| The silkworm(Bombyx mori)is an important economic insect.The silk gland acts as a silk-producing organ and carries out a special cell cycle,the endomitosis,which results in a constant increase in the amount of DNA in the silkgland cells and a significant increase in the ability to synthesise proteins,while the number of cells remains the same,eventually forming tissues containing polyploid cells.BmZFP67 is a zinc finger transcription factor that plays an important role in the mitosis-endomitosis transition of silk gland cells,but the regulatory network of BmZFP67 is unclear.The study of the transcriptional binding of BmZFP67 with candidate interacting genes and the regulation of cell cycle by both genes will provide a theoretical basis for the interpretation of cell cycle and the mitotic mechanism of silkworm silk gland.In this study,we verified the transcriptional regulation relationship between BmZFP67 and the candidate interaction gene BmCdc25,analyzed the sequence characteristics and expression pattern of BmCdc25,and explored the role of BmCdc25 on cell cycle and mitosis in the nucleus of silk gland of silkworm.The main research results and conclusions are as follows:1.Identification of candidate genes for transcription regulation of BmZFP67In order to find the transcriptional binding gene of BmZFP67,we identified the key homologous gene in Bombyx mori with reference to the study of mitotic and intra nuclear cycle transition in Drosophilous follicular cells.It was predicted that the upstream promoter region of Cdc25,Su(H),Hnt and Delta genes could bind to BmZFP67 in JASPAR website.By chromatin immunoprecipitation,BmCdc25 gene was identified to bind to BmZFP67,and the binding site was sna.The dual luciferase reporting system showed that BmZFP67 could reduce the promoter activity of BmCdc25 gene,and quantitative analysis showed that BmZFP67 had transcriptional inhibitory effect on BmCdc25 gene.The gel migration experiment proved that BmZFP67 protein could directly bind to BmCdc25 gene.Combined with the above results,we hypothesized that BmZFP67 affects the cell cycle through the transcriptional regulation of BmCdc25 gene.2.Analysation of the sequence and expression characteristics of BmCdc25 geneIn order to investigate whether BmCdc25 gene has homology with Cdc25 gene of other species,we analyzed the sequence of BmCdc25 gene by combining silkworm silk DB 3.0,silk Base and NCBI database,and found that its CDS sequence was 2097 bp,encoding 698 amino acids in total.The domain prediction of BmCdc25 gene was performed using SMART,and only one RHOD domain of the Cdc25 family was found in this protein.A phylogenetic tree was constructed using Clustal X1.83 and Mega 6.0software.The results showed that the vertebrate Cdc25 homologous proteins were clustered in one branch.The homologous proteins of Cdc25 gene in yeast and nematode were clustered into one branch.Cdc25 of Bombyx mori was clustered into a branch with other insects,indicating that the protein was conserved in the evolutionary process of each species.Tissue expression profile showed low expression of BmCdc25 gene in the fifth and third instar homologous silk glands.Further analysis of the expression characteristics of the embryonic silk gland stage showed that BmCdc25 gene was highly expressed during the mitosis stage of embryonic development,while low expression was found during the transition stage and the mitosis stage.At larval stage,the expression of BmCdc25 gene was low in the silk glands at feeding stage,high in the silk glands at molting stage,and continued to be low after the fifth instar.The results of quantitative analysis showed that BmCdc25 gene was highly expressed mainly in G2/M phase.These results indicate that BmCdc25 gene has homology with Cdc25 gene of other species.BmCdc25 gene mainly plays a role in the G2/M phase of the cell cycle,and its expression is inhibited during the vigorous mitosis.3.Regulation of BmCdc25 gene on cell cycle of Bombyx moriTo study the function of BmCdc25 gene in silkworm cell cycle.We overexpressed and interfered with BmCdc25 gene in Bm N-SWU1 cells,respectively.CCK-8proliferation assay,flow cytometry,Ed U labeling and q RT-PCR were used to analyze the changes of cell cycle.The results showed that overexpression of BmCdc25 gene promoted cell proliferation and significantly up-regulated the relative expression levels of cycle regulators in S and M phases,but had no significant effect on cell cycle phases.Interference with BmCdc25 gene would inhibit cell proliferation and block cell cycle in the G2/M phase,reduce cells in the DNA replication phase,and downregulate the cycle regulator BmCyclin E and BmCDK2 in the S phase,but not significantly affect the cycle regulator in the M phase.In order to explore the function of BmCdc25 gene in silk gland cells,we dissected silk gland tissues of the fourth age and two days for in vitro culture and transfection.Ed U labeling results showed that overexpression of BmCdc25 gene inhibited DNA replication in silk gland cells.After clarifying the function of BmCdc25 gene,we investigated the effect of BmZFP67 transcription binding to BmCdc25 on the cell cycle.BmCdc25 gene was overexpressed and interfered with after knocking out BmZFP67 gene in cells.Immunofluorescence analysis showed that interference with BmCdc25 gene could save the abnormal cytokinesis caused by knocking out BmZFP67 gene. |
