酿酒酵母TAP42基因在细胞壁应激反应中的作用

Roles of Gene TAP42 in the Cell Wall Stress Response of Saccharomyces cerevisiae

研究蛋白质磷酸酶2A调节亚单位Tap42p(type 2A associated protein-42kD)在酵母细胞壁应激反应中的作用。采用一步基因置换法构建TAP42基因缺失(tap42Δ)酵母菌株;观察在细胞壁应激反应条件下tap42Δ菌株的克隆形成能力;通过全自动生长曲线分析仪检测细胞分裂增殖活力;RT-PCR检测细胞壁应激反应通路中转录因子Swi4p、Swi6p、Ssd1p和Mpt5p的表达水平。在正常培养条件下,与野生型酵母细胞(BY4743)比较,tap42Δ菌株形成较小克隆,细胞增殖活力较差;在细胞壁应激反应条件下,tap42Δ菌株的克隆大小和增殖活力都与BY4743菌株相似,菌株对应激反应诱导剂有一定的抗性;且缺失TAP42基因上调SWI4、SWI6、SSD1和MPT5等基因的转录表达水平。因此,缺失TAP42基因影响酵母细胞的分裂增殖能力,增强对细胞壁应激反应的抵抗性,上调细胞壁应激反应通路中转录因子的表达水平。

We herein aim to explore the roles of the protein phosphatase 2A(PP2A)regulatory subunit Tap42p(type 2A associated protein-42kDa)in the cell proliferation of Saccharomyces cerevisiae.TAP42-deficient strain(tap42Δ)was constructed through polymerase chain reaction(PCR)-mediated one-step gene disruption.The colony-forming ability was observed on YPD(yeast extract peptone dextrose)culture plate under cell wall stress condition,and cell proliferation assay of the tap42Δstrain was performed using a Bioscreen C MB instrument.The expression levels of transcription factors including Swi4p,Swi6p,Ssd1p and Mpt5p in the cell wall stress response signaling pathway were determined by RT-PCR.The tap42Δformed smaller clones and had poor proliferation compared with wild-type S.cerevisiae BY4743 under the physiological condition.The clone-forming ability and proliferation of the tap42Δstrain presented a similarity to those of the BY4743 under cell wall stress condition,indicating that the tap42Δstrain showed a weak resistance to cell wall stress response.And TAP42 deficiency enhanced the transcriptional expression levels of SWI4,SWI6,SSD1 and MPT5.Conclusively,TAP42 deficiency impairs the ability of yeast cell proliferation,and enhances the resistance to the cell wall stress response and the transcriptional expression levels of SWI4,SWI6,SSD1 and MPT5 in the cell wall stress response signaling pathway.