PCR amplified from genomic DNA and almost completely sequenced using the dideoxy dye-terminator sequencing approach and automated DNA analysis. The primary structure was compared to the homologous molecule from 160 representatives of 37 genera of the order Actinomycetales . No higher similarity than 91.7% was found and the phylogenetic tree shows this morphologically unique organism to from an individual line of descent outside the main radiation of spore- and mycelium-forming organisms.     

一种新的抗真菌几丁质酶基因的分离及其植物表达载体的构建

以西瓜尖镰孢菌诱导、提纯的豇豆抗真菌 I类几丁质酶 N端前 1 0个氨基酸序列测定的基础上 ,设计合成了引物 ,运用 PCR等分子生物学技术 ,从豇豆基因组中分离克隆了该特异几丁质酶成熟蛋白基因 ,测定分析了其全序列。该新基因全长 894bp,无内含子 ;具 Aat I、Aat II、Bgl I、Dpn I、Dpn II、Eco R II、Hae I、Hae II、Hae III、Hinf I、Hpa II、Mae II、Mae III、Nba I、Oxa I和 Sst IV酶切位点 43个 ;豇豆、Vigna unguiculata、菜豆、豌豆、烟草、小麦、水稻的同源性依次递减。扩增克隆了菜豆几丁质酶信号肽基因 ,并将其与豇豆几丁质酶成熟蛋白基因连接 ,再与 p BI1 2 1重组 ,成功构建了特异几丁质酶基因的植物表达载体 ,为进一步培育抗真菌病转基因西瓜新品种打下了坚实基础。     

Rapid detection and identification of the metabolically diverse genus Gordonia by 16S rRNA-gene-targeted genus-specific primers

The importance of the emerging genus Gordonia in industrial and environmental biotechnology is evidenced by the recent increase in associated publications and patents. But, investigations into potentially valuable Gordonia members are restricted by the limitations of current isolation and detection techniques. This motivated us to design a genus-specific oligonucleotide primer pair which could assist in rapid detection of species of the genus Gordonia by means of PCR-specific amplification. The Gordonia-specific 16S rDNA fragment (829 bp) was successfully amplified for all the reference Gordonia species with the designed primer pair G268F/G1096R. No amplification was noted for closely related species from other genera. The genus specificity was validated with 47 strains including wild-type isolates. Interestingly, two strains assigned earlier as Gordonia nitida (DSM 777) and Gordonia rubripertinctus (ATCC 21930) failed to produce a Gordonia-specific fragment with this primer pair. Further analysis of these two isolates based on 16S rDNA sequencing and phylogenetic analysis classified them to the genus Rhodococcus. Preliminary screening of soil samples with the Gordonia-specific primers was successful in terms of the rapid detection of nine Gordonia wild-type isolates.     

一株[艹屈]高效降解菌的分离鉴定及其降解特性

以多环芳烃[艹屈] (Chrysene)为选择培养基的碳源, 从焦化污泥中筛选出一株[艹屈]的高效降解菌SQ-1, SQ-1可在以[艹屈]为唯一碳源的无机盐培养基中生长, 经过电镜形态学观察、生理生化和16S rDNA序列分析, 并基于16S rDNA序列结果, 构建了该菌株的系统发育树。最终确定菌株SQ-1为木糖氧化无色杆菌(Achromobacter xylosoxidans)。又考察了[艹屈]的初始浓度、投菌量、pH值对SQ-1菌株降解[艹屈]效果的影响, 确定了最佳降解条件。结果表明, 该菌对水中[艹屈]具有良好的降解特性, 在[艹屈]浓度为40 mg/L、接种量10% (V/V)、pH 7.0~7.5、温度30°C条件下, 接种5 d后对[艹屈]的降解效率达到80%以上。     

拟诺卡氏菌16S rRNA,gyrB,sod和rpoB基因的系统发育分析

为了更好地了解拟诺卡氏菌属(Nocardiopsis)各物种间的系统发育关系,该属现有有效描述种的gyrB,sod和rpoB基因的部分序列被测定,结合16S rRNA基因,对拟诺卡氏菌属进行了系统发育重建。研究发现拟诺卡氏菌属gyrB,sod和rpoB基因的平均相似性分别为87.7%、87.3%和94.1%,而16S rRNA基因的平均相似性则达到96.65%,3个看家基因均比16S rRNA具有更高的分歧度。比较基于不同基因的系统树发现,由gyrB基因得到的系统树拓扑结构与16S rRNA得到的结构在亚群上基本一致。因此,gyrB基因在拟诺卡氏菌属的系统分类上比16S rRNA基因更具优越性。     

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。     

Evidence that the novel microorganism 'Z' may belong to a new genus in the family Chlamydiaceae

Abstract The purpose of this study was to investigate possible phylogenetic relationships of a new microorganism called 'Z'. The organism was previously shown to be similar to chlamydia in its growth cycle and some of its metabolic requirements, but different in others and in its major outer membrane protein. In this study we report the sequencing of 'Z's 16S ribosomal DNA and comparison of the sequence with that of other microorganisms, including chlamydia and rickettsiae. While chlamydial species have 95.5% sequence identity among themselves, 'Z' had 83% identity with them, and 73% identity with certain rickettsia-like organisms. Based on the sequence analyses and taking into account physiologic considerations, we believe that 'Z' may belong to a novel genus in the family Chlamydiaceae.     

Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis

The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L. acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%). Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples.     

应用16S rDNA克隆文库技术研究长期稻草还田对水稻土细菌多样性的影响

利用中国科学院桃源农业生态试验站施肥制度长期定位试验田对照(CK)和稻草还田(OM)施肥处理的土壤样品,应用16S rDNA克隆文库技术直接提取土壤微生物总DNA,分别构建细菌16S rDNA克隆文库,并进行序列测定和分析。结果表明,与对照(CK)相比,稻草还田(OM)后土壤细菌群落结构发生了显著改变,土壤细菌多样性和均匀度均有所降低。对照(CK)和稻草还田(OM)两个施肥处理的优势种群均为变形菌,酸杆菌次之;稻草还田减少了变形菌、疣微菌、绿弯菌和绿菌的分布,而增加了硝化螺旋菌的分布。16S rDNA系统发育分析则表明,稻草还田对酸杆菌群落结构影响最大,其次是疣微菌和δ-变形菌。     

Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)

AIMS: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. METHODS AND RESULTS: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were < or =2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were < or =2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. CONCLUSIONS: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.     

一株多功能芽孢杆菌的鉴定

【摘 要】 目的 从土壤筛选所得1株芽孢杆菌并对该菌进行鉴定.方法 通过表型特征、生理生化特性和16S rDNA序列同源性分析（Gen Bank登录号为: JN609382）。结果 形态学观察：菌落呈圆形,白色微黄,中央凸起,半透明,长时间培养容易形成褶皱;革兰染色为阳性;芽孢中生,周生鞭毛,细长且多。生理生化特征：参照《常见细菌手册》和《伯杰氏细菌鉴定手册》,其中接触酶反应、V-P测定、卵磷脂以及革兰染色呈阳性;能够利用D-葡萄糖、D-木糖、D-甘露醇产酸,并能使淀粉水解;不能利用丙酸盐,不能形成吲哚,不能水解酪氨酸,生理生化试验结果显示该芽孢杆菌与枯草芽孢杆菌的特征一致。分子生物学鉴定：在Gen Bank上经过BLAT分析,其与枯草芽孢杆菌的相似性最高（99％）。结论 鉴定该菌为枯草芽孢杆菌。     

内蒙古呼伦贝尔地区传统发酵乳中乳酸菌的多样性分析

【目的】对内蒙古呼伦贝尔地区传统发酵乳制品中乳酸菌资源的生物多样性进行研究。【方法】采用纯培养和16S rRNA基因序列分析法对内蒙古呼伦贝尔地区传统发酵乳中的乳酸菌进行多样性分析。【结果】从8份传统发酵乳制品(6份酸牛奶和2份酸马奶)样品中分离到24株乳酸菌,通过16S rRNA基因序列分析和系统进化关系分析将24株乳酸菌鉴定为2株Lactobacillus kefiranofaciens、2株Lactobacillus kefiri、5株Lactobacillus paracasei、3株Lactobacillus plantarum、1株Lactobacillus rhamnosus、6株Lactococcus lactis subsp.lactis、2株Leuconostoc mesenteroides subsp.dextranicum、2株Streptococcus thermophilus和1株Enterococcus faecium。【结论】Lactococcus lactis subsp.lactis为内蒙古呼伦贝尔地区传统发酵乳制品的优势菌种,占总分离株的25%,其次为Lactobacillus paracasei,占总分离株的20.83%。     

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.     

斑点叉尾鮰一株致病菌的分离鉴定及系统发育分析

从发生急性流行性传染病的斑点叉尾肝、肾分离到一高致病性的菌株(CCF00024),经人工感染实验证实其为该病的病原菌。对该菌的形态、生理生化及16S rDNA序列分析结果表明,其为非发酵型,严格需氧,革兰氏阴性杆菌,极生多鞭毛,对除麦芽糖和甘露糖以外的多种糖类不能利用产酸,氧化酶阴性,DNA酶、蛋白酶、脲酶、赖氨酸脱羧酶阳性,MR阴性。在以该菌16S rDNA序列(GenBank登录号AY970826)和GenBank及RDP数据库内同源性较高的细菌16S rDNA序列构建的系统发育树中,分离菌CCF00024与嗜麦芽寡养单胞菌(Stenotrophomonasmaltophilia)聚在一簇,特别是与S.maltophiliaM5-1的同源性最高,其序列相似性达99.6%,结合形态和生理生化特点将其鉴定为嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)。     

Endosymbiotic Microflora of the Vestimentiferan Tubeworm (<Emphasis Type="Italic">Lamellibrachia</Emphasis> sp.) from a Bathyal Cold Seep

Gutless vestimentiferan tubeworms are known to harbor endosymbiotic bacteria in a specialized tissue, the trophosome, which consists of lobules. The endosymbionts of vestimentiferans inhabiting sulfide-rich hydrothermal vents are monospecific for their host. In contrast, previous studies suggest that vestimentiferas of methane-rich seeps may host multispecific symbionts. Phylogenetic analysis and dot-blot hybridization of 16S ribosomal RNA genes (16S rDNA) detected 4 operational taxonomic units (OTUs) in the trophosome of the vestimentifera Lamellibrachia species from a bathyal methane-seep. The OTUs were closely related to 16S rDNA of the species belonging to -Proteobacteria (Sulfitobacter), -Proteobacteria (Janthinobacterium), and -Proteobacteria (Acinetobacter and Pseudomonas). Localizations of the 4 OTUs within the trophosome were confirmed by in situ hybridization (ISH). ISH signals of the -proteobacterial OTU were observed in the innermost zone of the trophosome lobules. In contrast, ISH signals of the - and -proteobacterial OTUs were observed at the periphery of the lobules; however, whether they occur inside or outside the lobules remains unclear. These results support the possibility that the studied methane-seep tubeworm has a microflora composed of multispecific endosymbionts. Present address (Yukimasa Higashide): Noto Marine Center, 3-47 Uchiura, Suzu, Ishikawa 927-0552, Japan Present address (Hiroyuki Kimura): Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 6 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki 305-8566 Japan     

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.