对硫磷处理下果蝇细胞应激基因的高通量分析

RNA-Seq(quantitative) analysis of the stress gene in Drosophila cells treated with parathion

【目的】通过对硫磷处理黑腹果蝇Drosophila melanogaster(William)的S2细胞,研究果蝇细胞在有机磷杀虫剂作用下应激基因的表达及其表达量的变化。为进一步分析昆虫应激反应及分子毒理提供研究依据。【方法】利用CCK-8试剂盒和细胞计数板检测不同浓度对硫磷处理下细胞的毒性及细胞存活率,应用基因表达谱(RNA-Seq)和实时荧光定量PCR(q RT-PCR)检测用药处理后细胞内基因的差异表达及表达量的变化。【结果】在35μg/m L对硫磷浓度处理下细胞活力与其他浓度相比呈现一定程度的增强,选用35μg/m L对硫磷处理的细胞做高通量基因表达谱分析显示:35μg/m L对硫磷处理细胞后扩增表达的基因有481个,差异显著表达的基因有52个,包括氧化应激基因、解毒基因和免疫相关基因的扩增或差异表达。q RT-PCR验证基因表达谱中一部分差异显著基因的表达变化与表达谱变化趋势一致。【结论】对基因的差异表达和细胞毒性检测进行了研究,为对硫磷新的作用靶标和抗性机制研究奠定了基础。

[Objectives] To investigate stress gene expression, and quantitative changes in the expression of this gene, in Drosophila $2 cells treated with parathion, and provide a basis for further analysis of insect stress reactions and molecular toxicology. [Methods] The cytotoxicity and cell survival rate of Drosophila S2 cells were tested using CC-8 reagents and a cell count plate. Application of RNA-Seq and qRT-PCR to detect differentially expressed genes and quantitative changes in gene expression. [Results] Cells exposed to 35 μg/mL parathion had high survival rates. Analysis of changes in gene expression with RNA-Seq revealed that 481 genes were up-regulated and that 52 displayed significant changes. These included oxidative stress genes, detoxification genes and immune related genes. [Conclusion] Identifying genes that are differentially expressed in response to cytotoxicity lays the foundation for analyzing the acting targets of parathion and the mechanism underlying resistance to this pesticide.