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对粗糙脉孢菌进行功能基因组学、注释和表达分析，这种丝状真菌已经成为超过250,000种非酵母真菌的组合模式。大多数脉孢菌基因在酵母菌中没有同源基因，近40%的基因在任何生物体中到目前为止都没有很强的同源基因，这表明对这些基因的功能的研究将是新颖和有意义的。脉孢菌是真核生物基础研究的重要模型，与脉孢菌相关的真菌包括重要的动植物病原菌和产生抗生素、化学物质、酶和药物的工业菌株。 这个子项目将通过有针对性的基因替换、这些菌株的初步表型以及它们在整个科学界的分布来追求基因的系统性破坏。43Mb的脉孢子虫基因组已经完全测序，使用针对脉孢子虫基因的程序进行自动注释可以预测10,082个蛋白质。表型现在与其中约10%相关，大约一半的基因在其他生物体中没有很强的序列同源性；因此，没有关于它们功能的线索(S)。此外，一些基因的功能很可能与基因具有序列相似性 在其他生物中会有轻微或很大的修饰，反映了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(S)，而项目1的五个具体目标的近期目标是促进这一点。在特定的目标#1中，我们将使用聚合酶链式反应、酵母中的同源重组和长程聚合酶链式反应来产生可用于敲除脉孢子菌所有基因的结构(敲除盒)。在特定的目标#2中，我们将在脉孢子虫中进行基因替换，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特征分析。在具体目标#4中，我们将把新菌株存放在真菌遗传学库存中心，该中心将维持库存并分发 他们对整个科学界做出了贡献。这一努力将有助于在真菌王国锚定基因组探索。

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.