The Research Of Expressional Regulation Of Ecdysone Receptor And Ultraspiracle Genes In Silkworm, Bombyx Mori

Insects rely mainly on the titer changes of ecdysone to regulate post embryonicdevelopment of larvae exuvial, pupa metamorphosis and adult differentiation. Theecdysone receptor (EcR) and ultraspiracle (USP) are the nuclear receptors found in insects;they are the molecular target of the steroid hormone, ecdysone. The reserch of mode ofaction and regulation mechanism of EcR and USP with the ecdysone has theoreticalsignificance and application value.We used a dual spike-in qPCR technique to measure their transcriptional levels in theMalpighian tube, fat body and midgut of Bombyx mori5thinstar larvae fed on mulberryleaves that were immersed in2×10-3μg/μL ecdysone solution. Through the transcriptionlevels comparison between the tissues, analysis the the role of silkworm larvae tissues andthe EcR and USP gene in molting hormone metabolic process and expression regulationcharacteristics.Our results show that the mRNA transcription levels of two EcR isoforms inthe Bombyx mori, BmEcR-A and BmEcR-B1, changed notably, and was highest in the fatbody; other tissues had relatively lower gene expression. The mRNA transcriptionalactivity of BmUSP also changed markedly in all tissues; highest activity was in the fat body,lower in other tissues. In addition, under normal conditions, the mRNA expression levels ofBmEcR-A, BmEcR-B1and BmUSP of the4thinstar molting silkworm, and the5thinstartelophase were higher relative to other periods. The expression levels in the fat body weremuch higher compared to other tissues. Anyway, the high expression of BmEcR-A,BmEcR-B1, and BmUSP in the fat body, reveals the importance of this tissue in theecdysone metabolic process.We found that, the expression level of BmEcR-A was higher than BmEcR-B1insilkworm larvae, and the expression level of BmUSP was significantly higher than the BmEcR-A and BmEcR-B1, BmEcR-A and BmUSP from express time perspective earlierthan BmEcR-B1, this may indicate BmEcR-A prior to BmEcR-B1play physiologicalfunction, this study also showed that, in the silkworm BmEcR-A may play a moreimportant role than BmEcR-B1.Restructuring AcNPV as used for insects’ training cells of eukaryotic expressionvectors, infecting part of silkworm breeds, symptoms of infection lighter than BmNPV,silkworms can live a longer time, and don’t spread through the mouth. For this reason usingit as a kind of instantaneous expression vector to study the function of the gene expressionand regulation has certain advantage.In order to research the EcR and USP gene expression regulation mechanism, we usedthe double fluorescence quantitative expression vector system which rAcMNPV as agenetic mediate vector to analyze the function of the promoter region. Constructing aFHNLuc-A3RL double fluorescence plasmid, of which the Fireflies luciferase (FLuc) geneis activated by EcR and USP gene promoter, and the Renilla luciferase (FLuc) gene isactivated by a A3promoter of silkworm as the reference. After the deletion of EcR andUSP gene promoter, is inserted before the FLuc gene, and then analysis the luciferasectivity of FLuc and RLuc.First of all, constructing a double fluorescence donor plasmid, in Proper Sequencedeletion of BmEcR-A, BmEcR-B1and BmUSP gene promoters (2000bp,1600bp,1200bp,800bp and400bp or so), and then obtain the different length of promoter sequences bypolymerase chain reaction (PCR)amplification. The promoter sequences individually inertin double fluorescence plasmid FHNLuc-A3RL, and successfully obtain restructuringtransfer vectors. Through the Bac-to-Bac system prepare restructuring baculovirus andblank control baculovirus too. These restructuring baculovirus were injected the silkwormfifth instar larva respectively, then detecting luciferase ctivity of FLuc and RLuc.The results showed that, by the induction of2×10-3μg/μL ecdysone the expression ofBmEcR-A, BmEcR-B1and BmUSP gene were upregulated. Whether in normal conditionsor induction, the activity of every gene promoter in fat body was the highest. It also reveals the importance of the fat body in metabolic process.The fat body is the most importanttarget organization of ecdysone; The637～274area of BmEcR-A gene promoter existpromoter components, is the main active area of this gene promoter; The area BmEcR-B1promoter of435～+1is the main active area of this gene promoter; the BmUSP genepromoter1134～615and615～281are the main active area of this gene promoter.In the fat body, The area of BmEcR-A gene promoter637～274exists relatedfactors with20E induction; The area of BmEcR-B1gene promoter435～+1area existsrelated factors with20E induction; The area of BmUSP gene promoter1134～615and615～281exist related factors with20E induction.The Markov pipe and otherorganizations BmEcR-A, BmEcR-B1and BmUSP gene promoter activity is similar with thefat body.In conclusion, our results show that fat body is the most important organization ofexuvial hormone metabolic process; in the silkworm, EcR-A plays a more important rolethan EcR-B1; BmEcR-A, BmEcR-B1and BmUSP gene promoter critical areas are notidentical. This study laid the foundation to understand the EcR and USP gene expressionregulation mechanism, and further interaction mechanism research between20E andEcR/USP.