真菌G蛋白信号调控蛋白的功能研究进展

G蛋白信号途径是真菌细胞信号转导网络的枢纽,在细胞的各种生物学调控过程中具有重要作用。G蛋白信号调控蛋白(Rgulators of G protein signaling,RGS)是一类重要的G蛋白信号调控因子,能通过促进G蛋白α亚基(Gα)偶联的GTP水解,使Gα和Gβγ亚基发生聚合,导致G蛋白失活,从而迅速关闭与G蛋白偶联的信号途径。自从第一个RGS蛋白在酿酒酵母中被鉴定以来,目前已经有30多个RGS蛋白在重要的模式真菌中被报道,包括构巢曲霉、绿僵菌、稻瘟病菌、玉米赤霉菌、轮枝镰孢菌、新型隐球菌和白色念珠菌等。RGS蛋白在真菌的营养菌丝生长、产孢、毒素和色素生产、致病性和有性生殖等过程中发挥着重要作用。本文对真菌中已报道RGS蛋白的功能进行了总结,对真菌RGS蛋白的结构特征和调控机制进行了评述。     

真菌基因组学

正化学工业出版社出版《真菌基因组学》(原著第二版)向读者介绍了多种真菌,包括单细胞真菌酿酒酵母、丝状真菌的模式菌粗糙脉胞菌、重要的工业真菌里氏木霉,以及动植物致病菌镰刀菌、白色念珠菌等的基因组学研究的最新进展,并以此延伸,对基因     

致病菌烟曲霉新基因Afu4g13170生孢致毒相关性初步研究

【目的】对烟曲霉Afu4g13170基因功能进行初步研究。【方法】利用Double-jointPCR方法和一步基因敲除技术,构建Afu4g13170基因缺失突变株。【结果】序列比对表明烟曲霉Afu4g13170蛋白与构巢曲霉Ani04163蛋白和新型隐球菌Gib2蛋白的氨基酸序列相似性为88.6%;表型分析表明基因破坏使突变株生长迟缓、梗基伸长、孢子分化能力下降,产孢推迟、产孢量减少,色素产生量降低;色谱分析显示基因缺失突变株的产毒能力下降。【结论】烟曲霉Afu4g13170基因可以作为控制曲霉致毒的一个靶位点。     

真菌中的脂质不对称调节和P4型ATP酶

脂质组分在双分子膜中不对称分布是广泛存在于真核细胞中的现象,有助于膜系统的出芽和融合,从而促进胞吞胞吐、蛋白分选运输等生理过程。多种蛋白参与维持脂质不对称,其中P4型ATP酶是重要的脂质转运体。P4型ATP酶又称脂质翻转酶,主要介导氨基磷脂的转位。在酿酒酵母、新生隐球菌、白念珠菌和构巢曲霉中,多种P4型ATP酶亚基的缺失导致蛋白运输失常,对唑类药物的敏感性升高,细胞壁完整性受损,毒力减低,在巨噬细胞中生存能力减弱等表型,提示脂质翻转酶的重要性及其作为抗真菌靶点的潜力。     

蒺藜内生真菌发酵液抗菌及抗肿瘤活性初步研究

【目的】筛选出具有抗菌和抗肿瘤活性的蒺藜内生真菌。【方法】采用牛津杯法、稻瘟菌模型及肿瘤细胞模型评价蒺藜内生真菌PDB和察氏培养基发酵产物的抗菌活性和肿瘤细胞毒性。【结果】PDB培养基发酵液和察氏培养基发酵液抑菌圈直径大于10 mm的菌株分别占总菌株数的19.05%和23.81%。PDB培养基发酵液和察氏培养基发酵液对稻瘟菌的最小抑制浓度(MIC)低于10%的菌株分别占总菌株数的19.05%和47.61%。对肿瘤细胞抑制率高于50%的PDB发酵产物占PDB发酵产物总数的52.38%, 而对肿瘤细胞抑制率高于50%的察氏发酵产物占察氏发酵产物总数的28.57%。【结论】部分蒺藜内生真菌的发酵产物具有抗菌和抗肿瘤活性。     

基因组水平预测稻瘟菌分泌蛋白组及富集分析

稻瘟菌分泌蛋白在稻瘟菌入侵植物过程中发挥着重要的作用。这些分泌蛋白中有很多是效应蛋白,这些效应蛋白可以干扰寄主的抗性、抑制寄主免疫反应。因此,对稻瘟菌分泌蛋白组的预测及功能分析就显得十分必要,也是目前植物和微生物分子互作研究领域的热点。使用SignalP、TMHMM及SecretomeP等软件,完成稻瘟菌分泌蛋白组的预测。同时,对含信号肽的经典分泌蛋白进行了GO功能富集、KEGG通路分析、结构域统计以及可降解植物成分的经典分泌蛋白预测等分析。结果显示,稻瘟菌含有约789个分泌蛋白,其长度多集中在100-500 aa;GO功能分析发现,这些分泌蛋白多富集在分泌途径及宿主互作中;KEGG分析显示,分泌蛋白在糖代谢途径中发挥着重要作用;大规模筛选预测到156个分泌蛋白具有降解植物细胞壁等成分的功能;同时还发现稻瘟菌中有可能存在大量不含信号肽的非经典分泌蛋白。通过设计的生物信息学流程,实现了稻瘟菌分泌蛋白组的预测;预测出经典分泌蛋白具有可降解植物细胞壁等成分以及参与糖代谢途径的功能;稻瘟菌中存在大量的无信号肽的非经典分泌蛋白。     

产黄青霉工业生产菌种基因报告系统的构建及启动子效率的评价

本研究以四种不同物种来源,不同代谢途径功能基因的启动子为材料,分别构建了以产黄青霉异青霉素N合成酶(IPNS)基因pcbC的启动子Pipns、构巢曲霉3-磷酸甘油醛脱氢酶基因gpdA的启动子PgpdA、构巢曲霉色氨酸合成基因trpC的启动子PtrpC、粗糙脉胞霉氨基酸合成交叉途径控制基因cpc的启动子Pcpc为启动子,腐草霉素(phleomycin)抗性基因为报告基因,构巢曲霉色氨酸合成基因trpC的终止子TtrpC为终止信号的丝状真菌转基因质粒,建立了产黄青霉工业生产菌种启动子筛选、评价体系,调查了这四种启动子的强弱。结果表明选择性标记基因受强启动子驱动可以提高产黄青霉工业生产菌种的转基因效率。研究也显示这四种启动子的强弱的顺序为PgpdA、Pcpc、Pipns和PtrpC。     

一种快速提取真菌染色体DNA的方法

介绍了一种适用于多种真菌染色体DNA的快速提取方法。该方法用石英砂振荡破壁 ,快速便捷地提取真菌染色体DNA ,提取时间仅用 1～ 2h。作者应用该方法成功地提取了粗糙脉胞菌 (Neurosporacrassa)、米曲霉 (Aspergillusoryzae)、羊肚菌 (Morchellaesculcnta)、酿酒酵母 (Saccharomycescervisae)等 4种不同真菌的染色体DNA ,所提DNA片段均大于2 0kb ,可直接用于限制性内     

多胺是植物生长发育的调节物

人们在1948年就发现多胺是副流感嗜血杆菌(Haemophyllus parainfluenzae)生长绝对需要的。后来在构巢曲霉、粗糙脉孢菌、酿酒酵母和大肠杆菌等微生物中也找到生长必需多胺的突变体。从六十年代开始,美国耶鲁大学 Galston实验小组对多胺作系统的研究,认识到多胺具有刺激生长和防止衰老等作用,并开始联系到农业生产实际。近十年来研究越来越深入。最先提出多胺可能是一类新的植物激素.但后来更多人认为可能     

稻瘟菌附着胞形成和发育的研究进展

附着胞是稻瘟菌侵染寄主的关键结构,cAMP、MAPK和Ca2+等信号途径参与其形成和发育,同时受寄主表面识别蛋白基因、黑色素合成基因、甘油合成基因、细胞自噬基因以及SNARE蛋白等因子调控。从以上几个方面综述了稻瘟菌附着胞形成与发育的研究进展。     

Exon-intron structure of genes in complete fungal genomes

Computer analysis of genes was performed for lower fungi Aspergillus fumigatus, Candida glabrata, Cryptococcus neoformans, Debaryomyces hansenii, Encephalitozoon cuniculi, Eremothecium gossypii, Kluyveromyces lactis, Magnaporthe grisea, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Ustilago maydis, and Yarrowia lipolytica. The content of genes with an exon-intron structure in their genomes varied from 0.7 to 97.0%. The exon-intron structure substantially changes with an increasing portion of intron-containing genes. Gene size and total exon length proved to linearly depend on the intron number in the A. fumigatus, C. neoformans, M. grisea, N. crassa, S. pombe, and U. maydis genomes.     

Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae

Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (Deltacmp1 Deltacmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5.     

Deciphering the model pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete fungal pathogen of humans that has diverged considerably from other model fungi such as Neurospora crassa, Aspergillus nidulans, Saccharomyces cerevisiae and the common human fungal pathogen Candida albicans. The recent completion of the genome sequences of two related C. neoformans strains and the ongoing genome sequencing of three other divergent Cryptococcus strains with different virulence phenotypes and environmental distributions should improve our understanding of this important pathogen. We discuss the biology of C. neoformans in light of this genomic data, with a special emphasis on the role that evolution and sexual reproduction have in the complex relationships of the fungus with the environment and the host.     

Introns and splicing elements of five diverse fungi

Genomic sequences and expressed sequence tag data for a diverse group of fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Neurospora crassa, and Cryptococcus neoformans) provided the opportunity to accurately characterize conserved intronic elements. An examination of large intron data sets revealed that fungal introns in general are short, that 98% or more of them belong to the canonical splice site (ss) class (5'GU...AG3'), and that they have polypyrimidine tracts predominantly in the region between the 5' ss and the branch point. Information content is high in the 5' ss, branch site, and 3' ss regions of the introns but low in the exon regions adjacent to the introns in the fungi examined. The two yeasts have broader intron length ranges and correspondingly higher intron information content than the other fungi. Generally, as intron length increases in the fungi, so does intron information content. Homologs of U2AF spliceosomal proteins were found in all species except for S. cerevisiae, suggesting a nonconventional role for U2AF in the absence of canonical polypyrimidine tracts in the majority of introns. Our observations imply that splicing in fungi may be different from that in vertebrates and may require additional proteins that interact with polypyrimidine tracts upstream of the branch point. Theoretical protein homologs for Nam8p and TIA-1, two proteins that require U-rich regions upstream of the branch point to function, were found. There appear to be sufficient differences between S. cerevisiae and S. pombe introns and the introns of two filamentous members of the Ascomycota and one member of the Basidiomycota to warrant the development of new model organisms for studying the splicing mechanisms of fungi.     

Comparison of the orotidine 5'-monophosphate decarboxylase sequences of eight species

Predicted amino acid sequences of the enzyme orotidine 5'-phosphate decarboxylase (EC 4.1.1.23) from eight different organisms are compared. The comparisons are made on the basis of primary structural differences, primary amino acid sequence, hydropathy profiles, and secondary structure predictions. The organisms compared are Mus musculus, Aspergillus nidulans, Neurospora crassa, Kluyveromyces lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Escherichia coli, and Salmonella typhimurium.     

Fungal small nuclear ribonucleoproteins share properties with plant and vertebrate U-snRNPs.

snRNAs with properties closely related to those of the major vertebrate U-snRNAs are present in the fungi Aspergillus nidulans, Neurospora crassa and Schizosaccharomyces pombe. These RNAs possess a tri-methyl guanosine cap structure and a subset cross-hybridizes with human U1 and U2 clones. In the form of snRNPs, snRNAs from these fungi as well as from Saccharomyces cerevisiae and pea plants are immunoprecipitated by human and anti-Sm or anti-(U1)RNP autoimmune antibodies. On micro-injection into the cytoplasm of Xenopus oocytes, the snRNAs are packaged into ribonucleoprotein particles and migrate into the nucleus. The results demonstrate a hitherto unsuspected degree of evolutionary conservation in snRNA structure, snRNP protein structure, and sites of RNA-protein interaction within snRNPs.     

Survey of simple sequence repeats in completed fungal genomes

The use of simple sequence repeats or microsatellites as genetic markers has become very popular because of their abundance and length variation between different individuals. SSRs are tandem repeat units of 1 to 6 base pairs that are found abundantly in many prokaryotic and eukaryotic genomes. This is the first study examining and comparing SSRs in completely sequenced fungal genomes. We analyzed and compared the occurrences, relative abundance, relative density, most common, and longest SSRs in nine taxonomically different fungal species: Aspergillus nidulans, Cryptococcus neoformans, Encephalitozoon cuniculi, Fusarium graminearum, Magnaporthe grisea, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Ustilago maydis. Our analysis revealed that, in all of the genomes studied, the occurrence, abundance, and relative density of SSRs varied and was not influenced by the genome sizes. No correlation between relative abundance and the genome sizes was observed, but it was shown that N. crassa, the largest genome analyzed had the highest relative abundance of SSRs. In most genomes, mononucleotide, dinucleotide, and trinucleotide repeats were more abundant than the longer repeated SSRs. Generally, in each organism, the occurrence, relative abundance, and relative density of SSRs decreased as the repeat unit increased. Furthermore, each organism had its own common and longest SSRs. Our analysis showed that the relative abundance of SSRs in fungi is low compared with the human genome and that longer SSRs in fungi are rare. In addition to providing new information concerning the abundance of SSRs for each of these fungi, the results provide a general source of molecular markers that could be useful for a variety of applications such as population genetics and strain identification of fungal organisms.     

The Cryptococcus neoformans STE11alpha gene is similar to other fungal mitogen-activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific

Partial sequence analysis of the Cryptococcus neoformans MATalpha mating type locus revealed the presence of a gene with substantial sequence similarity to other fungal mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) genes. The C. neoformans gene, designated STE11alpha, showed the highest degree of similarity to the Neurospora crassa nrc-1, Schizosaccharomyces pombe byr2 and Saccharomyces cerevisiae STE11 genes. A polymerase chain reaction-mediated sib-selection technique was successfully adapted for the purpose of disrupting STE11alpha. C. neoformans ste11alphaDelta mutants were found to be sterile, consistent with the phenotypes of ste11 and byr2 mutants in S. cerevisiae and S. pombe respectively. Haploid ste11alphaDelta mutants were also found to be unable to produce hyphae, suggesting that the C. neoformans gene is functionally conserved when compared with its S. cerevisiae MAPKKK counterpart. Comparison of the wild-type STE11alpha strain with a ste11alphaDelta disruptant for virulence using the mouse model showed that the ste11alphaDelta strain was less virulent, but the difference was only minor. In spite of some of the conserved functions of STE11alpha, linkage analysis showed that STE11alpha is only found in mating type alpha strains. These results demonstrate that, although functionally conserved, the mating pathway in C. neoformans has a unique organization.     

A complete inventory of fungal kinesins in representative filamentous ascomycetes

Complete inventories of kinesins from three pathogenic filamentous ascomycetes, Botryotinia fuckeliana, Cochliobolus heterostrophus, and Gibberella moniliformis, are described. These protein sequences were compared with those of the filamentous saprophyte, Neurospora crassa and the two yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Data mining and phylogenetic analysis of the motor domain yielded a constant set of 10 kinesins in the filamentous fungal species, compared with a smaller set in S. cerevisiae and S. pombe. The filamentous fungal kinesins fell into nine subfamilies when compared with well-characterized kinesins from other eukaryotes. A few putative kinesins (one in B. fuckeliana and two in C. heterostrophus) could not be defined as functional, due to unorthodox organization and lack of experimental data. The broad representation of filamentous fungal kinesins across most of the known subfamilies and the ease of gene manipulation make fungi ideal models for functional and evolutionary investigation of these proteins.