Function Analysis Of BmBR-C Z2 In Regulating Innate Immunity Of Silkworm,Bombyx Mori

The early gene Broad-Complex(BR-C)is an important ecdysone-induced transcription factor.The protein configuration of BR-C contains a highly conserved protein-protein interaction in different insects such as Drosophila and Bombyx mori.Different BR-C isoforms contain two conserved domains.One of which is a BTB(bric-a-brac,tramtrack and aBroad-Complex)domain for protein–protein interaction,another is a pair of C2H2 zinc fingers.Because of the relatively conserved structure in different insects,their BR-C genes also have the same function.The moulting hormone 20-hydroxyecdysone(20E)is important to complete metamorphosis insects,which plays a significant role in development,moulting,metamorphosis and reproduction.As a transcription factor induced by ecdysone,BR-C was involved in several important mechanisms during the development and metamorphosis.In recent years,some studies have shown that 20 E play an important function in immune regulation.Interestingly,BR-C also relates to immune response induced by 20 E in Drosophila melanogaster.The important ecdysone-induced transcription factor BR-C can be induced by 20 E to activate some AMPs independent of PGRP-LC(the peptidoglycan receptor)in the Drosophila.What is more,BR-C can activate Relish and then interacts with activated Relish to regulate the expression of AMPs in the Malpighian tubules.However,the reaserch of whether the gene have same function in silkworm is still undefined.The silkworm not only can be used as a model organism,but also is an important insect which can provides important economic value in silk production.However,the infection of silkworm has caused certain problems in this agricultural production.Therefore,it is important to investigate the immunoregulation mechanism to control the silkworm diseases.Compared with the immune response role of the BR-C gene in Drosophila,BmBR-C Z2 is likely to regulate immunity function in silkworm.For this reason,we explored the function of transcription factor BmBR-C Z2 and got the following conclus: 1.The expression analysis of BmBR-C Z2 in silkwormThe experimental materials of each tissue was dissected from 5th instar and 3rd day of silkworms.The blood was obtained from different periods of silkworms too.The RNA of these materials was extracted and reverse transcribed to obtain cDNA.The expression level of BmBR-C Z2 was analyzed by fluorescence quantitative PCR in each material.The results showed that BmBR-C Z2 was widely expressed in various tissues of silkworm body at 5th instar and 3rd day(L5D3).The materials of blood in each period were detected by quantitative PCR analysis.The results showed that mRNA level of BmBR-C Z2 was reached a peak at the moulting stage of 4th larvae(L4M)then decreased at 5th larvae.After L5D7,the expression level of BmBR-C Z2 was increased rapidly and reached the highest level at prepupa(PP2)larvae.This phenomenon might related to the inconsistent level of ecdysone in the silkworms at various stages.2.The expression level of immunity related genes can be regulated by BmBR-C Z2 or 20 E.After injecting 20 E into the silkworm or stimulating the BmE cell lines by 20 E,the expression level of BmBR-C Z2 was up-regulated by detecting with fluorescence quantitative PCR.In addition,the expression level of antibacterial genes was detected in cells after 20 E stimulation too.The results showed that some of these AMPs were up regulated,which included Lysozyme(LZM),Lebocin(LEB),Gloverins(GLO),and Cecropin(CEC).Then we obtain the full-length cDNA of BmBR-C Z2 after designing the PCR primers,and the cDNA production was connected into the pSL1180-A4-Dsred overexpression vector.The overexpression plasmid was transfected into the BmE cell line,the location of DsRed was detected in nucleus of cells.In order to further examine the function of BmBR-C Z2 in immune response,we detected the mRNA level of AMP genes.The results show that the expression level of a part of AMPs were increased.It mainly includes lysozyme LZM and antibacterial peptide genes such as LEB,GLO,CECB,and CECD.Furthermore,the expression of Lysozyme was remarkably up regulated.This results suggested that BmBR-C Z2 might also play a significant role in the immune response induced by 20 E.3.BmBR-C Z2 gene could regulat the antibacterial ability of silkwormIn order to evaluate the potential function of BmBR-C Z2 in immunity of silkworm,qPCR was used to analyze the expression changes of BmBR-C Z2 in hemocytes treated by PAMPs.S.ta,P.ae,LPS and PGN were injected into the larvae.The results of qRT-PCR showed that BmBR-C Z2 had a high response after injection especially at 30 min.Then the expression level of BmBR-C Z2 was decreased at 2h to 6h.But after 12 h,the mRNA level was increased again.According to these results,we speculate that BmBR-C Z2 was related to immune response of silkworm.To confirm the role of BmBR-C Z2 in immunity of silkworm,shRNA of BmBR-C Z2 was used to decrease its expression in SID-1 cell,shEGFP was used as a control.The mRNA level of these AMPs,including Lysozyme,Lebocin,Gloverins and Cecropin,was suppressed when BmBR-C Z2 decreased.The expression of Lysozyme was decreased significantly.Furthermore,we injected E.coli into the B.mori after shRNA injection,the survival rates of the silkworms treated with BmBR-C Z2 RNAi were significantly lower than the control group.Those results above depicted down-regulating BmBR-C Z2 could make the response of innate immunity weaken by down-regulating the expression of AMP genes.It is speculated that BmBR-C Z2 could improve the silkworm's ability to resist bacteria by regulating the expression level of antibacterial genes.4.BmBR-C Z2 gene could bind to the promoter of LZM gene to activate its expression in silkwormIn this study,a part of AMP genes were increased by over-expressing the BmBR-C Z2,and they could also decreased when the expression level of BmBR-C Z2 decreased.Lysozyme,the most obviously changed gene,was chosen to detect its promoter activity after simulating via over-expressing BmBR-C Z2.The prediction promoter sequence of this gene was downloaded from the silkworm gene database,and the promoter sequence product of different regions was obtained by PCR.The production was connected to the plasmid PGL-3 of the luciferase activity reporter system.The recombinant plasmid was co-transfected with the BmBR-C Z2 overexpression plasmid into the cell lines.After 48 h the activity of the dual luciferase in different promoter regions was determined.The results showed that compared with the control group,the enzyme activity of the recombinant luciferase recombinant plasmid was significantly higher than the experimental group.Then the promoter was truncated and tested again.The BmBR-C Z2 could activated the expression of Lysozyme by binding to its promoter sequence and the site was at the sequence from-1476 to +88.Thereby,the BmBR-C Z2 could participate in immune regulation and enhance the immunity of the silkworm.