Functional Identification Of The BmGeml And BmGem2 In The Silkworm Cells

Cell cycle is an extremely universal and complicated cascade regulation process, which is closely related to cell proliferation and differentiation, DNA repair, ontogenesis, organ formation, tissue regeneration and the diseases occurrence. In metazoan, there is a series of strict regulation in the proliferation and differentiation, in which several specific protein is crucial. Geminin is one of these proteins, which is a small and unstable nucleoprotein. In 1998, Geminin was first identified in Xenopus egg extracts. Geminin contains a special coiled-coil super-helix secondary structure. Studies have shown that geminin can regulate the proliferation and differentiation in the individual development via binding to cdtl and several transcription factors that participate in cell differentiation. Geminin is a key regulator in coupling cell proliferation and differentiation, and many researchers pay the close attention to it. Basing on the cloning and identification of the two Geminin genes and in Bombyx mori, BmGeml and BmGem2, and a DNA replication initiation factor BmCdtl, we used Co-IP, BIFC, interference and overexpression of gene technology and the yeast two hybrid system to study these genes function. Our study will lay a foundation for study on cell cycle regulation in Bombyx mori, and can enrich the theory of cell biology. The results we obtained are as follows:1. The interaction among BmGeml, BmGem2 and BmCdtlWe detected the interaction among BmGeml, BmGem2 and BmCdtl using BiFc and Co-IP. The results showed that BmGeml and BmGem2 can interacts with itself in in the nucleus, and also interacts with each other. In the nucleus, BmGeml can interact with BmCdt1, but BmGem2 cannot interact with BmCdt1. And BmGem2 can interact with BmCdt1 in the cytoplasm. In addition, the Co-IP results showed that When BmN-SWUl cells were co-transfected with BmGeml, BmGem2 and BmCdtl, the proteins of BmGem1 and BmGem2 could bind to BmCdt1 in the cytoplasm with no competition among the three proteins. And BmGeml can interact with BmCdt1 in the nucleus, but BmGem2 can only interact with BmCdt1 in the cytoplasm. These results showed that BmGem1 is the ortholog of Geminin in Bombyx mori, BmGem2 might participate in other regulatory mechanisms instead of combining with BmCdt1 in the cell cycle regulation.2. The effect of BmGem1 and BmGem2 RNAi on the silkworm cellsIn this study, we knock down the expression of the endogenous BmGem1 and BmGem2 from BmN4 cells by RNA interference. The results showed BmGem1 levels were significantly reduced to 0.39±0.01, and BmGem2 levels were increased to 1.28± 0.01 in BmN4 cells transfected with the BmGem1 RNAi compared with the control groups. When BmN4 cells were transfected with the BmGem1 RNAi, BmGem2 levels were reduced to 0.61±0.07 and BmGem1 levels were increased by 1.66±0.04 compared with the control groups. When BmN4 cells were transfected with the BmGem1&BmGem2 RNAi cells, BmGem1 levels were reduced to 0.78±0.05 and BmGem2 levels were reduced to 0.68±0.06. These results suggested These suggested that there is a dosage compensation between the two genes. And suggested that there are some related functions through different in binding to BmCdt1.In the BmGem1 RNAi BmN4 cells, microscope observation and photograph analysis results showed that the cell volume increased significantly. We define the cell is the enlarged cell which the cell volume were greater than or equal to 1.5 times of the normal cells. Statistical analysis showed that there are about 58% of the BmGem1 RNAi cells are the enlarged cells, and the enlarged cell only 5.24% in the control group. There are no significant changes in cell morphology in the BmGem2 RNAi group. However, In the BmGem1&BmGem2 RNAi cells, the percentage of the enlarged cells is 87.35% and was significantly higher than in the BmGeml RNAi cells. MTT results showed that the cell proliferation activity was decreased by about 36% in the BmGem1 RNAi cells. There are no significant changes in the BmGem2 RNAi cells. The cell proliferation activity was decreased by about 53% in the BmGem1&BmGem2 RNAi cells, and was lower than in the BmGem1 RNAi cells. In addition, flow cytometry analysis showed that the occurrence of DNA overreplication in the BmGem1 RNAi cells, but not in BmGem2 RNAi cells. More overreplication occurs in the BmGem1 & BmGem2 RNAi cells compared with the BmGem1 RNAi cells. We speculate that BmGem1 can directly interact with BmCdtl to regulate the initiation of DNA replication in the silkworm cells, DNA overreplication and the cell enlarge is induced by loss of BmGem1. Whereas BmGem2 cannot directly interact with BmCdtl, loss of BmGeml cannot lead to DNA overreplication. And more cells which cell proliferation were inhibited and DNA overreplication were induced by the loss of BmGem1 and BmGem2. Which hinted that BmGem2 is also involved in the regulation of cell cycle, however, the role of BmGem2 can be replaced by BmGeml in cell cycle regulation.3. The effect of BmGeml and BmGem2 overexpression on the silkworm cellsWhen BmGem1 and BmGem2 over-expressed in BmN4 cells respectively, the BmGeml levels were increased by 18.22% and the BmGem2 levels were increased by 78.21% compared with the control groups. In the BmGem1 over-expressed cells, the nucleus had no significant changes, while cell density decreased significantly, and the cell proliferation activity decreased by 40% compared with the control cells. However, the cell proliferation activity did not decrease in the BmGem2 over-expression cells.The number of cells in S was increased significantly and G2 phase was decreased significantly respectively, there is no significantly change in G1 phase in the BmGeml over-expressed cells. And the cell cycle is normal in the BmGem1 over-expression cells. We observed the cell DNA replication using Brdu labeling, and found that the number of ongoing DNA replication cells reduced by about 35% instead of increased compared with the control group cells in the BmGeml over-expressed cells. The results showed that the BmGeml over-expression will lead to the cells excessive accumulation in the S phase, and can not effectively into G2 phase.This suggested that the main function of BmGeml is the regulation of DNA replication origin through interacting with BmCdtl in BmN4 cells. There are some differences in the function of BmGeml and BmGem2, and the further research of BmGem2 is needed in Bombyx mori.4. Screening and identification of the BmGem2 interacting proteinsOur previous results showed that there was significant difference of BmGeml and BmGem2 proteins in the initiation of DNA replication and the cell cycle regulation. In order to explore the main function of BmGem2 in silkworm, We constructed a yeast two hybrid library of Bombyx mori using the total silkworm RNA from embryonic phase, day 3 of fifth instar, walking phase and pupal phase. We screened 34 positive clones, which encoded proteins interacting with BmGem2 using the yeast two hybrid system. 12 of positive clones have been identified, in which 4 of them have been cloned and identified with numbers:No.17, No.21, No.38, No.39. And we further validated that these proteins encoded by the four genes interacting with BmGem2 by co-immunoprecipitation.Bioinformatics analysis found that the No.17 and No.39 protein are the specific proteins in the silkworm, and they had not yet been reported. Sequence alignment and conserved domain forecast results showed that the protein of No.21 is the homolog of the potein Centrosomin in Drosophila melanogaster, so we named the No.21 protein as Bmcnn, which had conservative functional domains. Research shows that Centrosomin is involved in the regulation of cell skeleton and plays a very important role in the stability of chromosome structure. Moreover, Bmcnn highly expressed in testis and ovary which were undergoing vigorous mitosis, but almost no expression in the silk gland which were undergoing endomitosis in the day 3 of fifth instar. We speculated that BmGem2 may be related to the regulation of the chromosome structure.The protein of No.39 is the homolog of ribosome biogenesis and regulatory protein RRS1, so we named it BmRRS1, which had conservative functional domains. BmRRS1 expressed in all the tissues in the day 3 of the fifth instar silkworm. The expression level is higher in testis, ovary and silk gland, while lower in blood and epidermis. We assumed that BmGem2 may be involved in the regulation of ribosome biogenesis in Bombyx mori. These results indicated that the function of the BmGem2 has been divided, the function in the regulation of DNA replication and cell cycle progression have been weaken, and the new function may be involved in the regulation of chromosome structure stability and ribosome biosynthesis.