Screening And Functional Verification Of Key Genes Of Ecdysone Transport Based On Cell Level

Ecdysone is an important steroid hormone,which plays a key role in many physiological processes such as insect embryonic development,larval growth,molting and metamorphosis,pupal morphological reconstruction and adult reproduction through its interaction with nuclear receptors.Compared with the extensive literature describing the biosynthetic pathways and signaling pathways of ecdysone steroid hormones,there are few studies on how these hormones enter and exit cells through the lipid bilayer of cell membranes.Ecdyssteroids,as lipophilic hormones,are generally thought to regulate various physiological processes by simply diffusing into and out of cells.In recent years,a series of studies have shown that membrane transporters have key functions in transport across cell membranes,challenging the simple classical diffusion model of steroid hormone transport.ABC transporter(ATP-binding cassette transporter)is a membrane transporter that can use the energy released by ATP hydrolysis to transport hormones,lipids and other substrates in a reverse concentration gradient.In Drosophila melanogaster,endocrine release of the ecdysone precursor E has been shown to be accomplished by DmAtet in the ABC protein G subfamily mediated by calcium ion signaling.The ABC family gene TcABCG-8A has also been confirmed to play a role in the metabolism of ecdysone in the study of rubesimilaptor.Therefore,in this paper,the S2 cells of Drosophila melanogaster were used as the main research material to screen key genes of ecdysone transport based on the ABC transporter G subfamily genes(DmABCGs)at the cellular level,and a variety of experimental techniques were used to verify the function of the screened genes,in order to find new effective targets for the development of insect growth regulators.The main results of this study are as follows:1.Bioinformatics analysis of DmABCGsIn this chapter,the DmABCGs sequence was obtained from NCBI,and then the sequence molecular characteristics,protein structure characteristics and phylogenetic evolution of 15 ABC transporter G subfamily members of Drosophila melanogaster were analyzed by bioinformatics technology.The results showed that all 15 DmABCGs contained the structural motifs of the typical ABC transporter family,and the nucleic acid binding domain and transmembrane domain were arranged in reverse,which only existed in the G subfamily and the H subfamily.Based on this,it is speculated that the G subfamily genes may have similar physiological functions to the H subfamily.2.Cloning of DmABCGs and screening of 20E transport key genesIn this chapter,the recombinant plasmids of 15 DmABCGs were constructed by PCR and homologous recombination.The constructed recombinant plasmid was transfected into Drosophila S2 cells to overexpress the corresponding genes,and the key genes of 20E transport were screened by ELISA.The results showed that DmCG3327(DmE23)was the most likely to play an important role in extracellular 20E.3.Analysis and verification of DmE23 gene functionIn this chapter,after overexpression of DmE23 in Drosophila S2 cells and 20E stress,semi-lethal concentration analysis was performed by CCK-8,20E response activity analysis was performed by Dual-luciferase reporter assay,and intracellular 20E titer analysis was performed by ELISA assay.Through the above three experiments,the function of DmE23 gene was analyzed and verified in different dimensions.The results showed that DmE23 may indeed be involved in the efflux of cell 20E.4.Screening of DmE23 Interacting proteinsIn this chapter,after co-overexpression of DmE23 and 15 DmABCGs in Drosophila S2 cells,the homologous or heterologous dimer interaction proteins of DmE23 were screened by bimolecular fluorescence complementation.The results showed that DmE23 can not only form homodimer,but also form heterodimer with DmAtet to perform its normal 20E transport function.