八肋游仆虫含绿色荧光蛋白基因的大核人工染色体的构建

为研究八肋游仆虫(Euplotes octocarinatus)相关基因的功能，构建了八肋游仆虫大核人工染色体(macronuclear artificial chromosome of E. octocarinatus，EoMAC-G)，其两端为克隆自八肋游仆虫大核β2-微管蛋白基因的5′和3′非编码区和两侧的端粒序列，中间为多克隆位点和密码子优化后的增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP-Eo) 报道基因. 用脂质体转染方法将携带有EoMAC－G的pBTub-Tel载体转入八肋游仆虫大核，分析EGFP-Eo基因在八肋游仆虫细胞中的表达. 荧光显微镜观察发现,EGFP-Eo产生的荧光均匀分布于八肋游仆虫细胞质中. 在细胞进行有丝分裂的情况下,荧 光可持续20 d以上. 相比pEGFP-N1质粒转化的游仆虫，人工染色体中的EGFP-Eo基因表达的荧光亮度强、稳定且持续时间长. Western 杂交分析进一步证实,外源EGFP-Eo基因在细胞中过量表达. 通过细菌喂食法进行纤毛虫RNA干扰实验，抑制了外源EGFP-Eo基因在八肋游仆虫细胞中的表达. 利用构建的人工染色体不仅可以在八肋游仆虫细胞内表达外源基因，对目的蛋白质进行活细胞实时动态的定位分析，还可通过RNA干扰的方法调控外源基因在纤毛虫细胞中的表达，便于进一步分析目的蛋白质的功能.

Construction of an Euplotes octocarinatus Macronuclear Artificial Chromosome Harboring Codon-optimized EGFP Gene

We have developed a macronuclear artificial chromosome (EoMAC－G) of E.-octocarinatus for studying the functions of its important genes. Codon-optimized EGFP and the multiple cloning sites were taken from the pEGFP-N1 plasmid (EGFP-Eo), the 5′and 3′non-coding regions of β2-tubulin gene flanked by telomeres were cloned from macronuclear genome to generate a minichromosome of E. octocarinatus.The constructed vectors carrying the EoMAC－G were introduced into Euplotes cells with liposomes. We found that the EGFP-Eo fluorescence was distributed throughout the cytoplasm and could be detected among the descendants after continuous cell divisions at least 20 days. Compared with the pEGFP-N1 plasmid transfection, the EGFP-Eo fluorescence appeared to be more intense and stable in E. octocarinatus. The over expressed EGFP was verified by Western blot, and could be knocked down by bacteria feeding with RNA interference reagents in live E. octocarinatus cells. The EoMAC－G could be adopted in realtime dynamic localization studies of exogenous expressed target proteins in E. octocarinatus, potentially be useful to investigate the function of a specific gene.