Abstract: 【OBJECTIVE】 The aim is to construct a gene targetting vector suitable to Tetrahymena thermophila and integrate it into the genome of T. thermophila. The characteristics of transformants were analyzed. 【METHOD】 A gene targetting vector was constructed, in which existed a neo (neomycin) gene fragment encoding paromomycin resistance used as the selected marker, as well as the 5’ and 3’ untranslated region of histone H4-I gene flanking the neo gene in the insert.The 5’ flanking region had the initiation codon ATG, and the 3’ flanking region initiated with the termination codon TGA of H4-I gene. This vector was transformed into T. thermophila by electroporation and integrated into the genome of Tetrahymena by homologous recombination. The integration of neo gene had been identified through the paromomycin selection and PCR amplification. Morphology of the transformants was investigated under optical and scanning electron microscope. [RESULTS] A gene targetting vector suitable to Tetrahymena thermophila was successfully constructed. The growing speed of the resistant cells was faster than that of the original ones, and the sizes of formers were only 1/20~1/30 of the latters. The surface of resistant cells became smooth, while a large amount of cilia were discovered on the outer surface of the original ones. 【CONCLUSION】A transformation vector suitable to Tetrahymena were constructed. The neo gene in this vector was integrated into the genome of cells by homologous recombination. The study will be helpful for developing a high efficient expression system and investigating heterologous gene expression.

Key words: Tetrahymena thermophila, gene targetting vector, H4-I, neo gene, homologous recombination