Cotton Chloroplast Rna Editing Sites, Measurement, Analysis, And Its Mechanism

RNA editing is one of the post-transcriptional process in which the nucleotide sequences of transcripts are changed by substitution, insertion or deletion of nucleotides, and then alter the genetic information on DNA sequences. RNA editing phenomenon is found in a variety of biological organisms. In higher plants, this process is mainly occurred in chloroplasts and mitochondria, which is one of ways of gene regulation in chloroplast. In this study, we investigated the RNA editing sites of protein-coding genes of Gossypium hirsutum using PCR, RT-PCR and sequencing methods. Bioinformatics were used to analyze protein secondary and three-dimensional (3D) structures before and after editing of each transcript. We found some editing sites that may play an important role in their proteins structure and function. Meanwhile, we identified and compared all RNA editing sites in four CRRI taxa chloroplast protein-coding genes, and investigated the RNA editing events of 10 plastid protein-coding genes of V1, which is a mutant of cotton with yellow cotyledons and green true leaves. In addition, we cloned a new PPR gene from cotton and analyzed its expression profiles in different tissues or under kinds of stress treatments. The main results are as follows:1. In 78 chloroplast protein-coding genes in cotton,58 RNA editing events have been predicted in 29 transcripts. Actually, using PCR, RT-PCR and sequencing technique,54 editing sites are identified in 27 of 29 transcripts, which is the highest number of RNA editing sites existed in angiosperms to date. All the editing sites are C-to-U conversion.47 sites are in the second position of codon,6 sites are located in the first position, and only 1 site occurs in the third position. Editing obviously prefers to an uridine_adenine (U_A) context, increased the proportion of hydrophobic amino acids and the protein conservation.2. Bioinformatics are used to analyze the effect of editing events in Coker310FR on protein secondary and three-dimensional (3D) structures. Among of 54 editing sites,21 sites could affect protein secondary structures, three sites of them would affect protein's 3D structures. Four editing sites would not change protein secondary structures, but may affect corresponding protein 3D structures. These results imply that total 25 editing sites in cotton chloroplast transcripts may play an important role in those proteins forming correct structure and executing function.3. 55 editing sites are identified in 27 transcripts in 4 CRRI taxa. All positing of the editing sites are similar besides some sites with different editing frequency. In Gossypium hirsutum buds yellow mutant V1,34 editing sites are identified in 10 plastid protein-coding genes, from cotyledons and leaves, respectively. All the 34 editing sites are the same, but 6 sites have different editing frequency.4. A novel PPR gene, name as Ghppr1, has been cloned from CRRI 24 using Genomic-Walking method. Full sequence of the gene is 2199 bp, including 2088 bp ORF, which encoding 695 amino acids. Ghppr1 has typical characteristics of PPR protein and DYW motif. The gene might locate in the nuclear because of predicted nuclear localization signal. In mRNA level, we analyze the expression of Ghppr1 in different tissues, roots, stems, leaves, flowers, ovules and fibers in CRRI 24. The results show that leaves have the highest expression, followed by ovules. In other tissues, no expression can be observed. We also detect the expression of Ghppr1 under different abiotic stress treatments. The results imply that Ghppr1 would rather response to salt, ABA, low and high temperature than to drought.