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Construction of Green Fluorescent Protein Expression Vector pXS75-GFP and Application in Tetrahymena thermophila
Liang Haixia, Wang Wei*
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China
Abstract: To construct a vector for studying the localization of protein in Tetrahymena thermophila， GFP expression vector pXS75-GFP was constructed by ligating GFP with Cd2+-inducible metallothionein (MTT1) promoter and terminator sequences. The target gene––GFP fusion gene can integrate into the MTT1 locus through homologous recombination and resistance screening. Expression of the target protein in-fusion with the C-terminal GFP tag was controllable by Cd2+. The recombinant plasmid pXS75-GFP-ATU1 was constructed and biolistically transformed into Tetrahymena. The expression of α-tubulin-GFP was analyzed by Western blot. Confocal microscopy showed that α-tubulin-GFP localized at cortex in living and fixed Tetrahymena cells. The results revealed that pXS75-GFP can be used for studying the subcellular localization of proteins in Tetrahymena thermophila.
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