Preliminary Study On The Involvement Of ABC Transporters In The Secretion Of 20-hydroxyecdysone

The growth and development of insects are regulated by many hormones.Among them,ecdysone 20-Hydroxyecdysone(20E),as an important regulator of cell proliferation,differentiation and apoptosis,plays an important role in the whole life cycle of insects.Ecdysone belongs to steroid hormones.For the transport mechanism of steroid hormones,the traditional view is to enter and exit cells through simple diffusion or active transport.At present,some studies have found that the transport of steroid hormones requires the participation of transporters.Atet in in the ABC transporter is involved in the efflux of the20 E precursor substance E.E is translocated into vesicles with the assistance of Atet and released into the hemolymph through exocytosis regulated by calcium signaling.Other studies have shown that SLC transporter Ec I is related to the absorption of steroid hormones E and 20 E into target cells.The efflux of 20 E precursor E is involved in the Atet of the ABCG family.However,the involvement of the ABC transporter in the efflux of 20 E also is still unknow.In this study,RNAi technology and 20 E rescue experiments were used to screen ABC superfamily genes that may involve in 20 E efflux,and functional analysis of the selected genes was performed to provide a reference for the study of the relationship between 20 E transport and ABC family.The main results are as follows :1.Screening of genes affecting the growth and development of Drosophila in ABC superfamily.In this chapter,we first used the Gal4/UAS system to systematically silence 50 genes in 8 subfamilies of ABC transporters in Drosophila melanogaster to observe their growth and development process,and found 17 ABC transporters that could affect the growth and development of Drosophila melanogaster after RNA silencing.The 17 ABC transporter genes were further silenced and then rescued by feeding a certain concentration of 20 E feed.The results showed that the genes that could be rescued by 20 E were obtained,and the ABC transporters that might be related to the role of 20 E were screened.2.Functional verification of 20 E transport-related genes in DrosophilaThe temporal and spatial expression patterns of DmE23 gene in Drosophila melanogaster were detected by RT-qPCR technique and the changes of gene expression level after 20 E induction.The experimental results showed that DmE23 gene could be significantly induced by exogenous 20 E.The Gal4 / UAS system was used to systematically silence the DmE23 gene,and the 20 E titer and the expression of 20 E downstream genes in the hemolymph after DmE23 silencing were detected.Compared with the control,after knocking down DmE23,the 20 E titer in the hemolymph decreased significantly,and the downstream gene expression of 20 E was significantly down-regulated.Then,we predicted that there was an Ec R / USP binding site in the promoter region of DmE23.The sequence of the relevant region was cloned and recombined into the p GL3-basic plasmid.The bimolecular luciferase assay showed that the promoter activity of the recombinant plasmid was significantly increased.The above proves that DmE23 gene may involve in the efflux of 20 E in Drosophila.3.Functional analysis of DmE23 homologous gene TcABCG-8A in Tribolium castaneumFirst of all,the expression pattern of TcABCG-8A gene in the last instar larvae of Tribolicum castaneum was detected by RT-qPCR,and the 20 E induction experiment was conducted.The results showed that the expression of TcABCG-8A gene was significantly increased after feeding 20 E.Using RNAi technology to knock out TcABCG-8A gene,the mortality rate of T.castaneum increased significantly.It was found that 20 E could rescue the death phenotype caused by TcABCG-8A gene silencing by adding different contents of 20 E to flour.It is speculated that TcABCG-8A may also be involved in 20 E efflux,and the genes involved in 20 E efflux are functionally conserved.