四膜虫基因打靶载体的构建及其在虫体内的转化

Construction of Gene Targeting Vector of Tetrahymena Thermophila and Its Transformation in the Parasite

【目的】构建一个适于嗜热四膜虫(Tetrahymena thermophila)的基因打靶载体,并分析转化四膜虫的特性。【方法】以嗜热四膜虫组蛋白H4-I基因作为启动子,利用H4-I基因的5′和3′侧翼区作为同源臂,并且保留H4-I基因编码区的起始密码子ATG和终止密码子TGA,中间嵌入巴龙霉素抗性标记基因neo,从而构建一个基因打靶载体。将该载体电击转化到嗜热四膜虫接合株体内,使其同源重组到四膜虫H4-I基因上。通过巴龙霉素抗性筛选、PCR扩增目的基因对抗性虫株进行鉴定。光学和电子显微镜观察抗性虫株形态变化。【结果】成功构建了一个适于嗜热四膜虫的基因打靶载体,将其电击转化到四膜虫体内,抗性虫株的生长速度明显高于普通四膜虫,显微镜观察抗性虫株形态变小,大约是正常形态的1/20～1/30,抗性虫株表面光滑,未见纤毛。【结论】利用基因打靶载体将neo基因定向整合到四膜虫H4-Ⅰ基因内部,为今后在嗜热四膜虫体内表达外源蛋白奠定了基础。

【Objective】 The aim is to construct a gene targeting vector suitable to Tetrahymena thermophila and integrate it into the genome of T. thermophila. The characteristics of transformants were analyzed. 【Method】 A gene targeting vector was constructed, in which neo gene encoding paromomycin resistance was flanked by 5’ a?nd 3’ untranslated regions of histone H4-I gene. The 5 ?flanking region kept H4-I gene promoter and the initiation codon ATG, and the 3’ flanking region contained 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 was identified by paromomycin selection and PCR amplification. Morphology of the transformants was investigated under optical microscope and scanning electron microscope. 【Result】 A gene targeting vector suitable to T. 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 latter’s. 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 is helpful for developing a highly efficient expression system and investigating heterogonous gene expression.