Cloning And Identification Of The Splice Isoform Of CCNB2 Gene And Its Function In Taihang Chicken

Cyclin B2(CCNB2)plays an important role in cell cycle regulation in animals through its combination with cell cycle activators(CDK)to form Maturation-Promoting Factor(MPF),which promotes transcriptional activation,cytoskeletal construction,and chromatin remodeling,and regulates the expression of cell cycle regulatory genes.In this study,different splicing isoforms of the CCNB2 gene were cloned in Taihang chicken.Bioinformatics analyses were conducted and the expression levels of CCNB2 in ovaries and follicles at different developmental stages were determined.A pc DNA3.1-CCNB2 overexpression vector was constructed,and the effects of the different splicing isoforms on the proliferation and cell cycle of chicken follicle granule cells were investigated to elucidates the mechanism by which CCNB2 regulates follicle development in the Taihang chicken.(1)The CCNB2 expression levels in ovaries,follicles,and granule and follicular cells at different stages were measured.The results showed that CCNB2 expression in small yellow follicles was significantly higher than that in large yellow follicles(P <0.05).CCNB2 expression was significantly higher in granulocytes than that in follicular membrane cells(P < 0.05).(2)Three splicing isoforms(CCNB2-AS1,CCNB2-AS2,and CCNB2-AS3)of the CCNB2 gene were successfully cloned in the Taihang chicken.The length of the open reading frame(ORF)of CCNB2-AS1 was 1173 bp,encoding 390 amino acids,while the ORF of CCNB2-AS2 was 1152 bp in length and encoded 383 amino acids,and the CCNB2-AS3 ORF was 1206 bp,encoding 401 amino acids.The three isoforms were found to be strongly hydrophilic with low hydrophobicity,and were water-soluble.None of the three isoforms had signal peptides and were thus not secreted proteins.Both the three-dimensional structures and functions of the proteins were found to be similar.(3)The results showed that the expression levels of CCNB2-AS1 in F6 follicles were significantly higher than those in the ovaries(P < 0.05),while the expression levels in small follicles(P < 0.05)were significantly higher than those in Rhubarb follicles(P < 0.05),and the expression levels in Rhubarb follicles were significantly higher than those in Rhubarb follicles and F5-F4(P < 0.05).CCNB2-AS2 was strongly expressed in large white follicles,significantly higher than in small white follicles,small yellow,and large yellow follicles(P < 0.05),as well as being significantly higher in F6 follicles(P < 0.05),and significantly higher in F6 follicles compared with F5-F1follicles(P < 0.05).The levels of CCNB2-AS3 in follicles at different times showed the opposite trend to CCNB2-AS1.(4)The results showed that cellular activity was significantly higher 72 h after transfection with the CCNB2-AS1 overexpression vector than in the control group(P <0.05),while there was no significant change in cellular activity after transfection of the CCNB2-AS2 and CCNB2-AS3 overexpression vectors(P > 0.05).Flow cytometry was used to evaluate the cell cycle in granule cells 72 h after transfection of the CCNB2-AS1 overexpression vector,and the results showed reduced numbers of cells in the G0/G1 phase(P < 0.05)while the numbers of cells in the S and G2/M phases increased significantly(P < 0.05).The expression of ISL1,CDK1,and CCNB1-related genes was upregulated(P < 0.05),while expression of JAG1 and P21 was significantly downregulated(P < 0.05).In summary,this study identified three splicing isoforms of the CCNB2 gene in the Taihang chicken,and predicted their biological functions.Only CCNB2-AS1 was found to promote cell proliferation and could regulate the expression of cell cycle-related genes.These findings lay a foundation for the in-depth study of the molecular mechanism by which CCNB2 regulates the cell cycle during the follicle selection process,and also provides new ideas for improving the reproductive rate of the Taihang chicken.