The Function Of BmSu(H) Gene In Cell Cycle Regulation Of Bombyx Mori

Notch signaling pathway is an evolutionarily highly conserved intracellular pathway that plays an important role in cell cycle regulation,cell proliferation and apoptosis.Transcriptional inhibitor Su(H)(Suppressor of Hairless)plays a unique role as a molecular switch of downstream target genes of Notch signaling pathway.In recent years,studies based on insect Notch signaling pathway have been very active.Bombyx Mori,as an important model of lepidopteran insects,has both mitosis and endoreplication in the cell cycle during organogenesis and development.However,the regulation of Notch signaling pathway on the cell cycle of Bombyx mori remains unclear.Therefore,in this study,Notch signaling pathway and Bm Su(H)gene were used as entry points to explore their effects on the cell cycle of Bombyx mori,and to analyze the regulation mechanism of Notch signaling pathway on the cell cycle.First,DAPT,a specific signaling pathway inhibitor,was used to block the activation of Notch signaling pathway,analyze the effects of Notch signaling pathway on mitosis and endoreplication of silk gland,and detect the expression changes of molecular switch Bm Su(H)gene.Furthermore,the effect of Bm Su(H)gene on the cell cycle of Bombyx mori and its regulatory network were further investigated,and Bm Su(H)specific silk gland knockout lines were constructed to explore the effect of Bm Su(H)gene on the endoreplication of the silk gland.In this study,we elucidated the effects of Notch signaling pathway and Bm Su(H)gene on the cell cycle of Bombyx mori,providing new theoretical support for improving the regulation mechanism of cell cycle,and providing molecular targets for genetic improvement of silk gland.The main results and conclusions of this study are as follows:1.Functional analysis of Notch signaling pathway in Bombyx mori cell cycleIn order to analyze the effects of Notch signaling pathway on the cell cycle of Bombyx mori,Bm N-SWU1 cells were cultured with specific signaling pathway inhibitor DAPT to block Notch signaling pathway.Firstly,by observing the cell morphology and detecting the effect of the inhibitor,the optimal inhibition conditions with the concentration of 60μmol/L and the time of 48 h were selected.Subsequently,cells were treated according to the screened DAPT inhibition conditions,and it was found that inhibition of Notch signaling pathway resulted in decreased cell proliferation ability through CCK-8 proliferation activity detection.Flow cytometry results showed that the cell cycle was blocked in G2/M phase after the addition of inhibitor.q RT-PCR and Western Blotting showed that the expression level of Bm Cyclin B was not significantly changed,and the expression level of Bm CDK1 was significantly increased.The results showed that inhibition of Notch signaling pathway promoted the DNA replication of silk gland cells.QRT-PCR results showed that the expression levels of DNA replication related cycle regulators Bm Cyclin A,Bm Cyclin E and Bm CDK2 were significantly increased.At the same time,the expression of Bm Su(H)gene was detected,and the expression level of Bm Su(H)gene was significantly decreased in both cells and tissues by inhibiting Notch signaling pathway,indicating that the regulation of Notch signaling pathway on cell cycle requires the participation of Bm Su(H)gene.2.Cloning and expression analysis of Bm Su(H)geneTo identify Bm Su(H)gene and explore its expression characteristics,the sequence of Bm Su(H)gene was analyzed with NCBI database,Silk DB 3.0 and Silk Base genome database.Through gene cloning and sequence characteristics analysis,it was found that the full length of CDS sequence of Bm Su(H)gene was 1473 bp.A total of 490 aa were encoded,and the predicted protein size was 54.99 k Da.Bm Su(H)protein domain was predicted by online website SMART,and it was found that Bm Su(H)protein contained LAG1 and BTD,which had typical DNA binding sites.LAG1 could bind to target gene promoter sequence to activate expression,and BTD was the binding site of Bm Su(H)gene to intracellular domain NICD.Multi-species sequence alignment using Clustalx1.83 software revealed that the two functional domains of Bm Su(H)protein were highly conserved in different insects.MEGA6.0 software was used to construct phylogenetic tree analysis,and the homologous proteins of vertebrates clustered in one branch,invertebrates clustered in another branch,Bm Su(H)proteins and Lepidoptera clustered in one branch,which were most similar to Manduca sexta,indicating that they were closer in evolutionary relationship.The expression of Bm Su(H)gene in silk gland tissues of Bombyx mori was analyzed.It was found that Bm Su(H)gene was highly expressed in mitosis and transition stage of embryonic silk gland,and low expressed in endoreplication stage.In larval stage,Bm Su(H)gene was low expressed in the silk gland during prefeeding stage and high expressed in the silk gland during molting stage.Finally,the nuclear localization signal of Bm Su(H)protein at 180aa-182 aa was predicted through the website,and immunofluorescence analysis also confirmed that the protein was located in the nucleus.3.Analysis of the function and regulation mechanism of Bm Su(H)geneIn order to better study the function of Bm Su(H)gene in the cell cycle of Bombyx mori,the overexpression vector and knockout vector of Bm Su(H)gene were constructed,and verified the overexpression and knockout efficiency.Cell cycle changes were analyzed by CCK-8 proliferation activity assay,Ed U labeling,flow cytometry,q RT-PCR and Western Blotting.The results showed that overexpression of Bm Su(H)gene inhibited cell proliferation and blocked cell cycle in G1 phase.The expression of Bm Cyclin D and Bm CDK4 in G1 phase was up-regulated,while the expression of Bm Cyclin A and Bm CDK2 in S phase was down-regulated at transcription level and protein level.After Bm Su(H)gene was knocked out,cell proliferation and DNA replication were promoted,and the cell cycle was blocked in S phase,and the expression levels of cycle regulators Bm Cyclin A and Bm CDK2 in S phase were significantly increased at the transcriptional level and protein level.Furthermore,the regulatory mechanism of Bm Su(H)protein as a transcription factor was explored.The binding sites of Bm Su(H)protein were predicted in the upstream promoter regions of Bm Cyclin A,Bm Cyclin E and Bm CDK1 in JASPAR website.The dual-luciferase reporter system proved that Bm Su(H)decreased the promoter activity of Bm Cyclin A and Bm Cyclin E genes,and increased the promoter activity of Bm CDK1 gene.QRT-PCR showed that Bm Su(H)protein could inhibit transcription of Bm Cyclin A and Bm Cyclin E,and promote transcription of Bm CDK1.Combined with the above results,we speculated that Bm Su(H)protein could affect the cell cycle through the transcriptional regulation of cell cycle factors,and its regulatory network may be divided into two pathways: one is to regulate the expression of Bm Cyclin A and Bm Cyclin E and affect the S-phase cycle;The second is to regulate the expression of Bm CDK1 and control the speed of cell cycle entering M phase.4.Creation and analysis of Bm Su(H)silk gland specific knockout strainIn order to study the effect of Bm Su(H)gene on the endoreplication of Bombyx mori,a line with specific knockout of Bm Su(H)silk gland was constructed,and the knockout efficiency was up to 70%.By observing the cocoon size,cocoon layer weight and cocoon layer rate,we found that after deletion of Bm Su(H)gene in silk gland,the cocoon became larger,and the cocoon layer weight and cocoon layer rate were significantly higher than those of control.Scanning electron microscopy(SEM)was used to observe the surface and inner surface of the cocoons.It was found that the arrangement of cocoon silk on the surface of cocoons of the knockout strain was more disordered than that of the control strain.Phenotypic observation of silk glands in different days of the fifth age showed that on the third day of the fifth age,the middle silk glands of the knockout lines became significantly longer and heavier,while there was no significant difference between anterior and posterior silk glands.By Ed U labeling,we found that Bm Su(H)gene knockout could promote DNA replication and improve the efficiency of endoreplication.QRT-PCR was used to detect the expression of target genes in the knockout lines.The results showed that the changes of target genes were consistent with the results of Bm Su(H)gene knockout in cells,and it was found that the change of target gene was consistent with the result of Bm Su(H)gene knockout at the cellular level.