THE ALTERNATIVE OXIDASE IS ENCODED IN A MULTIGENE FAMILY IN SOYBEAN

The copy number of the alternative oxidase gene, Aox, was investigated in soybean (Glycine max L.) using a Polymerase chain reaction (PCR) a pproach to amplify fragments from soybean genomic DNA. The primers use d were based on absolutely conserved regions of Aox cDNA clones from a variety of plant species and the yeast Hansenula anomala. After subcl oning of the 170-bp PCR products, 12 individual colonies were sequence d. Eleven plasmids yielded inserts representing three sequences in the ratio 4:3:4(Aox1-3). The sequence of Aox1 was 100% identical at the n ucleic acid level to the published full-length cDNA from soybean. The other two sequences were 60-75% identical to Aox1 and to each other at the nucleic acid and protein levels. Similar analysis of Nicotiana ta bacum L. revealed an additional gene copy with high homology to the so ybean Aox2 sequence. Genomic DNA from soybean cut with Hind III and pr obed with the full-length Aox1 yielded a single positive band of 6.5 k b; when the same genomic blot was probed with a mixture of all three P CR fragments, three bands of 9 kb, 6.5 kb and 3 kb were detected. Reve rse transcription-PCR performed on total RNA from various soybean tiss ues, followed by hybridisation with the three Aox sequences individual ly, revealed differential expression of the Aox genes between cotyledo ns and leaves. It is suggested that soybean contains a multigene Aox f amily. The implication of this for the understanding of alternative ox idase expression and regulation in plant tissues is discussed.