构巢曲霉与30种丝状子囊菌的基因组比较

为探讨丝状子囊菌的序列同源性,文章利用公开发表的真菌基因组序列构建本地基因组数据库,设置E值统计阈值为0.1,将构巢曲霉(Aspergillus nidulans)基因组的10560个注释基因分别与30种丝状子囊菌基因组比较。结果表明,同源匹配基因数量的多少可反映子囊菌之间的进化关系。构巢曲霉基因组的924个基因与这30种子囊菌基因组同时存在匹配序列,其中E值在10-5～0.1、10-30～10-5、10-100～10-30、0～10-100范围内都存在匹配序列的基因分别为6个、3个、6个和6个。ClustalX多序列比对分析显示,E值10-5～0.1的6组序列和E值10-30～10-5的3组序列均显示变异性过大而E值0～10-100的6组序列过于保守,E值介于10-100～10-30之间的6组同源序列可用于本研究的31种子囊菌系统学分析。     

从真菌全基因组中筛选丛赤壳科的DNA条形码

将粗糙脉孢菌(Neurospora crassa)的注释基因分别与30 种丝状子囊菌基因组比较, 根据E值大小预测同源序列, 从中选择Hsp90, AAC, CDC48和EF3作为候选基因, 以丛赤壳科(Nectriaceae)13 个属34 个概念清晰的种为材料, 对215个序列片段采用不同方法进行分析, 筛选适合于该科的DNA 条形码. 将种内与种间序列差异以及序列获得的难易程度作为评价指标. 结果表明,Hsp90 和AAC基因虽然具有较高的 PCR 扩增与测序成功率, 但种内、种间距离频率分布存在重叠, 可能导致部分种的鉴定错误;CDC48基因可以恰当地区分种内与种间差异, 但扩增与测序成功率相对较低; EF3基因不存在种内、种间遗传距离重叠, 序列容易获得, 适合作为该科真菌的DNA 条形码.     

子囊菌较担子菌具有更快的进化速率和更高的物种多样性

研究表明, 在一些进化分支或有机体之间存在着核苷酸或氨基酸替代速率差异. 越来越多的证据表明, 种群内的中性分子突变与物种多样化相关. 超过98%的陆生真菌属于子囊菌门和担子菌门, 而且前者的物种多样性明显多于后者. 获得了地衣型真菌红脐鳞的21种蛋白编码基因序列, 并应用这些及GenBank中的相关序列进行了随后的分析. 建立了3组矩阵: (1) 13种真菌, 包括105种蛋白编码基因; (2) 9种真菌, 包括21种蛋白编码基因; (3) 299种真菌的nu LSU rDNA序列. 应用这些数据, 检测了子囊菌门与担子菌门以及子囊菌内部主要纲之间的基因替代速率. 蛋白质数据和nu LSU rDNA数据分析显示, 子囊菌的基因替代速率显著快于担子菌; 而且在子囊菌内部, 物种丰富的粪壳菌纲进化速率最快, 物种数量较少的锤舌菌纲进化最慢. 结果提示, 子囊菌的快速进化不是得益于互惠共生、生态条件、无性繁殖、代谢速率或者短世代时间, 而可能是由奠基者效应引起的. 这是物种数量与进化速率相关的又一证据, 与奠基者效应是导致物种丰富的分支进化速率较快的主要原因的假说相吻合.     

产毒真菌基因组研究进展

真菌毒素是产毒真菌产生的次生代谢产物,以木霉、曲霉、毛霉、青霉和根霉为主的丝状真菌是农产品中常见的产毒真菌。阐明农产品中产毒致病微生物的基因组序列信息是揭示真菌特殊遗传性状的基础。截至2013年12月,共有子囊菌门中204个种和担子菌门中62个种的基因组序列已经测序或公布,它们的基因组大小大部分在30-40 Mb。整理了部分已完成基因组测序的具有产毒能力或致病力的真菌基因组信息,并对真菌测序方案、主要产毒真菌及其比较基因组学的研究进展进行了综述。     

过量表达OsUgp2基因提高紫芝多糖含量

尿苷二磷酸葡萄糖焦磷酸化酶（UDP-glucose pyrophosphorylase,UGPase）是多糖生物合成过程中重要的酶,水稻基因组中存在两个UGPase同源基因分别命名为OsUgp1和OsUgp2。构建了由构巢曲霉3-磷酸甘油醛脱氢酶基因启动子驱动OsUgp2表达的真菌过量表达载体,并通过农杆菌介导法将OsUgp2基因转入紫芝中,获得了潮霉素抗性的转化菌株。PCR和Southern杂交结果显示OsUgp2基因成功整合到受体紫芝基因组中。半定量RT-PCR检测结果显示外源基因OsUgp2在紫芝转     

广陈皮外源真菌的组成及产毒真菌分析

【背景】广陈皮为药食同源中药材,在高温、高湿且贮存不当的条件下容易发霉,从而产生毒素,严重威胁陈皮的质量安全。【目的】分析广陈皮表面外源真菌的组成及其产生毒素的真菌。【方法】采用平板稀释法分离广陈皮表面外源真菌,利用分生孢子形态特征及DNA序列分析进行真菌鉴定,采用高效液相色谱-三重串联四极杆质谱联用技术对青霉属和曲霉属进行产毒检测。【结果】从广陈皮表面分离外源真菌共132株,鉴定为子囊菌亚门（Ascomycota,98.48%）和毛霉菌亚门（Mucoromycota,1.52%）,包括散囊菌纲（Eurotiomycetes,95.45%）、座囊菌纲（Dothideomycetes,3.03%）和毛霉纲（Mucoromycetes,1.52%）。分离活动曲霉属（Aspergillus spp.）菌株共77株,为广陈皮药材外源真菌的优势菌,青霉属（Penicillium spp.）次之。毒素检测筛选出1株产毒真菌,鉴定为黄曲霉（Aspergillus flavus） JXCP1-3。该菌株产生黄曲霉毒素B₁ （aflatoxin B₁,AFB<...     

瑞氏木霉表达黑曲霉葡萄糖氧化酶

利用高表达分泌纤维素酶的真菌瑞氏木霉表达重组的黑曲霉葡萄糖氧化酶。在大肠杆菌DH5α中构建瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子pUC19(命名为pCBHGOD)质粒,线性化后用瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子(命名为CBHGOD)核酸片段转化瑞氏木霉QM9414原生质体。用PCR扩增方法筛选出同源重组葡萄糖氧化酶基因的瑞士木霉突变株。用麦杆诱导瑞氏木霉突变株,生产黑曲霉葡萄糖氧化酶,Westernblot分析重组的葡萄糖氧化酶分子量与Sigma公司的天然黑曲霉葡萄糖氧化酶一致,生产的重组酶活性25umL,相当于Sigma公司葡萄糖氧化酶标准品的产量为0.5gL。瑞氏木霉可用于生产黑曲霉葡萄糖氧化酶。     

烟曲霉rRNA基因ITS区的克隆测序分析

对烟曲霉rRNA基因内转录间区(ITS区)进行了克隆测序,并将之与其他几种常见曲霉的相应序列进行了比较.发现3株烟曲霉的ITSⅠ区完全相同,而其中1株烟曲霉的ITSⅡ区与一条已知相应序列仅有2个碱基的变异.提示烟曲霉rRNA基因的两个ITS区序列均十分保守,而且与黄曲霉、黑曲霉、土曲霉及构巢曲霉的相应序列相比较,均有一定程度的变异.     

一株Monacolin K高产菌株生理特性

根据丝状真菌的常用鉴定方法,对实验室保存的一株Monacolin K高产菌株进行形态和生理特性实验。结果表明该菌株属于真菌门(Eumycophyta)、子囊菌纲(Asomycetes)、真子囊菌亚纲(Eurotiaceae)、曲霉目(Eurrotiales)、曲霉科(Eurotiaceae)、红曲霉属(Monascus)。     

应用实时荧光PCR技术检测构巢曲霉的初步研究

目的 根据构巢曲霉（Aspergillus nidulans）3-磷酸甘油醛脱氢酶（Glyceraldehyde-3-phosphate dehydrogenase,GAPDH）基因特异位点设计并合成Taqman探针及引物,建立构巢曲霉实时荧光 PCR检测方法。方法 应用lasergene7.1软件对构巢曲霉与13种常见曲霉主要包括黑曲霉（A.niger）、烟曲霉（A.fumigatus）、杂色曲霉（A.versicolor）、土曲霉（A.terrus）、黄曲霉（A. flavus）、温特曲霉（A.wentii）、寄生曲霉（A. parasiticus）、泡盛曲霉（A.awamori）、米曲霉（A. oryzae ）、棒曲霉（A.cavatus）、赤曲霉（A.ruber ）、亮白曲霉（A.ochraceus）及赭曲霉（A.ochraceus）GAPDH基因序列比对分析,在特异位点设计引物和探针,建立构巢曲霉实时荧光 PCR检测方法,并对该方法进行特异性及敏感性分析。结果 用曲霉属22种41株不同曲霉及其他属的12株病原真菌验证实验表明,所建立的荧光PCR方法特异性强;检测灵敏度可达4.03×10－12μg/ml的模板DNA。 结论 应用实时荧光PCR技术能够有效检测构巢曲霉,该方法具有特异、灵敏、快速等特点,可在实际工作中应用。     

Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes.

We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.     

A phylogenomic approach to reconstructing the diversification of serine proteases in fungi

Using a phylogenomic approach with 10 fungi of very different virulence and habitat, we determined that there was substantial diversification of subtilase-type proteases early in ascomycete history (with subsequent loss in many lineages) but with no comparable diversification of trypsins. Patterns of intron loss and the degree of divergence between paralogues demonstrated that the proliferation of proteinase K subtilases and subtilisin type subtilases seen in pathogenic ascomycetes (Metarhizium anisopliae, Magnaporthe grisea, Fusarium graminearum) occurred after the basidiomycete/ascomycete split but predated radiation of ascomycete lineages. This suggests that the early ascomycetes had a lifestyle that selected for multiple proteases, whereas the current disparity in gene numbers between ascomycete lineages results from retention of genes in at least some pathogens that have been lost in other lineages (yeasts, Aspergillus nidulans, Neurospora crassa). A similar prevailing trend towards lineage specific gene loss of trypsins in saprophytes and some pathogens suggests that their phylogenetic breadth will have been much wider in early fungi than currently.     

Unique regulatory mechanism for D-galactose utilization in Aspergillus nidulans

This study describes two novel regulators, GalX and GalR, that control d-galactose utilization in Aspergillus nidulans. This system is unique for A. nidulans since no GalR homologs were found in other ascomycetes. GalR shares significant sequence identity with the arabinanolytic and xylanolytic regulators AraR and XlnR, but GalX is more distantly related.     

Neurospora crassa ve-1 affects asexual conidiation

The velvet factor of the homothallic fungus Aspergillus nidulans promotes sexual fruiting body formation. The encoding veA gene is conserved among fungi, including the ascomycete Neurospora crassa. There, the orthologous ve-1 gene encodes a deduced protein with high similarity to A. nidulans VeA. Cross-complementation experiments suggest that both the promoter and the coding sequence of N. crassa ve-1 are functional to complement the phenotype of an A. nidulans deletion mutant. Moreover, ve-1 expression in the heterologous host A. nidulans results in development of reproductive structures in a light-dependent manner, promoting sexual development in the darkness while stimulating asexual sporulation under illumination. Deletion of the N. crassa ve-1 locus by homologous gene replacement causes formation of shortened aerial hyphae accompanied by a significant increase in asexual conidiation, which is not light-dependent. Our data suggest that the conserved velvet proteins of A. nidulans and N. crassa exhibit both similar and different functions to influence development of these two ascomycetes.     

Discovery of a novel superfamily of type III polyketide synthases in Aspergillus oryzae

Identification of genes encoding type III polyketide synthase (PKS) superfamily members in the industrially useful filamentous fungus, Aspergillus oryzae, revealed that their distribution is not specific to plants or bacteria. Among other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus), A. oryzae was unique in possessing four chalcone synthase (CHS)-like genes (csyA, csyB, csyC, and csyD). Expression of csyA, csyB, and csyD genes was confirmed by RT-PCR. Comparative genome analyses revealed single putative type III PKS in Neurospora crassa and Fusarium graminearum, two each in Magnaporthe grisea and Podospora anserina, and three in Phenarocheate chrysosporium, with a phylogenic distinction from bacteria and plants. Conservation of catalytic residues in the CHSs across species implicated enzymatically active nature of these newly discovered homologs.     

Proteins involved in microbody biogenesis and degradation in Aspergillus nidulans

Fungal microbodies (peroxisomes) are inducible organelles that proliferate in response to nutritional cues. Proteins involved in peroxisome biogenesis/proliferation are designated peroxins and are encoded by PEX genes. An autophagy-related process, termed pexophagy, is responsible for the selective removal of peroxisomes from the cell. Several genes involved in pexophagy are also required for autophagy and are collectively known as ATG genes. We have re-analysed the Aspergillus nidulans genome for the presence of PEX and ATG genes and have identified a number of previously missed genes. Also, we manually determined the correct intron positions in each identified gene. The data show that in A. nidulans and related fungi the basic set of genes involved in peroxisome biogenesis or degradation are conserved. However, both processes have features that more closely resemble organelle formation/degradation in mammals rather than yeast. Thus, filamentous fungi like A. nidulans are ideal model systems for peroxisome homeostasis in man.     

G-protein and cAMP-mediated signaling in aspergilli: a genomic perspective

We have carried out an in silico exploration of the genomes of Aspergillus nidulans, Aspergillus fumigatus, and Aspergillus oryzae, and identified components of G-protein/cAMP-mediated signaling. Putative G-protein coupled receptors (GPCRs) were distributed over nine classes. The GPCRs within classes were well conserved among aspergilli but varied in other ascomycetes. As previously observed in A. nidulans and other fungi, three Galpha, one Gbeta, and one Ggamma subunits of G proteins were identified in A. fumigatus, whereas an additional likely non-functional Galpha subunit was present in A. oryzae. While most fungal species had five proteins containing the regulator of G-protein signaling (RGS) domain predicted to participate in attenuation of G-protein signaling, A. fumigatus and A. oryzae had an additional RGS protein (RgsD) related to RgsA of A. nidulans. Genes encoding adenylate cyclase, a regulatory subunit and two catalytic subunits of the cAMP-dependent protein kinase, were also identified in the three aspergilli. Finally, regulators of cAMP signaling including low- and high-affinity phosphodiesterases were identified. Taken together, our data indicate a striking diversity at the GPCR level, but little diversity of components at the G-protein and cAMP-signaling level. This may reflect the abilities of these fungi to adapt to various ecological niches and to integrate diverse environmental cues into highly conserved cellular processes.     

Regulatory genes controlling fatty acid catabolism and peroxisomal functions in the filamentous fungus Aspergillus nidulans

The catabolism of fatty acids is important in the lifestyle of many fungi, including plant and animal pathogens. This has been investigated in Aspergillus nidulans, which can grow on acetate and fatty acids as sources of carbon, resulting in the production of acetyl coenzyme A (CoA). Acetyl-CoA is metabolized via the glyoxalate bypass, located in peroxisomes, enabling gluconeogenesis. Acetate induction of enzymes specific for acetate utilization as well as glyoxalate bypass enzymes is via the Zn2-Cys6 binuclear cluster activator FacB. However, enzymes of the glyoxalate bypass as well as fatty acid beta-oxidation and peroxisomal proteins are also inducible by fatty acids. We have isolated mutants that cannot grow on fatty acids. Two of the corresponding genes, farA and farB, encode two highly conserved families of related Zn2-Cys6 binuclear proteins present in filamentous ascomycetes, including plant pathogens. A single ortholog is found in the yeasts Candida albicans, Debaryomyces hansenii, and Yarrowia lipolytica, but not in the Ashbya, Kluyveromyces, Saccharomyces lineage. Northern blot analysis has shown that deletion of the farA gene eliminates induction of a number of genes by both short- and long-chain fatty acids, while deletion of the farB gene eliminates short-chain induction. An identical core 6-bp in vitro binding site for each protein has been identified in genes encoding glyoxalate bypass, beta-oxidation, and peroxisomal functions. This sequence is overrepresented in the 5' region of genes predicted to be fatty acid induced in other filamentous ascomycetes, C. albicans, D. hansenii, and Y. lipolytica, but not in the corresponding genes in Saccharomyces cerevisiae.