细菌双杂交验证古菌UDG与TRX相互作用的研究

Interaction between Uracil-DNA Glycosylase and Thioredoxin in Archaeon Using Bacterial Two-hybrid System

目的验证别府硫化叶菌(Sulfolobus tokodaii,S.tokodai)i DNA碱基切除修复中重要的酶-尿嘧啶-DNA糖基化酶(uracil-DNA glycosylase,StoUDG)与硫氧还蛋白(thioredoxin,StoTRX)之间的相互作用,方法将StoUDG和StoTRX的基因分别克隆到细菌双杂交载体上,共转化至报告菌株,从筛选平板上检测StoUDG和StoTRX相互作用的强弱.结果 (1)pBT-TRX与pTRG-UDG等细菌双杂交质粒共转化至报告菌株,可使报告菌株在含氯霉素、四环素和卡那霉素的LB平板上生长;(2)含pBT-TRX与pTRG-UDG质粒的细菌双杂交报告菌株可在筛选平板上缓慢生长,且不发生自激活.结论 StoUDG与StoTRX存在较弱的相互作用,提示StoTRX参与依赖于StoUDG的DNA碱基切除修复过程,

Objective To investigate the protein-protein interaction （PPI） between an important DNA base excision repair enzyme uracil-DNA glycosylase （StoUDG） and thioredoxin （StoTRX） in hyperthermophilic archaeon Sulfolobus tokodaii. Methods The bacterial two-hybrid reporter strain was co-transformed by pBT recombined with StoUDG gene and pTRC recombined with StoTRX gene. The strength of PPI was detected by observing the bacterial colony in selective plating medium. Results （1） The co-transformation of bacterial two-hybrid plasmids enabled reporter strain to resistant to chloramphenicol, tetracycline and kanamycin. （2） The reporter strain which contained pBT-TRX and pTRG-UDC plasmids grew slowly in selective plating medium without self-activation. Conclusion The results showed a weak interaction between StoUDG and StoTRX, which indicated that StoTRX played a role in UDG-dependent DNA base excision repair.