Project description:RNAs associating with PIWI proteins were Immunoisolated from BmN4 cells. Sequence libraries were generated with NEBNext Small RNA Library Prep Set for Illumina(NEB). Libraries were sequenced using Illumina MiSeq (single-end, 51 cycles).
Project description:Insect NF-κB-like factor, Relish, is activated by viral infection and induces the production of antiviral proteins. In this study, we performed a transcriptomic analysis of BmE cells expressing the active form of BmRelish (BmRelishact) and identified BmVago as the most strongly-induced secreted-protein. Expression of Bmvago was specifically triggered by Bombyx mori nucleopolyhedrovirus (BmNPV) infection and regulated by BmSTING-BmRelish pathway. Incubating the fresh culture of cells with supernatant medium of BmVago-expressing cells or recombinant BmVago protein (rBmVago) significantly increased antiviral resistance. On the contrary, reducing the expression of Bmvago by RNA interference (RNAi) in BmE cells as well as in silkworm larvae impaired antiviral response. Furthermore, we constructed transgenic silkworm line over-expressing BmVago (BmVagoOV) and found they had markedly lower viral load and higher survival rate after BmNPV infection compared with the wild-type control. Co-immunoprecipitation assay showed Bmintegrin β1 interacts with BmVago and it was involved in BmVago-mediated antiviral response. Finally, we found the expression level of signaling molecules in Jak-Stat pathway increased in rBmVago-treated cells and BmVagoOV silkworm larvae but decreased in RNAi-treated cells. In summary, our research uncovered an inducible antiviral response in silkworm mediated by cytokine BmVago, which is the downstream effector of BmSTING-BmRelish pathway and functions as an antiviral cytokine.
Project description:A mass spectrometry-based proteomics analysis was performed to study the protein binders of GTSF1L in germ cell culture (BmN4; ovary-derived) of Bombyx mori. Anti-HA pull-downs were performed in BmN4 cells transfected with the HA-BmGtsf1L plasmid or the HA-eGFP control plasmid. Proteins were digested in-gel by trypsin. The resultant peptides were then dimethyl labelled and combined. Peptides were measured on a Q Exactive Plus Orbitrap mass spectrometer.
Project description:RNAs associating with PIWI proteins were Immunoisolated from BmN4 cells. Sequence libraries were generated with NEBNext Small RNA Library Prep Set for Illumina(NEB). Libraries were sequenced using Illumina MiSeq (single-end, 51 cycles).
