Characterization of atpA and orf220 genes distinctively present in a cytoplasmic male-sterile line of tuber mustard

Abstract

SummaryIn the present investigation, the atpA (Accession Number: AY211265, AY211266) gene, encoding the a subunit of F1-ATP synthase, as well as the open reading frame orf220 (Accession Number: AY208898), presumably encoding a hydrophobic membrane-associated polypeptide with deduced 220 amino acids, were found to be distinctively present in a cytoplasmic male-sterile (cms) line of tuber mustard. They were preferentially cloned by using mitochondria-specific primers and were partially characterized. The atpA gene cloned from cms and its fertile maintainer (m) lines were sequenced to be 1,454 and 1,073 bp-sized in fragments, respectively. They had a high similarity of approximately 70% in terms of nucleotide constituents. However, alignment of these two genes suggested that the atpA gene from the cms line had an insertion of 383 bp-sized fragment in 5’-flank spanning from 141th to 523th base site, compared with that of its m line. Besides, the open reading frame, orf220, was found to be exclusively present in the cms line of tuber mustard. It was deduced to possibly encode a hydrophobic N-terminus membrane-associated polypeptide with a deduced molecular weight of 26 kD. By Southern hybridization, it was further shown that orf220 existed only in the cms line and was absent from the fertile m line with no hybridization signal detected. Although the atpA gene was blotted in either cms or m line, they were of different fragment sizes when blotted with DNA digested with Hind III and Xba I, respectively. Both atpA and orf220 genes were supposed to be low coped in mitochondria genomes. In addition, by phylogenetic analysis, it was manifest that the orf220 gene had a closer genetic relationship with orf222 in Brassica nap cms than both of orf224 in Brassica pol cms and orf240 in Arabidopsis thaliana cms. The direct evidence of the involvement of these two genes in the expression of cms of tuber mustard needs further investigation.