尽管在丝状真菌中广泛进行了与增强绿色荧光蛋白(EGFP)融合的蛋白质的亚细胞定位分析,对编码EGFP融合蛋白的信使RNA(mRNAs)的定位知之甚少。在这项研究中,我们使用egfp探针进行了单分子荧光原位杂交(smFISH),以同时在丝状真菌米曲霉的菌丝细胞中显示EGFP融合蛋白及其mRNA。我们研究了编码细胞质EGFP的mRNA的亚细胞定位,用EGFP标记的肌动蛋白标记蛋白Lifeact,和几个EGFP融合蛋白AoSec22,AoSnc1,AoVam3和AoUapC,它们位于内质网(ER),顶端囊泡簇Spitzenkörper,液泡膜,和质膜,分别。通过smFISH对这些mRNA的可视化表明,每种mRNA表现出不同的定位模式,可能取决于mRNA序列。特别是,我们揭示了编码Lifeact-EGFP的mRNA,EGFP-AoSec22、EGFP-AoVam3和AoUapC-EGFP,但不是细胞质EGFP和EGFP-AoSnc1,优先定位于顶端细胞,提示某些机制来调节这些转录物在菌丝区域之间的存在。我们的发现提供了米曲霉菌丝细胞中每种mRNA的独特定位信息。
Although subcellular localization analysis of proteins fused with enhanced green fluorescence protein (EGFP) has been widely conducted in filamentous fungi, little is known about the localization of messenger RNAs (mRNAs) encoding the EGFP-fused proteins. In this study, we performed single-molecule fluorescence in situ hybridization (smFISH) using an egfp probe to simultaneously visualize EGFP-fused proteins and their mRNAs in the hyphal cells of the filamentous fungus Aspergillus oryzae. We investigated the subcellular localization of mRNAs encoding cytoplasmic EGFP, an actin marker protein Lifeact tagged with EGFP, and several EGFP-fused proteins AoSec22, AoSnc1, AoVam3, and AoUapC that localize to the endoplasmic reticulum (ER), the apical vesicle cluster Spitzenkörper, vacuolar membrane, and plasma membrane, respectively. Visualization of these mRNAs by smFISH demonstrated that each mRNA exhibited distinct localization patterns likely depending on the mRNA sequence. In particular, we revealed that mRNAs encoding Lifeact-EGFP, EGFP-AoSec22, EGFP-AoVam3, and AoUapC-EGFP, but not cytoplasmic EGFP and EGFP-AoSnc1, were preferentially localized at the apical cell, suggesting certain mechanisms to regulate the existence of these transcripts among hyphal regions. Our findings provide the distinct localization information of each mRNA in the hyphal cells of A. oryzae.