Research On The Expression Regulation Of Ecdysone Receptor And Ultraspiracle Genes And Their Relative Function In Bombyx Mori

Ecdysone 20-hydroxyecdysone (20-hydroxyecdysone,20E) is an important regulator of insect hormones during development. Ecdysone receptor (Ecdysone receptor, EcR) and ultravalve protein (Ultraspiracle, USP) can form a heterodimer which is the target of insect 20E. With the help of heterodimer,20E can induce expression of many downstream target genes and passes 20E signal.In this study, we detected the gene expression levels of ecdysone receptor EcR and ultraspiracle protein USP at different times of the silk glands by real-time quantitative PCR in vivo and vitro. Ecdysone receptor domain gene EcR-B1D and ultraspiracle domain gene USPD were cloned by PCR using cDNA derived from silkgrand of silkworm as template.Then we linked them with the prokaryotic expression vector pET-28a(+), transferred the vectors to Escherichia coli BL21 cell, collected the expression products and purificated them; We detected the conjugation reaction between EcR-B1D and E75-EcRE(or deletion mutants of E75A-EcRE). The main research achievements are as follow:1.Analysis on the expression characteristics of BmEcR-A, BmEcR-B1 and BmUSP in silkworm silk grands induced by 20EThe expression level of BmEcR-A, BmEcR-B1 and BmUSP had a increase in middle silk gland and posterior silk gland induced by 20E, indicating that 20E can promote the expression of the three genes. We found that the exresssion characteristics of BmEcR-A, BmEcR-B1 and BmUSP are different in middle silk gland and posterior silk gland.2.Analysis on the expression characteristics of BmEcR-A, BmEcR-B1 and BmUSP in silkworm silk grands induced by 20EUnder standard in vitro culture, the expression level of BmEcR-A, BmEcR-B1 and BmUSP had a increase in middle silk gland and posterior silk gland induced by 20E, which was consistent with the results of in vivo experiments.When induced by 20E with JH, the expression level of BmEcR-A, BmEcR-B1 and BmUSP increased significantly, especially in the middle silk gland the performance was more significant,which indicated that JH was also possible to promote the expression of BmEcR-A, BmEcR-B1 and BmUSP under certain conditions.3.Prokaryotic expression of ecdysone receptor domain protein BmEcR-B1D and ultraspiracle domain protein BmUSPDIn this study, we cloned silkworm ecdysone receptor domain gene EcR-B1D and ultraspiracle protein domain gene USPD by PCR.The sequence analysis showed that the EcR-B1D gene has a length of 786 bp and its calculated molecular mass is 29.66 kDa;the USPD gene has a a length of 798 bp and its calculated molecular mass is 29.76 kDaWe connected BmEcR-B1D and BmUSPD into the expression vector pET-28a (+), transferred to Escherichia coli BL21 (DE3) cell after digestion identification and DNA sequencing.Protein expression was induced by 1 mmol/L IPTG. SDS-PAGE analysis showed that pET28a-BmEcR-B1D and pET2%a-BmUSPD recombinant bacteria can be detected specific bands in the 30 kDa position, indicating BmEcR-B1D and BmUSPD were successfully expressed. Both of them were subsequently purificated.4.Isothermal titration calorimetric study on binding interactions between BmEcRD protein with BmE75A-EcRE or its deletion mutantsUnder in vitro conditions we used isothermal titration calorimetry technology (ITC) to obtain a series of thermodynamic parameters of the binding interaction between BmEcR-B1D protein and BmE75A-EcRE or its deletion mutants including the affinity constant (K), reaction heat (△H) and entropy (△S). The results showed that the BmEcR-B1D protein can be combined with BmE75A-EcRE and found BmE75-EcRE core conserved region TCTTC, the region was most likely the exact site where BmEcR-B1D protein binded with BmE75A-EcRE.In this study, with 20E induced in vivo and in vitro we found 20E can upregulate the expression levels of the three genes under two conditions; Through prokaryotic expression of BmEcR-B1D and BmUSPD protein and the use of ITC technology we found the core conserved regions of the BmE75A-EcRE. These studies provide some clues to further exploring the mechanism of 20E signaling pathway, function of nuclear receptor and interaction between nuclear receptor gene.