Interaction network of the ribosome assembly machinery from a eukaryotic thermophile

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre‐ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre‐ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (～180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in‐depth analysis of their protein–protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2‐hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein–protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre‐ribosome factors forming the ctUTP‐A and ctUTP‐B modules, and the Brix‐domain containing assembly factors of the pre‐60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.     

Mycoparasitism of Rhizoctonia solani by Endophytic Chaetomium spirale ND35: Ultrastructure and Cytochemistry of the Interaction

The interaction between endophytic biocontrol agent Chaetomium spirale ND35 and the soil‐borne plant pathogen Rhizoctonia solani was studied by light microscopy and transmission electron microscopy (TEM), as well as further investigated by gold cytochemistry to assess the potential role of cell wall degrading enzymes (CWDEs) during the mycoparasitic process. Macroscopic observations of fungal growth in dual cultures revealed that pathogen growth inhibition occurred soon after contact with the antagonist, followed by the overgrowth of C. spirale on the colony of R. solani. The coiling of C. spirale around R. solani and intracellular growth of the antagonist in its host occurred frequently. Moreover, in advanced stage of interaction between the antagonist and the pathogen, The growth and development of C. spirale were associated with highly morphological changes of the host fungal cell, characterized by retraction of plasma membrane and cytoplasm disorganization. Further, TEM investigations through localization by gold immunocytochemistry showed that contact between the two fungi was mediated by an amorphous β‐1,3‐glucan‐enriched matrix originating from cell wall of the antagonist C. spirale and sticking to its host surface. At the same time, the hemispherical wall appositions which were intensely labeled by the antibodies of β‐1, 3‐glucan in cell wall of R. solani were induced to form at sites of potential antagonist entry. However, the antagonist was capable of penetrating this barrier, indicating that β‐1,3‐glucanases were produced during the mycoparasitic process. Localization of N‐acetylglucosamine residues (chitin) with the gold‐labelled wheat germ agglutinin (WGA) implicated that chitinases might be involved in the CWD of R. solani in this antagonistic process as well. This report is the first evidence about mechanisms of the interactions between C. spirale and R. solani in ultrastructural and cytochemical aspects.     

Fungi in sediments of the sea of Japan and their biologically active metabolites

The most abundant marine fungi encountered in various regions of the Sea of Japan belong to the generaPenicillium, Aspergillus, Wardomyces, Trichoderma, Chrysosporium, andChaetomium. Facultative marine fungi of the generaScytalidium, Verticillium, andOidiodendron and obligate marine fungi of the genusDendryphiella are much less abundant. The composition of marine sediments and the anthropogenic load on them were found to influence the abundance and species diversity of fungi, as well as the occurrence of fungal strains producing hemolytically active substances. The biodiversity of mycobiota and the abundance of hemotoxin-producing fungi in marine sediments may be used to evaluate the anthropogenic load on marine biocenoses. Hemolytic compounds were produced by 57% of the fungi isolated from marine sediments. The hemolytic activity ofChaetomium spiculipilium was revealed in the fraction of the culture liquid containing extracellular fatty acids and pigments. The fatty acid composition of this marine fungus was determined.     

微生物菌剂对楸树幼苗生长及根际土细菌群落结构的影响

为探究球毛壳ND35微生物菌剂对楸树幼苗生长及土壤肥力的作用机制,本研究楸树幼苗为研究对象,采用室内盆栽试验,设计0（CK）,10（T1）,15（T2）,20（T3）4种微生物菌剂施用量,测定幼苗生长情况、土壤微生物组成结构、土壤酶和土壤养分等特征。研究结果如下：（1）球毛壳ND35微生物菌剂可显著促进楸树幼苗的生长,株高、地径、地上及地下生物量显著提高（P<0.05）,T2处理下促生效果最好。（2）施用球毛壳ND35微生物菌剂可显著提高土壤中有机质、硝态氮、铵态氮含量及脲酶、磷酸酶、蔗糖酶活性（P<0.05）。（3）球毛壳ND35微生物菌剂可显著影响土壤细菌群落组成,提高细菌群落的丰富度和多样性,使土壤中β-变形菌纲（Betaproteobacteria）、γ-变形菌纲（Gammaproteobacteria）的相对丰度显著下降,α-变形菌纲（Alphaproteobacteria）、δ-变形菌纲（Deltaproteobacteria）的相对丰度呈显著提高,可使土壤中鞘氨醇单胞菌属（Sphingomonas）的相对丰度显著提高21.88%-103.56%（P<0.05）,芽孢杆菌属（Bacillus）的相对丰度提高66.28%-65.97%（P<0.05）,酸杆菌属（Acidibacter）的相对丰度提高12.76%-38.06%。（4）冗余分析（RDA）结果表明,土壤硝态氮、铵态氮、有机质是影响土壤细菌群落分布和多样性的重要环境因子,土壤细菌群落结构的改变会显著影响土壤脲酶、蔗糖酶、碱性磷酸酶的活性。因此,施用球毛壳ND35微生物菌剂可通过影响植物根际土壤的化学性质及生物性质,促进楸树幼苗的生长。这一研究结果为楸树繁育提供了新的指导方向,亦为将其用于困难立地及退化生态系统植被恢复提供基础理论指导。     

角毛壳菌几丁质酶基因chi58多点突变及在毕赤酵母中的高效表达

应用重叠延伸PCR技术(gene splicing by overlap extension PCR,gene SOEing),简称SOE-PCR对角毛壳菌(Chaetomium cupreum)的几丁质酶基因chi58进行多点突变.依据毕赤酵母密码子偏爱性,将毕赤酵母中编码Arg使用频率几乎为0的密码子CGC突变为使用频率高的AGA,构建了含有正确突变的酵母表达载体pPIC9K-chi58A,电转化毕赤酵母GS115,获得的重组酵母株在诱导120 h酶活力最高,平均可达101.71 U/mL±3.33 U/mL;其活力比未优化重组酵母株(31.83 U/mL±4.85 U/mL)提高了约3倍,且经10代传代培养后遗传稳定性良好.表达产物的SDS-PAGE分析表明,酶蛋白分子大小为58 kD.     

球毛壳菌组蛋白H3基因克隆与特性分析

构建了球毛壳菌菌丝的cDNA文库,并获得了1410条ESTs序列,用里氏木霉(Hypocrea jecorina,AAM76068)和粗糙脉胞菌(Neurospora crassa,CAA25761)的组蛋白H3基因(Histone H3)蛋白序列对球毛壳菌(Chaetomium globosum)ESTs序列本地数据库进行tBlastn检索,获得了球毛壳菌组蛋白H3cDNA序列。cDNA序列全长739bp,开放阅读框411bp,编码136个氨基酸组成的多肽,蛋白分子量为15.4kD。BlastP同源性分析表明该基因与里氏木霉同源性最高为100%;与地钱(Marchantia polymorpha)同源性最低为95%。三级结构预测表明,该蛋白C端为球状结构域,而N端结构对其发挥调控作用起重要作用。该基因的cDNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY669068,AAT74576)。     

川乌植物内生真菌抗菌活性的研究

从川乌（Radix aconiti）植物的根和茎中分离获得内生真菌61株,经显微形态观察进行内生真菌的分类鉴定,同时采用纸片法进行抗菌活性筛选。结果有29株内生真菌显示抗菌活性,占菌株数的47．54％;毛壳菌属是抗菌活性菌株的优势种属;活性高且抗菌谱广的菌株有3株,主要分布在毛壳菌属和束丝菌属。结果表明：川乌植物内生真菌中广泛分布着有抗菌活性的菌株。     

银杏内生菌Chaetomium globosum ZY-22次生代谢产物分离鉴定

采用柱层析方法从银杏叶内生真菌Chaetomium globosum ZY-22的培养菌丝体提取物中分离得到脑苷脂B(1)、脑苷脂C(2)、尿囊素(3)、9(11)-去氢麦角甾醇过氧化物(4)以及4,6,8,22-四烯-3-酮-麦角甾烷(5)和球毛壳甲素(6)共6个次生代谢物;经波谱分析确定了6个化合物的结构,其中脑苷脂B、脑苷脂C和尿囊素是首次从内生真菌中得到;海虾致死试验结果显示,化合物1～6在10 μg/mL浓度下对丰年虾的致死率分别为1.6%、4.2%、7.4%、16.9%、12.8%、83.6%、表明球毛壳甲素对海虾表现出很强的毒性作用.     

嗜热毛壳菌内切β-葡聚糖酶的分离纯化及特性

探讨了液体发酵嗜热毛壳菌（Chaetomium thermophile)产生的内切β－葡聚糖酶的分离纯化及特性。粗酶液经硫酸铵分级沉淀,DEAE－Seplharose Fast Flow阴离子层析,Pheny1-Sepha-rose疏水层析,Sephacry1 S-100分子筛层析等步骤便可获得凝胶电泳均一的内切β－葡聚糖酶,经12．5％SDS－PAGE和凝胶过滤层析法分离纯化酶蛋白的分子量约为67．8kD的69．8kD。该酶反应的最适温度和pH分别为60℃和4．0－4．5在pH5.0条件下,该酶在60℃下稳定：70℃保温1h后,仍保留30％的活性;在80摄氏度的半衰期为25min,金属离子内切β－葡聚糖酶的活性影响较大,其中Na^ 对酶有激活作用;Fe^2 ,Ag^ ,Cu^2 ,Ba^2 ,Zn^2 等对酶有抑制作用。该酶对结晶纤维素有没水解能力。     

Phylogeography and palaeodistribution modelling in the Patagonian steppe: the case of Mulinum spinosum (Apiaceae)

Aim An integrative study of the endemic, yet ubiquitous, Patagonian shrub Mulinum spinosum (Apiaceae) was performed: (1) to assess the historical processes that influenced its geographical pattern of genetic variation; (2) to test hypotheses of its survival in situ or in glacial refugia during glacial cycles; and (3) to model its extant and palaeoclimatic distributions to assess support for the phylogeographical patterns recovered. Location Chilean and Argentinian Andean region and Patagonian steppe. Methods Chloroplast DNA sequences, trnH–psbA, trnS–trnG and 3′trnV–ndhC, were obtained for 314 individuals of M. spinosum from 71 populations. The haplotype data matrix was analysed using nested clade analysis (NCA) to construct a network. Analysis of molecular variance (AMOVA), spatial analysis of molecular variance (SAMOVA) and neutrality tests were also used to test for genetic structure and range expansion in the species. The present potential geographical distribution of M. spinosum was modelled and projected onto a Last Glacial Maximum (LGM) model. Results Amongst the 29 haplotypes observed, one was widely distributed, but most were restricted to either northern or southern regions. The populations with highest haplotype diversity were found in southern Patagonia, the high Andean region, and northern Patagonia. AMOVA and SAMOVA showed latitudinal structure for Argentinian populations. NCA implied patterns of restricted gene flow or dispersal but with some long‐distance dispersal and also long‐distance colonization and/or past fragmentation. Neutrality tests did not support range expansions. The current distribution model was a fairly good representation of the extant geographical distribution of the species, and the distribution model for the LGM did not show important shifts of the extant range to lower latitudes, except for a shift towards the palaeoseashore. Main conclusions Based on agreement amongst phylogeographical patterns, distribution of genetic variability, equivocal evidence of putative refugia and palaeodistribution modelling, it is probable that glaciations did not greatly affect the distribution of Mulinum spinosum. Our results are consistent with the in situ survival hypothesis, and not with the latitudinal migration of plant communities to avoid adverse climate conditions during Pleistocene glaciations. It is possible that populations of northern Patagonia may have been isolated from the southern ones by the Chubut and Deseado basins.