Studies On The Relationship Between 4 Subunits Genes Of F₀-ATPase And Male Sterility In Tobacco

To uncover the relation between cytoplasmic male sterility in tobacco and subunit genes(atp9,atp6,orf25,orfB)of F0-ATPase in tobacco mitochondrial.The experiments were carried out to study differences of these genes sequences,RNA editing,all level structure of protein and interaction of proteins by coded target genes with three different species of tobacco male sterility lines(MS Zhongyan90,MS Yunyan85,MS K326)and their corresponding maintainer lines(Zhongyan90,Yunyan85,K326).The main results are as follows:At the genetic level.The target genes sequences were sequenced after in three different species materials,which were aligned in male sterility lines and their corresponding maintainer lines.The alignment result display that there are mutations in atp9,atp6,orf25,orfB gene sequences in tobacco male sterility lines compare with their corresponding maintainer lines.Otherwise,the mutations in each target gene were consistent in sequenced nine samples of male sterility lines.Thereinto,there are respectively 1 base mutation in atp9 and orf25 gene,the mutation of atp6 gene has occurred 6 nucleotide sites,there are 2 base mutation in orfB gene.In these mutation sites,they are silent mutations in the 126th site of atp9 gene and in the 253th and786th sites of atp6 gene.Except above silent mutation sites,the remaining mutation sites in four target gene resulted in the change of amino acid coded by these sites.So,except atp9,it is speculated that one,several or all of the mutations among the other three subunit genes in FO-ATP synthase of mitochondrial are likely to become one of the important causes of tobacco cytoplasmic male sterility in tobacco male sterility.At the level of RNA editing.To obtain RNA editing sites,the target genes cDNA sequences were sequenced after in three different species materials,which were aligned with their corresponding DNA sequences.The alignment result showed that there are 8 editing sites in the conservative regions of atp9 gene transcripts in male sterility lines.The 8 editing sites are identical with atp9 gene transcripts in maintainer lines.However,there are missing 2 editing sites for atp9 gene in male sterility lines compare with maintainer lines.RNA editing of atp6 gene transcripts occurred in 6 sites for maintainer lines,but RNA editing of atp6 gene transcripts not occurred in male sterility lines.There are 10 and 4 editing sites occurred correspondingly in transcripts of orf25 and orfB gene for male sterility lines,what a more,these editing sites are same with the sites occurred in maintainer lines.The mode of RNA editing occurred in target gene is that the C base replaced by U base.Most of the editing sites occurred at the first or second position of codon and caused alteration of amino acid coded by them.Nevertheless,the editing sites occurred at third position with no change of amino acid type.So we can draw the conclusion:the difference of gene editing site between atp9 and atp6 in mitochondrial FO-ATP synthase of the tobacco male sterile line and maintainer line has big relations to the causes of tobacco cytoplasmic male sterility;However,there is no difference in the gene editing sites between orf25 and orfB of sterile line and maintainer line,therefore,from the consideration just on the RNA editing,gene editing of the two gene may have no direct relationships with tobacco cytoplasmic male sterility.At the protein level.All level structure of proteins coded by target gene in male sterility lines and maintainer lines was analyzed with the method of Bioinformatics.The study result showed that,in the primer structure,the hydrophilic of amnio acid of protein by coded separately atp9,atp6,orf25,orfB in male sterility lines is better strong than its corresponding maintainer lines.The molecular weight and isoelectric point of target gene coding protein has different difference between male sterility lines and maintainer lines.On the second structure,comparing with their corresponding maintainer lines,the quantity of a-helix and β-turn were decreased and the quantity of random coil and β-sheet were increased for atp9 gene coding protein in male sterility lines,the quantity of a-helix and random coil were increased and the quantity of β-turn and β-sheet were decreased for atp6 gene coding protein in male sterility lines,the quantity of β-sheet were decreased and the quantity of random coil were increased and the quantity of a-helix and β-turn were not changed for orf25 gene coding protein in male sterility lines,the quantity of a-helix and random coil were increased and the quantity of β-turn and(3-sheet were decreased for orfB gene coding protein in male sterility lines.In the predicted domain and tertiary structure,except a small part of a-helix is missed in N-terminal for atp6 gene coding protein in male sterility lines comparing with maintainer lines,the predicted domain and tertiary structure of target gene coding protein has no obvious change between male sterility lines and their corresponding maintainer lines.In addition,the protein-protein interactions were predicted for protein coded by atp9,atp6,orf25 and orfB genes in tobacco mitochondrion,the prediction results show that the protein-protein interactions may have happened in coded proteins by atp9 and atp6 gene.So,we think that the difference in protein’ s primary structure and the secondary structure coded by the four subunit genes in mitochondrial FO-ATP synthase of the tobacco male sterile line and maintainer line lead to the interaction changes of protein coded by atp9 and atp6,even has influence on the performance of FO-ATP synthase,thus leading to a important reason of tobacco cytoplasmic male sterility.