The Molecular Mechanism Of CYCLINB Gene In The Regulation Of Cell Cycle In Stylonychia Lemnae

Stylonychia lemnae,a model species of ciliates with a large body size,a high number of splice types,and nuclear amphipathism,has been widely studied as a single-cell biological model in genetics,genomics,developmental biology and molecular biology.In our work,we analyzed the transcriptomic data of S.lemnae division and proliferation under epidermal growth factor(EGF)pro-division conditions at 96 h,108 h and 120 h,and found that the gene family containing the B family of cell cycle proteins and their kinase-dependent analogs is closely related to cell cycle regulation.Cell cycle regulation mechanism has always been one of the important scientific questions in biology research.In this study,three StyCYCB genes were obtained by RT-PCR cloning using S.lemnae as the experimental model,and bioinformatics analysis of gene sequences and encoded proteins were performed,combined with qRT-PCR technique to quantify wild-type S.lemnae the gene expression patterns of three StyCYCB genes during asexual proliferation.Construction of a targeted RNA interference expression vector for the StyCYCB2 gene to achieve silencing of StyCYCB2 gene expression through a stable interference system.and the effects of StyCYCB2 gene silencing on the asexual division cycle of S.lemnae cells were analyzed in combination with changes in gene expression,localization of fluorescent protein expression and changes in nuclear behavior.Meanwhile,the molecular mechanism of cell cycle protein B family genes regulating the proliferation of S.lemnae cells were analyzed using single cell transcriptome sequencing technology.The main results were obtained as follows:1.Cloning,sequence analysis of S.lemnae CyclinB family genesThe cDNA sequences of StyCYCB1,StyCYCB2 and StyCYCB3 were obtained by RT-PCR,and the length of the coding regions were 801 bp,1452 bp and 1662 bp,respectively.Bioinformatics analysis revealed that all were hydrophilic proteins mainly localized in the nucleus,and the tertiary structure corresponded to the secondary structure with multiple conserved sites.Quantitative fluorescence expression analysis revealed that the three StyCYCB genes were expressed with significant temporal specificity during the nutritional reproduction period of S.lemnae,with different expression patterns in interphase and metaphase.It is hypothesized that it has an important role in initiating the asexual reproduction process,and positions 806-1132 of the StyCYCB2 sequence are COG5024 Super family Cyclin,which can be involved in biological processes such as cell division and chromosome partitioning.2.Study on construction of StyCYCB2 gene RNA interference system and control function of cell cycleThree pairs of shRNA sequences targeting StyCYCB2 gene were designed and synthesized,and recombined into YSH-RP005 vector to construct interference expression vector.The transfection system was established by bacterial solution feeding method,and gene interference experiments were conducted on the cells at the five periods from the beginning to the end of S.lemnae asexual bifurcation.The changes of body length,body width and gene expression and the incidence of malformation of cells in each period under the action of shRNA were observed and counted,and the StyCYCB2-shRNA2 with the highest interference efficiency was identified for subsequent study.S.lemnae cells in the continuous observation experimental group(C6h and C18h)and the rehydration observation experimental group(A6h and A18h)under the StyCYCB2-shRNA2 treatment condition were photographed morphologically.In addition,the expression of DNA content and the fluorescence expression intensity of StyCYCB2 protein in the interference group and the control group were different in different periods.The decrease of StyCYCB2 mRNA expression level caused the change of cell morphology and structure,which in turn affected the growth and development of the S.lemnae,resulting in the inability of the cells to divide normally and the lethal effect,indicating that the StyCYCB2 gene is an important regulatory gene in the process of cell division and proliferation of S.lemnae.3.Analysis of the molecular mechanism of StyCYCB2 gene on cell cycle based on single-cell transcriptome sequencing(1)Single-cell transcriptome sequencing analysis of the StyCYCB2-shRNA2 experimental groupSingle-cell transcriptome sequencing techniques were used on four shRNA interference-treated cells in the one-time shRNA2 transfection groups C6 h and C18 h and the intermittent supplemental shRNA2 transfection groups A6 h and A18 h,and a total of 54.68 Gb Clean Data were obtained,with an average of 6.835 Gb per sample.the differentially expressed genes obtained were GO-classified by gene expression pattern analysis note that at 6h of interference treatment(interphase),it was found that the construction of cell membranes,organelles and cytoskeleton were mainly affected at18 h of interference treatment(terminal phase),it further deeply affected the function of ribosome,and also affected protein kinase activity,ion transport and the normal life activities of S.lemnae were seriously affected.Moreover,the treatment time increased the degree of damage to S.lemnae and caused DNA damage,which led to the finallysis and death of the cells.The KEGG enrichment analysis showed that these genes were involved in multiple cellular signal transduction pathways,including PI3K-Akt,AMPK and mTOR metabolic pathway,and there were several genes affected by StyCYCB2-shRNA2 interference in this pathway.In addition,the comprehensive GO annotation analysis results of C6h/CKC6 h and C18h/CKC18 h groups found that S.lemnae cell division and proliferation processes have a dose-effect relationship with the StyCYCB2-shRNA2 interference expression vector,suggesting that the regulation of StyCYCB2 transcriptional expression is not a unidirectional programmed process..(2)Regulation pattern of StyCYCB2 gene networkAnalysis of the significantly enriched signaling pathways revealed that StyCYCB2 is associated with the following important metabolic pathways.Firstly,it can interact with the StyCDK2 gene and participate in the regulation of the S.lemnae cell cycle through the phosphorylation process.Secondly,it interacts with Cyclin A in the M phase and is rapidly degraded through the ubiquitination pathway,resulting in the loss of CDKl activity and the cell transformation from the M phase.The StyCYCB2 gene can also participate in the regulation of the actin cytoskeleton together with the mTORC2 complex.Thus,the StyCYCB2 gene is a key regulator of the S.lemnae G2/M cell cycle and forms a cell cycle regulatory network with StyCDK2,Cyclin A and mTORC2 complex.