Mabashi Yuka, Kikuma Takashi, Maruyama Jun-Ichi, Arioka Manabu, Kitamoto Katsuhiko
Department of Biotechnology, The University of Tokyo, Japan.
Biosci Biotechnol Biochem. 2006 Aug;70(8):1882-9. doi: 10.1271/bbb.60052. Epub 2006 Aug 1.
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.
我们在此报告一种基于MultiSite Gateway™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质粒。该系统允许以确定的顺序和方向同时连接插入到入门克隆中的三个DNA片段以及一个目的载体。我们制备了多种入门克隆和目的载体，它们包含启动子、编码载体蛋白和融合标签的基因以及选择标记，这使得针对每个靶蛋白能够生成80种表达质粒。使用该系统，生成了用于表达与柠檬酸合酶（AoCit1）的线粒体靶向信号融合的EGFP的质粒。在表达AoCit1-EGFP融合蛋白的转化体中观察到了线粒体的管状结构。这种质粒构建系统使我们能够无需繁琐的DNA操作就制备大量表达质粒，这将有助于对米曲霉进行分子生物学研究。
