Strongylus asini (Nematoda, Strongyloidea): Genetic relationships with other strongylus species determined by ribosomal DNA

Genomic DNA was isolated from adult Strongylus asini collected from zebra. The second ribosomal transcribed spacer (ITS-2) was amplified and sequenced using polymerase chain reaction (PCR) based techniques. The DNA sequence was compared with previously published data for 3 related Strongylus species. A PCR-linked restriction fragment length polymorphism method allowed the 4 species to be differentiated unequivocally. The ITS-2 sequence of S. asini was found to be more similar to those of S. edentatus (87.1%) and S. equinus (95.3%) than to that of S. vulgaris (73.9%). This result confirms that S. asini and S. vulgaris represent separate species and supports the retention of the 4 species within 1 genus.     

The Aspergillus niger carbon catabolite repressor encoding gene, creA

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.     

Amplification and methylation of an esterase gene associated with insecticide-resistance in greenbugs, Schizaphis graminum (Rondani) (Homoptera: Aphididae)

The greenbug aphid, Schizaphis graminum (Rondani) has developed resistance to organophosphorus insecticides by the over-production of esterases that have been classified as Type I and Type II. The first twenty N-terminal amino acids of the Type I esterase were determined and used to design an oligonucleotide, which in conjunction with an active site primer derived from conserved sequences of other insect esterases and two internal primers specific for esterases from another aphid species resulted in a 0.85 kb genomic DNA fragment from resistant greenbugs. This was extended by 5′ RACE which provided approximately 1.2 kb of the 5′ end of the esterase gene. The 5′ DNA sequence corresponded to 19 of the 20 known amino acids of the Type I esterase, with the last needing only a one base change (probably resulting from a PCR artifact). Furthermore, the sequence showed very close similarity to the amplified E4/FE4 esterase genes of Myzus persicae (Sulzer). A comparison of sequences suggested that the S. graminum gene has introns in the same positions as the first two introns of E4/FE4, with the second intron being considerably larger in S. graminum. Probing of Southern blots with the 0.85 kb esterase fragment showed that the gene encoding the Type I esterase is amplified 4- to 8-fold in resistant S. graminum and that the amplified sequences contain 5-methylcytosine at MspI/HpaII sites, again in agreement with previous findings for M. persicae genes.     

共培养条件下绿木霉对褐环乳牛肝菌产酶的诱导效应

为了探讨绿木霉与褐环乳牛肝菌的互作机理,在体外共培养条件下,采用光学显微镜和扫描电镜对二者生长重叠部分进行体外观察,发现二者生长无相互影响,在营养生长方面几乎不存在竞争关系。为进一步揭示绿木霉与褐环乳牛肝菌的互作机制,采用体外诱导和生物化学等方法,向褐环乳牛肝菌发酵液中加入灭活绿木霉菌丝诱导物,每天对发酵液中的多酚氧化酶、几丁质酶、漆酶和中性蛋白酶等酶活性进行检测。试验结果表明,绿木霉诱导褐环乳牛肝菌产漆酶能力最强;在整个共培养过程中,多酚氧化酶和漆酶活力始终处于较高水平,在诱导培养第6天,这两种酶活性升至最高,分别达到25.2U/mL和1 580U/mL;灭活绿木霉菌丝对褐环乳牛肝菌几丁质酶的诱导具有“瞬时性”,在诱导培养第2天即检测到较高的几丁质酶活性;中性蛋白酶的活性变化基本上呈先上升后下降的规律,且能增大褐环乳牛肝菌中性蛋白酶的固有产量,形成“叠加效果”。综上所述,绿木霉对褐环乳牛肝菌几乎不存在营养竞争关系,但其灭活菌丝体对褐环乳牛肝菌发酵液的多种酶活性存在诱导增效作用。     

Cloning and characterization of femA and femB from Staphylococcus epidermidis

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.     

Phylogenetic identification of Sparganum proliferum as a pseudophyllidean cestode

Sparganum proliferum is characterized by continuous branching and budding, the resulting progeny invading all tissues of the human body, causing fatal sparganosis. Its life cycle, definitive hosts and the route of infection to humans have not yet been disclosed. Because its morphology is similar to Spirometra erinacei, the phylogeny of S. proliferum has been thought to be identical to or closely related to S. erinacei. However, the taxonomy of S. proliferum has not been established up to present due to the lack of definitive observations. In order to clarify the phylogenetic relationship between S. proliferum and S. erinacei, nucleotide sequences of mitochondrial NADH dehydrogenase subunit 3 gene (ND3) and four mitochondrial tRNA coding genes of S. proliferum and other pseudophyllidean cestodes were analyzed. The sequences of S. proliferum showed high similarity to those of S. erinacei, although they were clearly different from each other, indicating that the phylogeny of S. proliferum and S. erinacei is distinct. This is the first report showing the phylogenetic relationship among S. proliferum and other pseudophyllidean cestodes at the DNA sequence level.     

Direct sequencing of baculovirus genomic DNA: sequence determination of the engineered respiratory syncytial virus chimeric FG gene.

Primer-directed enzymatic sequencing has proven to be an efficient and effective method for sequencing various size double-stranded DNA templates. We previously developed a primer-directed sequencing procedure for using double-stranded cosmid (50 kb) DNAs as template. We are interested in using this method to directly sequence larger DNA templates. Towards this goal we applied this method to directly sequence an engineered gene that had been transferred and integrated into the 130-kb baculovirus genome. Both crudely prepared and CsCl gradient-banded baculovirus DNAs were tested and reasonable sequencing ladders were obtained for both types of DNA templates. As little as 3 micrograms of gradient-banded baculovirus DNA were found to be sufficient to obtain film exposure times similar to those observed for cosmid size templates, 24 to 48 h. Effectiveness of the described method was demonstrated by obtaining the complete sequence of the engineered respiratory syncytial virus chimeric FG gene (2.5 kb in length) directly from the recombinant baculovirus "Baculo-FG" genome. Thus, our results demonstrate first, that double-stranded DNA templates as large as 130 kb can be sequenced directly and second, that the nucleotide sequence of engineered genes integrated within the baculovirus genome can be determined without the use of any intermediate steps of procedures.     

Meiotic recombination between paralogous RBCSB genes on sister chromatids of Arabidopsis thaliana

Paralogous genes organized as a gene cluster can rapidly evolve by recombination between misaligned paralogs during meiosis, leading to duplications, deletions, and novel chimeric genes. To model unequal recombination within a specific gene cluster, we utilized a synthetic RBCSB gene cluster to isolate recombinant chimeric genes resulting from meiotic recombination between paralogous genes on sister chromatids. Several F1 populations hemizygous for the synthRBCSB1 gene cluster gave rise to Luc+ F2 plants at frequencies ranging from 1 to 3 x 10(-6). A nonuniform distribution of recombination resolution sites resulted in the biased formation of recombinant RBCS3B/1B::LUC genes with nonchimeric exons. The positioning of approximately half of the mapped resolution sites was effectively modeled by the fractional length of identical DNA sequences. In contrast, the other mapped resolution sites fit an alternative model in which recombination resolution was stimulated by an abrupt transition from a region of relatively high sequence similarity to a region of low sequence similarity. Thus, unequal recombination between paralogous RBCSB genes on sister chromatids created an allelic series of novel chimeric genes that effectively resulted in the diversification rather than the homogenization of the synthRBCSB1 gene cluster.     

美洲大蠊内生菌几丁质酶基因的克隆、表达及其活性研究 *

Objective: To clone the chitinase gene ChiA from the endophytes of Periplaneta americana soluble expression of the protein and to identify its function.Methods:The chitinase gene ChiA was amplified by PCR from the DNA of Serratia marcescens,which was separated from the gut of Periplaneta americana and obtained by subcloning. The expression plasmid ChiA/pET21b was constructed and analyzed by bioinformatics. The plasmid was transformed into E. coli BL21(DE3) and the postive strains were induced by IPTG at 20℃ for 20h. The bioactivity of the protein was determined by small punch test.Results:The cloned sequence was associated with Serratia marcescens ChiA gene of GenBank and their homology was 99%. The sequence encoded a protein containing of 571 amino acids and expressed stably in prokaryotic system.SDS-PAGE/Western blot show that the soluble target protein was obtained. The small punch test suggested that the target protein had the activity of decomposing chitin and was stronger than that of the Serratia marcescens.Conclusion:The chitinase gene ChiA of the Serratia marcescens from the gut of Periplaneta americana was cloned successfully. The soluble chitinase that shows marked bioactivity was attained by prokaryotic expression system, which has provided theoretical basis for its further application.     

基于转录组测序分析甜菜夜蛾卵巢细胞离体培养成系进程

【目的】本研究旨在分析甜菜夜蛾Spodoptera exigua蛹卵巢细胞建立细胞系的整个过程,探究细胞由体内到体外培养过程中其基因表达在转录水平的变化,为昆虫体外培养模型的建立提供理论基础。【方法】利用Illumina Hiseq测序平台对甜菜夜蛾蛹卵巢细胞离体培养过程中各阶段的细胞分别进行转录组测序,对获得注释的差异表达基因及其相关信号通路进行分析;通过荧光定量PCR对部分细胞周期相关基因(cycd和cdk4)、调控基因(cdc20,apc1,skp2和mad1)、增殖相关分子标志物(mcm4和pcna)在甜菜夜蛾卵巢细胞离体培养过程中的转录进行验证。【结果】甜菜夜蛾蛹卵巢细胞离体培养过程包括5个阶段:解剖获得离体的卵巢组织,卵巢组织贴壁培养后游离出原代细胞,细胞转化重新具备增殖能力,成功首次传代,以及能够连续传代15代以上建立细胞系。上述5个阶段的细胞经转录组测序、数据组装后共获得46 796条unigenes序列,组装得到序列长度完整性好;转录本unigenes序列拼接长度分布合理,样本碱基Q30均在94%以上。通过KEGG数据库获得注释的unigenes有1 473条,参与细胞过程紧密相关的20条信号通路,其中有92条unigenes在细胞周期信号通路中获得注释。聚类分析表明,在体内处于快速发育状态的卵巢细胞与同样处于增殖状态的细胞系基因表达模式非常接近。原代细胞由短暂停滞生长至成簇细胞的转化关键期,筛选到差异表达基因619个,cdk4在离体培养期表达量显著降低,cycd在细胞转化关键期之后表达量显著升高,cdc20,apc1,skp2和mad1在卵巢组织和细胞系的表达量显著高于原代细胞、转化关键期细胞和首次传代细胞的。从原代细胞至传代后,cycd的表达显著升高8.7倍,显著高于mcm4和pcna的变化水平。【结论】甜菜夜蛾卵巢细胞离体培养过程中5个阶段的细胞转录组测序获得的序列质量符合数据分析的基本要求。筛选获得了甜菜夜蛾蛹卵巢细胞经离体培养过程中的差异表达基因。原代细胞逆转增殖可能与cdk4,cycd,skp2和mad1等细胞周期调控基因表达有关。另外,cycd可作为原代细胞具备传代能力的标志物。     

Molecular cloning and expression of a mammalian homologue of a translationally controlled tumor protein (TCTP) gene from Penaeus monodon shrimp

We previously observed secretion of native-type Streptomyces mobaraensis transglutaminase (MTGase) in Corynebacterium glutamicum by co-expressing the subtilisin-like protease SAM-P45 from S. albogriseolus which processes the pro-region. In the present study, we have used a chimeric pro-region consisting of S. mobaraensis and Streptomyces cinnamoneus transglutaminases for the production of MTGase in C. glutamicum. As a result, secretion of MTGase using the chimeric pro-region is increased compared to that using the native pro-region.     

Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

内蒙古草原3种针茅属植物不同组织水平的生物量生殖分配

植物资源的生殖分配是链接进化生态学和功能生态学的纽带。该文从4个组织水平上研究了针茅属(Stipa) 3种植物克氏针茅(S. krylovii)、大针茅(S. grandis)和贝加尔针茅(S. baicalensis)的生物量生殖分配以及株丛和种群水平上可育散布体的数量和生物量。结果表明: 1) 3种针茅属植物在不同组织水平上的生物量生殖分配呈现明显分异。在株丛水平上, 克氏针茅和大针茅的株丛生物量分配到生殖枝的比例分别为44.3%和47.9%, 均显著高于贝加尔针茅的35.7%。在生殖枝水平, 克氏针茅的生殖枝生物量分配到穗器官的比例为30.3%, 显著低于大针茅的42.9%和贝加尔针茅的48.4%。在穗器官水平, 大针茅穗生物量分配到散布体的比例(63.9%)最高, 克氏针茅(49.9%)次之, 贝加尔针茅(39.1%)最低。在散布体水平, 贝加尔针茅的可育散布体生物量占散布体总生物量的比例为92.3%, 显著高于克氏针茅的67.2%和大针茅的71.3%。2) 尽管3种针茅属植物在不同组织水平上的生物量生殖分配存在显著差异, 但从最终可育散布体占株丛生物量的比例看, 克氏针茅为6.1%, 贝加尔针茅为6.3%, 大针茅为9.5%; 三者在生物量生殖分配上表现出明显的趋同效应。3) 3种针茅属植物生物量生殖分配的限制性环节存在显著差异。生殖枝向穗的生物量分配是克氏针茅和大针茅生殖分配的限制性环节, 株丛向生殖枝的生物量分配或穗器官向散布体的分配是贝加尔针茅生物量生殖分配的限制性环节。从可育散布体的数量和个体生物量看, 克氏针茅采取了倾向于拓展空间的增加散布体数量的策略, 而大针茅和贝加尔针茅逐步进化出了趋向于提高个体竞争能力的增加散布体个体生物量的策略。     

Rapid and reliable identification of Staphylococcus equorum by a species-specific PCR assay targeting the sodA gene

Rapid and reliable identification of Staphylococcus (S.) equorum was achieved by species-specific PCR assays. A set of primers targeting the manganese-dependent superoxide dismutase (sodA) gene of S.equorum was designed. Species-specificity of the primer set was evaluated by using a total of 112 strains (including 27 reference strains of the DSM collection), representing 26 different species of the genus Staphylococcus, 3 species of the genus Kocuria, and different strains of Macrococcus caseolyticus. By using primers SdAEqF and SdAEqR the expected PCR fragment was obtained only when DNA from S. equorum strains was used as template. The rapidity (about 4 h from DNA isolation to results) and reliability of the PCR procedures established suggests that the method may be profitably applied for specific detection and identification of S. equorum strains.     

喜马拉雅-横断山区优越虎耳草谱系地理学研究

利用叶绿体DNA非基因编码区rpl20-rps12和trnL-trnF作为分子标记,对喜马拉雅-横断山区优越虎耳草13个居群151个个体进行谱系地理学研究,旨在揭示优越虎耳草现有的遗传结构及其演化历程。共检测到19个单倍型,其中63%的单倍型为居群特有单倍型。研究还发现,优越虎耳草居群总的遗传多样性较高（H_t=0.868）,居群内平均遗传多样性较低（H_s=0.466）。分子变异分析（AMOVA）表明,优越虎耳草居群57.37%的遗传变异来自居群内,居群间遗传变异为42.63%。居群遗传分化系数N_st大于G_st（N_st=0.463,G_st=0.438,P>0.05）,但不显著,表明优越虎耳草在其整个分布范围内没有明显的谱系地理结构。中性检验结果表明,Tajima's D为负值（-1.348 32,P>0.05）而Fu's F_s*为正值（18.915 72,P>0.05）,但均不显著,结合歧点分布分析发现该物种在整个分布范围内未经历过居群扩张。此外,在本研究中优越虎耳草遗传多样性和核苷酸多样性较高的居群及大量特有单倍型在整个分布范围内随机分布,符合"微型避难所"假说。优越虎耳草居群在冰期可能随气候波动而发生分布范围的不断变化,最终在相互隔离的"高山岛屿"中发生异域分化,导致大量特有单倍型产生。因此,推测优越虎耳草与其生境中的乔木和灌木可能具有相似的谱系地理历史,它们可为优越虎耳草提供微型避难所而使之在冰期时保留下来。     

红花变豆菜叶绿体基因组结构及同属种间关系研究

红花变豆菜（Sanicula rubriflora F. Schmidt）是有药用价值的植物,全株干燥后与其他药用同属植物易混淆,种间关系存在争议,通过高通量测序技术对红花变豆菜叶绿体基因组测序,利用生物信息学方法对测序数据进行拼接、注释,首次报道红花变豆菜叶绿体基因组结构及特点,利用叶绿体基因组数据,提供种间分类新证据,并且分析相关类群的进化关系。S. rubriflora叶绿体基因组序列的长度为155 721 bp,其中包括一个85 981 bp的大单拷贝区（large single copy,LSC）和一个17 060 bp的小单拷贝区（small single-copy region,SSC）,它们被两个26 340 bp的反向重复区（inverted repeat sequence,IRs）隔开。红花变豆菜叶绿体基因组GC含量为38.20%,包含129个基因,其中84个蛋白质编码基因,37个tRNA基因和8个rRNA基因。红花变豆菜叶绿体基因组结构具有高度保守性,其中编码基因共有51 907个密码子,最多编码5 095个亮氨酸,最少编码689个色氨酸,简单重复序列分析共发现32个位点,大多数是单碱基重复的A/T类型。叶绿体基因组聚类结果支持天胡荽亚科（Hydrocotyloideae）是伞形科（Umbelliferae）内比较原始的类群;变豆菜亚科（Saniculoideae）和芹亚科（Apioideae）为姊妹类群,是伞形科较进化的类群;变豆菜属植物是一个相对自然的类群;红花变豆菜与黄花变豆菜（S. flavovirens）为近缘姊妹种,但是两者形态和地理分布差异较大。该研究结果为变豆菜属属下种间鉴定及其种间演化奠定基础。