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

利用曲霉对丝状真菌的液泡功能进行分子生物学研究

• 批准号: 12460041
• 负责人: KITAMOTO Katsuhiko
• 金额: $ 9.09万
• 依托单位: THE UNIVERSITY OF TOKYO
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12460041/
• 关键词: koji mold; vacuoles; A. nidulans; EGFP; carboxy peptidase; VPS mutants; A. oryzae; A.nidulans; カルボキシペプチダーゼ; GFP

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.

在酿酒酵母中，液泡在pH和渗透调节、蛋白质降解和氨基酸、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，液泡在分子水平上的功能知之甚少。从细粒曲霉中分离到与酵母菌VPS （VPS1、VPS45和VPS21）和VAM （VAM4和VAM6）同源的基因，分别命名为vpsA、vpsB、vpsC、avaA和avaB。这些基因破坏实验表明，空心拟南芥生长不良，含有破碎的液泡。这些结果表明所分离的基因与液泡的发生和功能有关。此外，还分离到了cpyA（羧基肽酶Y同源物）基因，并将其作为一种液泡标记酶。将cpyA基因与EGFP （Enhanced Green Fluorescent Protein，增强绿色荧光蛋白）基因融合，并将融合后的基因分别导入到A. nidulans和A. oryzae中。通过表达CPY-EGFP，在荧光显微镜下可以清晰地看到活菌中的液泡。利用FACS（荧光激活细胞分选器）或荧光微板阅读器从表达CPY-EGFP为亲本菌株的a . oryzae中分离出18个hfc（分生孢子中高EGFP荧光）突变体、20个dfc（分生孢子中较暗荧光）突变体和22个hfm（培养基中高EGFP荧光）突变体。Hfc-1突变体在碱性培养基中生长很差。在hfc-3、hfc-4、dfc-14和hfc-4突变体中观察到与酵母中B类vps突变体相似的破碎液泡。在碱性培养基中，dfc-14和hfm-4突变体在液泡膜附近积聚了小的球形结构，与E类vps突变体相似。