医学真菌基因组学的研究进展

酿酒酵母基因组测序的完成是基因组学史的一个里程碑,其重要性已远远超出酵母本身,促进了人类及其他生物基因组学的发展.但迄今为止,真菌基因组学的发展仍落后于细菌,而医学真菌基因组的研究则更为滞后,这与真菌基因组对真核生物和人类医学所做的贡献不相匹配.近年来,随着免疫受损人群的增长,相关致病真菌得以重视,其基因组学的研究取得一定进展.目前有超过30种的真菌基因组测序正在进行或已完成,其中包括致病真菌的主要代表菌种[1],现概述如下.     

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。     

串联重复序列在琴叶拟南芥基因组中的特征探究

串联重复序列广泛存在于真核生物的基因组中,它通过影响染色质的空间结构及基因表达从而影响生物的遗传与进化.本研究以琴叶拟南芥(Arabidopsis lyrata)基因组为材料,分析了1～50 bp重复单元的串联重复序列特征.研究发现串联重复序列在基因的5'UTR和启动子区域密度最高(8757 bp/Mb,8430 bp/Mb),而编码区CDS的密度最低(2406 bp/Mb).基因组中重复模体最高的为单核苷酸重复的T/A碱基,5'UTR中包含大量的二核苷酸重复模体,而在CDS中主要是三核酸重复模体.串联重复序列特征在琴叶拟南芥基因组不同区域的差别,显示其与基因表达和调控功能相适应.本研究深入探讨了串联重复序列在植物基因组中的特征及作用,为重复序列调控基因表达及植物基因组进化提供借鉴.     

真菌基因组de novo测序组装的方法与实践

真菌基因组较其他真核生物基因组结构简单,长度短,易于测序、组装与注释,因此真菌基因组是研究真核生物基因组的模型。为研究真菌基因组组装策略,本研究基于Illumina HiSeq测序平台对烟曲霉菌株An16007基因组测序,分别使用5种de novo组装软件ABySS、SOAP-denovo、Velvet、MaSuRCA和IDBA-UD组装基因组,然后通过Augustus软件进行基因预测,BUSCO软件评估组装结果。研究发现,5种组装软件对基因组组装结果不同,ABySS组装的基因组较其他4种组装软件具有更高的完整性和准确性,且预测的基因数量较高,因此,ABySS更适合本研究基因组的组装。本研究提供了真菌de novo测序、组装及组装质量评估的技术流程,为基因组<100 Mb的真菌或其他生物基因组的研究提供参考。     

植物病原生物基因组测序进展

随着基因组学的迅猛发展,越来越多的生物全基因组序列已经测定完成或正在进行之中。植物病原生物基因组序列的测定为理解植物与病原物互作分子机制有重要意义,并为植物病理学的发展作出了重要贡献。目前已有9种病原细菌和1种病原真菌的基因组序列彻底完成,另外还有更多的基因组草图正在组装或测序工作正在进行之中。对NCBI上主要的植物病原真菌和细菌全基因组测序进展作了整理和概述。     

真菌生物信息学研究概况

真菌是真核生物的一大类群,主要包含酵母、霉菌之类的微生物以及最为人所熟知的菇类,无论是在生态、生命周期以及形态都有很大的差别.然而,到目前为止,对于真菌界的了解还很少,预估大约有150万个物种,之中已知种约占5％.1986年,美国科学家Thomas Roderick提出了基因组学概念,1990年代几个物种基因组计划的启动,揭开了历史性的一页.随着生物实验技术和信息处理技术的迅速发展与提高,生物信息学的概念应运而生,利用数学、信息学、统计学和计算机科学的方法研解决生物学的问题.介绍真菌的测序技术,概述了真菌基因组学、转录组学、蛋白质组学等方面的进展,并对真菌生物信息学的未来研究内容进行展望.     

东乡野生稻内生放线菌Streptomyces sp. PRh5的全基因组测序及序列分析

【目的】Streptomyces sp. PRh5是从东乡野生稻(Oryza rufipogon Griff.)中分离获得的一株对细菌和真菌都具有较强抗菌活性的内生放线菌。为深入研究PRh5菌株抗菌机制及挖掘次级代谢产物基因资源,有必要解析PRh5菌株的基因组序列信息。【方法】采用高通量测序技术对PRh5菌株进行全基因组测序,然后使用相关软件对测序数据进行基因组组装、基因预测与功能注释、直系同源簇(COG)聚类分析、共线性分析及次级代谢产物合成基因簇预测等。【结果】基因组组装获得290 contigs,整个基因组大小约11.1 Mb,GC含量为71.1%,序列已提交至GenBank数据库,登录号为JABQ00000000。同时,预测得到50个次级代谢产物合成基因簇。【结论】将为Streptomyces sp. PRh5的功能基因组学研究及相关次级代谢产物的生物合成途径与异源表达研究提供基础。     

黄曲霉毒素生物合成代谢相关的基因组信息挖掘和转录调控因子研究进展

黄曲霉毒素生物合成调控机制的研究已成为研究真核生物次级代谢过程的模式系统,真菌基因组学及其他组学技术的快速发展为我们挖掘基因组信息、获取基因表达谱继而为黄曲霉毒素生物合成调控网络及其他真菌次级代谢机制的研究提供基础。真菌次级代谢基因的表达需要不同类型的转录因子进行调控,包括通路特异性转录因子、全局性转录因子及应答各种环境信号的广泛转录因子等,对这些转录因子功能的研究加快了对黄曲霉毒素生物合成代谢调控机制的认识。     

基于三代测序的大秃马勃的rDNA ITS鉴定及其基因组生物信息学分析

【背景】马勃是著名的传统可食用药用真菌,具有多种药理作用,包括抗菌、抗炎、止咳、抗肿瘤和抑制细胞增殖等。目前关于马勃的研究多集中在活性物质的分离提取及其药理药效上,而对马勃基因组遗传信息知之甚少。【目的】获得马勃真菌的基因组遗传信息,挖掘潜在具有工业应用价值的功能基因。【方法】联合使用Illumina二代测序技术和PacBio三代测序技术进行基因组测序,并综合使用多种数据库进行基因组结构分析和基因注释,包括GO注释。基于基因组信息,使用rDNAITS序列进行马勃真菌物种鉴定,并使用dbCAN和antiSMASH进行碳水化合物活性酶和次级代谢物基因簇的鉴定。【结果】测序获得大小为46.04 Mb基因组,有11 903个蛋白编码基因,包括6 634个GO注释基因。基于基因组中的rDNA ITS序列信息将该菌鉴定为大秃马勃(Calvatia gigantea),分析了麦角甾醇和萜类合成相关基因25个,鉴定出317个碳水化合物酶和18个次级代谢物生物合成基因簇,并发掘出1个潜在的、可分泌的抗丹宁α-淀粉酶。【结论】获得一株马勃真菌基因组,并发掘出潜在的食品工业应用价值的抗丹宁α-淀粉酶和多个新的次级代谢物合成基因簇。这将为马勃真菌遗传进化、功能基因组学研究,以及其在食品工业应用和次级代谢物的药用价值开发提供重要的遗传信息基础。     

乳酸菌基因组学研究进展

伴随着高通量测序技术的快速发展和测序成本的降低,越来越多的微生物基因组全序列测定得以实现,从基因组学的层面了解乳酸菌的遗传结构和组成,进而分析和掌握其生物学功能已经逐渐成为可能.迄今为止已经有22株乳酸菌的基因组完成测序并公开发表,还有至少12株测序工作仍在进行中.本文在分析相关文献和生物信息数据基础上,从乳酸菌基因组特点、代谢多样性、进化及共线性四个方面对乳酸菌基因组学研究进展进行了总结,旨在为乳酸菌研究和应用提供参考.     

Comparison of the Yeast Proteome to Other Fungal Genomes to Find Core Fungal Genes

The purpose of this research was to search for evolutionarily conserved fungal sequences to test the hypothesis that fungi have a set of core genes that are not found in other organisms, as these genes may indicate what makes fungi different from other organisms. By comparing 6355 predicted or known yeast (Saccharomyces cerevisiae) genes to the genomes of 13 other fungi using Standalone TBLASTN at an e-value <1E-5, a list of 3340 yeast genes was obtained with homologs present in at least 12 of 14 fungal genomes. By comparing these common fungal genes to complete genomes of animals (Fugu rubripes, Caenorhabditis elegans), plants (Arabidopsis thaliana, Oryza sativa), and bacteria (Agrobacterium tumefaciens, Xylella fastidiosa), a list of common fungal genes with homologs in these plants, animals, and bacteria was produced (938 genes), as well as a list of exclusively fungal genes without homologs in these other genomes (60 genes). To ensure that the 60 genes were exclusively fungal, these were compared using TBLASTN to the major sequence databases at GenBank: NR (nonredundant), EST (expressed sequence tags), GSS (genome survey sequences), and HTGS (unfinished high-throughput genome sequences). This resulted in 17 yeast genes with homologs in other fungal genomes, but without known homologs in other organisms. These 17 core, fungal genes were not found to differ from other yeast genes in GC content or codon usage patterns. More intensive study is required of these 17 genes and other common fungal genes to discover unique features of fungi compared to other organisms.Reviewing Editor: Prof. David Gottman     

A consistent phylogenetic backbone for the fungi

The kingdom of fungi provides model organisms for biotechnology, cell biology, genetics, and life sciences in general. Only when their phylogenetic relationships are stably resolved, can individual results from fungal research be integrated into a holistic picture of biology. However, and despite recent progress, many deep relationships within the fungi remain unclear. Here, we present the first phylogenomic study of an entire eukaryotic kingdom that uses a consistency criterion to strengthen phylogenetic conclusions. We reason that branches (splits) recovered with independent data and different tree reconstruction methods are likely to reflect true evolutionary relationships. Two complementary phylogenomic data sets based on 99 fungal genomes and 109 fungal expressed sequence tag (EST) sets analyzed with four different tree reconstruction methods shed light from different angles on the fungal tree of life. Eleven additional data sets address specifically the phylogenetic position of Blastocladiomycota, Ustilaginomycotina, and Dothideomycetes, respectively. The combined evidence from the resulting trees supports the deep-level stability of the fungal groups toward a comprehensive natural system of the fungi. In addition, our analysis reveals methodologically interesting aspects. Enrichment for EST encoded data-a common practice in phylogenomic analyses-introduces a strong bias toward slowly evolving and functionally correlated genes. Consequently, the generalization of phylogenomic data sets as collections of randomly selected genes cannot be taken for granted. A thorough characterization of the data to assess possible influences on the tree reconstruction should therefore become a standard in phylogenomic analyses.     

The importance of sourcing enzymes from non-conventional fungi for metabolic engineering and biomass breakdown

A wealth of fungal enzymes has been identified from nature, which continue to drive strain engineering and bioprocessing for a range of industries. However, while a number of clades have been investigated, the vast majority of the fungal kingdom remains unexplored for industrial applications. Here, we discuss selected classes of fungal enzymes that are currently in biotechnological use, and explore more basal, non-conventional fungi and their underexploited biomass-degrading mechanisms as promising agents in the transition towards a bio-based society. Of special interest are anaerobic fungi like the Neocallimastigomycota, which were recently found to harbor the largest diversity of biomass-degrading enzymes among the fungal kingdom. Enzymes sourced from these basal fungi have been used to metabolically engineer substrate utilization in yeast, and may offer new paths to lignin breakdown and tunneled biocatalysis. We also contrast classic enzymology approaches with emerging ‘omics’-based tools to decipher function within novel fungal isolates and identify new promising enzymes. Recent developments in genome editing are expected to accelerate discovery and metabolic engineering within these systems, yet are still limited by a lack of high-resolution genomes, gene regulatory regions, and even appropriate culture conditions. Finally, we present new opportunities to harness the biomass-degrading potential of undercharacterized fungi via heterologous expression and engineered microbial consortia.     

Fungal genome sequencing: basic biology to biotechnology

The genome sequences provide a first glimpse into the genomic basis of the biological diversity of filamentous fungi and yeast. The genome sequence of the budding yeast, Saccharomyces cerevisiae, with a small genome size, unicellular growth, and rich history of genetic and molecular analyses was a milestone of early genomics in the 1990s. The subsequent completion of fission yeast, Schizosaccharomyces pombe and genetic model, Neurospora crassa initiated a revolution in the genomics of the fungal kingdom. In due course of time, a substantial number of fungal genomes have been sequenced and publicly released, representing the widest sampling of genomes from any eukaryotic kingdom. An ambitious genome-sequencing program provides a wealth of data on metabolic diversity within the fungal kingdom, thereby enhancing research into medical science, agriculture science, ecology, bioremediation, bioenergy, and the biotechnology industry. Fungal genomics have higher potential to positively affect human health, environmental health, and the planet’s stored energy. With a significant increase in sequenced fungal genomes, the known diversity of genes encoding organic acids, antibiotics, enzymes, and their pathways has increased exponentially. Currently, over a hundred fungal genome sequences are publicly available; however, no inclusive review has been published. This review is an initiative to address the significance of the fungal genome-sequencing program and provides the road map for basic and applied research.     

Fungal evolution: diversity,taxonomy and phylogeny of the Fungi

The fungal kingdom comprises a hyperdiverse clade of heterotrophic eukaryotes characterized by the presence of a chitinous cell wall, the loss of phagotrophic capabilities and cell organizations that range from completely unicellular monopolar organisms to highly complex syncitial filaments that may form macroscopic structures. Fungi emerged as a ‘Third Kingdom’, embracing organisms that were outside the classical dichotomy of animals versus vegetals. The taxonomy of this group has a turbulent history that is only now starting to be settled with the advent of genomics and phylogenomics. We here review the current status of the phylogeny and taxonomy of fungi, providing an overview of the main defined groups. Based on current knowledge, nine phylum‐level clades can be defined: Opisthosporidia, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Zoopagomycota, Mucoromycota, Glomeromycota, Basidiomycota and Ascomycota. For each group, we discuss their main traits and their diversity, focusing on the evolutionary relationships among the main fungal clades. We also explore the diversity and phylogeny of several groups of uncertain affinities and the main phylogenetic and taxonomical controversies and hypotheses in the field.     

Ecological constraints limit the fitness of fungal hybrids in the Heterobasidion annosum species complex

The ability of two closely related species to maintain species boundaries in spite of retained interfertility between them is a documented driving force of speciation. Experimental evidence to support possible interspecific postzygotic isolation mechanisms for organisms belonging to the kingdom Fungi is still missing. Here we report on the outcome of a series of controlled comparative inoculation experiments of parental wild genotypes and F(1) hybrid genotypes between closely related and interfertile taxa within the Heterobasidion annosum fungal species complex. Results indicated that these fungal hybrids are not genetically unfit but can fare as well as parental genotypes when inoculated on substrates favorable to both parents. However, when placed in substrates favoring one of the parents, hybrids are less competitive than the parental genotypes specialized on that substrate. Furthermore, in some but not all fungus x plant combinations, a clear asymmetry in fitness was observed between hybrids carrying identical nuclear genomes but different cytoplasms. This work provides some of the first experimental evidence of ecologically driven postzygotic reinforcement of isolation between closely related fungal species characterized by marked host specificity. Host specialization is one of the most striking traits of a large number of symbiotic and parasitic fungi; thus, we suggest the ecological mechanism proven here to reinforce isolation among Heterobasidion spp. may be generally valid for host-specialized fungi. The validity of this generalization is supported by the low number of known fungal hybrids and by their distinctive feature of being found in substrates different from those colonized by parental species.     

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.     

Ecological Constraints Limit the Fitness of Fungal Hybrids in the Heterobasidion annosum Species Complex

The ability of two closely related species to maintain species boundaries in spite of retained interfertility between them is a documented driving force of speciation. Experimental evidence to support possible interspecific postzygotic isolation mechanisms for organisms belonging to the kingdom Fungi is still missing. Here we report on the outcome of a series of controlled comparative inoculation experiments of parental wild genotypes and F₁ hybrid genotypes between closely related and interfertile taxa within the Heterobasidion annosum fungal species complex. Results indicated that these fungal hybrids are not genetically unfit but can fare as well as parental genotypes when inoculated on substrates favorable to both parents. However, when placed in substrates favoring one of the parents, hybrids are less competitive than the parental genotypes specialized on that substrate. Furthermore, in some but not all fungus × plant combinations, a clear asymmetry in fitness was observed between hybrids carrying identical nuclear genomes but different cytoplasms. This work provides some of the first experimental evidence of ecologically driven postzygotic reinforcement of isolation between closely related fungal species characterized by marked host specificity. Host specialization is one of the most striking traits of a large number of symbiotic and parasitic fungi; thus, we suggest the ecological mechanism proven here to reinforce isolation among Heterobasidion spp. may be generally valid for host-specialized fungi. The validity of this generalization is supported by the low number of known fungal hybrids and by their distinctive feature of being found in substrates different from those colonized by parental species.