Transcriptional Regulation Of 20E Signal Pathway By Bmmyc In Bombyx Mori Posterior Silk Gland

Bombyx mori is both an important economic insect and a suitable model organism to study the metamorphosis development and genetic evolution of insects. The mechanism complex that coordinates the insulins and ecdysone is of great significance for the growth and development of insects. The Myc proto-oncogene is known to participate in many cellular functions and have effects on cell growth, the transcription factor which acts Insulin and 20E signaling pathway influence Myc mRNA and protein levels, and control the tissues growth and organismal size. Bombyx mori Myc (named BmMyc) plays a critical role in regulating the development of posterior silk gland and improving the silk yield.This study focus on the transcriptional regulation of 20E signaling pathway genes by BmMyc. Combined RNA-Seq based transcriptomic analysis of Ras1CA-overexpressed PSG and quantitative Real-time PCR (qRT-PCR), verification of differentially expressed genes regulated by Rasl, including BmMyc and 20E genes were carried out. The sequence of BmMyc was analyzed by using bioinformatics software and online database. The spatial and temporal transcriptional profiling of BmMyc was detected by Reverse Transcription PCR. Gene expression profile of 20E signaling pathway resulted from BmMyc regulation were detected by qRT-PCR in vitro and vivo. This study proposed that BmMyc is able to surpress 20E genes transcriptional expression which determines the timing of developmental progression. The mainly achievements are as below:1. RNA-Seq technology based transcriptomic analysis of transcriptional regulation by Ras1CA-overexpression in Bombyx mori PSG.The transcriptomic data indicated that there are several differentially expressed genes in cell cycle and 20E signaling pathway, as well as BmMyc, were regulated by Ras1CA-overexpression in transgenic silkworm PSG. qRT-PCR verification of the selected genes was consistent with the transcriptomic data (FRKM). In cell cycle pathway, cdkl, cdk4/6, cdk2, cdcDl, cycD2, cdc7, cdhl and dp-1,2 genes encode cyclin dependent kinase (CDK), cyclin, cell division cycle (Cdc/Cdh) and transcriptional factors were selected and they were significantly upregulated by Ras1CA, the difference of cycD2 reached very signigicant level. BmMyc was significantly upregulated by Ras1CA.20E signal pathway genes EcR-B, USP, E75, E74, BrC, EcR-A, Ftz-fl and Hr3 were significantly downregulated by Ras1CA.2. The bioinformatics analysis and time space expression profile of BmMyc gene and protein.BmMyc gene coding sequence was cloned successfully based on the NCBI gene bank data. The full-length sequence of BmMyc gene is 1970bp with an 1130bp open reading frame (ORF) encoding a 42.7kD protein which is composed of 376 amino acids. The isoelectric point (pI) of BmMyc protein is 6.69, instability index is 70.44, aliphatic index is 69.97, grand average of hydropathicity (GRAVY) is-0.888 which indicates its hydrophilicity. The protein has no signal peptide and is forcasted to be a non-secretory protein. Subcellular localization prediction showed nuclear localization of the protein with a 0.88 coefficient. A helix-loop-helix (HLH) domain was identified in BmMyc. This region is highly conserved compared to the homologous sequence.BmMyc is widely expressed in various tissues, including head, epidermis, hemolymph, malpighian tubule, midgut, trachea, fat body, silk gland, testis and ovary. Furthermore, there is a high expression in silk gland and low in testis and ovary. Expression of BmMyc has been detected broadly during developmental stages from embryo to adult. It has a gradual rise from 1th to 5th instar, and has a decrease from prepupa to adult. From day 2 of 4th instar to prepupa, the expression of BmMyc reachs high peak at 4th molting stage, and decreases during 5th instar to the minimum amount at wandering stage, but increases slightly at day 2 of prepupa.3. Transcriptional regulation of 20E singal pathway genes caused by BmMyc overexpression in Bm12 cells.By constructing expression vector to overexpress BmMyc in Bm12 cells, the gene has a very significant upregulation, about 6000 fold. EcR-B, USP, E75, E74, BrC, EcR-A, Hr3 and E93 were significantly repressed by BmMyc overexpression. The promoters of 20E genes and BmMyc were co-transfected into Bm12 cell line, the dual luciferase assay revealed that BmMyc reduced the transcriptional activities of EcR but not USP, E75 and Brc, indicating that BmMyc had binding sites in the promoter of EcR genes, but still had no directly evidence on the binding sites. The expression of EcR-B1 and E75 were rescued after 20E treatment even when BmMyc overexpressed.4. Transcriptional regulation of 20E singal pathway genes caused by BmMyc overexpression and RNAi in silkworms.By constructing vector to express recombinant baculoviruses of BmMyc in Sf9 cells, injection of P2-generation baculovirus carried out on day 3 of 5th instar. Skin lesion of larva was observed 72 hours later after the injection. Results revealed that BmMyc overexpression delay the developmental rate during wandering and pre-pupation stage after 96 hours and 126 hours of injection. The BmMyc expression in PSG has been detected and showed significant upregulation over hundred fold.20E genes were repressed by BmMyc overexpression, the difference of BrC and EcR-A is significant, EcR-B, USP, E75, E74, Hr3 and E93 are very significant. In order to confirm the hypothesis that BmMyc has an inhibitory effect on 20E genes, this study subsequently blocked BmMyc by RNA mediated interference (RNAi) on day 7 of 5th instar. The BmMyc expression was significantly suppressed in PSG and fat body about 50%, while usp, BrC and EcR-B were steady upregulated in both PSG and fat body. The difference of usp was very significant, BrC and EcR-B were significant in PSG, usp and EcR-B were very significant and BrC was significant in fat body. The results indicate that BmMyc is able to surpress 20E genes expression combined the overexpression and RNAi.Overall, the results indicate that BmMyc is able to surpress 20E genes expression and participate in the transcriptional regulation of insect molting and metamorphosis.