Autophagy-related Gene Expression In Silkworm(bombyx Mori) And BmCPV Infection Model

As a defended and stress-regulated mechanism of the organism, autophagy can clear various biological macromolecules that have lose the function of cells such as denatured proteins, nucleic acids and other cellular components, in order to maintain cell homeostasis, and in favor of cell survival. Therefore, autophagy mechanisms have generally been preserved, among species and various cell types, showing a high degree of conservatism. Recent studies have shown that autophagy also involved in the innate and the adaptive immune response, in terms of the biological response to environmental stress, and the crucial role to prevent certain diseases such as cancer, myopathy, and to resist pathogens infection.Silkworm is in order of Lepidoptera, drawed the autophagy researchers’ attentions due to the metamorphosis characteristic. In the metamorphosis period, a large number of silkworm larvae tissues are degradated by autophagy and other proteolytic enzymatic, and then are used for the synthesis of body’s new tissues and organs. In this experiment, the expression of main autophagy-related genes in silkworm(Bombyx mori) was analyzed in the gene transcription level and protein translation level. In addition, since autophagy researchs on silkworm pathology have not been reported, silkworms were infected by Cytoplasmic Polyhedrosis Virus(Bm CPV), and the relationship between autophagy-related gene expression and viral infections was analyzed.1. The expression patterns of three autophagy-related genes(Bm Atg8, Bm Atg5 and Bm Atg7) silkworm in different tissues were detected by q RT-PCR method. The Bm Atg genes had highest relatively expression in silk gland, secondly in the fat body and midgut, while had relatively low expression in malpighian tubules, muscle and skin.2. The recombinant plasmid p ET32a-Bm Atg8 was constructed, and Bm ATG8 polyclonal antibodies were obtained. The expression of endogenous silkworm Bm ATG8 and Bm ATG8-PE proteins were detected using the WB method. It found that Bm ATG8 and Bm ATG8-PE protein bands were located on 14 k Da, 12 k Da position. The protein expression patterns in different silkworm tissues were consistent with real-time quantitative test results. Bm ATG8-PE / Bm ATG8 ratio showed significantly changes at 1-8 days in 5th instar silkgland.3. Phylogenetic tree was constructed based on the amino acid sequence of ATG8 protein.It suggested that silkworm ATG8 protein had a closer genetic distance with other species associated proteins, which showed the evolutionarily conserved characteristic of autophagy mechanism.4. Using q RT-PCR and Western blot technique, Bm Atg gene and Bm ATG8 protein expression in silkworm midgut at the 5th instar day 2 were detected after the silkworms were treated by starvation, rapamycin, high temperature and low temperature. It found a significant increase of Bm Atg8 gene expression at 24 h after starvation, and the WB strips also showed synchronization increased protein expression of Bm ATG8. Rapamycin treatment did not significantly change the 3 Bm Atg gene and Bm ATG8 protein expression. Bm ATG8 conversion efficiency decreased significantly after keeping for 1 h at 37°C, while no significant change was found at 4°C.5. The typical symptoms of infected silkworms were analysed. Viral RDRP gene proliferation throughout the course of the infection was determined using RT-PCR technique. RDRP gene can be detected at 24 h after infection, and highly expressed at 72 h. Using hematoxylin- eosin staining(HE) method, CPV infection process was observed on the midgut paraffin. A large number of virus polyhedra were found at 72 h after infection, then increased gradually, and the columnar cells showed severe nuclear enrichment and cell rupture. Genomic DNA degradation of Silkworm midgut was present at 120 h after infection.6. It was confirmed using immunohistochemical techniques that the endogenous Bm ATG8 / Bm ATG8 PE proteins of silkworm were widely distributed in the cytoplasm. Typical autophagic features of silkworm midgut tissue were observed using a transmission electron microscope(TEM), showing double membrane structure.7. Autophagy-related gene and protein expression in silkworm midgut were detected after CPV infection. Bm Atg8 gene expression decreased at 4 h after CPV infection on silkworm at the beginning of 4th instar, while increased at 24 h. Bm Atg5 expression trends were similar to Bm Atg8. Bm Atg7 gene expression increased after CPV infection. The transformation efficiency from Bm ATG8 to Bm ATG8 PE increased after CPV infection on silkworm at the beginning of 5th instar, which was in accordance with Bm Atg7 gene upregulation on transcription level. It suggested that antagonism occurred between the CPV infection and midgut autophagy.8. The recombinant plasmid p ET28-S10 was obtained by gene cloning methods, and recombinant polyhedrin was expressed by inducing the host bacteria E.coli Rosseta. Phylogenetic tree was constructed based on the amino acid sequence of polyhedrin, it proposed the polyhedrin was relatively conservative in CPV virus evolution. The CPV virus owns the characteristics of cross-infection, in a manner similar to "Trojan horse" approach.9. The characteristics of the CPV polyhedra were analyzed after alkaline buffer processing. If polyhedra solution were treated with alkaline solution and then used to infect silkworms, the pathogenic time would be in advance by 1.2 days than the original polyhedra solution. The polyhedra dissociated after the alkaline solution treatment in vitro. However, when the alkaline environment was removed and polyhedrin was added into the dissociated polyhedra solution, virus particles can not be re-entrapped to form polyhedra.