Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria
Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria
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DOI：
10.1271/bbb.60052
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发表时间：
2006-08-01
影响因子：
1.6
通讯作者：
Kitamoto, Katsuhiko
中科院分区：
文献类型：
--
作者：
Mabashi, Yuka;Kikuma, Takashi;Kitamoto, Katsuhiko
我们报道了一种基于多站点网关(TM)的通用质粒构建系统的开发，该系统适用于快速、高效地制备米曲霉表达载体。该系统允许以规定的顺序和方向将插入条目克隆中的三个DNA片段与目的载体同时连接。我们准备了多种进入克隆和目的载体，包括启动子、编码载体蛋白和融合标签的基因以及可选择的标记，这使得为每个目的蛋白产生80个表达质粒成为可能。利用该系统构建了融合了柠檬酸合成酶线粒体靶向信号(AoCit1)的绿色荧光蛋白表达载体。在表达AoCit1-EGFP融合蛋白的转化子中可见线粒体的管状结构。这种构建体系使我们无需费力的DNA操作就可以制备大量的表达载体，这将为稻曲霉的分子生物学研究提供便利。
We report here a development of the MultiSite Gateway (TM)-based versatile plasmid construction system applicable for, the rapid and efficient preparation of Aspergillus oryzae expression plasmids. This system allows the simultaneous connection of the three DNA fragments inserted in entry clones along with a destination vector in a defined order and orientation. We prepared a variety of entry clones and destination vectors containing promoters, genes encoding carrier-proteins and fusion tags, and selectable markers, which makes it possible to generate 80 expression plasmids for each target protein. Using this system, plasmids for expression of the EGFP fused with the mitochondrial-targeting signal of citrate synthase (AoCit1) were generated. Tubular structures of mitochondria were visualized in the transformants expressing the AoCit1-EGFP fusion protein. This plasmid construction system allows us to prepare a large number of expression plasmids without laborious DNA manipulations, which would facilitate molecular biological studies on A. oryzae.