Cotton Fiber And Ovule Mitochondrial Gene RNA-seq Sequencing And RNA Editing Site Determination

RNA editing is a post-transcriptional process that alter the nucleotide sequence of RNA molecules to differ from the gene sequence,which includes insertion substitution or deletion of nucleotide.In 1986,initial discovery of the U addition/deletion system in protozoon trypanosome mitochondrial cox2 was reported.Later research found that the RNA editing phenomenon widely existed in a variety of biological organisms,such as virus,fungus,plant and and animals.The frequent replacement of cytidine bases by uridines is main RNA editing type in higher plant mitochondria and chloroplasts,with less amount of U insertion and U-C convesion.Some recent study has demonstrated that RNA editing makes significant biological sense.Cotton is an important textile fiber.Our group previously has already completed determination of RNA editing sites in the cotton chloroplast protein-coding genes,the results showed that the cotton chloroplast transcripts have more RNA editing sites.But the cotton mitochondrial RNA editing sites are rarely reported.This study,using Gossypium hirsutum Xuzhou 142(WT)and the Fuzzless-Lintless mutants of ovule(fl)as materials,employing high-throughput sequencing and RT-PCR amplification sequencing technologies,we investigated fiber and ovule mitochondrial gene RNA editing site.The main research results are as follows:1.The RNA-seq shows a total of 626 RNA editing sites that exist in the mitochondrial gene and distribute in coding region and non-coding region.Of the all RNA editing sites,560 RNA editing sites have been identified in the wildtype Xuzhou 142 fiber,and 543 in mutation.479 RNA editings sites are shared by fiber and ovule,79 is fiber specific RNA editing sites,64 is ovule specific.The overall editing efficiency of samples fiber is higher than samples ovule;In Ovule,The coding region RNA editing frequency is less than wild type by 4.8%,that of intergenic region less by 3.0%;2.All the editing sites are C-to-U and U-to-C conversion.Of the 14 U-to-C editing events,except for rp116-362 and atp4-362,the rest of the 12 editing sites locate in intergenic region.Among them,7 editing sites shared by the fiber and ovule,6 editing sites for fiber specific.tRNA editing phenomenon was bothly detected in the sample fiber and ovule,located in the genomes position of 37077,corresponding to the 33rd bases of 5' end of tRNA-Asn.3.Analysis of the two cotton mitochondrial genes transcription abundance shows ovule has four genes that raise significantly,four genes downregulate significantly.Significant increase genes include atp4,atp8,ccmFN and rp110,reduce genes include atpl,ccmFC,nad3 and rps12.4.Protein-coding region gene editing sites were futher tested by use of cloning sequencing method.The reslts showed that high troughput sequencing results could cover 78%of RNA editing sites identified by clone sequencing.Total of 685 RNA editing sites were identified by filtering redundant editing,of which,617 editing sites were identified in fiber,and 598 editing site in ovule.In samples fiber,protein coding regions specific RNA editing sites number which obtained from high-throughput sequencing are 56,from clone sequencing are 60,more than that of sample ovule,13 and 1,respectively.5.There are 26 different editing sites in protein-coding genes,distributed in nad4,sdh4,atp1,rpl10,rps4,ccmFN and matR.The 9 different editing sites were identified in ccmFN.5 sites detected in atp1.Sdh4,matR,rps4 and rp110,each have two editing sites.Only 1 different RNA editing sites identified in nad4 and nad9.21 RNA editing sites are fiber specific except that 5 RNA editing sties in nad4,sdh4 and matR are ovule specific.11 of 21 different fiber specific RNA editing sites could increase the amino acids conservation.6.In cotton mitochondrial protein-coding genes,RNA editing mainly occurs in the first and second codon position,accounting for 86%of the coding region editing site numbers.395 RNA editings sites amino acid change analysis showed that:63 editing site is silence editing,332 edit sites can cause the conversation of the amino acids,of which 155 sits were significantly affect the nature of the amino acids.Among them,the serine conversion into hydrophobic amino acids accounted for 76.1%.Most of the mitochondrial editing site does not restore amino acids conservatism.Therefore,unlike plant chloroplast,abundant mitochondrial RNA editing sites may be evolution in neutral manner.