Functional Analysis of Filamentous Fungi

该计划项目中四个相互依赖的项目的总体目标是进行粗糙脉孢菌的功能基因组学，注释和表达分析，该丝状真菌已成为25万多种非酵母真菌集合的模型。大多数脉孢菌基因在酵母中没有同源物，近40%的基因在任何生物体中没有强同源物，这表明对这些基因功能的研究既新颖又有意义。脉孢菌是真核生物基础研究的重要模式，与脉孢菌相关的真菌包括重要的动物和植物病原体以及产生抗生素、化学品、酶和药物的工业菌株。 该子项目将通过靶向基因替换、这些菌株的初步表型分析以及它们在整个科学界的分布来系统地破坏基因。43 Mb脉孢菌基因组被完全测序，并且使用在脉孢菌基因上训练的程序进行自动注释预测10，082种蛋白质。表型现在与其中约10%相关，大约一半的基因在其他生物中没有强序列同源物;因此，没有关于其功能的线索。此外，可能与基因共享序列相似性的一些基因的功能 在其他生物体中的基因将被轻微或极大地改变，反映出丝状真菌的新生物学。我们的长期目标是在所有这些基因中创建基因敲除，作为确定每个基因功能的第一步，项目1的五个具体目标的近期目标是促进这一点。在特定目标#1中，我们将使用PCR、酵母中的同源重组和长距离PCR来产生可用于敲除脉孢菌中的所有基因的构建体（敲除盒）。在具体目标#2中，我们将在脉孢菌中进行基因置换以产生明确的功能敲除。在具体目标#3中，我们将对所有突变体进行基本的表型表征。在具体目标#4中，我们将把新菌株存款在真菌遗传学储备中心，该中心将维持储备并分发 向广大科学界。这一努力将有助于锚基因组探索的王国真菌。

The overall goal of the four interdependent projects in this Program Project is to carry out functional genomics, annotation, and expression analyses of Neurospora crassa, the filamentous fungus that has become a model for the assemblage of over 250,000 species of non-yeast fungi. Most Neurospora genes have no homologs in yeasts and nearly 40% have no strong homologs in any organism to date, suggesting that examination of the functions of these genes will both novel and informative. Neurospora is an important model for basic research in eukaryotes, and fungi allied to Neurospora include significant animal and plant pathogens and industrial strains yielding antibiotics, chemicals, enzymes, and pharmaceuticals. This subproject will pursue the systematic disruption of genes through targeted gene replacements, preliminary phenotyping of these strains, and their distribution to the scientific community at large. The 43 Mb Neurospora genome is completely sequenced, and automated annotation using programs trained on Neurospora genes predict 10,082 proteins. Phenotypes are now associated with about 10% of these, and roughly half of the genes have no strong sequence homologs in other organisms; thus, there are no clues to their function(s). Furthermore, it is likely that functions of some genes sharing sequence similarity with genes in other organisms will be modified slightly or greatly, reflecting the novel biology of filamentous fungi. Our long term goal is to create gene knockouts in all of these genes as a first step to determining each gene's function(s), and the immediate goal of the five Specific Aims of Project #1 is to facilitate this. In Specific Aim #1, we will use PCR, homologous recombination in yeast, and long range PCR to generate constructions (knockout cassettes) that can be used to knockout all of the genes in Neurospora. In Specific Aim #2, we will carry out gene replacements in Neurospora to generate unambiguous functional knockouts. In Specific Aim #3, we will carry out a basic phenotypic characterization of all the mutants. In Specific Aim #4, we will deposit the novel strains in the Fungal Genetics Stock Center which will maintain the stocks and distribute them to the scientific community at large. This effort will help to anchor genomic exploration in the Kingdom of the Fungi.