| Bombyx mori is a silk-spinning insect of Bombycidae,Lepidoptera.C2H2 zinc finger protein is an important transcriptional regulator in Bombyx mori,which can bind to specifical nucleic acid,combine with different target gene sequences to play different biological functions,and participate in versatile physiological processes.In this study,c DNA cloning,prokaryotic expression,protein purification and antibody preparation were performed on Bombyx mori zinc finger protein 525(BmZFP525/BMSK0013623),and its expression patterns were analyzed.Chromatin immunoprecipitation(ChIP-Seq)was performed,and the experimental data were analyzed by GO and KEGG.The main results obtained are as follows:1.The open reading frame sequence of BmZFP525 gene is 975 bp,which encoded protein sequence is 37.6 k Da,and the isoelectric point is 8.73.It is located on chromosome 23 of silkworm,and has 8 C2H2 type domains at the C terminal.It is a typical C2H2 type zinc finger protein.The multiple sequence alignment indicated that BmZFP525 had high homology with other Lepidoptera C2H2 zinc finger proteins and was conserved in evolution.2.The recombinant vector p ET28a-BmZFP525 was constructed,converted to E.coli(DE3),and induced by IPTG of different concentration gradients,purified the recombinant protein,and polyclonal antibodies were prepared.3.Total proteins and total RNA of silkworm were separately extracted from different tissues,fat body at different development stages and silk glands at different development stages,Western Blot and q RT-PCR detection indicated that the expression of BmZFP525 gene was spatiotemporal specific.4.According to ChIP-Seq results,8270 peaks were obtained,Peaks were mainly located in the intron region and intergenic region,and 6.8% peak was located in the promoter region.Moreover,12 immune-related genes which enriched in related pathways were obtained by GO annotation,indicating that BmZFP525 is likely to participate in immune regulation.Motif analysis showed that there were 5 potential target genes with functional annotation for BmZFP525,probable tubulin polyglutamylase TTLL1,sphingomyelin synthase-related 1,leishmanolysin-like peptidase,sodium-dependent phosphate transporter,acylphosphatase-1-like,as a transcription factor binding target gene promoter,BmZFP525 regulates gene transcription and participates in a variety of biological processes,affecting immunity,metamorphosis and development of silkworm.5.In order to investigate how BmZFP525 participates in and affects the immune process of Bombyx mori,four different types of pathogenic microorganisms were used to perform immune stimulation on the fifth instar larvae of silkworm,and the induced expression pattern of BmZFP525 in fat body was detected.The results showed that BmZFP525 was up-regulated to different degrees in transcription and protein levels after immune stimulation,and the upregulation degree was different according to the types of different pathogenic microorganisms,indicating that BmZFP525 played an important role in the immune response of Bombyx mori. |
