荧光标记米曲霉细胞器菌株构建及细胞器形态观察

Construction of Aspergillus oryzae strains with organelles labeled with fluorescence and observation of organelle morphology

摘要:

多核丝状真菌米曲霉(Aspergillus oryzae)的细胞器具有多态性。为了观察米曲霉细胞器的形态以及为米曲霉中未知蛋白的定位分析和一系列生物反应通路的揭示提供参考，将不同的亚细胞定位信号与荧光蛋白融合，得到不同亚细胞定位的荧光蛋白表达载体，再利用农杆菌介导转化法(Agrobacterium tumefaciens-mediated transformation, ATMT)将构建好的载体转入米曲霉中，通过显微观察证明成功构建了米曲霉荧光标记细胞核、线粒体、内质网、液泡、脂滴、过氧化物酶体和高尔基体的报告菌株。通过小分子特异性染料对线粒体、细胞核和脂滴荧光定位报告菌株染色进行共定位，对报告菌株进行了进一步验证。本研究对这些荧光标记的细胞器在不同生长时期和不同培养条件下的分布和形态进行了动态观察。报告菌株的构建为研究米曲霉的细胞器形态提供了工具，同时也为米曲霉中未知目的蛋白的定位以及亚细胞层面上的研究提供了工具。

关键词:米曲霉;细胞器;融合荧光蛋白;共定位

Abstract:

The organelles in the multi-nucleated filamentous fungus Aspergillus oryzae present polymorphism. To observe the organelle morphology in A. oryzae and provide references for the localization prediction of unknown proteins and the disclosure of biological reaction pathways in A. oryzae, we fused different subcellular localization signals with green fluorescent protein (GFP) to obtain different subcellular localization vectors, which were then transferred into A. oryzae by Agrobacterium tumefaciens-mediated transformation. The A. oryzae reporter strains with fluorescence-labeled nuclei, mitochondria, endoplasmic reticulum, vacuole, lipid droplets, peroxisome, and Golgi apparatus were successfully constructed. Furthermore, staining with small-molecule specific dyes was carried out to validate the co-localization of fluorescence-labeled mitochondria, nuclei, and lipid droplets in the reporter strains, which further confirmed that the reporter strains were successfully constructed. The distribution and morphology of fluorescence-labeled organelles were observed at different growth stages and under different culture conditions. The constructed reporter strains provide basic tools for studying the organelle morphology, localization of unknown target proteins, and subcellular localization in A. oryzae.

Key words:Aspergillus oryzae;organelles;fusion fluorescent protein;co-localization