| The cell cycle is a highly conserved and strictly regulated biological process,which is essential for tissue development,homeostasis maintenance and genetic information transmission.As an important model of lepidopteran insects,the silkworm,is of great significance to the study of cell cycle regulation mechanisms during its development.The regulation of cell cycle involves the involvement of many cytokines,among which the E2F transcription factor is an important regulator in the cell cycle regulatory network and plays an important role in the cell cycle process.Studying the function of the B.mori E2F（BmE2F）genes in the process of cell cycle regulation can provide a theoretical basis for the interpretation of the silkworm ontogeny and the unique endomitotic mechanism of silk glands.This study analyzed the sequence characteristics and expression patterns of the BmE2F genes,and explored the effect of the BmE2F4 gene on the silkworm cell cycle and endomitotic cycle,and then the interacting proteins were screened and identified by immunoprecipitation and mass spectrometry.The main research results and conclusions obtained are as follows:1.Cloning and characteristic analysis of BmE2F geneIn combination with SilkDB,SilkBase and NCBI databases,it was found that there were 3 E2F genes in silkworm through biological information analysis.Among them,the CDS sequence of BmE2F1 gene is 1443 bp,consisting of 11 exons and 10introns,and the encoded protein contains 481 amino acid residues;The CDS sequence of BmE2F4 gene is 783 bp,consisting of 7 exons and 6 introns,and the encoded protein contains 261 amino acid residues;The CDS sequence of BmE2F7 gene is 1008bp,consisting of 3 exons and 2 introns,and the encoded protein contains 336 amino acid residues.The online tool SMART was used to predict the protein domain of the BmE2F.The results showed that the BmE2F protein contains the conserved E2F-TDP domain of the E2F family,and BmE2F4 also has a dimerization domain.The E2F amino acid sequences of multiple species were obtained by homologous search of the full-length amino acid sequence of the BmE2F protein.The software Clustalx1.83 and MEGA6.0 were used to construct a phylogenetic tree.The results showed that all lepidopteran insects,including the silkworm,the E2F protein first clustered into one group,and then clustered with vertebrates,indicating that the BmE2F protein is conserved among different species.Semi-quantitative experiments were carried out to detect the expression of BmE2F gene in different tissues of 3rd day of 5th instar of silkworm.The results showed that the BmE2F gene was expressed in silk gland tissues,and the expression of BmE2F4 gene in silk gland was higher and more specific.Further analysis of the expression of the BmE2F gene in the silk glands at different developmental stages showed that the BmE2F4 gene was expressed at low levels in the full-feeding period,increased in the dormant period,and reached the highest expression in the fourth instar dormant period.The expression level gradually decreased,but the BmE2F1 and BmE2F7 genes showed no obvious change trend.2.The effect of BmE2F4 gene on the cell cycle of Bombyx moriAfter overexpression and RNAi of BmE2F4 gene,flow cytometry,EdU labeling,CCK-8 proliferation activity detection and analyzing the changes of cell cycle by qRT-PCR technology.The results showed that overexpression of the BmE2F4 gene in BmN-SWU1 blocked the cell cycle in the G1 phase,and significantly inhibited DNA replication and cell proliferation.RNAi of BmE2F4 gene increased the proportion of S-phase cells,markedly increased EdU labeling rate and cell proliferation activity,indicating that RNAi of BmE2F4 gene would promote cell DNA replication and promote cell proliferation.The qRT-PCR technique was used to detect the relative expression of each cycle regulatory factor after overexpression and RNAi of BmE2F4 gene.The results showed that after overexpression of the BmE2F4 gene,the expression of cyclins BmCyclin A and BmCyclin E and the cyclin-dependent kinases BmCDK1 and BmCDK2amount is significantly reduced.After RNAi of BmE2F4 gene,the expression of cyclins BmCyclin A,BmCyclin B and BmCyclin E and cyclin-dependent kinases BmCDK1and BmCDK2 were significantly up-regulated.3.The effect of BmE2F4 gene on DNA replication in silk gland tissue of Bombyx moriThe relative expression level of BmE2F4 gene in silk glands of stage 24,stage 25and stage 26 was analyzed by qRT-PCR technology.The results showed that BmE2F4gene expression was higher in stage 24 silk gland tissues undergoing mitosis,During the transition phase and the 25 and 26 phases of endomitotic,the expression level was significantly reduced.Through in vitro culture and Brdu labeling the silk gland tissue of 3rd day of 4th instar silkworms,the results showed that the BmE2F4 gene inhibited DNA replication of silk gland cells.4.Preliminary study on the mechanism of BmE2F4 gene regulating the cell cycle of Bombyx moriIn order to explore the mechanism of the BmE2F4 gene regulating the cell cycle of the silkworm,this study used the BmE2F4 protein as the bait protein to perform immunoprecipitation and mass spectrometry analysis,and screened 9 proteins that may interact with BmE2F4 were screened,namely the prohibitin protein,Bm14-3-3ζprotein.Furthermore,the interaction between Bm14-3-3ζprotein and BmE2F4 was verified by immunoprecipitation technology and BmE2F4 gene can inhibit Bm14-3-3ζexpression.Further use flow cytometry and CCK-8 cell proliferation viability detection to analyze the effect of overexpression of Bm14-3-3ζgene on the cell cycle.The results of flow cytometry showed that overexpression of Bm14-3-3ζblocked the cell cycle at G1 period.CCK-8 cell proliferation viability test results showed that overexpression of Bm14-3-3ζinhibited cell proliferation. |
