MOLECULAR AND CELL-BIOLOGICAL ANALYSIS OF VACUOLAR FUNCTION IN FILAMENTOUS FUNGI

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、小离子和聚磷酸盐的聚集等方面起着非常重要的作用。虽然已知丝状真菌中的液泡具有高度的多形性，其中一些液泡，如管状泡泡，与细胞内和细胞间的运输有关，但在分子水平上对液泡的功能和形态动力学知之甚少。在本研究项目中，我在分子和细胞生物学水平上对液泡的功能进行了阐明。利用粘粒基因组文库对已分离到的一株曲霉菌突变株水稻空泡蛋白错配突变株进行了转化。并成功分离到与VPS突变互补的DNA片段，由于其与酵母DNF3基因同源，将其命名为Aodnf3。接下来，我从稻曲霉菌中分离出两个基因Aovps10和Aovam3。融合了Aovps10基因的绿色荧光蛋白在米曲霉中的表达表明，在空泡和泡前隔室中观察到荧光。在一株表达EGFP-Aovam3融合蛋白的菌株中，激光共聚焦显微镜可以清楚地观察到空泡膜的动态变化。该系统能够以较高的分辨率在活细胞中观察到空泡的形态，包括大的球形空泡、管状空泡和可能的前泡状腔。

Vacuoles play very important roles in pH and osmotic regulation, protein degradation and strage of amino acids, small ions as well as polyphosphate in yeast S.cerevisiae. Although it is known that vacuoles in filamentous fungi are highly pleiomorphic and some of them, e.g. tubular vauoles, are implicated in intra-and intercellular transport, little is known about vacuolar functions and morphology dynamics at the molecular level. In this research project I have carried out elucidation of vacuolar function at the molecular and cell-biological level. One of Koji mold A.oryzae vps (Vacular Protein Missorting) mutants, which had been isolated previously, was transformed with cosmid genome library. And the DNA fragment complement the vps mutation was successfully isolated and designated it Aodnf3 due to its homology with yeast DNF3 gene. Next I isolated two genes Aovps10 and Aovam3 from A.oryzae. Expression of EGFP fusion of Aovps10 gene in A.oryzae showed that fluorescence was observed in vacuoles and prevacuolar compartments. In a strain expressing EGFP-Aovam3 fusion, dynamics of vacuolar membranes was clearly observed by confocal laser microscopy. This system enabled to observe vacuole morphology including large spherical vacuoles, tubular vacuoles, and putative prevacuolar compartments at higher resolution level in living cells.