Abstract：
A sporeless strain is an important breeding target in the mushroom industry. However, basidiospore production in the oyster mushroom Pleurotus ostreatus has been shown to be impaired by single-gene mutations in only two meiosis-related genes, mer3 and msh4. This study proposed a strategy for identifying the genes essential for basidiospore formation after meiotic division to determine new targets for molecular breeding. RNA-seq analysis was performed to identify P. ostreatus genes that are specifically expressed in the gill tissue of fruiting bodies, where basidiospore formation occurs. Transcriptome data during fruiting development of Coprinopsis cinerea, in which the meiotic steps progress synchronously, were then used to identify genes that are active in the postmeiotic stages. Based on these comparative analyses, five P. ostreatus genes were identified. Plasmids containing expression cassettes for hygromycin B-resistance screening, Cas9, and single-guide RNA targeting each gene were introduced into the protoplasts of dikaryotic strain, PC9×#64, to generate dikaryotic gene disruptants. Among the obtained transformants, three dikaryotic pcl1 disruptants and two cro6c disruptants did not produce basidiospores. Microscopic analyses indicated that spore formation was arrested at particular stages in these gene disruptants. These results indicate that these two genes are essential for mature spore formation in this fungus.
摘要：
无烂菌株是蘑菇行业的重要育种目标。然而,平菇平菇中的担子孢子生产已被证明仅受到两个减数分裂相关基因的单基因突变的损害，mer3和msh4。这项研究提出了一种策略，用于鉴定减数分裂后担子孢子形成所必需的基因，以确定分子育种的新靶标。进行RNA-seq分析以鉴定在子实体的g组织中特异性表达的平菇基因。发生担子孢子形成的地方。灰黄连翘结果发育过程中的转录组数据，减数分裂步骤同步进行，然后用于鉴定在减数分裂后阶段活跃的基因。基于这些比较分析，鉴定了5个平菇基因。含有用于潮霉素B抗性筛选的表达盒的质粒，Cas9和针对每个基因的单向导RNA被引入到原核菌株的原生质体中，PC9×＃64，以产生原核基因破坏物。在获得的转化体中，三个双原核pcl1破坏剂和两个cro6c破坏剂不产生担子孢子。显微镜分析表明，在这些基因破坏物中，孢子的形成在特定阶段被阻止。这些结果表明，这两个基因对于该真菌中成熟孢子的形成至关重要。
