摘要
为了进一步了解2价Mg2+和1价Na+存在与否的情况下,多核酶系统对底物RNA的切割效率,构建了pGEM-Coat′A,pGEM-Coat′A196Rz质粒和pGEM-MDR1靶质粒.通过用SP6/T7转录试剂盒在体外转录RNA,在无细胞系统进行切割反应,反应产物通过6%变性聚丙烯酰胺凝胶电泳,干胶、X光片曝光自显影,利用ImageJ生物图像分析软件分析.结果表明,多核酶系统的切割效率依赖于二价Mg2+的浓度,切割产物随Mg2+浓度的增加而增加,而且具有反应时间的依赖性.在Na+浓度低于200mmol/L且单独存在时,没有切割产物生成.相反,在Na+和Mg2+共存时,表现出Na+抑制Mg2+诱导的切割活性,切割效率明显低于Mg2+单独存在时的结果.这些结果提示,在生理环境下,Mg2+对于多核酶系统对底物的切割反应是必需的,而Na+则不是.
To better understand the cleavage efficiency of multiribozyme system on its RNA substrate in the presence and absence of divalent magnesium and monovalent sodium ions, we constructed pGEM-Coat' A, pGEM-Coat'A196Rz plasmids and pGEM-MDR1 target plasmid. They were applied to transcribe RNAs with SP6/T7 transcription kit. Cleavage reactions were carried out in cell-free system and reaction products were analyzed by electrophoresis on 6% denaturing polyacrylamide gels in TBS buffer. The gels were dried and exposed to X-ray films for autoradiography. The Image J software was employed to analyze the dried gels. The results indicated that the cleavage efficiency of the multiribozyme was dependent on the concentration of divalent Mg^2+ . The cleavage products increased with the concentrations of divalent Mg^2+ and were Mg^2+- concentration and time dependent. No cleavage product was obtained in the presence of lower than 200 mmol/L Na^+ alone. On the contrary, monovalent Na^+ inhibited the Mg^2+ -induced cleavage reaction in Na^+ and Mg^2+ coexistance. The cleavage rate was significantly lower than that observed with divalent Mg^2+ alone. These results suggested that divalent Mg^2+ was required for multiribozyme on substrate cleavage reaction in the physical condition, whereas monovalent Na^+ was not.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2008年第7期612-618,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
深圳市科技局项目支柱(20008)
T.J.Martell Foundation for AIDS,Leukemia and Cancer Research~~