To construct a bait vector for S-adenosyhomocystine hydrolase( SAHH ) of Dunaliella salina and to e-valuate its self-activation activity and toxic effects in yeast two-hybrid system. Methods: The full-length SAHH gene was amplified by RT-PCR and confirmed by sequencing. A fragment of the gene was then subcloned into the vector SAHH of pGBKT7 to construct the bait vector. The constructed bait vector pGBKT7-SAHH was transformed into yeast strains AH109 and Y187 by PEG/LiAc method and its self-activation was tested by the phenotype assay. Results: The constructed bait vector of pGBKT7-SAHH was successfully transformed into yeast strains Y187 and AH109, and the fusion proteins were not toxic to yeast cells; the reporter genes HIS3 and ADE2 were not self-activated, but MEL1 was self-activated. Conclusion: The pGBKT-SAHH could act as a bait to screen interaction proteins of SAHH in yeast two-hybrid system.%目的:构建杜氏盐藻S腺苷高半胱氨酸水解酶(SAHH)的酵母双杂交诱饵载体pGBKT7-SAHH,并检测其对酵母细胞的毒性和自激活作用.方法:应用RT-PCR扩增杜氏盐藻的SAHH cDNA,测序分析后与酵母双杂交诱饵载体pGBKT7连接,构建诱饵载体pGBKT7-SAHH.用PEG/LiAc法将诱饵载体转入AH109和Y187酵母中,通过表型筛选检测诱饵蛋白对酵母有无毒性和自激活作用.结果:成功构建了诱饵载体pGBKT7-SAHH并成功转化到酵母细胞AH109和Y187中,表达的融合蛋白对宿主酵母细胞无毒性.在AH109和Y187酵母细胞中诱饵载体均未激活报告基因HIS3和ADE2,但是激活了报告基因MEL1.结论:诱饵载体pGBKT7-SAHH可用酵母双杂交方法筛选与SAHH相互作用的蛋白.
展开▼
