54. Cloning and comparison of the atpa gene in mtDNA between male-sterile and normal rice lines

Institute of Genetics, Academia Sinica, De-Sheng-Men-Wai, Bei-Sha-Tan Bldg. 917, Beijing 100012, China

ATP-synthase complex is located on the mitochondrial inner membrane in plants. It plays an essential role in the terminal step of energy production. The enzyme complex consists of two portions, F1 and FO. The F1 portion located outside the membrane is composed of five subunits (a, b, c, d and e), and is responsible for the synthesis of ATP. In fungal and animal cells, all five subunits of the F1 component are encoded by the nuclear genome. In contrast, in higher plants, the a-subunit is encoded by the mitochondrial genome (Boutry et al. 1983; Hack and Leaver 1983). It was reported that in maize fertile lines had two copies of the atpa gene, but cytoplasmic male-sterile lines had only one copy (Isaac et al. 1985). In order to know the situation in rice, we have cloned the atpA gene and compared mtDNAs between cytoplasmic male-sterile and normal lines.

Mitochondrial DNA (mtDNA) was isolated from Qiuguang B, a maintainer line, and was digested completely with two restriction endonucleases, BamHI and HindIII. After electrophoresis on 1% agarose gel, the DNA fragments were transferred onto nitrocellulose membrane, and were hybridized with the maize atpA gene probe. A single hybridized band was found in each of the BamHI and HindIII patterns. The band size in the BamHI pattern was about 3.5 kb, which was slightly smaller than that in the HindIII pattern. This suggests that the mitochondrial genome of Qiuguang B contains only one copy of the atpA gene, which is located on the BamHI or HindIII 3.5 kb fragments. Then, 3.5 kb BamHI DNA fragments were recovered from the gel by electroelution and cloned into

D. Qiuguang A mtDNA hybridized with atpA gene probe, showing two hybridization bands.

E. Qiuguang B mtDNA hybridized with atpA gene probe, showing one hybridization band only.

PBR322. Since the host bacterial DNA had homologous sequences to the rice mitochondrial atpA gene, we could not identify recombinant plasmids by colony hybridization. Therefore, plasmids were isolated and recombinant DNA contain- ing the atpA gene was identified by dot-blot hybridization. The recombinant plasmid DNA was confirmed by Southern hybridization.

Subsequently, mtDNAs prepared from both Qiuguang A, a cytoplasmic male- sterile line of BT type, and Qiuguang B were restricted with BAMHI and subjected to Southern hybridization analysis using rice atpA gene probe. As shown in Fig. 1, two hybridized bands (3.5 kb and 2.9 kb) were found for Qiugang A, but only one hybridized band (3.5 kb) for Quiguang B. Since these bands showed nearly the same intensity on X-ray film, we consider that the mitochondrial genome of Qiuguang A has two copies of the atpA gene and that of Qiuguang B has one copy only.

It is known that the mitochondrial genome of plants is much larger and more complex than those of animals and microorganisms. The plant genome contains a lot of repeated sequences, in which recombination is frequent. Gene multiplication may have arisen by recombinational events between repeated sequences. Actually, the atpA gene, coxI gene, coxII gene, 18s rRNA and 26s rRNA genes were found to have several copies in the mitochondrial genomes of plants (Morikami and Nakamura 1987; Stern and Palmer 1984). In these cases, these genes were located at or near repeated sequences.

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Hack, E. and C. J. Leaver, 1983. The a-subunit of the maize F1-ATPase is synthesised in the mitochondrion. EMBO J. 2: 1783-1789.

Isaac, P. G. et al., 1985. The mitochondrial genome of fertile maize (Zea mays L.) contains two copies of the gene encoding the a-subunit of the F1-ATPase. Curr. Genet. 10: 321-328.

Morikami, A. and K. Nakamura, 1987. Structure and expression of pea mitochondrial F1-ATPase a-subunit gene and its pseudogene involved in homologous recombination. J. Biochem. 101: 967-976.

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