Molecular cloning of the Restorer gene for Cytoplasmic male-Sterility in Rice

细胞质雄性不育和育性恢复系统是杂交水稻发展不可缺少的基础。为了阐明BT型CMS的分子机理，我们决定利用图位克隆的方法鉴定BT型CMS的恢复基因（Rf-1）。用6104个分离子进行的Rf-1基因附近的DNA标记的连锁分析将Rf-1基因位点限定在22.4 kb的基因组区域。在该区域，我们发现了两个编码PPR蛋白的ORF（Rf-1A和Rf-1B）。我们还从每个ORF中克隆了cDNA。序列分析表明，非恢复基因型的两个ORF中均存在插入/缺失，导致产生无功能的蛋白。Rf-1A基因编码的蛋白几乎完全由10个重复的PPR基序组成。另一方面，编码蛋白质的Rf-1B基因缺乏线粒体的信号肽。认为PPR蛋白参与细胞器RNA的加工，Rf-1基因参与线粒体atp 6基因结构域mRNA的加工。结果表明，在Rf-1基因座上发现了两个ORF中的一个，命名为Rf-1A，它编码的Rf-1蛋白可能参与线粒体atp 6基因mRNA的加工。

Cytoplasm male sterility and the fertility restoration system are indispensable to development of hybrid rice. In order to clarify the molecular mechanism of the CMS system, we decided to identify the restorer (Rf-1) gene for BT type CMS in rice by using a map-based cloning strategy. Linkage analysis with the DNA marker near the Rf-1 gene using the 6104 segregants delimited the Rf-1 gene locus to a 22.4-kb genomic region. In this region, we found out two ORF (Rf-1A and Rf-1B) encoding PPR proteins. We also cloned cDNA from each ORF. Sequence analysis revealed that several ins/del had occurred in both ORF of non-restoring genotype, which resulted in a production of nonfunctional protein. Rf-1A encodes a mitochondrially targeted protein almost entirely comprised 10 repeats of PPR motif. On the other hands, the Rf-1B gene encoded the protein lacked the signal peptide to a mitochondria. It is thought that PPR protein participates in processing of RNA of an organella and Rf-1 gene participate mRNA of the atp6 gene domain of a mitochondria in processing. It was concluded that one of the two ORF had been found in the Rf-1 locus, named as Rf-1A, encodes the Rf-1 protein which may be involved in the processing mRNA of the mitochondrial atp6 gene.