A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5' splice site of the first intron
1998
Isshiki, M. | Morino, K. | Nakajima, M. | Okagaki, R.J. | Wessler, S.R. | Izawa, T. | Shimamoto, K.
In cultivated rice two wild-type alleles, Wx(a) and Wx(b), predominate at the waxy locus, which encodes granule-bound starch synthase. The activity of Wx(a) is 10-fold higher than that of Wx(b) at the level of both protein and mRNA.Wx(b) has a +1G to T mutation at the 5' splice site of the first intron. Sequence analysis of Wx(b) transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5' splice sites of both Wx(a) and Wx(b), fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wx(a) reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wx(b) restored GUS activity and the level of spliced RNA to that of wild-type Wx(a). These results demonstrated that the low level expression of Wx(b) results from a single base mutation at the 5' splice site of the first intron. It is of interest that the Wx(b) allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation.
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