Molecular Function Of Bombyx Mori TGF-β Family Members Dpp And Daw

Transforming growth factor β（TGF-β） signaling pathway is an important intracellular signal transduction pathway, is very conservative in the process of biological evolution. The studies in humans, mice, Drosophila showed that TGF-β protein is a multifunctional cell signaling proteins. It plays an important role in cell proliferation, identification, apoptosis, differentiation and specificity of extracellular matrix synthesis. A large number of studies have shown that TGF-β plays a critical role in Drosophila and mammal. However, little research reports of genes and molecular mechanism relative to TGF-β signaling pathway is known in Silkworm, one typespecies of Lepidoptera. In this study, we cloned Bmdpp and Bmdaw genes in the silkworm TGF-β signaling pathway, detected expression level of Bmdpp and Bmdaw genes in different tissues of the silkworm and the subcellular localization of Bmdpp and Bmdaw in Bm N cells, investigated the immune responses of Bmdpp and Bmdaw to pathogenic microorganisms, showing the regulation of expression of Bmdpp and Bmdaw genes influence the proliferation of Bm NPV and Bm CPV. Explores the relevance between TGF-β signaling pathway and Toll, Imd, JAK / STAT, RNAi pathways. The interacting Bm N cell proteins of Bmdpp and Bmdaw were screened by Co-IP and the candidates were identified by mass spectrometry. These results can help us clearly understand the molecular functions of Bmdpp and Bmdaw, expecting to clarify the mechanism of the TGF-β signaling pathway in silkworm innate immune and provide new clues for the study of silkworm disease resistance. The main results obtained in this study are as follows:1. Cloning and sequencing analysis of Bmdpp and Bmdaw genesIn order to study the function of the TGF-β signaling pathway of silkworm, we refer Drosophila dpp（serial number: NM₀57963.5） and daw（Serial Number: NM₀01273038.1）genes sequences compare in the database of silkworm（http://www.silkdb.org/silkdb/） than obtaining silkworm Bmdpp and Bmdaw gene sequences.Designed gene-specific primers according to the existing EST sequences for RT-PCR cloning. The results showed that the open reading frame of Bmdpp was 1110, encoding 370 aa. The open reading frame of Bmdaw was 1086, encoding 362 aa.Structural analysis showed that the secondary structure of Bmdpp protein were mainly random coil and α helix, while Bmdaw protein were random coil primarily. The TGF-β domains were detected in Bmdpp and Bmdaw, suggesting that they were endowed with TGF-β protein activity and they showed high homology between silkworm and fly.2. The expression profiling of Bmdpp and Bmdaw genes of silkwormIn order to investigate the expression of Bmdpp and Bmdaw genes, real-time quantitative PCR was performed to analysis the expression levels of Bmdpp and Bmdaw genes in diverse tissues（testes, ovaries, fat bodies, heads, silk glands, malpighian tubules, hemocytes, midgut and trachea） in the 3rd day of 5th instar. The results showed that the Bmdpp and Bmdaw genes were expressed highest in blood cells while in a low volume in other tissues.3. The preparation of polyclonal antibody and subcellular localizationIn order to investigate the cellular localization features of Bmdpp and Bmdaw, Bmdpp and Bmdaw genes were cloned into the prokaryotic expression vector p ET-28a（+） respectively. The recombinant plasmids were transformed into E.coli and inducible expression. The mice were immunized with Bmdpp and Bmdaw recombinant protein respectively and polyclonal antibody was preparaed. Western blotting analysis showed that polyclonal antibody can be used for the detection of Bmdpp and Bmdaw because of its specificity.Cellular localization results showed that Bmdpp and Bmdaw protein were mainly localized in the cytoplasm and membrane of cells,4. Responses of Bmdpp and Bmdaw to pathogenic microorganismsIn order to explore immune response of Bmdpp and Bmdaw in challenge of pathogenic microorganisms, 4 instar of silkworms and Bm N cells were infected with Bm NPV, Bm CPV, inactivated Bab,use LPS infected Bm N cells and inactivated E.coli infected 4 instar of silkworms. Taken the cells and tissuses of experimental group and the control group, extracted the total RNA. The real-time PCR results showed that expression level of Bmdpp gene had significantly been inhibited and the expression level of Bmdaw gene was up-regulated after infection of LPS, Bab and Bm CPV.After Bm NPV infection, the expression level of Bmdpp increased, the level of Bmdaw decreased. After 4 instar of silkworms infected Bm NPV 48 h and 72 h, the transcription level of Bmdpp were 4.4 and 45.9-fold,the tanscription level of Bmdaw were 0.5 and 0.02 respectively.Induced by Bm CPV 48 h and 72 h, the transcription level of Bmdpp in midgut was increased to 34-fold and than decreased, but there are no significant changes of transcription levels in midgut of Bmdaw.5. Regulation of expression of Bmdpp and Bmdaw genes influence the proliferation of Bm NPV and Bm CPVTo further identify the function of Bmdpp and Bmdaw genes, this study designed and synthesized three kinds of sequence-specific si RNA according to the sequence of Bmdpp and Bmdaw genes, and investigated the effect of silencing Bmdpp and Bmdaw genes in cultured Bm N cells. The Bmdpp and Bmdaw were cloned into the vector p IZT/ V5-His respectively, then transfected the recombinant plasmids in Bm N cells, normal cultured and screened by zecion month obtain stable transgenic cell lines genetically. The results showed that the relative expression levels of Bm NPV and Bm CPV were reduced when Bmdpp gene was overpression. The relative expression levels of Bm NPV was down-regulated and the relative expression levels of Bm CPV had no significant changes when overpression Bmdaw gene. After silencing Bmdpp and Bmdaw genes by transfecting si RNA in Bm N cells, the relative expression levels of Bm NPV and Bm CPV were increased, indicating that regulation of expression of Bmdpp and Bmdaw gene may influence the proliferation of Bm NPV and Bm CPV.6. The correlation analysis of TGF-β signaling pathway and other signaling pathways in silkwormIn insect, innate immune pathways were mainly involved in Janus kinase（JAK） /signal transducer and activator of transcription（STAT） cascade transduction,Toll, immune deficiency（Imd） and RNAi pathway. In order to investigate the regulation of TGF-β pathway in silkworm,we chose the key genes in the four immune pathways（spz,PGRP-LB,PGRP-LE,stat,SOCS2,SOCS6,ago2,drc2）.The results showed, after overpression the Bmdpp gene,the relative expression levels of Bm SOCS2 and Bm PGRP-LB were increased 14-fold and 13-fold respectively, the expression of Bmdcr2 gene was also up-regulated. However, when overexpression Bmdaw gene, the expression of SOCS2 was decreased 97%,and the xpression of Bm STAT gene was also down-regulated.7. Screening and identification of the proteins interacting with Bmdpp and BmdawIn order to study the interaction proteins with Bmdpp and Bmdaw, the proteins of overpression Bmdpp or Bmdaw in Bm N cells were used by experiment of co-immunoprecipitation.By analysis of the mass spectrometry, we found 10 kinds of proteins.Information analysis showed that these proteins were involved in signal transduction, cell proliferation differentiation, ion channels, ovarian development, nucleotide degradation, immune response, cell cycle, cell apoptosis and other aspects.In summary, we cloned Bmdpp and Bmdaw which belong to TGF-β signaling pathway in silkworm, investigated the main features and expression patterns of Bmdpp and Bmdaw.Cleared the response of Bmdpp and Bmdaw in challenge of pathogenic microorganisms, found that regulated the expression of Bmdpp and Bmdaw gene can influence the proliferation of Bm NPV and Bm CPV. Co-immunoprecipitation identified 10 species of proteins in the silkworm Bm N cells which may interact with Bmdpp and Bmdaw proteins. These results not only can help us clarify the mechanism of the TGF-β signaling pathway in silkworm immune, but also provide a new clue for the study of silkworm innate immune.