Department of Biotechnology, The University of Tokyo, Tokyo, Japana; Bio’c Co., Ltd., Toyohashi, Japan.
Appl Environ Microbiol. 2012 Apr;78(8):2819-29. doi: 10.1128/AEM.07034-11. Epub 2012 Feb 10.
The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of a complementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed, including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MAT genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1 expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed.
通过研究 MAT 基因的存在和功能，评估了米曲霉的有性生殖潜力。先前的基因组研究已经在参考菌株 RIB40 中鉴定出了 MAT1-1 基因。我们现在报告存在互补的 MAT1-2 基因，并对来自米曲霉菌株 AO6 的同形区域进行了测序。这使得开发了一种 PCR 诊断检测方法，可以在 180 个检测的菌株中检测到 MAT1-1 和 MAT1-2 基因型的分离株，包括工业清酒曲分离株。用于酿造清酒和味增的菌株显示出 MAT1-1 和 MAT1-2 交配型的接近 1:1 比例，而用于酱油生产的菌株则显示出 MAT1-2 交配型的明显偏向。然后通过对驻留同形物的遗传操作创建了 MAT1-1 和 MAT1-2 同基因菌株，并通过 DNA 微阵列和定量实时 PCR（qRT-PCR）方法在表达 MAT 基因的条件下比较了基因表达。发现有 33 个基因在 MAT1-1 宿主菌株或 MAT1-2 基因替换菌株中相对于彼此上调了 10 倍以上，表明 MAT1-1 和 MAT1-2 基因在米曲霉中以交配型依赖的方式在功能上调节基因表达，这是首次对假定的无性真菌进行的报告。MAT1-1 表达特异性地上调了一个α-信息素前体基因，但大多数受影响基因的功能尚不清楚。结果与米曲霉中的异宗配合繁殖系统一致，并讨论了发现有性循环的前景。
