Retrotransposon insertion-site context (RISC) typing: a novel typing method for Aspergillus fumigatus and a convenient PCR alternative to restriction fragment length polymorphism analysis

Retrotransposon(-like) sequences in Aspergillus fumigatus have been used as typing targets through restriction fragment length polymorphism (RFLP)/Southern blotting approaches. Differences in fingerprints between unrelated isolates are the result of variations in copy-number and differences in the regions flanking the retrotransposon elements. Here, we present retrotransposon insertion-site context (RISC) typing as a novel and convenient PCR-based typing alternative to the RFLP approach. RISC typing aims at amplifying the sequences flanking the retrotransposon-like sequences in A. fumigatus and allows large numbers of isolates to be analyzed in a timely fashion with excellent discriminatory power.     

压力应激影响烟曲霉致病力的研究进展

烟曲霉是环境中广泛存在的腐生真菌,是重要的机会致病菌。近年来随着免疫受损人群的增多,烟曲霉引起侵袭性曲霉病不断增加,在深部丝状真菌感染中居于首位。由于宿主体内环境与自然环境存在诸多差异,使得烟曲霉孢子适应体内环境条件是其能够生存并致病的前提。研究发现烟曲霉适应宿主环境的能力,如温度、氧化应激、渗透压、缺氧、营养缺乏等,与其致病力密切相关。对压力应激与致病力关系的研究有助于解析烟曲霉的致病机制,可为研发烟曲霉感染诊治的新途径提供理论基础。本文对烟曲霉的压力应激与其致病力的关系进行了综述。     

烟曲霉细胞壁的组分、组装及其功能概述

环境中普遍存在的腐生丝状真菌烟曲霉Aspergillus fumigatus在免疫功能低下或缺陷的人群中可引起多种急慢性疾病,包括致死率很高的侵袭性曲霉病。细胞壁作为真菌的细胞外骨架结构不仅起维持细胞形状、保护细胞抵抗外界压力等作用,在病原真菌极性生长、入侵新的生态域、启动宿主免疫反应中也起重要作用。细胞壁组分还是真菌感染的分子诊断基础和开发抗真菌药物的理想靶标。近几十年来烟曲霉细胞壁的遗传、生物化学及免疫学方向的研究使其成为研究真菌细胞壁的模式真菌。本文主要概述烟曲霉细胞壁的组分、分子组装机制及其在真菌生存和感染中的作用,并对未来研究方向提出了展望。     

Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus

Polymerase chain reaction using degenerate primers was used to identify genes encoding proteins of the ATP-binding cassette superfamily in Aspergillus fumigatus and Aspergillus flavus. In A. fumigatus, two genes (AfuMDR1 and AfuMDR2) encoding proteins of the ATP-binding cassette superfamily were identified. One gene (AflMDR1) was isolated from A. flavus and is the apparent homologue to AfuMDR1. AfuMDR1and AflMDR1 encode proteins of molecular weights 148 000 and 143 000, respectively, each containing 12 putative transmembrane regions and two ATP-binding sites. These proteins are arranged in two homologous halves, each half consisting of a hydrophobic region (encoding six putative transmembrane domains) and an ATP-binding site. The AfuMDR1 and AflMDR1-encoded proteins bear a high degree of similarity to the Schizosaccharomyces pombe leptomycin B resistance protein and to human MDR1. The second gene identified in A. fumigatus, AfuMDR2, encodes a protein of molecular weight 85 000, containing four putative transmembrane domains and an ATP binding domain. The encoded protein is similar to those encoded by MDL1 and MDL2, two MDR-like genes of Saccharomyces cerevisiae. Expression of AFUMDR1 in S. cerevisiae conferred increased resistance to the antifungal agent cilofungin (LY121019), an echinocandin B analog.     

Purification, N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.     

Major surface protein 1a effects tick infection and transmission of Anaplasma marginale.

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

我国黄曲霉遗传多样性与产毒特性

黄曲霉(Aspergillus flavus)是一种广泛分布的腐生真菌, 是黄曲霉毒素B (aflatoxin B, AFB)和圆弧偶氮酸(cyclopiazonic acid, CPA)的主要产生菌, 也是动植物的条件致病菌。全球的玉米、花生和棉籽均不同程度地遭到黄曲霉及其毒素的污染。黄曲霉菌株间在形态学、遗传学和产毒特性上变异较大, 且其居群遗传结构也尚不明确。为了揭示黄曲霉居群遗传结构及其产毒素特性的规律, 本研究选取了从我国26省区(包括大小兴安岭)不同环境中分离的黄曲霉88株, 结合模式菌株和国际权威菌株9株, 基于钙调蛋白基因(CaM)和β-微管蛋白基因(benA)进行多基因序列分型(multi-locus sequence typing, MLST), 使用MEGA 6.0和Structure 2.3.4软件进行系统发育学分析和居群结构推导, 并结合菌株的产毒特性(AFB和CPA)进行比较分析。结果显示本研究的97株黄曲霉可分为3个居群, 即黄曲霉居群I、黄曲霉居群II和米曲霉居群, 该97株黄曲霉共有17个序列型(sequence type, ST), 其中我国的88株菌分布于15个序列型。米曲霉居群均不产AFB, 黄曲霉居群I和II的菌株绝大多数都产AFB和CPA, 其产毒特性只具有菌株特异性, 与居群和序列型无关。黄曲霉菌株产毒特性与地理分布或农作物类型间存在一定关系。我国东北玉米产区、西北干旱棉花产区和南方花生产区的黄曲霉居群I和II菌株均产AFB和CPA, 我国青海可可西里和四川阿坝地区的黄曲霉仅产CPA而不产AFB, 不产AFB的米曲霉居群大部分来自我国气候和地理环境多样的华北地区, 该地区也是我国农村传统酿造黄豆酱的地区。     

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.     

Phylogeny and phylogeography of Arenaria section Pseudomoehringia

Phylogeographic and evolutionary studies are necessary to establish solid taxonomic treatments and to implement effective conservation programs. Unfortunately, a well‐argued and well‐founded taxonomic framework is still lacking for some Mediterranean taxa. This is the case of Arenaria section Pseudomoehringia, a group that currently comprises three species endemic to the Iberian Peninsula (A. funiculata, A. suffruticosa, and A. tejedensis) and A. glochidisperma, which is restricted to the Rif mountains, North Africa. However, the taxonomic boundaries and phylogenetic relationships among these species are still uncertain. To explore the evolutionary history and phylogeographic patterns within this section, analyses based on nuclear (amplified fragment length polymorphism and internal transcribed spacer) and plastid DNA markers (psbA‐3’ trnK‐matK and rps16) were performed. Our study has confirmed the monophyly of the section, in which two species are clearly identified (A. funiculata and A. glochidisperma) and an additional species complex (“A. suffruticosa + A. tejedensis complex”) is also supported as a monophyletic clade. The phylogeographic results point toward a dispersal event of a common ancestor of the group from the Iberian Peninsula, giving rise to A. glochidisperma in North Africa. Moreover, A. funiculata and A. glochidisperma are identified as closely related species edaphically differentiated across the Strait of Gibraltar. Our study indicates low levels of a recent gene flow among populations of the “A. suffruticosa + A. tejedensis complex,” which are genetically highly structured and suggest an isolation by distance pattern, probably due to a combination of ecological and geographic barriers. Furthermore, the taxonomic and conservation status of taxa included in Arenaria section Pseudomoehringia has been reviewed.     

黑木耳复合群中种类学名说明

黑木耳在我国已经有1 000多年栽培历史,是我国最重要的栽培真菌之一,多年来我国黑木耳的学名一直使用模式产地为欧洲的Auricularia auricula-judae。最近的研究表明A. auricula-judae实际为一复合种,该复合种在全球范围内有5个种。其中A. auricula-judae仅分布于欧洲;美洲有2个种,生长在针叶树上的美洲木耳A. americana和生长在阔叶树上的尚未命名的木耳。中国该类群有3个种：自然分布和栽培最广泛的黑木耳的学名应为A. heimuer,此外,短毛木耳A. villosula在我国东北也广泛分布,并有少量栽培,生长针叶树上的木耳为美洲木耳A. americana,主要分布于中国东北和华北。     

Loss of innovative traits underlies multiple origins of Aquilegia ecalcarata

Traits are basic attributes of organisms that form the basis for speciation and diversity. The floral nectar spur is a classic example of a key innovative trait. Differences in nectar spur morphology can lead to pollinator specialization and thereby promote reproductive isolation between species. Despite its importance, the nectar spur has been lost in some members of the columbine genus (Aquilegia), resulting in a new spurless trait, and the evolutionary influence of this trait has become a topic of scientific interest. Aquilegia ecalcarata is an important representative columbine species that lacks spurs. Here, we resequenced the genomes of 324 individuals from A. ecalcarata and four related species. We found that A. ecalcarata was divided into three groups based on the phylogenetic relationships and population genetic structures. Topology weighting analysis revealed that A. ecalcarata has multiple origins, and Patterson′s D statistic showed that the spurless trait may have one origin. Floral morphological analysis revealed significant differences between A. ecalcarata and its spurred sister groups, and the floral phenotypes of the three A. ecalcarata groups have identical or similar floral phenotypes. Our results confirmed that the spurless trait not only produced the phenotype of A. ecalcarata but also contributed to the emergence of the A. rockii phenotype. Moreover, the spurless trait promoted the divergence between A. ecalcarata and its close, spurred relatives. Our research shows that the loss of key innovative traits can play a very important role in speciation and species diversity.     

Daily Rhythms of Body Temperature in Acornys russatus: The Response to Chemical Signals Released by Acomys cahirinus

Two species of spiny mice of the genus Acomys—the golden spiny A. russaturs and the common spiny A. cuhirinus—are syrnpatnc in the and and hot parts of the Rift Valley in Israel. The coexistence of these two species is due to exclusion of A. russatus mice by A. cuhirinus mice from nocturnal activity. The aim of this research was to study if odor signals released by A. cahirinus mice can play a role in the exclusion of A. russatus mice. A. russatus mice with an implanted transmitter recording body temperature (T_b) were kept alone in a metabolic chamber under constant conditions of ambient temperature (27°C) and photoperiod (12 h light: 12 h dark). After 5 days of recording, chemical signals from an A. cuhirinus mouse were added through the air tube going into the metabolic chamber of the A. russatus mice. This treatment caused a shift of ∼ 2 h inT_b daily rhythm of the naive tested A. russutus mice, whereas no shift was observed in A. russatus mice that had been kept in the same room with the A. cahirinus mouse before measurements. These results strongly support the idea that chemical signals released by A. cahirinus mice can entrain the T_b rhythms of A. russatus mice. Therefore, it may be assumed that the exclusion of A. russatus mice from nocturnal activity by A. cuhirinus mice could be achieved through the odor released by the latter.     

鲟鱼分子鉴定方法的整合应用

鲟形目物种是国家重点保护水生野生动物和CITES附录物种。其人工养殖种群数量众多, 种类丰富, 产品贸易量大, 但种类鉴定困难。本文在厘清当前鲟鱼商业类群的基础上, 通过分析现有种类鉴定方法, 整合了线粒体DNA遗传分析、SNP分析和微卫星DNA分析的鉴定方法, 探讨其鉴定国际贸易所涉鲟鱼的可行性。结果表明: 上述3种方法整合应用可在11种纯种鲟鱼及其正反杂交产生的杂交鲟范围内进行盲检。当前共有贸易鲟鱼36种, 其中杂交鲟14种, 杂交鲟的亲本共涉及9种鲟鱼。整合方法可准确鉴定小体鲟(Acipenser ruthenus)、达氏鳇(Huso dauricus)、施氏鲟(Acipenser schrenckii)、欧洲鳇(Huso huso)、闪光鲟(Acipenser stellatus)、高首鲟(A. transmontanus)两两杂交所产生的杂交鲟, 小体鲟为母本与纳氏鲟(Acipenser naccarii)或富氏鲟(A. fulvescens)或中华鲟(A. sinensis)产生的杂交鲟, 纯种的达氏鳇、高首鲟、富氏鲟和中华鲟, 但无法准确鉴定纯种的西伯利亚鲟(Acipenser baerii)和俄罗斯鲟(A. gueldenstaedti)以及父母本涉及此两种鲟鱼的杂交鲟。由于已开发的分子标记仍有限, 上述结果是对当前CITES贸易所涉鲟鱼鉴定的最大范围, 可以满足一些鲟鱼野生种群保护、贸易产品检测、种质资源管理等情景下的鉴定需求。     

Fluorinated carbohydrates. IV. 4-deoxy-4-fluoro-D-galactose

An analytical study has been made of gum specimens from Acacia deanei, A. filicifolia (three specimens), A. leucoclada, A. parramattensis (two specimens), A. parvipinnula, A. silvestris, A. terminalis, and A. trachyphloia, which are species belonging to Series II ({Botryocephalae}) in Bentham's classification of the genus. The three specimens from A. filicifolia are all closely similar, but the specimens from A. parramattensis differ appreciably in parameters other than their sugar ratios. Several of the analytical values reported increase considerably the range of values established so far for Acacia gum exudates. The Botryocephalae species give gum exudates of at least 2 chemically distinct types. Group A species (A. deanei, A. parramattensis, A. parvipinnula, and {A. trachyphloia}) have low galactose-arabinose ratios (<2:1) but have strongly negative rotations, high intrinsic viscosities and molecular weights, and relatively high nitrogen, methoxyl, uronic anhydride, and rhamnose contents. Group B species (A. filicifolia, A. leucoclada, and A. terminalis) have high galactose-arabinose ratios (> ) but low negative or positive rotations, low intrinsic viscosities and molecular weights, and relatively low nitrogen, methoxyl, uronic anhydride, and rhamnose contents.     

烟曲霉FADB基因参与菌株耐药的潜在机理

FAD结合的氧化还原酶编码基因FADB(GenBank ID:Afu4g14630)的编码产物在烟曲霉中为一种与E4D结合的氧化还原酶,可能参与真菌的呼吸.为了探究其具体功能,本研究通过克隆烟曲霉E4DB基因,构建烟曲霉FADB基因的敲除株,了解该基因对烟曲霉药物敏感性、渗透压、氧化压力物质敏感性的作用机理.采用高通量...     

黑曲霉中生物合成赭曲霉毒素A的非核糖体肽合成酶基因的鉴定

赭曲霉毒素A(ochratoxin A,OTA)是国际癌症研究机构认定的"2B"类致癌物。黑曲霉Aspergillus niger是美国食品药品监督局认可的食品安全菌。然而近年来陆续发现某些黑曲霉菌株能够产生OTA,这会对人类健康构成潜在威胁。阐明黑曲霉生物合成OTA的关键基因有助于理解OTA生物合成机制,这对OTA污染的防控具有重要意义。本研究克隆了产OTA黑曲霉中非核糖体肽合成酶(NRPS)编码基因(An15g07910),并对其进行了生物信息学分析,在此基础上采用同源重组的方法敲除了该基因,获得了一株性能稳定的敲除突变株Δnrps。与野生株相比,Δnrps突变株的表型在CYA培养基中并无明显改变,但在7d培养期间完全失去了合成赭曲霉毒素α(ochratoxinα,OTα)和OTA的能力,而赭曲霉毒素β(ochratoxinβ,OTβ)的合成不受影响。在野生株培养过程中,该nrps基因前4d表达量逐渐增大,并在第4天达到最高,随后基因表达量逐渐下降并趋于稳定,这与OTA的含量变化基本一致。结果表明该nrps基因(An15g07910)参与OTA的生物合成,其编码的NRPS可能负责催化苯丙氨酸部分和二氢异香豆素部分的交联。     

Induction of tannin acyl hydrolase (EC 3.1.1.20) activity in some members of fungi imperfecti

The potential of gallotannin, methyl gallate, gallic acid, and pyrogallol to induce tannin acyl hydrolase (EC 3.1.1.20) activity in Aspergillus niger, Aspergillus fischerii, Fusarium solani, and Trichoderma viride has been investigated. The maximum induction ratios recorded were A. fischerii (26.7), F. solani (26.1), and T. viride (40.7) when the fungi were induced with gallotannin, gallic acid, and methyl gallate, respectively. A. niger was devoid of basal enzyme activity. The gallotannin tolerance limits for A. niger, A. fischerii, F. solani, and T. viride were determined by progressively increasing the gallotannin concentration and were found to be 20, 4, 3, and 3%, respectively. F. solanii induced with 3% gallotannin has been recommended for the production of tannase enzyme.     

北京地区鼠类群落结构的研究

在生态系统中,啮齿动物为食物链的主要环节之一,研究鼠类群落结构及作用是评价北京地区环境生态的重要内容。有关鼠类群落的研究,国外已有较多报道(Hafner,1977;Grant和Birney,1979),近年来也引起我国动物生态学工作者的注意(刘季科,1979;钟文勤,1981;周庆强,1982)。而北京地区尚无这方面的报道。 根据我们1982年8月到1983年底在北京地区的调查,兽类区系中啮齿类约占34%,其中约一半种类为不同环境中的主要种类,现将鼠类群落结构的研究报道如下。     

Comparison between a native and exotic adelgid as hosts for Laricobius rubidus (Coleoptera: Derodontidae)

Laricobius rubidus LeConte, native to eastern North America whose primary host is Pineus strobi Hartig, has been observed in association with Adelges tsugae, an exotic and lethal pest of hemlock trees in eastern North America. Pineus strobi and A. tsugae were used to evaluate host preference and suitability of this predatory beetle to determine in part its suitability as a potential biological control agent of A. tsugae. In a paired-choice oviposition test, L. rubidus preferred to oviposit in P. strobi ovisacs over those of A. tsugae. However, in the no-choice oviposition test, there was no significant difference in the mean number of eggs laid by L. rubidus females in P. strobi or A. tsugae ovisacs. There were no significant differences in larval developmental time or survivorship for L. rubidus reared on a diet of either P. strobi or A. tsugae. Laricobius rubidus completed development to the adult stage on A. tsugae indicating that it is a suitable host and therefore has the potential to contribute to biological control of A. tsugae in the eastern United States. The introduction of a congener, Laricobius nigrinus to eastern North America may result in competition with L. rubidus on A. tsugae, but the inability of L. nigrinus to complete development on P. strobi suggests that the two congeners will not compete on the primary host of L. rubidus.