Cloning And Functional Analysis Of Cell Division Cycle 48 Gene In Wheat

Cell division cycle is one of the basic physiological activities of lives.Cell division cycle protein?CDC48?participate in cell division by activating cyclin-dependent kinase?CDK?.Rebuilding of cell division ability is closely related to somatic embryogenesis obtaining and dedifferentiation state maintaining of the plants'callus.In this research,we cloned the cell division cycle gene of wheat TaCDC48 by homologous alignment for the first time.We analyzed its'molecular characteristics and biological functions through transgenic technology.We hope to reveal the impact of cell division cycle gene on somatic embryogenesis and its role in improving cell division efficiency of plant.The main results of this study are as follows:1.The molecular cloning of TaCDC48 in wheat and its'expression pattern analysis:We cloned the TaCDC48 gene from wheat by homologous alignment.Tissue specific expression analysis by real time quantitative PCR showed that TaCDC48 has higher expression level in panicle and embryogenic callus which grow vigorously.In addition,TaCDC48 expression could be induced at the early stage of callus induction and differentiation.2.Interaction identification of TaCDC48 and TaSERK1:Using yeast two-hybrid system and bimolecular fluorescence complementation?BiFC?assay,we proved the interaction relationship between TaCDC48 and TaSERK1,and their interaction occurs in the cytoplasm.Our results showed that TaCDC48 may participate in somatic embryogenesis by forming complex with TaSERK1.3.Functional analysis of TaCDC48:We constructed the over-expression vector of TaCDC48 and transformed it into Arobidopsis thaliana by agrobacterium-mediated method.Analysis of transgenic plants showed that TaCDC48 would promote the germination rate of seeds at 4°C,but not at 21°C.Compared to the wild type,TaCDC48 can promote the DNA replication efficiency in transgenic Arobidopsis thaliana plants and leading to early bolting.4.Obtaining of transgenic wheat plant:We constructed over-expression vector of TaCDC48 and transformed the recombinant plasmid into callus of wheat by particle bombardment method.After selection and regeneration,we obtained the regenerated plants.Identifying of the T₀ generation plants by PCR,3 transgenic wheat plants were achieved.In conclusion,cell division cycle protein?CDC48?of wheat can interact with somatic embryogenesis receptor kinase?SERK1?and participate in somatic embryogenesis process.Analysis of transgenic Arobidopsis demonstrated that TaCDC48 could promote seeds'germination and affect the bolting time.We speculate that it's the cell cycle regulation of TaCDC48 caused this phenotype.