Function Of BmDredd On Programmed Cell Signaling Pathways Of Degeneration And Apoptosis In Cells And Silk Glands Of The Silkworm Bombyx Mori L

The silkworm Bombyx mori is an important economic insect and model organism.The economic importance of silkworms is dependent on the functionality of the silkgland,a specialized organ that synthesizes silk proteins in the silkworm.The silkgland undergoes massive degeneration during larva to pupa stage,which involves in cell apoptosis.At present,the apoptosis mechanism was investigated relatively clearly in mammals,while little was known in silkworm.Dredd was the homologous protein of the caspase family.In this study,we cloned Bombyx mori Dredd?BmDredd?and studied its functions in cellular and animal levels respectively.We also investigated the interaction between BmDredd and BmFadd.Simultaneously,the high-sequence technology was used to detect the expression of mRNA,lncRNA and miRNA from silkglands of day 3 in the fifth instar larva and the spinning 36 h.We analyzed the GO functions of target genes of the differential expressed lncRNAs and miRNAs.Finally,we applied the interaction network analysis among BmDredd,lncRNAs and miRNAs.All of the above researches made us understand more about the apoptosis mechanisms in silkworm cells and silkglands.The main results are as follows:1)We successfully cloned the BmDredd gene from the silkworm and analyzed its biological information.The ORF of BmDredd was 1632 bp,coding 543 amino acids.The molecular weight was about 63 KDa with an N-terminal long prodomain and a C-terminal CASc domain.2)The functions of BmDredd at the celluar level were studied.The BmN cells were induced with UVC or ecdysone and also treated with dsRNA.We found the expression of BmDredd was elevated in cells undergoing apoptosis and the dsRNA interference was able to decrease the apoptosis ratio and improve the number of normal cells.Furthermore,the N-terminal,C-terminal and ORF overexpression vector of BmDredd were constructed respectively.Overexpression of BmDredd increased the caspase-3/7 activity.When cells in apoptosis,BmDredd migrated from the cytoplasm to the nucleus.Its nuclear localization fragment was neither an N-terminal long prodomain sequence,nor a C-terminal CASc domain sequence.It was suggesting that the alignment sequence might be between 376 bp and 987 bp.Moreover,we quantified the transcriptional expressions of apoptosis-related genes including BmDredd,BmDaxx,BmCide-b,BmFadd and BmCreb in BmN cells with dsRNA interferences,the results demonstrated that there are complex interactions among these apoptosis-related genes.3)The functions of BmDredd in silkgland were studied.We detected the expression of BmDredd in silkglands at different developmental stages and different tissues of silkworm.The results showed that BmDredd has an important effect on silkgland apoptosis.When silkworms were feed with ecdysone,the BmDredd expression increased.We also injected the silkworm with caspase inhibitor which reduced the expression of BmDredd.Moreover,dsRNA-BmDredd injection could delay the apoptosis of silkgland and overexpression of BmDredd increased the caspase-3/7 activities.All of the results showed that BmDredd participated and induced the silkgland apoptosis.4)Fadd can bind with Fas,recruit procaspase-8 forming the DISC to induce cell apoptosis.We expressed and purified the GST-BmFadd,and then used it to pull down its interacting proteins from apoptosis cell lysate.After mass spectrometry,we found there's no interaction between GST-BmFadd and BmDredd.5)We utilized the high-sequence technology to investigate the regulations among mRNA,lncRNA and miRNA.mRNAs those belonged to the Death process showed no significant differences in transcript levels.10947 lncRNAs were detected in silkgland and the index number TCONS₀0023629?TCONS₃5829?TCONS₂8940?TCONS₂8943?TCONS₃094???lncRNAs could regulate the apoptosis of silkgland.In addition,there were 344 miRNAs could regulate 285 mRNAs involved in cell death.The results indicated that miRNAs play a more extensive and important role in the molecular regulation of silkgland apoptosis compared with lncRNAs.Finally,we screened 746 lncRNAs and 20 miRNAs that might interact with BmDredd,and drew an interaction network between them.