SRO9基因参与调控酿酒酵母内质网应激反应

SRO9 is involved in the endoplasmic reticulum stress response in Saccharomyces cerevisiae

摘要:

【目的】研究酵母SRO9基因在内质网应激(Endoplasmic reticulum stress，ERS)中的作用。【方法】利用PCR介导的同源重组方法构建SRO9基因缺失菌株，检测其在内质网应激诱导剂衣霉素处理条件下的克隆形成能力；通过比色法检测细胞内的H2O2含量，超氧化物歧化酶SOD活性和细胞增殖能力；通过实时荧光定量PCR检测内质网应激靶基因和超氧化物歧化酶编码基因SOD1及SOD2的转录水平。【结果】相对于野生型酵母菌株，SRO9基因缺失酵母菌株对内质网应激诱导剂衣霉素的抗性增强，参与内质网应激反应的靶基因转录上调；细胞内H2O2含量下降，SOD1、SOD2转录水平降低，总SOD活性降低；对氧化剂CHP和VK3的抵抗性减弱，复制寿命明显缩短。【结论】SRO9基因缺失酵母细胞对内质网应激诱导剂衣霉素的抗性增强，原因可能是由于SRO9基因缺失激活了细胞的内质网应激反应。

Abstract:

[Objective] To explore the role of SRO9 in the endoplasmic reticulum stress (ERS) in saccharomyces cerevisiae. [Methods] The SRO9-deletion yeast strain was made by PCR-mediated homologous recombination in wild-type yeast. Colony-forming ability of SRO9-deletion and wild-type strains was analyzed under the tunicamycin-treated ERS condition. The cell proliferation assay was performed using the Microbial viability assay kit. The intracellular H2O2 levels and total SOD activity were detected using the colorimetric method according to the assay kit. The expression levels of endoplasmic reticulum stress target genes, SOD1 and SOD2 were determined by quantitative RT-PCR (qRT-PCR). [Results] We observed significantly higher resistance to ER stress in the SRO9-deletion cells than in wild-type cells. The mRNA expression levels of endoplasmic reticulum stress target genes were up-regulated in the SRO9-deletion strain. The intracellular H2O2 levels, total SOD activity, SOD1 and SOD2 mRNA expression levels were down-regulated in the SRO9-deletion strain. Further more, the SRO9-deletion cells show increased sensitivity to oxidant CHP and VK3, the replicative lifespan also reduced in SRO9-deletion cells. [Conclusion] SRO9 deficiency enhances the resistance ability of the strain to ERS, and these might due to the ER stress responses that triggered by SRO9-deficiency.