杜氏盐藻寡糖基转移酶亚基stt3a酵母双杂交诱饵载体的构建及自激活和毒性检测  

Construction of yeast two-hybrid bait plasmid for stt3a of oligosaccharyltransferase from Dunaliella salina and detection of self-activation and toxicity of stt3a

作　　者：侯永杰[1] 李杰[1] 李庆华[1] 王建人[2] 薛乐勋[1] 

机构地区：[1]郑州大学生物工程系细胞生物学研究室,郑州450001 [2]河南中医学院生理教研室,郑州450008

出　　处：《郑州大学学报（医学版）》2011年第6期835-838,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：科技部国际科技合作基金资助项目2007DFA01240;国家自然科学基金资助项目30700014

摘　　要：目的:观察杜氏盐藻寡糖基转移酶亚基stt3a的酵母双杂交诱饵载体表达产物对酵母细胞有无毒害作用并检测其自激活作用。方法:应用PCR方法获得stt3a可溶端的基因片段,将基因片段按正确的方向插入到酵母表达质粒pGBKT7中,经限制性内酶切鉴定正确后,用PEG/LiAc法转化到酵母菌株Y187中,并通过表型筛选检测诱饵蛋白有无毒性和自激活作用。结果:成功获得了stt3a可溶端的基因片段,其表达的蛋白对酵母菌株Y187无毒害,报告基因-半乳糖苷酶活性没有被诱导。结论:酵母双杂交GAL4系统可以被用来研究杜氏盐藻中与stt3a相互作用的蛋白。Aim：To observe whether expression products of oligosaccharyltransferase subunit stt3a bait plasmid affect the growth of yeast cells and its self-activation.Methods：The cDNA fragments of stt3a were obtained by PCR and then inserted into the plasmid pGBKT7 to construct a recombinant bait plasmid pGBKT7-stt3a.Subsequently,the bait plasmid was transformed into the yeast strain Y187 by PEG/LiAc method.Results：The cDNA fragments of stt3a were amplified successfully.All expression productions were not toxic to Y187 cells and activity of the reporter geneβ-galactosidase was not induced.Conclusion：Yeast two-hybrid GAL4 system can be utilized to study proteins interacted on stt3a in Dunaliella salina.

关 键 词：杜氏盐藻 stt3a 酵母双杂交 鞭毛再生 

分 类 号：Q782[生物学—分子生物学]