出芽酵母蛋白激酶TOS3的过量表达及其在热胁迫应答中的作用

OVerexpression of protein kinase TOS3 and its role in heat stress response in budding yeast Saccharomyces cerevisiae

出芽酵母SNF1蛋白激酶参与葡萄糖阻遏和细胞胁迫应答.TOS3可通过磷酸化激活SNF1,参与SNF1调控的信号途径.本研究利用PCR方法扩增tos3基因的蛋白质编码序列,克隆到多拷贝表达载体pYES2/NTA上构建真核表达载体,转化酵母细胞并诱导TOS3过量表达.带HIS6标签的重组TOS3蛋白通过免疫印迹得以鉴定.进一步研究了过量表达TOS3对细胞热胁迫耐受性的影响,发现在热胁迫处理条件下,TOS3过量表达可恢复△_(sn)f1突变体细胞的生长缺陷,表明TOS3可能通过不依赖SNF1的其他信号途径参与细胞对热胁迫应答的调控.

In Saccharomyces cerevisiae,SNF1 protein kinase is required for transcription of glucose-repressed genes and general stress responses.TOS3 activates SNF1 by phosphorylation of its activationloop threonine.In this study,the ORF of tos3 was amplified by PCR,and cloned into multicopy expression vector pYES2/NTA to construct recombinant expression plasmid pYES2/NTA-tos3.The recombinant plasmid was transformed into yeast cells and TOS3 protein was overexpressed.The His6 tagged fusion protein was analyzed by immunoblot.The effect of TOS3 overexpression on cell growth under heat stress treatment was also studied.The results showed that the growth deficiency of △snf1 under heat stress treatment could be compensated by TOS3 overexpression.Our data indicate that TOS3 may regulate heat stress response through a pathway independent of SNF1.