Nucleotide sequence and transcription analyses of the rapeseed (Brassica napus L.) mitochondrial F1-ATPase ?-subunit gene

Abstract

The mitochondrial F~F o ATPase complex (ATP synthase) plays an essential role in the energy production by oxidative phosphorylation, which comprises at least ten different polypeptides. In higher plants, subunits 6 and 9 of the membrane-bounded Fo domain are encoded by mitochondrial genes: atp6 and atp9, respectively. The ~-subunit of catalytic F~ component of higher plants is also encoded by mitochondrial gene, atpA, which is in the nucleus in other organisms (reviewed in Mulligan and Walbot [6]). In sunflower, rearrangements of mitochondrial genome is detected within or near atpA, which is also the only mitochondrial gene whose transcripts differ between the fertile and cytoplasmic male-sterile (cms) lines [7]. In this paper, to understand the mechanism of biogenesis of plant F 1 ATPase and relationship between the cms trait and mitochondrial genes, we performed analyses of the nucleotide sequence of the rapeseed (Brassica napus L.) atpA and transcriptional patterns of atpA in the fertile and cms lines. Rapeseed mitochondria were isolated from 8week-old plants. Mitochondrial DNA (mtDNA) was extracted as described by Handa et al. [3], and mitochondrial RNA (mtRNA) was isolated by the procedure of Stern and Newton [ 8 ], except that no sucrose step gradient was used for the purification of mitochondria. A Hind III library of mtDNA of the cms line having 'Polima' cytoplasm was screened for the presence of clones containing the atpA gene by colony hybridization with digoxigenin-labelled pea atpA clone [5] as probe. Positive clone contained a 5.6 kb Hind III fragment was isolated, and its physical map was constructed (Fig. la). We determined the nucleotide sequence of the rapeseed atpA gene by the dideoxy chain-termination method, and deduced amino acid sequence according to the universal codons and assuming no mRNA editing (Fig. lb). The coding frame of rapeseed atpA extends continuously for 1521 bp and shares nucleotide sequence homology with the atpA genes from pea (93.8~o) [5] and maize (91.7~) [4]. The rapeseed ~-subunit deduced