Function On BHLH Transcription Factors,Bmsage And Bmdimm, Expressed Specially In The Silk Gland Of Silkworm, Bombyx Mori

The Bombxy mori silk gland is a specifically differentiated silk-producing organ composed of the anterior (ASG), middle (MSG) and posterior (PSG) silk gland. The ASG is responsible for silk spinning, the MSG secretes sericin and the PSG produces fibroin. Fibroin is composed of three main protein components, fibroin heavy chain (fib-H), light chain (fib-L) and P25, encoded by the corresponding genes, which forms a hexameric structure with a fib-H/fib-L/P25ratio of6:6:1. In molting of silkworm larvae, only little or no silk proteins are synthesized. When entering fifth instar period, fibroin and sericin genes transcription levels are greatly improved and silk proteins are greatly synthesized. Previous studies showed that the expression of silk protein genes is mainly regulated at the level of transcription. A number of transcription factors have been identified in silk glands, which widely involved in the process of whole life, such as the development of silk gland, the transcription of silk proteins genes and silk gland cell apoptosis. After the silkworm genome database and microarray data were accomplished, it provides a information platform for us to study the specific transcription factors in B.mori silk glands. By screening the microarray data, it was found two transcription factors expressed specifically in silk glands which contain bHLH domain and named as Bmsage and Bmdimm. It is hypothesized that Bmsage and Bmdimm may be involved in the transcriptional regulation of silk protein genes. Therefore, its biological evolution and expression pattern from the transcription level and protein level were analyzed. Meanwhile, its role in the transcriptional regulation of silk protein genes and the effect of juvenile hormone on silk proteins synthesis were discussed from cell level and molecular level, the main results are as follows:1、Prokaryotic expression and expression pattern analysis of Bmsage and Bmdimm Phylogenetic analysis revealed that Bmsage has high similarity with Drosophila sage and Mouse Mesp; Bmdimm has high similarity with Drosophila dimmed and Mouse Mist. Together, we think that Bmsage belongs to the Mesp subfamily of the bHLH family and Bmdimm belongs to the atonal subfamily as the same with Mist. In addition, we cloned and expressed the Bmsage and Bmdimm recombinant proteins. Among them, Bmsage is expressed in the form of inclusion body at37℃, Bmdimm is not expressed in full length in many conditions, but expressed the soluble protein in the first short form (187-636aa). Two New Zealand rabbits were immunized with purified recombinant proteins to prepare polyclonal antibody. The titers of polyclonal antibodies reached512K and the purity were more than95%which fitting for further experiments. The transcription factors of bHLH family have important regulatory functions in each stage of biological process. From the level of transcription, Bmsage and Bmdimm were expressed in silk glands, and Bmdimm have a small amount of expression in the midgut that is consistent with the data of silkworm microarray. Further analysis showed that it can be detected the transcription of Bmsage and Bmdimm in ASG, MSG and PSG. However, Bmsage and Bmdimm only existed in MSG and PSG from the protein level. Bmdimm protein in PSG was significantly higher than in MSG. RT-PCR analysis revealed that the expression of Bmsage and Bmdimm begun from the late4th molting, gradually increased in5th intermolting and reached the highest at6-7day, and then gradually reduced in the spinning stage. The expression pattern of Bmsage and Bmdimm is similarity to that of fib-H. qRT-PCR analysis showed that Bmsage and Bmdimm expression in PSG of872were significantly higher than in Dazao (Dz), and Bmdimm gene in PSG is also higher than that of MSG. The transcription of fib-H in PSG of872is about3-bold in Dz. Thus, we speculate that Bmsage and Bmdimm may be involved in the transcriptional regulation of fib-H gene.2、Transcriptional regulation of Bmsage on fib-HTranscription factors generally work with other factors and regulate the downstream target gene. Western blot analysis showed that the fork head factor SGF1existed in MSG and PSG as the same with Bmsage. To determine whether or not Bmsage interacts with SGF1, the far-western blot and ELISA assays were preformed. The results showed that Bmsage can interact with SGF1in vitro using the recombinant protein. To further verify the results, immunostaining and immunoprecipitation (co-IP) assays were preformed in embryo cell line of B. mori. The results showed that it was consistent with the results in vitro. EMSA assay showed that SGFl can bind to the A and B sites of the fib-H promoter by interacting with Bmsage after incubation in vitro. The fib-H promoter deletion experiments showed that A and B sites are the regulation of SGF1sites on fib-H, indicating that Bmsage and SGF1proteins are able to form complexes and bind to A and B sites involved in the regulation of the expression of fib-H.3、Transcriptional regulation of Bmdimm on fib-H For the analysis of the mechanism of transcriptional regulation of fib-H gene,865bp upstream regions from the transcription start site of fib-H gene from the silkworm genome database was download and predicted, the result showed that except A, B, C, D and E sites, there are transcription binding sites in the promoter region, such as E-box, βFTZ-F1and POU. Chromatin immunoprecipitation assay (ChIP) results showed that Bmdimm can bind to the E-box (CAAATG) element in the promoter region of fib-H. It was further verified by EMSA assay. Besides binding to CAAATG element, Bmdimm can also be interacting with other forms of E-box sites, such as CAATTG, CAACTG, suggesting that the binding sites of Bmdimm are not the only, implying that Bmdimm may be involved in the transcriptional regulation of other genes, such as fib-L and P25. The results of site-directed mutagenesis showed that the conserved arginine (113rd) in the basic regions of Bmdimm is the key site binding to CAAATG element. The results of promoter truncation suggested that the promoter activity deleted the E-box site of the fib-H promoter has no change after over-expressing Bmdimm, but in the normal promoter activity was significantly increased, suggesting that Bmdimm can regulate the expression of fib-H gene by the E-box site. The bHLH transcription factor via its bHLH domain form the homodimer or heterodimer each other, binding to specific DNA sequences by a conservative basic region and regulating the expression of target gene. To determine whether or not Bmdimm interacts with Bmsage, the far-western blot and ELISA assays were preformed. The results showed that Bmdimm can interact with Bmsage in vitro. To further verify the results, immunostaining and immunoprecipitation (co-IP) assays were preformed in embryo cell line of B. mori. The results showed that Bmdimm can interact with Bmsage in BmE cells. In order to analyze the regulation network of Bmdimm, about2000bp upstream region of the Bmdimm transcription start site was analyzed. The results revealed that there are fork head response elements, suggesting that SGF1may play a regulatory role in the expression of Bmdimm gene. EMSA assay showed that SGF1could bind to the fork head response elements, and Bmdimm promoter activity was increased by over-expressing SGF1in BmE cells. qRT-PCR showed that the expression of Bmdimm in cells was also increased.4、Regulation of juvenile hormone to fibroin H chain geneThe bio-informatics showed that there are many insect hormone response elements in the upstream region of Bmdimm, including E-box, BR-C, Kr, E74A and βFTZ-F1. It have been confirmed that these elements are important transcription factor binding element of insect hormone signal transduction pathway in regulating the expression of downstream target genes. Therefore, it implys that Bmdimm may be involved in hormone signal transduction pathway. With the treatment of the early fifth instar of silkworm by juvenile hormone analogue (JHA), it was found that the stage of larvae prolonged2-3days compared to control (treatment with acetone). qRT-PCR results showed that the expression of fib-H gene and its regulatory factor Bmdimm was decreased in the early stage, then increased gradually along with the development, and the expression was delayed compared to control.Meanwhile, the expression of BmKr-hl which is early response genes of JH was also increased in the early stage, and then decreased with the decline of JH receptors expression, and the expression was delayed in the late of stage. In the silkworm cell lines, it was found that Bmdimm has dose and time-dependent to JHA induced. BmKr-hl is located in the downstream of JH receptor BmMet2in response to induced expression of JH. The expression of Bmdimm was increased by over-expressing BmKr-hl protein, but the expression of ecdysone primary response gene Brc (Brc-Z2and Brc-Z4) was inhibited. It indicated that there is the hormone signal transduction pathway in B. mori cell lines. Furthermore, Bmdimm and fib-H no longer response to induced by JH after RNAi with dsBmKr-h1in BmE cells, indicating that BmKr-h1located in the upstream of Bmdimm and regulated its expression. Torgether, our results provided clues to further clarify the regulation of insect hormones on silk proteins synthesis.