Influence of zinc ions on protein secretion in a heavy metal tolerant strain of the ericoid mycorrhizal fungas Oidiodendron maius

A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium. We have investigated some of the specific molecular responses of this fungal strain to the presence of increasing concentrations of zinc ions in the growth medium. In particular, we show that zinc ions induce a general change in the array of secreted proteins, with a shift towards the production of more basic, low molecular weight polypeptides. Some of these proteins were microsequenced and identified through homology search in databases. Among them are hydrolytic enzymes (nuclease, proteinase, lysozyme) and two superoxide dismutase isoforms. The latter are antioxidant enzymes known to play a role in heavy metal response in plants, animals and microorganisms.     

Specific regions in the Sod1 locus of the ericoid mycorrhizal fungus Oidiodendron maius from metal-enriched soils show a different sequence polymorphism

The huge diversity of fungi may reflect both the heterogeneity of the niches they occupy and the diverse stresses they must cope with. In order to investigate the genetic and functional diversity in the ericoid mycorrhizal fungus Oidiodendron maius subjected to heavy metal stress, we isolated O. maius strains from a serpentine site naturally enriched by heavy metals. Despite the high Cr and Ni soil concentrations, a high level of diversity was found in the serpentine fungal community. The growth of these isolates in the presence of different metal contaminants identified some tolerant strains, suggesting a site-specific adaptation. To investigate within-species gene divergence in stressful environments, we then compared the sequence polymorphism of a neutral (internal transcribed spacer) and a functional (Cu,ZnSOD) gene in O. maius isolates derived from the serpentine site, from a site heavily polluted with industrial wastes and from unpolluted sites. For all isolates tested, the polymorphism was higher in the nucleotide sequence of the functional gene. However, when compared with isolates from the serpentine area, isolates from industrially polluted sites showed a significantly higher polymorphism in the Cu,ZnSOD promoter region, suggesting that environmental stress may influence the rate of mutations in specific regions of the Sod1 locus.     

基因表达分析方法及其研究进展

近几年来,随着功能基因组学研究的兴起,基因表达研究的分析方法也在不断发展,主要有：差减杂交、差异显示、表达序列标签、基因表达的序列分析、微阵列杂交等。简要评述这五种方法的原理、优缺点等。     

Gene expression and role in cadmium tolerance of two PLAC8-containing proteins identified in the ericoid mycorrhizal fungus Oidiodendron maius

Fungi living in heavy metal polluted soils have evolved different cellular and molecular systems to adapt and survive in these harsh environments. Oidiodendron maius Zn is an ericoid mycorrhizal fungus previously shown to be highly tolerant to zinc thanks to antioxidative enzymes and membrane transporters. A novel gene, OmFCR1, was recently identified from this fungus because it conferred strong cadmium tolerance when expressed in yeast. OmFCR1 codes for a protein belonging to the PLAC8 family and physically interacts in yeast with the mismatch repair system, involved in DNA damage repair. The O. maius Zn genome also contains another gene – named OmFCR2 – that codes for a protein sharing with OmFCR1, the PLAC8 domain and other sequence similarities.     

EST-library construction using spore RNA of the arbuscular mycorrhizal fungus Gigaspora rosea

RNA was extracted from activated spores of the arbuscular mycorrhizal fungus Gigaspora rosea. Double-stranded cDNA was synthesised, digested and cloned into the vector lambda-ZAP express. Of the 1,500 clones obtained, 1.5% carried inserts of the rRNA gene cluster. After excision, inserts from 50 randomly selected clones were sequenced. Database searches revealed that 62% of the clones had similarities to already known sequences. These mainly code for proteins involved in translation and protein processing, replication and the cell cycle and cell signal transduction. One fragment probably belonged to a metallothionein-encoding gene which may be involved in heavy-metal binding. The method presented is an easy and rapid way to obtain short fragments of coding regions for expressed sequence tag libraries. Accepted: 28 November 2000     

Identification of heavy metal-induced genes encoding glutathione S-transferases in the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi are able to alleviate the stress for plants caused by heavy metal contamination of soil. To analyze the molecular response of arbuscular mycorrhizal fungi to these pollutants, a subtractive cDNA library was constructed using RNA from Glomus intraradices extraradical hyphae of a root organ culture treated with a mixture of Cd, Zn, and Cu. Screening by reverse Northern blot analysis indicated that, among 308 clones, 17% correspond to genes up-regulated by heavy metals. Sequence analysis of part of the clones resulted, amongst others, in the identification of six genes putatively coding for glutathione S-transferases belonging to two different classes of these enzymes. Expression analyses indicated that the genes are differentially expressed during fungal development and that their RNA accumulation dramatically increases in extraradical hyphae grown in a heavy metal-containing solution.     

Analysis of acid invertase and comparison with acid phosphatase in the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf and Kernan

Fractions of acid invertase and acid phosphatase of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan were compared by column chromatography and polyacrylamide gel electrophoresis. Acid invertase levels were measured during the exponential phase after 14 days growth in pure culture. Most acid invertase was wall associated (50%) with 41% forming an extracellular fraction and 9% a soluble, cytoplasmic fraction. The wall-bound fraction was partially solubilized by 1 M NaCl, bulked with the extracellular fraction and separated by gel filtration into two acid invertase activity peaks. These peaks corresponded closely to two acid phosphatase activity peaks measured in the same eluates. Anion exchange chromatography under a continuous salt gradient separated the invertase and phosphatase isoforms from each other. Non-denaturing polyacrylamide gel electrophoresis demonstrated that the more active isoforms of each enzyme have different electrophoretic properties and are high mannose-type glycoproteins with a high affinity for the lectin, concanavalin A. The results are discussed in terms of the functional aspects of the two enzymes and their cytochemical localization.     

Wall texture in the spore of a vesicular-arbuscular mycorrhizal fungus

Summary InGlomus epigaeum Daniels and Trappe, a vesicular-arbuscular mycorrhizal fungus, the mature spore has a complex multi-layered wall containing a regular pattern of wall subunits.The outer wall (2–4 m thick) consists of a simple layer of parallel microfibrils. The inner wall (5–6 m thick) is built from two layers possessing different organization. The innermost layer, near the plasmalemma has a texture of apparently dispersed fibrils, whereas the second layer is regularly organized with an arced texture. Ten to twelve bundles of fibrils connected by apparently bow-shaped fibrils are consistently observed. The appearance of this arced organization depends on the section plane and on the angle of observation in the electron microscope as confirmed by tilting experiments. Wall subunits are evident as straight electron transparent fibrils; particularly well-defined in negatively stained frozen sections: their diameter is about 3.5nm.The regular pattern of wall subunits in this fungal cell wall is compared with the textures shown by cellulose fibrils in algae or higher plants and by chitin fibrils in arthropod cuticle.Research work supported by CNR, Italy. Special grant I.P.R.A.—Sub-project 1. Paper No. 55.     

cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. The full-length cDNA of Zhikong scallop Chlamys farreri HSP90 (designated CfHSP90) was cloned by EST and rapid RACE techniques. It was of 2710 bp, including an open reading frame (ORF) of 2181 bp encoding a polypeptide of 726 amino acids with all the five HSP90 family signatures. BLAST analysis revealed that the CfHSP90 gene shared high similarity with other known HSP90 genes. Fluorescent real-time quantitative RT-PCR was used to examine the expression pattern of CfHSP90 mRNA in haemocytes of scallops exposed to Cd²⁺, Pb²⁺ and Cu²⁺ for 10 and 20 days, respectively. All the three heavy metals could induce CfHSP90 expression. There was a clear dose-dependent expression pattern of CfHSP90 after heavy metals exposure for 10 days or 20 days. Different concentrations of the same metal resulted in different effects on CfHSP90 expression. The results indicated that CfHSP90 responded to various heavy metal stresses with a dose-dependent expression pattern as well as exposure time effect, and could be used as a molecular biomarker in a heavy metal polluted environment.     

应用大规模测序技术和生物信息学研究造血干/祖细胞的基因表达及新基因的识别和克隆

从新生儿脐血和成人骨髓中分选出造血干／祖细胞（ＨＳＣ／ＨＰＣ）,构建成ｃＤＮＡ文库,对其进行大规模表达序列标签（ＥＳＴ）测序,通过生物信息学等手段分析基因表达谱,并进行新基因的全长ｃＤＮＡ克隆。在所测的１０５１２条可分析ＥＳＴ序列中,有９８６６条来自脐血ＣＤ３４＋细胞,其中４６９７条（４７６％）为已知基因,２６０３条（２６４％）为已知ＥＳＴ,１４１５条（１４３％）代表未知ＥＳＴ。在已知基因中,８２％基因与造血相关,２２７％涉及细胞代谢、结构和迁移,１３０％与细胞分裂和防御相关,２６２％与ＲＮＡ、蛋白质的合成相关,１０６％和细胞信号传递有关。对一些已知和未知的ＥＳＴ,综合测序、生物信息学等方法,进行全长克隆,已获得２３个新基因的全长ｃＤＮＡ。     

大片吸虫成虫cDNA表达文库的构建及其表达序列标签初步分析

为发现潜在的抗大片吸虫病的候选疫苗分子,控制大片形吸虫病,本研究以大片吸虫成虫为材料,应用SMARTTcDNA文库构建技术,构建了以表达载体λTriplEx2为基础的大片吸虫成虫cDNA表达文库。经测定,库容量为1·08×106PFU/ml ,重组率为96·6 %,扩增后的文库滴度为2·41×109PFU/ml ,插入片段平均大小约为1 000 bp;经大肠杆菌BM25·8质粒化后,从文库中随机挑选40个重组克隆测序,获得32条有效ESTs ;经BLASTX和BLASTn程序检索和分析,发现有9条ESTs代表已知基因, 16条ESTs相似性较低或无匹配,列为新基因。9条已知基因代表了半胱氨酸蛋白酶、卵壳蛋白、钙连接蛋白等三类功能蛋白,其余新基因也暗示与信号传导、蛋白合成、免疫刺激等基因相关,具有潜在的研究价值[动物学报51 (5) : 879 -883 , 2005]。     

Carbohydrate oxidases in ericoid and ectomycorrhizal fungi: a possible source of Fenton radicals during the degradation of lignocellulose

Isolates of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf et Kernan, and the ectomycorrhizal fungi Suillus variegatus (Swartz ex Fr.) and Pisolithus tinctorius (Pers.) Coker & Couch, along with a Cortinarius sp. and the white rot Phanerochaete chrysosporium Burdsall were examined for the ability to oxidize carbohydrates to their corresponding lactones and to excrete the H₂O₂ produced thereby. All except Phanerochaete chrysosporium were found to express cellobiose oxidase (cellobiose dehydrogenase, EC 1.1.19.88) and glucose oxidase (β- d -glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) when grown on cellobiose and glucose respectively. Production of extracellular H₂O₂ was visualized during growth on both substrates using ABTS as the chromogen. According to the Fenton reaction, H₂O₂ will react with hydrated or chelated Fe(II) in the environment to produce hydroxyl (Fenton) radicals, HO^·. Mycelial extracts from each of the mycorrhizal fungi produced HO^· in the presence of cellobiose and Fe(II), presumably mediated by H₂O₂ produced by cellobiose oxidase activity in the extracts. Conditions favourable to HO^· production were shown to exist in Modified Melin–Norkrans medium, and the data discussed in relation to previously observed lignin degradation by mycorrhizal fungi.     

When the ribosomal DNA does not tell the truth: The case of the taxonomic position of Kurtia argillacea, an ericoid mycorrhizal fungus residing among Hymenochaetales

The nuclear ribosomal DNA (nuc-rDNA) is widely used for the identification and phylogenetic reconstruction of Agaricomycetes. However, nuc-rDNA-based phylogenies may sometimes be in conflict with phylogenetic relationships derived from protein coding genes. In this study, the taxonomic position of the basidiomycetous mycobiont that forms the recently discovered sheathed ericoid mycorrhiza was investigated, because its nuc-rDNA is highly dissimilar to any other available fungal sequences in terms of nucleotide composition and length, and its nuc-rDNA-based phylogeny is inconclusive and significantly disagrees with protein coding sequences and morphological data. In the present work, this mycobiont was identified as Kurtia argillacea (= Hyphoderma argillaceum) residing in the order Hymenochaetales (Basidiomycota). Bioinformatic screening of the Kurtia ribosomal DNA sequence indicates that it represents a gene with a non-standard substitution rate or nucleotide composition heterogeneity rather than a deep paralogue or a pseudogene. Such a phenomenon probably also occurs in other lineages of the Fungi and should be taken into consideration when nuc-rDNA (especially that with unusual nucleotide composition) is used as a sole marker for phylogenetic reconstructions. Kurtia argillacea so far represents the only confirmed non-sebacinoid ericoid mycorrhizal fungus in the Basidiomycota and its intriguing placement among mostly saprobic and parasitic Hymenochaetales begs further investigation of its eco-physiology.     

铜胁迫对丛枝菌根真菌群落功能的影响

过去20年来,对不同来源丛枝菌根真菌(AMF)的功能开展了大量研究,但多数基于对单个AMF功能的比较,群落水平的研究较少,且很多研究混淆了AMF来源和群落结构对其功能的影响,忽略了宿主的重要作用.本研究通过两个短期温室盆栽试验,分析了铜胁迫对AMF群落结构的影响,比较了不同AMF群落的功能差异.结果表明:铜胁迫显著改变了AMF群落的孢子丰度和组成.以根内球囊霉和幼套球囊霉为优势种的AMF群落有效减缓了铜胁迫对玉米生长和部分生理特性的抑制作用.