酿酒酵母CMK2在氧化胁迫应答中的作用

The role of CMK2 in oxidative stress response in Saccharoyces cerevisiae

CMK2是酵母中和哺乳动物钙调蛋白激酶CaM kinasesⅡ同源的一种丝氨酸/苏氨酸蛋白激酶,具有重要调控作用.本文利用PCR方法扩增含内源启动子的CMK2基因序列,克隆到pRS316上并在C末端连接6个组氨酸标签,构建真核表达载体,转化不同酵母进行表达,并通过免疫印迹鉴定.发现氧化胁迫后细胞中CMK2发生磷酸化.生长实验结果显示cmk2缺失的细胞对氧化胁迫非常敏感,而△cmk2中转入pRS316-cmk2-HIS6可以部分恢复△cmk2细胞对双氧水的敏感性.表明CMK2参与细胞氧化胁迫应答途径,并发挥正调控作用.

CMK2 is a serine/threonine dependent protein kinase, homologous to mammalian CaM-dependent kinase-2. It plays a key role in signal transduction. The ORF and endogenous promoter of cmk2 was amplified by PCR, and coloned into expression vector pRS316 to construct recombinant expression plasmid, moreover, six Histidines（HIS6） tag was added to the C terminus of crnle2 gene. The recombi- nant plasmid was transformed into yeast. Expression of cmk2 was verified by western blot. The results showed that CMK2 was phosphorylated under oxidative sti-ess. △cmk2 strain was hypersensitive to oxidative stress, but this sensitivity can be partially suppressed if recombinant plasmid （pRS316-cmk2- HIS6） was transformed into △cmk2 cells. In conclusion, CMK2 as a positive factor involved in the regulation of oxidative stress response pathway.