| As a transcription factor, Bm OVO plays an important role in regulating the development of ovary. In order to explore the transcriptional regulatory activity of four Bm OVO spliced isoforms and different domains, p Fast Bac TMDual was selected as basic vector, and then four luciferase expression vectors p Fast-potu-Luc-ie1-ovo were constructed. In these vectors, four spliced isoforms of Bmovo gene(Bmovo-1, 2, 3, 4) are controlled by the Bombyx mori baculovirus ie-1 promoter, while luciferase luc gene is controlled by the promoter of Bombyx mori ovarian cancer gene otu. The dual-luciferase reporter assay was used to investigate how Bm OVO regulates the activity of otu promoter by co-transfecting both the luciferase luc gene expression plasmid and reference plasmid p RL-TK into Bm N cells. The results show that Bm OVO-2, Bm OVO-3 and Bm OVO-4 have a positive regulation on otu promoter activity, of which Bm OVO-2 has the highest transcriptional activation. When low doses of p Fast-potu-Luc-ie1-ovo1 was transfected into Bm N cells, the transcriptional activation of Bm OVO-1 for otu promoter was not obvious, but with the doses of p Fast-potu-Luc-ie1-ovo1 increased, otu promoter activity was enhanced. Additionally, this study also explored when different combinations of luciferase gene expression vectors were co-transfected into Bm N cells, how otu promoter activity changed by dual-luciferase reporter assay. The result showed when p Fast-potu-Luc-ie1-ovo1 was respectively co-transfected with p Fast-potu-Luc-ie1-ovo2, p Fast-potu-Luc-ie1-ovo3 and p Fast-potu-Luc-ie1-ovo4, otu promoter activity was significantly increased compared with single transfection of p Fast-potu-Luc-ie1-ovo. This result indicated that Bm OVO-1 might interact with Bm OVO-2, Bm OVO-3, Bm OVO-4 to improve the transcriptional activity of otu promoter.Bm OVOs all have C2H2 type zinc finger at the C-terminal, while different at the N- terminal. In order to investigate the function of each domain which is located in the N-terminal of the protein Bm OVO-1, Bmovo-1 gene was truncated based on the sequence analysis of Bm OVO-1, and the luciferase expression vectors with different truncated Bmovo-1 expression cassettes were constructed to transfect into Bm N cells. The results of dual-luciferase reporter assay showed that the first acidic domain A1(A1,13-25aa) and third acidic domain A3(A3, 180-191aa) at the N-terminal had transcriptional inhibition on otu promoter; the second acidic domain A2(A2, 30-51aa) and the first basic domain(B1, 253-311aa) had inconspicuous transcriptional regulation; the forth acidic domain A4(A4, 339-352aa) and the fifth acidic domain A5(A5, 362-372aa) might play a role in transcriptional activation. Thus, when the domain A1 fused with the N-terminal of transcriptional activator Bm OVO-2, the transcription activation of fusion protein declined sharply compared with Bm OVO-2. This result further illustrated that A1 could inhibit transcription of target genes.Furthermore, whether Bmovo can interact with other genes to regulate the expression of target genes is still unknown. Previous studies have shown that small peptides encoded by the Drosophila can regulate the function of Shavenbaby, but whether Bombyx mori has similar regulation with Drosophila is unclear. Transcript of Bombyx mori tal-like gene has four coding reading frames(A1-A4 area) and a B area. In this study, gene tal-like c DNA sequence(Tal),containing 5 ’non coding sequence and it’s downstream A1-A4 and B district c DNA sequence(5A1-4+B), containing A1-A4 and B district c DNA sequence(A1-4+B),containing B district and its downstream sequence(B) were respectively cloned into the insect cell expression vector p IZT-V5/His to construct the tal-like expression vector p IZT/V5-His-tal, p IZT/V5-His-5A1-4+B, p IZT/V5-His-A1-4+B, p IZT/V5-His-B.Dual-luciferase reporter gene system assay results showed that otu promoter activity was significantly reduced in the cells transfected by above tal-like expression plasmids, suggesting that the Bombyx mori Tal-like peptide can down regulate otu promoter activity. The results of tal-like gene expression vector and p Fast-potu2-2-Luc-ie1-ovo1 co-transfection experiments showed that the Tal-like peptide(Tal, 5A1-4+B and A1-4+B) down regulation on otu promoter could be rescued by Bm OVO-1 to a certain extent. Bm OVO-1 protein N terminal(28aa) and red fluorescent protein(Ds Red) fusion expression vector p IZT/V5-His-ovods RED and tal-like gene expression vectors were co-transfected into Bm N cells, by fluorescent microscopy and Western blotting assay, Tal-like peptide(5A1-4+B and A1-4+B) can stimulate hydrolysis of the N-terminal of Bm OVO-1.Additionally, the dual-luciferase reporter assay also showed that TGF- beta family members Dpp and Daw, Wnt signal pathway regulatory factor-receptor tyrosine kinase-like orphan receptor(Ror2), signal transduction and transcription activation of transcription(STAT) and insulin BBx-B8 had an antagonistic effect on the transcription activation of Bm OVO-2. These results provide a clue for us to understand the precise regulation of the expression of target genes by Bm OVO.To identify the target binding sites of Bm OVO on otu promoter, according to the conserved sequence of Drosophila OVO target site(TTACMGTTACA, M = A / C), we analyzed and predicted the potential Bm OVO binding sites on the otu promoter(BABH01009636) by JASPAR CORE(http://jaspar.genereg.net/cgi-bin/jaspar_db.pl) and further concluded that there are three candidate binding sites, GTACCGTTGTA(CE1,20496-20506 region), AGGCCGTTAAG(CE2, 20598-20608 region), CCTGAACTACA(CE3,20728-20738 region). The results of Electrophoretic Mobility Shift Assays(EMSA) showed that C terminal recombinant protein of Bm OVO-2(400-577 aa) could bind with otu promoter at CE1 site, suggesting that Bm OVO can directly regulate the expression of otu by binding with its promoter. What’s more, we also found otu promoter could also bind with other nucleoprotein. The second base T mutated to C(T2->C) at site CE1, causing its ability to bind with the nucleoprotein to be weakened, while the eleventh base A mutated to G(A11-> G), leading to the binding capacity being enhanced. Different truncated otu promoter controlling luciferase gene expression, the results of dual-luciferase reporter assay showed that deletion of CE1 and CE2 had an inconspicuous effect on the activity of otu promoter regulated by Bm OVO-1; With deletion of CE1, Bm OVO-2- mediated regulation of otu promoter activity plummeted, while otu promoter activity did not change significantly with deletion of CE1 and CE2; when CE2 was deleted, Bm OVO-3-mediated regulation of otu promoter activity decreased, but the change of otu promoter activity is not obvious with deletion of CE1. These results indicated otu promoter activity was not only affected by the cis-regulatory element, but also by transcription factor Bm OVO and other trans-acting regulatory factors.The dual-luciferase reporter assay of otu promoter with different mutation / deletion of CE1 site showed that when T2 mutated to G or C, Bm OVO-2-mediated regulation of otu promoter activity was enhanced. However when T2 mutated to A, there was no significant change to otu promoter activity; when A3 mutated to T, G or C, Bm OVO-2-mediated regulation of otu promoter activity was weakened, while A11 mutated to G, otu promoter activity increased; when T2, A3 and A11 was deleted respectively, there was no significant change to otu promoter activity; interestingly, otu promoter activity was enhanced with deletion of all of these bases. All of these results gave molecular clues for us to understand the precise regulation of the expression of target genes by BmOVO. |
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