Cloning, Characterization And Functional Analysis Of Genes In The Hedgehog Signaling Pathway In The Intestine Of Silkworm,Bombyx Mori

Hedgehog (Hh) signaling pathway was first discovered in Drosophila, and plays a vital role in drosophila embryonic patterning and development. In mammals, Damage to components of the Hh signal-transduction pathway can lead to birth defects and cancer. Hh pathway plays a key role in a variety of processes, such as embryogenesis, maintenance of tissue homeostasis, tissue repair and carcinogenesis. Hh pathway was widely present in the invertebrates and vertebrates, and highly conserved in evolution. There was a high degree of homology between drosophila and other species. Recently studies have shown that Hh may be involved in the repaire of intestinal epithelial damage and homeostatic of intestinal stem cell.The role of Hh in silkworm had not been reported.Silkworm was a modle of lepidopteral insects, and also an important economic insects. In recent years, the field of stem cell research has become the focus, however, there were a few report on slikworm stem cell. Scince silkworm midgut has a high cell turnover, We carry out research of the regulatory mechanisms of intestinal stem cells in the silkworm. Based on the the silkworm genome detailed map of the silkworm genome database, this research analyzed and studied the the members of the Hh pathway homologous genes, and discuss its function. The main results are as follows:1. Full-length cDNA clone of the Hh signaling pathway homologous genes in silkworm.On the basis of the analysis by the biological information, we identified the homologous genes of Hh, Ptch, Smo and Ci in silkworm, and named BmHh, BmPtch, BmSmo and BmCi respectively. By RACE techniques, we obtained5’RACE or3’RACE sequence of BmHh, BmPtch, BmSmo and BmCi gene. The full-length cDNA of BmHh gene is3172bp, on the chromosome9. The gene ORF length is1149bp, encoding382amino acids, the molecular weight of BmHh is42.4kDa, and the isoelectric point is8.70. BmPtch is on the chromosome3, the ORF length is3702bp, encoding1233amino acids, the molecular weight of BmPtch is137.4kDa. The full-length cDNA of BmSmo gene is3009bp, on the chromosome3. The ORF length is2613bp, encoding857amino acids, molecular weight of BmSmo is97.3kDa, and the isoelectric point is9.40. The full-length cDNA of BmCi gene is6285bp, on the chromosome27. The ORF length is4716bp, encoding857amino acids, molecular weight of BmCi is170.3kDa, and the isoelectric point is7.17. The phylogenetic tree of homologues from different species showed that Hh、Ptch、Smo、Ci were conserved from inverterbrates and verterbrates. There is one gene in every insect species. During moleclar evolution, there are three homologous genes GLI1, GLI12and GLI3, three homologous genes Shh, Ihh and Dhh, two homologous genes Ptchl and Ptch2in mammal species. We confirm that there is Hh signaling pathway in silkworm.2. The analysis of BmHh subcellular location and identification of BmCi nuclear localization signal(NLS)Analyzed self cleavage sites of BmHh, we found that cleavage site was on cysteine185, which were highly conserved from insects to mammals. Transient expression vector containing the complete CDS of BmHh was constructed. BmE-SWU3cells were transfected with the transient expression vector by liposome-mediated method. After72hours, we found BmHh distribued in the cytoplasm like network by the fluorescence observation. Detected by qRT-PCR, The expression level of BmHh raised more than100times. BmCi as transcription factors must be targeted to nucleus. By sequences analysis, four aa at512-515amino acids site was predicted to be typical NLS motif. This motif with DsRed was together cloned into transient expression vector, and expressed in BmE-SWU3cell lines, then each of the four aa were mutated respectively and constructed into vector in the same method. The results showed that the predicted NLS motif was able to target all DsRed to nucleus, but the NLS, which were mutated could not target any DsRed to nucleus.3. Expression pattern of BmHh, BmPtch, BmSmo and BmCi geneWe extracted RNA from silkworm midgut at developmental stages and different tissue at day3of5th instar.By RT-PCR techniques, we analyzed expression pattern of BmHh, BmPtch, BmSmo and BmCi gene. The results showed that BmHh, BmPtch, BmSmo and BmCi gene are expressed in the midgut throughout the developmental stages of silkworm,and the exression level are higher before each molting period. The expression lavel of BmHh, BmPtch and BmCi increased significantly at three days of I instar, I instar molting stage, II molting stage and Ⅳ molting stage, but, BmSmo had a little changes. BmHh, BmSmo, BmPtch and BmCi were expressed in all tissues of silkworm, and had a different expression pattern in different tissues.4.The pathogens induced Hh signaling(1) After fed Escherichia coli (E. coli) and Bacillus thuringiensis (Bt) on silkworm at day1of Ⅳ instar, We detected the expression of BmHh gene in the silkworm midgut by qRT-PCR. The results showed that BmHh expression in midgut increase significantly, compared with the control group after feeding E. coli at3-9h. With the time increased, the expression of BmHh consistent with the control group at12h. BmHh expression in midgut alsos increased after Bt. Different to E. coli, the expression level of BmHh also increased at3h and9h, but low compared with E. coli, then increased significantly at12h. This results show that Hh signaling may be involved in the repaire of intestinal epithelial damage. After injected Hh signaling pathway inhibitor, the expression level of BmHh, BmPtch, and BmCi were declined. We speculated that BmHh may response to intestinal injury, which may be involved in regulating intestinal stem cell proliferation and differentiation, and intestinal repairing after injuryed.(2) Cyclopamin is a small molecule compounds, which can bind to Smo inhibiting Hh signal transduction. After injeced a dose of cyclopamin, we found the expression level of BmHh and BmPtch reduced significantly from3to9hour, the expression level of BmSmo reduced significant from6to9hour; but the expression level of BmCi increased at3h, then reduced from6to9. We Speculate cyclopamin may also play a role in inhibiting Hh signaling in silkworm midgut.