与杜氏盐藻驱动蛋白Ⅱ动力亚基C端相互作用的新蛋白的酵母双杂交筛选

Screening of novel proteins that interact with FLA8-C terminal from Dunaliella salina using yeast two-hybrid system

目的:用酵母双杂交的方法筛选与杜氏盐藻驱动蛋白Ⅱ动力亚基(FLA8)C端相互作用的蛋白。方法:设计特异性引物(上下游分别引入EcoRⅠ和BamHⅠ酶切位点),扩增FLA8C端(433个氨基酸)cDNA,与穿梭载体pGBKT7连接以构建诱饵表达载体,然后转化酵母菌株Y187和AH109,Westernblot法检测诱饵蛋白在转化酵母菌株内的表达。通过自激活实验和毒性实验检测诱饵表达载体是否适合利用酵母双杂交的方法筛选相互作用蛋白。转化诱饵质粒的酵母菌株Y187与AH109(文库菌)杂交,待三叶形合子形成后用营养缺陷型培养基和α-半乳糖苷酶活性实验筛选阳性克隆,并对阳性克隆进行测序。结果:成功构建了pGBKT7-FLA8-C双杂交诱饵载体;经检测该载体的表达产物对Y187和AH109均无自激活和毒性作用,可用于酵母双杂交实验;杂交后筛选得到2个阳性克隆,测序结果显示为鞭毛结合蛋白107(FAP107)和含蛋白结合结构域的预测蛋白。结论:成功筛选得到了与杜氏盐藻驱动蛋白Ⅱ动力亚基C端相互作用的蛋白,分别为FAP107和预测蛋白。

Aim：To isolate proteins that interact with C-terminal region of FLA8 which is the motor subunit of kinesin Ⅱfrom Dunaliella salina.Methods：The coding sequences of FLA8-C（433 aa）was cloned into the pGBKT7 as bait and transformed into Y187 and AH109 yeast strains,and then the transcriptional activation and toxicity were tested.Yeast two-hybrid method was performed to screen the Dunaliella salina cDNA expression library.The positive clones were screened by auxotroph culture and assayed for α-galactosidase activity according the manufacturer’s instructions.The cDNA fragments of prey proteins from positive colonies were detected by colony PCR.Results：The results showed that the FLA8-C is suitable for two-hybrid library screening.Sequence analysis on positive colonies showed that they were flagellar associated protein 107（FAP107）and predicted protein（containing protein binding domain）.Conclusion：FAP107 and predicted protein interact with C-terminal of FLA8 have been screened successfully.