摘要
通过RT-PCR获得番木瓜eIF4E和eIFiso4E基因的编码区,将其分别克隆到pBD-GAL4载体中,构建酵母双杂交系统的诱饵载体pBD-GAL4-eIF4E,pBD-GAL4-eIFiso4E。测序正确后,将重组质粒导入YRG-2酵母菌株,检测其表达产物对酵母细胞有无毒性及对报告基因有无激活作用。结果表明,获得了正确的番木瓜eIF4E,eIFiso4E基因编码区,并成功克隆到pBD-GAL4诱饵载体中,且转化有诱饵载体的YRG-2在SD/-Trp营养缺陷平板上生长良好,在SD/-His-Trp营养缺陷平板上不能生长,说明表达产物对酵母细胞无毒性,对报告基因也无自激活作用,这为下一步利用酵母双杂交系统检测番木瓜eIF4E,eIFiso4E蛋白与病毒的相互作用奠定了基础。
The coding regions of papaya eIF4E and eIFiso4E were amplified by RT-PCR and then fused with pBD-GAIA vector. After confirmation with sequence analysis, the plasmid was transformed into the yeast cell, YRG-2, and the toxicity and transcriptional autoactivation of expressed protein were tested. The results indicated that the coding regions were successfully amplified and subcloned into pBD-GAL4, and the YRG-2 transformed with bait plasmids grew well on SD/trp plate but not on SD/-his-trp. These data showed that the bait vectors, pBD-GAL4-eIF4E and pBD-GAL4-eIFiso4E, were expressed correctly without toxicity, and could not autoactivate the transcription of reporter gene alone in yeast two hybrid systems, and could be used for analyzing the interaction between papaya eIF4E, eIFiso4E protein and virus.
出处
《热带生物学报》
2010年第1期50-54,共5页
Journal of Tropical Biology
基金
国家自然科学基金项目(30760134)
中央级公益性科研院所基本科研业务费资助项目(ITBBZD0732)
