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育不可缺少的环节。为了阐明水稻BT型CMS的分子机制，我们决定采用定位克隆的方法，鉴定水稻BT型CMS的恢复基因（Rf-1）。使用6104个分离片段与Rf-1基因附近的DNA标记进行连锁分析，将Rf-1基因位点划分为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。序列分析显示，非恢复性基因型的两个ORF中都存在多个ins/del，导致非功能性蛋白的产生。Rf-1A编码的线粒体靶向蛋白几乎完全由10个重复PPR基序组成。另一方面，编码该蛋白的Rf-1B基因缺乏通往线粒体的信号肽。认为PPR蛋白参与细胞器RNA的加工，Rf-1基因参与线粒体atp6基因域mRNA的加工。结果表明，两个ORF中有一个位于Rf-1位点，命名为Rf-1A，其编码的Rf-1蛋白可能参与线粒体atp6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.