自噬相关基因ATG8在酵母细胞时序性寿命调控中的作用

The autophagy-related gene ATG8 regulates the chronological lifespan of yeast cells

摘要:

【背景】自噬在调控细胞寿命和衰老过程中起着至关重要的作用，本课题组前期研究发现缺失自噬相关基因ATG8缩短酵母细胞复制性寿命，但有关其在寿命调控中的作用机制尚未明确。【目的】研究自噬相关基因ATG8在酵母细胞寿命调控中的作用及可能机制。【方法】基于基因同源重组原理构建ATG8基因过表达酵母菌株(ATG8 OE)，检测ATG8基因缺失酵母菌株(atg8Δ)和ATG8 OE酵母菌株的时序性寿命(chronological lifespan, CLS)，采用生长曲线仪检测各菌株的增殖速率，采用流式细胞仪检测各菌株的活性氧(reactive oxygen species, ROS)含量，采用实时荧光定量PCR (real-time quantitative PCR, RT-qPCR)检测各菌株的氧化应激相关基因转录水平，比色法检测各菌株的细胞凋亡关键酶胱天蛋白酶-3 (Caspase-3)活性。【结果】缺失ATG8基因缩短酵母时序性寿命，减弱细胞增殖速率；细胞内ROS含量增多，氧化应激相关基因表达下调，Caspase-3酶活性升高；ATG8 OE菌株时序性寿命和细胞增殖能力无明显变化，活性氧含量下降，氧化应激相关基因表达上调，Caspase-3酶活性无明显变化。【结论】自噬相关基因ATG8在调控酵母细胞时序性寿命和氧化应激反应中具有一定作用。

关键词:酿酒酵母;ATG8;氧化应激;时序性寿命

Abstract:

[Background] Autophagy plays a pivotal role in regulating the cell lifespan and aging. Our previous study showed that deletion of the autophagy-related gene ATG8 significantly decreased the replicative lifespan of yeast cells, while the specific mechanism remains to be explored. [Objective] To study the effect and potential mechanism of ATG8 in regulating the cellular lifespan of Saccharomyces cerevisiae. [Methods] The yeast strain overexpressing ATG8 (ATG8 OE) was constructed by homologous recombination. The chronological lifespans (CLS) of ATG8-deleted (atg8Δ) and ATG8 OE strains were measured. The proliferative rates of yeast cells were analyzed by a Bioscreen C MB instrument based on growth curves. The production of reactive oxygen species (ROS) was measured by flow cytometry. The expression levels of oxidative stress-related genes were determined by quantitative RT-PCR (RT-qPCR). The activity of the key apoptotic enzyme, Caspase-3, was measured by the colorimetric method. [Results] The deletion of ATG8 shortened the chronological lifespan and weakened the proliferation of yeast cells. Meanwhile, it increased the production of intracellular ROS, down-regulated the expression levels of oxidative stress-related genes, and enhanced the activity of caspase-3. The overexpression of ATG8 decreased the production of intracellular ROS and up-regulated the expression levels of oxidative stress-related genes in yeast cells. The ATG8 OE strain showed no significant change in the chronological lifespan, proliferation, or caspase-3 activity. [Conclusion] The autophagy-related gene ATG8 plays a role in regulating the chronological lifespan and oxidative stress response of yeast cells.

Key words:Saccharomyces cerevisiae;ATG8;oxidative stress;chronological lifespan