草地贪夜蛾Sf9细胞对饥饿诱导自噬的响应

为研究草地贪夜蛾对饥饿诱导自噬的响应。从草地贪夜蛾EST数据库中筛选到一段核苷酸序列,设计特异性引物,RT-PCR技术从草地贪夜蛾Sf9细胞中扩增该核苷酸序列,经过生物信息学分析该序列与昆虫自噬相关基因ATG8同源性最高,命名为SfATG8基因,并构建了pIEX-4-mCherry-EGFP-SfATG8载体转染Sf9细胞。Sf9细胞经饥饿(PBS)诱导4 h后,Lyso-Tracker染色、Western Blotting、共聚焦显微镜检测细胞对自噬的响应程度。结果表明,经饥饿(PBS)诱导4 h的Sf9细胞中出现Sf Atg8-PE的表达,共聚焦显微镜检测到Sf9细胞膜上的自噬小体。以上证据证实饥饿(PBS)能诱导Sf9细胞发生自噬,为深入研究Sf9细胞对自噬的响应奠定了基础。

The Response of Spodoptera frugiperda Sf 9 Cells to Starvation Induced Autophagy

In order to research the response of Spodoptera frugiperda to starvation induced autophagy, a nucleotide sequences were screened from the Spodoptera frugiperda EST database, then a pair of specific primers were designed, followed the nucleotide sequences were amplified by RT-PCR technique from Spodoptera frugiperda Sf9 cells.The bioinformatics analysis showed the nucleotide sequences were high homology with insect autophagy related gene ATG8, therefore designed as SfATG8, then constructed the pIEX-4-mCherry-EGFP-SfATG8 recombinant vector and transfected into Sf9 cells.After 4 h induction by starvation (PBS), Lyso-Tracker staining, Western Blotting, confocal microscope experiments detected autophagic response.The results showed that successfully expression of SfAtg8-PE in Sf9 cells after 4 h inducing by starvation (PBS), and the autophagosomes could be detected by confocal microscope.These evidences confirmed starvation (PBS) could induce autophagy in Sf9 cells and constructed the foundation for the deeply research the response of Sf9 cells to starvation induced autophagy.