Nowrousian Minou, Cebula Patricia
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, 44801 Bochum, Germany.
BMC Microbiol. 2005 Nov 3;5:64. doi: 10.1186/1471-2180-5-64.
The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog.
Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Deltatap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains.
The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.
丝状真菌大孢粪壳菌形成称为子囊壳的复杂三维子实体,这些子囊壳保护发育中的子囊孢子并确保其正常释放。在之前的微阵列分析中,已鉴定出几个在无菌突变体中与野生型相比下调的基因。这些基因中有tap1(与子囊壳发育相关的转录本),一个编码假定凝集素同源物的基因。
与导致子实体发育的条件相比,在仅允许营养生长的条件下分析野生型中tap1的转录水平,结果表明tap1不仅在发育突变体中下调,而且在子实体发育期间在野生型中也上调。我们克隆并测序了一个包含tap1开放阅读框和相邻序列的3.2 kb基因组DNA片段。包含tap1的基因组区域与其在密切相关的丝状真菌粗糙脉孢菌中的同源区域是同线的。为了确定tap1是否参与大孢粪壳菌的子实体发育,构建了一个敲除构建体,其中tap1开放阅读框被真菌调控区域控制下的潮霉素B抗性基因hph取代。用该构建体转化大孢粪壳菌野生型产生了一个tap1缺失菌株,其中tap1已被hph盒取代。与野生型相比,敲除菌株在营养生长和有性发育条件下没有表现出表型差异。在几个发育突变体背景中携带Δtap1等位基因的双突变体在表型上与相应的发育突变体菌株相似。
tap1转录本在大孢粪壳菌有性发育期间强烈上调;然而,对tap1敲除菌株的分析表明,tap1对大孢粪壳菌的子实体形成不是必需的。