杜氏盐藻S腺苷高半胱氨酸水解酶酵母双杂交诱饵载体的构建及自激活和毒性检测

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SAHHcDNA,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。

Construction of a bait vector for SAHH of Dunaliella salina and evaluation of its self-activation in yeast two-hybrid system

Aim:To construct a bait vector for S-adenosyhomocystine hydrolase(SAHH) of Dunaliella salina and to evaluate 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.