M. nakazono. y. Ito, D. saisho and A. hirai
Laboratory of Radiation Genetics, Graduate School of Agricultural and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113 Japan
A database search has shown that the deduced amino acid sequence of exon 1 of the Arabidopsis thaliana AOX1a gene (Saisho et al., in press) has significant similarity with the putative protein encoded by the EST clone from rice (cv. Nipponbare) calli. The partially sequenced EST clone was provided by the National Institute of Agrobiological Resources, Rice Genome Research Program. The full sequence of the cDNA clone was determined. There was a 1474-bp insert that encoded a complete 996-bp open reading frame (ORF) in the clone. The amino acid sequence of the ORF indicated high homology with the predicted AOX proteins of other plants. This AOX gene was designated as the AOX1a (lto et al. in press).
A genomic clone containing the AOX1a gene was also isolated. Southern blot analysis of the clone revealed that there is an additional AOX gene in the flanking region of the AOX1a gene. Therefore, we determined the complete nucleotide sequence of a genomic clone containing the two AOX genes. Interestingly, the additional AOX gene, designated as the AOX1b gene, was located about 1.9 kb upstream of the AOX1a gene.
To examine the specific expression of the AOX1a and AOX1b genes, we constructed a probe specific to AOX1a or AOX1b, which corresponds to the 3'-UTR of the gene.
Total RNA was extracted from etiolated seedlings of rice which had been grown at 28°C for six days, then transferred to 4°C and harvested on every day to day 7. Northern hybridization was performed using the probe specific to AOX1a or AOX1b (Fig. 1 ). We
did not detect any transcripts of AOX1a or AOX1b in etiolated seedlings grown at 28°C (Fig. 1, lanes 1 and 7). However, treatment with low temperature increased the amounts of transcripts of both of the AOX genes starting on the second day of treatment and the levels of each transcript reached a maximum after four days (Fig. 1, lanes 2-6 and lanes 8-12). We can rule out the possibility that these inductions were caused by senescence because increases in both of the AOX mRNAs were not observed when the seedlings were kept at 28°C for more than 6 days in the dark.
Although it is uncertain whether heat produced by the alternative pathway under low temperature directly protect plants (Moynihan et al. 1995), to our knowledge, this is the first evidence of AOX genes whose expressions at the mRNA level are induced by treatment with low temperature.
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