MOLECULAR-BIOLOGICAL RESEARCH ON VACUOLAR FUNCTION OF FILAMENTOUS FUNGI USING KOJI-MOLDS

在酵母S.在酿酒酵母中，液泡在pH和渗透调节、蛋白质降解以及氨基酸、小离子和多磷酸盐的储存中起着非常重要的作用。然而，在丝状真菌中，人们对液泡在分子水平上的功能知之甚少。从构巢曲霉（Aspergillus nidulans）中分离出与酵母VPS（VPS 1、VPS 45和VPS 21）和VAM（VAM 4和VAM 6）基因同源的几个基因，并将这些基因分别命名为vpsA、vpsB、vpsC、avaA和avaB。这些基因中断实验的A. nidulans表现出不良的生长，并含有破碎的空泡。这些结果表明，分离的基因参与液泡的生物发生和功能。此外，还分离到了作为液泡标记酶的cpyA基因。nidulans。将cpyA基因与EGFP（增强型绿色荧光蛋白）基因融合，并将所得融合基因导入A. nidulans和A.，分别。在荧光显微镜下，通过表达CPY-EGFP，在活真菌中可以清楚地观察到胞浆。从黑曲霉中分离到18个hfc（hyperEGFP fluorescenceinconidia）突变体、20个dfc（darkenfluorescenceinconidia）突变体和22个hfm（hyperEGFP fluorescenceinmedium）突变体。使用FACS（荧光激活细胞分选仪）或荧光酶标仪，将表达CPY-EGFP的质粒作为亲本菌株。HFC-1突变体在碱性培养基中显示非常差的生长。在hfc-3、hfc-4、dfc-14和hfm-4突变体中观察到类似于酵母中B类vps突变体的破碎空泡。dfc-14和hfm-4突变体在碱性培养基中观察到液泡膜附近的小球形结构的积累，这与E类vps突变体相似。

In yeast S. cerevisiae, vacuoles play very important roles in pH and osmotic regulation, protein degradation and storage of amino acids, small ions as well as polyphosphates. In filamentous fungi, however, little is known about vacuolar functions at a molecular level. Several genes homologous to yeast VPS (VPS1, VPS45, and VPS21) and VAM (VAM4, and VAM6) genes were isolated from Aspergillus nidulans and designated the genes as vpsA, vpsB, vpsC, avaA, and avaB, respectively. These gene disruption experiments of A. nidulans showed poor growth and contained fragmented vacuoles. These results suggested that the genes isolated are involved in vacuolar biogenesis and functions. In addition, cpyA (carboxy peptidase Y homologue) gene was also isolated as a marker enzyme of vacuoles from A. nidulans. The cpyA gene was fused with EGFP (Enhanced Green Fluorescent Protein) gene and the resulting fusion gene was introduced into A. nidulans and A. oryzae, respectively. Vacuoles are clearly visualized by a fluorescent microscope in the living fungi by expressing of CPY-EGFP. Eighteen hfc (hyper EGFP fluoresce in conidia) mutants, 20 dfc (darker fluorescence in conidia) mutants, and 22 hfm (hyper EGFP fluorescence in the medium) mutants were isolated from A. oryzae expressing CPY-EGFP as a parent strain using a FACS ( fluorescence activated cell sorter) or a fluorescent micro plate reader. hfc-1 mutant showed very poor growth in alkaline medium. Fragmented vacuoles, which are similar to class B vps mutants in yeast, were observed in hfc-3, hfc-4, dfc-14, and hfm-4 mutants. Accumulation of small spherical structure near vacuolar membrane was observed in dfc-14, and hfm-4 mutants in alkaline medium, which are similar to class E vps mutants.