Identification And Analysis Of RNA Editing Sites In Chloroplast Transcripts Of Aegilops Tauschii L

RNA editing changes original genetic information by the insertion, deletion, and base substitution of nucleotides within mRNA molecule, which is an important way to regulate gene expression at post-transcriptional level in the chloroplast of higher plants. Aegilops tauschii L. is the D genome progenitor of common wheat, and its chloroplast genome was sequenced in 2014. Our study fills a gap which has hitherto existed in RNA editing in the chloroplast of Ae. tauschii. For the first time, the RNA editing sites of 76 chloroplast transcrips of Ae. tauschii were identified with three methods--bioinformatics prediction, mapping RNA-Seq reads to chloroplast transcrips and molecular cloning. Besides, protein structures were analyzed before and after editing. Specifically, the RNA editing sites in chloroplast transcripts of Ae. tauschii were compared with those of common wheat, wheat A and B genome progenitors, as well as five other Poaceae species including barley, rye, rice, corn and sugarcane, so as to provide new information for better understanding of the expression and regulation of chloroplast gene as well as the origin and evolution of RNA editing sites in chloroplast transcripts of Poaceae species. The main results are as follows:1. In the 76 chloroplast transcripts of Ae. tauschii, 34 editing sites in 15 transcripts were predicted, all of which are C to U conversion. Among them, ndhB is the most edited gene with 9 sites. 88.24% of editing events occur at the second base of codon, and the remaining at the first. Amino acid conversions contain eight types, that is, Sâ†'L, Pâ†'L, Sâ†'F, Tâ†'M, Hâ†'Y, Pâ†'S, Lâ†'F and Tâ†'M.2. Mapping detected 5 tissue-specific editing sites in 3 transcripts. They are infA-183, rps7-323, rps7-387, rbcL-637 and rbcL-1173, all of which are C to U conversion. All the sites are partially edited except rps7-387. And all the editing events result in conversion of amino acid except infA-183 and rps7-387. Amino acid conversions contain three types, that is, Aâ†'V, Râ†'C, and Tâ†'S.3. Protein structure analysis indicated that all the editing events will change the secondary structure composition nearby apart from petB, rpoA, rps8 and infA. The editing of petB makes one transmembrane segment expand to C-terminus. After editing, One transmembrane segment of ndhA contracts and the other shifts. Besides, editing led to an increase in the number of transmembrane segments of ndhB.4. Comparative analysis of the RNA editing sites in chloroplast transcripts of Ae. tauschii with common wheat, wheat A and B genome progenitors--Triticum urartu L. and Aegilops speltoides L., as well as Triticum turgidum L. ssp. dicoccoides, showed that 33 sites are shared by these five species, suggesting that these sites are highly conserved during the evolution of common wheat. atpB-35 is edited in neither T. urartu nor Ae. tauschii, indicating that atpB-35 of common wheat may be derived from Ae. speltoides. After comparative analysis of the RNA editing sites in chloroplast transcripts of Ae. tauschii with five other Poaceae species including barley, rye, rice, corn and sugarcane, we found 12 highly conserved sites, which are completely or partially edited in these six species.5. 16 editing sites in 5 transcripts predicted by Prep-Cp were validated by PCR. A new site, rbcL-600, was also detected with this method. Combined with bioinformatics prediction and mapping result, we discussed the advantages and disadvantages of three methods, as well as the impact on the study.