A Preliminary Study On The Regulation Of BmEsr16 Expression By 20-hydroxyecdysone

Insects respond to the infection of pathogenic microorganisms mainly through the two classic signal transduction pathways of Toll and IMD.In recent years,it has been reported that 20 E can also participate in the regulation of the synthesis of antimicrobial peptides when there is no pathogen infection.There are many reports on the mechanism of 20 E regulation of the synthesis of antimicrobial peptides,but the specific regulatory mechanisms are still not clear.In our previous study,we found that the silkworm ecdysone regulatory protein(Esr16)can bind to lipopolysaccharide(LPS)and induce the expression of the antibacterial peptides Moricin and Cecropin and participate in innate immunity.So can BmEsr16 be regulated by 20 E to participate in the humoral immunity of silkworm?Based on this problem,we chose to conduct a preliminary study on lepidopteran insect silkworm as the representative of the following:(1)To investigate whether the silkworm BmEsr16 has a synergistic relationship with the titer of 20 E under natural development;(2)Exogenous 20 E treated with BmEsr16 in vivo,in vitro cultured fatbody and BmN cells of midgut and silkworm respectively.The expression of BmEsr16 was detected by Western blot and RT-qPCR,respectively.(3)Bioinformatics analysis of the structure of BmEsr16.And promoter sequences,looking for target transcription factors regulated by 20 E,and using double luciferase assay for promoter activity detection.Through the above experimental study,the following results have been obtained:(1)Bioinformatics analysis of BmEsr16 protein revealed that BmEsr16 protein has a domain similar to that of human Npc2 protein and belongs to the ML superfamily,and there are three potential cholesterol or lipid binding sites,suggesting that BmEsr16 may be involved in Silkworm lipid metabolism.(2)The expression of BmEsr16 in the fat body and midgut of the silkworm was detected by Western blot and RT-qPCR.It was found that the transcriptional activity of BmEsr16 showed the same trend as that of the 20 E titer,from the 5th instar 2d to the walking phase(W The expression level of BmEsr16 was up-regulated at both RNA and protein levels with increasing 20 E titer.(3)Different doses of 20 E were injected into 5th instar and 4d silkworms,and fat bodies and midgut were detected by Western blot and RT-qPCR.The expression of BmEsr16 in fat bodies and midgut was detected after 10?g/head 20 E injection for 12 h.The amount will be significantly raised.(4)Exogenous 20 E was used to treat the in vitro cultured silkworm fat bodies.When the 20 E concentration was 5 ?g/mL,the transcriptional expression of BmEsr16 was significantly up-regulated.(5)After treatment of BmN cells with 20 E,the 20 E primary response factor E75 A was significantly up-regulated,but there was no significant change in the transcriptional expression of BmEsr16.It is suggested that only BmEsr16 can respond to the induction of 20 E and increase its transcriptional activity at the level of living body of silkworm.(6)Analysis of the BmEsr16 upstream promoter region revealed 6 possible ecdysoneregulated transcription factor binding sites,namely BR-C Z1(1),BR-C Z2(1),and BR-C.Z4(2),E74A(1),EIP74EF(1)binding sites.After the full-length promoter sequence was obtained by PCR amplification,the truncated and deleted promoters were cloned by overlapping PCR technology and cloned into the luciferase vector pGL3-Basic to obtain the full-length type and the truncated type.Recombinant plasmids with mutant promoters.In summary,20 E treatment of silkworm in vivo and in vitro tissue culture will cause the up-regulation of BmEsr16 expression,laying the foundation for further research on the relationship between 20 E and innate immunity.