| Autophagy is an important intracellular starvation stress response and rescue mechanism, which including Cvt pathway and autophagy pathway. Autophagy is induced to phagocytosis and degradation on the part of the cytosol protein aggregates and organelles, it will provide renewable resources for cells and maintain cell homeostasis. If the rescue failed or insect was invaded by pathogens and in the development of metamorphism, autophagy might be in the high expressed levels. Then pathogens will be removed or trigger apoptosis caused irreversible cell death. In the development of metamorphosis, hormone secretion of insect inceresed very rapidly from last instar larvae to pupate, were molting increased activity, high levels of autophagy in multiple larvae organizations were induced, such as fat body, midgut, salivary glands and silk gland. During the innate immune response, autophagy can help the body clear intracellular bacteria, viruses and other microorganisms.In the process of autophagy, as the ubiquitin-like conjugation system,ATG5-ATG12was necessary for autophagosome formation, and ATG5protein could induce or mediate cell autophagy to apoptosis. Calpain-mediated cleavage of ATG5will be truncated to a24-kDa protein molecules, it could improve the sensitivity to apoptotic stimuli. Moreover, the ATG5protein play an important role as an essential component of autophagy in the innate immune process. In this sdudy, we obtained the autophagy-related gene Atg5from Spodoptera exigua and Chilo suppressalis, while ATG5protein expression and function evaluation were also investigated.(1) The full-length cDNA sequences of Spodoptera exigua and Chilo suppressalis were obtained by SMARTer RACE PCR. Open reading frame encode a264-amino acid polypeptide (32kDa). Domain structure analysis revealed that the deduced protein (from81to264) had a Pfm APG5conserved domain. The result about phylogenetic analysis demonstrated both of deduced amino acid sequences (SeATG5and CsATG5) in relationship were near with Lepidoptera. Then recombinant protein was purificated by prokaryotic expression, and western blot analysis was measured.(2) Semi-quantitative RT-PCR and RT-qPCR are employed to reveal the spacial expression pattern of Atg5from Spodoptera exigua and Chilo suppressalis. Atg5always expressed at a low level in various stages of larvae. And both of Atg5genes up-regulated from0day pupal instar to one day pupal instar. SeAtg5from Spodoptera exigua expressed at a high level in fat body and midgut during prepupa instar. (3) AcMNPV and SeNPV viral suspension (PBS as negative control) were injected to infect4th instar larva of S. exigua. results demonstrated expressed level SeAtg5up-regulated after6hours, After12hours, expressed level of SeAtg5was highest, and then decreased gradually. After18hours, expressed level of SeAtg5returned to normal. These results indicated that ATG5protein showed antiviral effect against both of insect virus.(4) Based on bioinformatics study on transcriptome of Chilo suppressalis, more autophagy-related genes including CsAtgl, CsAtg2b, CsAtg3, CsAtg6, CsAtg7, CsAtg8, CsAtg9a and CsAtg12were obtained. Different function of these genes will be study in the future.In conclusion, function of Atg5from Spodoptera exigua and Chilo suppressalis is similar to friutfly and silkworm. Atg5is not only important in the development of metamorphism, but also interacts with innate immunity. These studies will be useful in understanding the molecular regulating mechanisms in Lepidoptera pest including Spodoptera exigua and Chilo suppressalis, and insect immune theory will be further enriched, which would provide basic information and new idea in exploration of controlling strategies of this important vegetable pest. |
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