The Premilinary Identification Of DYW Protein With Deaminase Activity In Rice(oryza Sativa L.)

As a method of processing after the transcription,RNA editing includes the insertion or deletion of uridines and the single base conversion.Among them,the base conversion includes the substitution from the cytidine to uridine and the generation of flavin adenine from adenine.In flowering plants,the RNA editing in organelles mainly refers to the editing of cytidine in the transcript into uridine.The reduced or completely loss of the editing efficiency significantly influence the growth and development of the plant,and the lack of RNA editing in some sites may cause the lethal effect on the plants.Recently the gene editing technology make a fast pace,succeeding in the genome editing of lots of species and obtaining the creature with the ideal phenotype.Sequence-specific nucleases(SSN),including Zinc-finger nucleases(ZFNs),Transcription activator like effector nucleases(TALENs)and CRISPR/Cas9,consist of DNA binding domain and nuclease.By the alteration of DNA binding domain,the genome editing tool can change its specificity on DNA recognition,which will create the double-strand breaks(DSBs)in the target site and trigger the DNA repairment to perform the genome modification.Similar with these systems,there are also N-terminal nucleotide binding domain and C-terminal deaminase in the DYW-type PPR proteins.We can alter the binding sequence to achieve the conversion of the target site,which can be used to perform the deamination in the RNA strand.In this study,we mainly focus on the identification of the DYW protein with the deaminase activity,as well as the aim to develop a new RNA editing tool.Since we express transiently this system in the tobacco finally,the tobacco mitochondria gene atp1 is chosen as the target sequence.The PPR protein binding motif and RNA probe are designed according to the sequence 5bp~15bp upstream of the cytidine occupying the position 724 of the gene atp1,and we have verified the binding activity between the artificial PPR protein and RNA probe by the REMSA experiment in vitro.The result of Subcellular localization demonstrates that the artificial PPR protein locate extensively in the cell,by the way,the nuclear gene atp1 in tobacco also possesses the sequence with the same binding specificity of the PPR protein.After the addition of mitochondria target sequence to the 5'end of the artificial PPR protein,we can observe that this protein locates in the mitochondria.According to the analysis result,there are 105 candidate deaminases of DYW protein in the rice,and we choose 6 DYW proteins for the construction of editing system.With the transformation of recombination carrier into the tobacco protoplast,we can observe none of these 6 editing systems perform the C-U deamination activity in certain site.Due to no report on the construction of RNA editing system,and it involves in the direct ligation between the PPR motif and foreign DYW protein during the creation of this system,which we can't make sure if it will affect the deaminase activity.Further work for identification of the deaminase in the rice requires a deep research.