Study On The Mechanism Of BmZFP67 Regulating Mitosis-endomitosis Transformation In The Silk Glands Of Bombyx Mori

Endomitosis is a different form of cell cycle from mitosis.Only DNA replication occurs in the cells that undergoing endomitosis but no cytoplasmic separation,resulting in a multiplicative increase in the amount of genetic material in the mitotic tissue,while the number of cells remains the same,eventually forming a tissue that contains polyploid cells.The significance of endomitosis is that it can effectively increase the number of gene copies in the same number of cells and greatly improve the synthesis of related proteins,so as to perform specific functions in the biological process.Endomitosis cells are usually differentiated from mitotic cells.The mechanism of the transition of tissue cells from mitosis to endomitosis is unclear and the mechanism of endomitosis has always been the focus and difficulty of the study.As an economic insect of secreting silk,silkworm,Bombyx mori,experiences two processes of mitosis in early embryo and efficient endomitosis,which provide an ideal material for studying mitosis-endomitosis transformation mechanism.It is of great biological significance to understand the mechanism of endomitosis.In addition,the silk gland as one of the most special organs of important economic value in insect,its powerful protein synthesis ability not only to make it is of great economic value in silk production,better make it has become a new bioreactor.The mitosis-endomitosis transformation study of silk gland will provide the theoretical foundation to promote it as a bioreactor process and improve the economic value of silkworm.In this study,a key gene BmZFP67 be obtained,which may play a role in the process of mitosis-endomitosis switch through transcriptome sequencing screening.The expression characteristics and functions of BmZFP67 during cell cycle and silk gland development were analyzed by BrdU labeling,gene editing,transgenic,ChIP-qPCR and immunoprecipitation,and the regulatory network of BmZFP67 was constructed.This paper further explains the mitosis-endomitosis transformation mechanism,enriches and improves the theory of cell cycle regulation,and also provides the basic theoretical basis for the improvement of endomitosis efficiency and silk production.The main results and conclusions are as follows: 1.Analysis of mitosis-endomitosis transition period in silk glandIn order to screen for the key genes involved in mitosis-endomitotic transformation,firstly,the complete silk gland tissues of stage 24,25,and 26 embryos were obtained successfully through embryo dissection.Then,the expression of the cell cycle stage marker protein,Cyclin B and Cyclin E and cell cycle protein kinase dependent CDK1 and CDK2 in silk glands of stage 24,25 and 26 embryos were detected by real-time fluorescent quantitative PCR(qRT-PCR).The results showed that the expression of G2/M phase of the cell cycle related proteins Cyclin B and CDK1 is down-regulated.Cyclin B and CDK1 were highly expressed in stage 24 embryonic silk glands,significantly decreased in stage 25 embryonic silk glands,and unexpressed in stage 26 embryonic silk glands.The expression of G1/S phase cell cycle related proteins Cyclin E and CDK1 in stage 25 embryonic silk glands was significantly higher than that in stage 24 embryonic silk glands and stage 26 embryonic silk glands.It is speculated that the embryonic stage 25 is the transition period of mitosis-endomitosis of the silk gland cells of silkworm.The expression of Cyclin B in silk glands of embryo 24,25 and 26 stages was verified by immunofluorescence labeling.The results of immunofluorescence were consistent with those of qPCR.The Cyclin B fluorescence signal was obviously present in the whole silk glands at embryo 24 stage.A part of silk gland cells has signal at embryo 25 stage.There was no Cyclin B fluorescence signal in silk gland at embryo 26 stage.In summary,it is determined that the embryo 25 stage is the mitosis-endomitosis switch period of the silk gland cells of silkworm.2.Screening of genes involved in the regulation of mitosis-endomitosis transformation in silk glandsTranscriptome sequencing was performed on embryonic silk gland tissues at stages 24,25 and 26,and 6,786 differentially expressed genes were screened from three groups of samples.According to the Gene ontology(GO)classification,these genes mainly belong to molecular function and biological process,and relatively few belong to cellular component.KEGG clustering was found in 6,786 different genes has 679 genes were enriched in 131 signaling pathways,including DNA replication,mismatch repair pathways,MAPK signaling pathways,mTOR signaling pathways,Notch signaling pathway,and hormone signaling pathways have been shown in previous studies to be closely related to mitosis-endomitosis switch or endomitotic efficiency.By the transcriptome data and previously Geminin2 yeast two hybrid data in our laboratory,14 coexisting genes were found.Functional analysis of these 14 genes subsequently revealed that BmZFP67 is the only transcriptional regulator gene.Analysis the expression pattern of BmZFP67 genes in each silkworm tissues and silk gland development period found that BmZFP67 expression in silk gland was significantly lower than any other tissues which don’t undergo endomitosis,and the transcription level in the process of the transformation of silk gland mitosis-endomitosis showed a trend of downregulation,suggests the BmZFP67 may play a role in the transformation of silk gland mitosis –endomitosis.Finally,the transcription regulatory factor gene,BmZFP67,was chosen as the main study object.3.The function of BmZFP67 in cells and silk glands of Bombyx moriBioinformatics,molecular cloning and fluorescence quantitative real-time PCR were used to analyze and clone the BmZFP67 gene of silkworm,and to analyze its expression characteristics in different phases of cell cycle and embryonic silk gland.The results showed that the BmZFP67 gene encodes 518 amino acids and the protein size is about 59 kDa.Its homologous proteins are mainly found in lepidoptera.It is speculated that this gene may be related to the characteristics of silk spinning and cocooning in lepidoptera.BmZFP67 gene was highly expressed in the S and G2/M phases of cell cycle,and its expression faded during the transition from mitosis to endomitosis in the silk gland.This provides reference for its function studying.Gene editing technology,BrdU labeling technology,flow cytometry,MTS cell proliferation activity detection technology,fluorescence quantitative real-time PCR technology,cell staining and morphological observation were used to construct the BmZFP67 overexpression vector and knockout vector to determine the influence of BmZFP67 on the cell cycle of silkworm and determine the function of BmZFP67 at the cell level.The results showed that overexpression of BmZFP67 could inhibit cell proliferation,leading to a decrease in the proportion of cells in the S phase,an increase in the proportion of cells in the G2/M phase,and a significant increase in the transcription levels of CyclinB and CDK1 in the M phase.Knockdown of BmZFP67 can lead to abnormal cell division,which is similar to the switch from mitosis to endomitosis.The number of aneuploid cells increased,the cell volume increased,the proportion of cells in S phase increased,the proportion of cells in G2/M phase decreased,and the transcription levels of CyclinB and CDK1 in M phase were significantly down-regulated.The specific expression system of BmZFP67 gene in silk gland tissue was constructed by gene editing and transgenic technology.The effect of BmZFP67 gene on mitosis and endomitosis in silk gland tissue was determined by BrdU labeling technique.Immunofluorescence labeling,qRT-PCR and other techniques were used to detect the expression of cell cycle protein in different phases,and the function at the level of silk gland tissue of BmZFP67 gene in mitosis-endomitotic transformation was further determined.Studies have shown that knock out BmZFP67 leads the delay of mitosis-endomitotic transformation,endomitosis efficiency was significantly reduced,and the silk protein synthesis ability was significantly reduced compared with the large-scale production.4.Study on BmZFP67 gene regulation networkTarget genes for transcription regulation of BmZFP67 were identified by fluorescence quantitative real-time PCR,chromatin immunoprecipitation and double lucifer reporting system.The results showed that BmZFP67 had positive transcriptional regulation on cell cycle M-phase gene Cyclin B,and its transcriptional regulation site was sna site.BmZFP67 interacting protein was extracted by immunoprecipitation and fluorescence quantitative real-time PCR.The effect of the interaction was also studied.Results showed that 14-3-3ζ,Aurora-B kinase can promote transcriptional regulation of BmZFP67.Based on the above results,BmZFP67 is highly expressed in the G2/M phase of mitotic cell cycle,which promotes its transcriptional regulation of Cyclin B gene,the key regulatory gene in the G2/M phase of cell cycle,so as to realize the promotion of G2/M cell cycle.The expression of BmZFP67 was decreased during mitosis-endomitosis transformation in the silk gland,resulting in the down-regulated expression of its downstream gene Cyclin B,which made the silk gland cells unable to pass the G2/M checkpoint,hindered the M-phase process,and promoted the silk gland cells only to have DNA replication without cytoplasmic division.