| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We mapped the fragments with sequence (s) expressed in the rice callus onto a physical map for a 328-kb BAC contig around the rice waxy locus. Among the hybridized fragments, we attempted to characterize strongly hybridized fragments located in a region about 45 kb downstream of waxy. The fragments were found to contain two housekeeping genes encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and ribosomal protein small subunit 20 (rps20). Interestingly, these housekeeping genes are adjoined, with only a 303-bp spacer, and are directed in a tail-to-tail orientation. Nearly-full-length cDNA clones of the two genes were obtained by screening the callus cDNA library. A clone for the EPSPs gene possessed an open reading frame of 1683 bp encoding a deduced 561-amino-acid (aa) polypeptide that is the first expressed gene identified in rice using this method. EPSPs is a key enzynae on the shikimate pathway and is localized in the plastids of higher plants. This enzyme is known to be
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targeted by glyphosate, which has been employed as a nonselective herbicide. A gene for the rice rps20 peptide had previously been isolated, and our rps2O cDNA clone completely matched the registered sequence for this peptide. Northern blotting analyses were conducted using blots of total RNAs extracted from roots, seedlings, spikelets and anthers of Japonica and Indica rice strains. The 2.2-kb transcript of the EPSPs gene was easily detected in RNA samples from roots, seedlings and spikelets, but not the RNA from anthers. A 0.75-kb rps20 transcript was detected in all the tissues we examined, although the expression in roots from both rice strains was very weak. We have examined an extent of the transcript of either gene. These transcripts proceeded beyond the spacer ; the expression of the other gene might be modified by the antisense RNA from the opposite gene, resulting in double-stranded RNA, which might act as a trigger for RNA degradation in the mechanism called post-transcriptional gene silencing. The short spacer found here is uncommon in the nuclear gene organization in higher plants. This segment may have evolutionary implications about spacer expansion or contraction in the plant nuclear genome, and may play a role in gene regulation caused by the opposite gene transcript. Less
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