Molecular Characterization Of Atg6in Lepidoptera

Autophagy is commonly existed in eukaryotic cells; it degrades or clears the damaged cell structures, aging organelles and unwanted biological macromolecules through lysosomal acidic enzymes, and it also promotes the recycling of cellular components. Under favorable conditions, autophagy maintains cell survival, and plays a very important role in preventing against pathogenic microorganisms. However, when the level of autophagy is too high or too low, it will induce cell death. The autophagic process is very complex, and it is tightly regulated by a group of evolutionarily conserved genes, the atg (autophagy-related gene). The autophagic regulation gene Atg6is firstly discovered in yeast cells, and this gene is mainly involved in the formation of phosphatidylinositol3-kinase complex which would affect the formation of autophagosome. A key protein in autophagy signaling is Bcl-2-interacting protein-1(Beclin-1), which is the yeast Atg6homologous in mammalian. Some studies have shown that Beclinl is not only involved in the formation of autophagosome membrane, but also involved in apoptosis signaling pathways. The Beclinl mediates signaling pathway crosstalk between apoptosis and autophagy in certain circumstances. Thus, the majority of scholars began to study the relationship of apoptosis with autophagy, and the Beclinl that participats in the signaling pathway between the two processes became an exciting target of research. Meanwhile, in the insect cells, the homologous gene of Beclinl which was named Atg6has been found. Many Lepidoptera insects are important economic insects, and the study on function of Atg6gene in the representative Bombyx mori is required.Specific primers were designed to amplify the open reading frame sequence (ORF) of Atg6gene in silkworm cells. Following it, the research on the function of the silkworm Atg6gene was initiated. Through the establishment of eukaryotic expression vector which carried the reporter gene, green fluorescent protein (GFP), the localizition of Atg6protein was assayed. The results revealed that Atg6protein was distributed in both the cytoplasm and the nucleus, and they existed in granule form in the cytoplasm. Meanwhile, the punctate degree of Atg6protein expresses differently in different insect cell lines. In the Spodoptera litura cell (TH) cell, the Atg6protein doesn’t co-localazation nor with mitochondria, or with lysosome. On the other hand, via the establishment of prokaryotic expression vector, Atg6protein was expressed and purified, and the specific antibody (anti-Atg6) was prepared by immunizing mice. The western blot analysis showed that no cleavage occurred in Atg6proteins from the silkworm cells (Bme), the Spodoptera litura cells (TH) and the bollworm cells (Ha) after the treatment of actinomycin D on these cell lines using anti-Atg6antibody. We also studied the Atg6expression differences in various organs of fifth instar bollworm, and our results showed that the Atg6protein expressed in all the head, midgut, fat body and epidermis. Meanwhile, due to the Atg6gene belongs to the low-expressed genes, and the instability of transfection efficiency, the bac-to-bac expression system which highly expressed the Atg6protein was constructed, and it contributed to the studies on the Atg6protein molecular biology characteristics. The next step is to knock down the expression of Atg6by RNA interference, thereby to further study on Atg6protein function, and the effect of this protein on the interaction of autophagy with apoptosis.