Isshiki M, Morino K, Nakajima M, Okagaki R J, Wessler S R, Izawa T, Shimamoto K
Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Takayama, Japan.
Plant J. 1998 Jul;15(1):133-8. doi: 10.1046/j.1365-313x.1998.00189.x.
In cultivated rice two wild-type alleles, Wxa and Wxb, predominate at the waxy locus, which encodes granule-bound starch synthase. The activity of Wxa is 10-fold higher than that of Wxb at the level of both protein and mRNA. Wxb has a +1G to T mutation at the 5' splice site of the first intron. Sequence analysis of Wxb 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 Wxa and Wxb, 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 Wxa reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wxb restored GUS activity and the level of spliced RNA to that of wild-type Wxa. These results demonstrated that the low level expression of Wxb results from a single base mutation at the 5' splice site of the first intron. It is of interest that the Wxb 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.
在栽培稻中，蜡质基因座上有两个野生型等位基因Wxa和Wxb占主导地位，该基因座编码颗粒结合淀粉合酶。在蛋白质和mRNA水平上，Wxa的活性比Wxb高10倍。Wxb在第一个内含子的5'剪接位点有一个从G到T的突变。对Wxb转录本的序列分析表明，剪接发生在突变的AG/UU位点以及两个隐蔽位点：第一个是A/GUU，在原始位点上游一个碱基处，第二个是AG/GU，在突变剪接位点上游约100个碱基处。我们对Wxa和Wxb的5'剪接位点引入单碱基突变，与gus报告基因融合并将它们导入水稻原生质体。对GUS活性和转录本的分析表明，Wxa中的G到T突变降低了GUS活性和剪接RNA的水平。相反，Wxb的T到G突变将GUS活性和剪接RNA的水平恢复到野生型Wxa的水平。这些结果表明，Wxb的低水平表达是由第一个内含子5'剪接位点的单碱基突变导致的。有趣的是，携带内含子1的G到T突变的水稻Wxb等位基因在水稻栽培历史中一直保留下来，因为这种突变导致种子直链淀粉含量较低。
