Expression And Relationship With Diapause And Developmental Metamorphosis Of Ecdysone Receptor And Two Subtypes Of Ultraspiracle In Sitodiplosis Mosellana

The steroid hormone 20-hydroxyecdysone(20E)plays an important regulatory role in the life processes of insect development,reproduction and diapause.The ecdysone receptor(EcR)and the ultraspiracle(USP)are the targets of 20 E,which form a heterodimer interaction and play an important role in the signal transduction of 20E.Sitodiplosis mosellana(Géhin)is an important agricultural pest and a typical obligate diapause insect.Based on the prior transcriptome database of S.mosellana,we identified the EcR and USP subtypes of Saccharomyces cerevisiae and determined the transcript level of EcR and USP in different developmental stages of S.cerevisiae and primarily characterized the potential interaction of EcR and USP.The main results are as follows:1.Based on comparative genome sequence analysis,we identified the EcR and USP genes in 13 representative insect species from six Orders.Using the nucleotide and amino acid sequence of EcR and USP from other insects as the template,we diged the transcriptome database of S.mosellana and found that S.mosellana potentially encodes 1EcR and 2 USP(USP-A,USP-B)proteins.2.RT-PCR obtained the open reading frame of SmEcR,SmUSP-A and SmUSP-B,with the length of 1,563,1,377 and 1,314 bp,respectively,and encoded 520,458 and 437 amino acids,respectively.The molecular weight of the encoded protein was 59.6,51.6 and 49.2kDa,respectively.The theoretical isoelectric point was 5.51,9.00 and 8.78,respectively.Multiple amino acid sequences alignemt showed that the EcR and USP of S.mosellana have a similar domain organization as the other members of the nuclear receptor superfamily and contain the typical A/B domain(trans-activation domain),C domain((DNA binding domain),D domain(hinge region)and the E domain(hormone-ligand binding domain).The amino acid identity of USP-A and USP-B of S.mosellana is 76.42%.They contain a distinct N-terimal A/B domain with the length is 106 residues(USP-A)and 85 residues(USP-B),respectively.3.The expression trends of SmEcR,SmUSP-A and SmUSP-B in the diapause process of M.sinensis larvae were consistent and closely related to the diapause stage,that is,the expression level after diapause was significantly reduced,and the diapause maintenance period was always low.However,the expression of most larvae was significantly increased after the termination of diapause,and the expression of the early stage of the recovery of the larvae was significantly reduced.These results indicate that the expression of three genes is related to the initiation and termination of diapause and the maintenance of resting stage after diapause.Two SmUSPs may simultaneously form a heterodimer with SmEcR to play a role in diapause.4.The expression trends of SmEcR,SmUSP-A and SmUSP-B were also consistent during the development of adult larvae of adult larvae,that is,the expression of larvae in the late 3rd instar was significantly higher than that in other developmental stages,followed by the pre-expansion period;The expression of female adults was slightly higher than that of males.It is speculated that the three proteins may interact at the same time in the development of the larva to the metamorphosis of the pupa and the reproductive process of the adult.5.In order to clarify the response of SmEcR and SmUSPs to 20 E during the diapause of M.sinensis,the expression levels of three genes after treatment of diapause larvae at different concentrations(0.1～0.6 pg/nL)for 20 E were analyzed.The results showed that 20 E treatment induced the expression of the three genes,but the induction effect was closely related to the time and application concentration.That is,the induction after 3 h was stronger than 1 h;after 3 h,in the concentration range of 0.1-0.4 pg/nL,the expression levels of the three genes increased with the increase of concentration,that is,at 0.4 pg/nL.The induction is the strongest.6.We constructed the EcR and USP transient expression plasmids for bimolecular fluorescence complementation(Bi FC)and coimmunoprecipitation analyses.In transfected or cotransfected Spodoptera frugiperda Sf9 cells,we did not find the complemented m Cherry fluorescence and detect the expression of the separated m Cherry domains(Nm and Cm)fused EcR and USP by Western blotting.In transfected cells,we only detected the expression of HA-tagged USP-A and USP-B.The heterologus expression system(Lepidoptera Sf9 cells)or a larger tag fused at the N-termini of EcR and USP may affect the expression of these proteins.