Identification Of BmUSP Gene And Study On The Binding Activity Of Its Protein And Retinoic Acid RA In Silkworm,Bombyx Mori

The insect ultraspiracle(USP)gene encodes a nuclear receptor protein that belongs to the NR2 subfamily of the nuclear hormone receptor family and is homologous to vertebrate retinoid X receptor(RXR)protein.Previous studies have shown that the nuclear receptor(NR)heterodimer complex composed of insect USP and ecdysone receptor(Ec R)responds to the regulation of20-hydroxyecdysone(20E)and participates in insect Development process such as metamorphosis and molting.Carotenoids and their metabolites(retinol,retinoic acid,etc.)have various functions to regulate the body color,vision,immunity,reproductive development of animals.In mammals,retinoic acid(RA)can bind to the receptors RXR and RAR,which in turn stimulates downstream gene regulatory networks.Early research on the silkworm BmUSP mainly focused on the regulation of ecdysone.Whether it can combine with other substances and have other physiological functions has not been reported.In this study,based on genomic data,the structure and sequence of the BmUSP gene were analyzed in detail,and then the gene was cloned,identified,and prokaryotic expression.The m RNA and protein levels of the gene were detected by RT-PCR and WB Expression pattern,obtained active protein,and explored in vitro whether it is combined with retinoic acid,whether it is involved in the regulation of carotenoid and its metabolites in the growth and development of silkworm.The main results are as follows:1.Bioinformatics analysis and expression pattern detection of BmUSP gene in silkworm ProkaryoticAccording to the reported sequence information of Drosophila USP genes,the related sequences of BmUSP gene were obtained by comparing the silkworm genome database with NCBI.The analysis results show that the silkworm BmUSP consists of 12 exons and encodes 9 kinds of m RNAs;8 kinds of m RNAs encode the same protein,which is composed of 462 amino acids,the predicted molecular weight is 51.99 KDa,and the isoelectric point is 8.11;the other The m RNA(BmUSP-X9)is predicted to encode 408 amino acids,with a molecular weight of 45.95 KDa and an isoelectric point of 7.49.The transmembrane region and signal peptide sequence were not found in both proteins;RT-PCR detection results showed that BmUSP m RNA was highly expressed in the silkworm in its dormant period,and was low in the silkworm initiation period.Among the tissues dissected at the age of five days and three days,the expression level was highest in the midgut,while BmUSP?X4(462-amino acid protein)was higher than BmUSP?X9(408-amino acid protein)in ovaries and testes.The expression levels of the two proteins gradually increased in the pre-pupa stage,and gradually increased in the first three days of ovarian development in the pupal stage,but showed a trend of high expression to gradually decreasing in the testis.After the moths laid their eggs,we continued the detection of the egg stage and found that there was high expression of BmUSP in the first 5 days.2.Prokaryotic expression and protein purification of BmUSP gene in silkworm,Bombyx moriSuccessfully constructed prokaryotic expression vectors,transformed the constructed recombinant plasmid into Rosetta expression strain,successfully expressed the two proteins encoded by BmUSP in vitro,and obtained soluble proteins.The optimal conditions for expression of BmUSP recombinant protein in Rosetta are: adding 0.5 m M IPTG in the exponential growth phase,then shaking at 16 ° C,rotating at 135 rpm,and the best induction effect after 20 hours.By comparison,the content of BmUSP?X9 in the supernatant was significantly higher than that of BmUSP?X1 in the supernatant.The recombinant protein was purified using a pre-packed column containing Ni-NTA medium and washed with a washing buffer containing 10 m M.The gradient elution revealed that the optimal elution buffer had an imidazole content of 250 m M.After purification,we obtained abundant BmUSP?X9 protein with a concentration of 0.75 mg / m L and a purity of more than 90%.3.Preparation of Bombyx mori BmUSP Protein Antibody and Western Blot AnalysisWe used purified BmUSP?X9 to immunize rabbits for 10 weeks to prepare polyclonal antibodies with antibody titers greater than 1: 50 K.Because BmUSP?X1 and BmUSP?X9 have 16 identical epitopes,antibodies prepared using BmUSP?X9 can recognize both proteins encoded by BmUSP.Both proteins of BmUSP are expressed at high levels in the dormant phase.In five-three-year-old and three-day tissues,we found that both proteins of BmUSP are expressed in various tissues.BmUSP?X1 is highly expressed in the midgut,ovary,and testes.And the expression level is higher than BmUSP?X9.BmUSP?X9 is highly expressed in the head,blood,fat,and midgut.However,the expression of BmUSP protein gradually increased in the first three days of ovarian development in the pupa stage,and gradually stabilized in the later stage with lower expression.In the early testis,BmUSP was also expressed at high levels.During the moth egg development period,BmUSP was highly expressed from two hours after the spawning to the fifth day,and the WB results were basically consistent with the semi-quantitative results,which proved the reliability of the results.4.Study on the Function of BmUSP Protein of Silkworm(Bombyx mori)Binding to Retinoic Acid in VitroBy immunofluorescence of blood cells at the age of five and three days,it was found that the content of BmUSP is abundant,Changes in absorbance of retinal-like substances dissolved in PBS by DMSO in vitro.The relationship between BmUSP and them was explored in vitro through the change in absorbance of retinoid substances in aqueous solution;However,ROH is more stable in aqueous solution than RA and RAL.BmUSP protein has no effect on changes in absorbance of ROH;In the absorbance change experiments of BmUSP and RAL,the absorbance of the blank control group(containing only RAL)is indeed lower than that of the experimental group(mixed of USP and RAL),but the degradation rate is not significantly different.In the experiment with RA,the control There were significant differences in the degradation rates between the experimental group and the experimental group.In the immunoprecipitation experiment,BmUSP did not precipitate ROH,a small amount of RAL was precipitated,and a large amount of RA was precipitated.In the subsequent Native-page,we predict that BmUSP can carry RA to form a light yellow band in the separation gel,but RA cannot enter the gel.However,compared with the control group,the USP protein was not added to the blank,and RA ran out of the spotted well.We can further confirm that BmUSP binds to RA and makes RA reside in the well.