Functional Analysis Of A Cell Cycle-related Gene OsE2F2 In Rice

The endosperm of cereal crops is an important component of seed,and its development affects the yield and quality of crops.The development of endosperm is accompanied by the process of cell division,nuclear duplication and material accumulation.However,the mechanism of the development of endosperm cells and the regulation of cell cycle is not very clear.It has been reported that transcription factor E2 Fs is associated with the G1/S phase transition in the cell cycle,but its role in the division and development of rice endosperm cells has not been reported.In this study,RNAi interference was used to down regulate the expression of OsE2F2 gene.The function and mechanism of the gene in the development of rice endosperm were studied by means of cytology and molecular biology.The main results are as follows: 1.The results of q RT-PCR and in situ hybridization showed that OsE2F2 were expressed in root,stem and leaf,and different developmental stages of seed spikelets,but highly expressed in 5-7 days after pollination in the endosperm,suggesting that it may be involved in the development of the endosperm.2.Down-regulation of OsE2F2 leads to defects in seed development.The development of OsE2F2-RNAi T2 seeds showed that the seeds of transgenic plants had different degrees of shrinkage after 5 days of pollination,indicating that the plumpness of seeds was lower than that of wild type.Agronomic traits of OsE2F2-RNAi T2 statistical analysis showed that the OsE2F2 RNAi T2 seeds 1000 grain weight,grain width and panicle number was significantly lower than the wild type.The decrease of the expression of the gene was positively correlated with the severity of the seed defect phenotype,suggesting that the down-regulation of the expression of the gene leads to the defect of seed development.3.OsE2F2 is involved in the regulation of endosperm cell division and endoreduplication.The paraffin section of rice seeds at different developmental stages were observed.The results showed that compared with the wild type,the OsE2F2 RNAi family of 3 to 10 days after pollination,seed,the number of endosperm cell number appears to be reduced in different degree,may lead to shrinkage of seed production.Meanwhile,the OsE2F2 RNAi family also appeared abnormal embryo morphology.The results of flow cytometry showed that the proportion of 3C cells in the seeds of OsE2F2 RNAi lines was lower than that of wild type,which further proved that OsE2F2 was involved in the regulation of endosperm cell division.Flow cytometry results also showed that OsE2F2 RNAi lines of 5 DAP seeds with 6C and 12 C ploidy level of endosperm cells was significantly lower than that of the wild type,indicating that the gene may result in blocked replication of endosperm cell nucleus expression.The results of cytology and flow cytometry revealed that OsE2F2 regulates the development of rice seeds mainly through the control of endosperm cell division and endoreduplication.4.OsE2F2 controls the division of endosperm cells by regulating the expression of cell cycle related genes.q RT-PCR detection of the expression of related genes in 5 DAP seeds.The results showed that the cell cycle related genes OsCCS52A/B,OsPCNA,and the target gene E2 F gene expression,including OsMCM6-8,OsORC1-2 were decreased in the RNAi lines.The results indicated that OsE2F2 could affect cell cycle the operation of endosperm Cell cycle by regulated the expression of OsCCS52A/B and DNA replication related genes.