National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China.
J Exp Bot. 2012 Feb;63(3):1285-95. doi: 10.1093/jxb/err355. Epub 2011 Nov 16.
Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.
细胞质雄性不育（CMS）是高等植物中广泛存在的现象，有几项研究已经证实，这种母系遗传缺陷通常与线粒体突变有关。在十字花科植物中，大约有 10 个嵌合基因被鉴定与相应的 CMS 系统有关，但几乎没有直接证据表明这些基因导致雄性不育。在这项研究中，发现了一个新的嵌合基因（命名为 orf288），它位于 atp6 基因的下游，与 hau CMS 不育系中的该基因共转录。Western blot 分析表明，这个预测的开放阅读框（ORF）在雄性不育植物的线粒体中被翻译。此外，ORF288 的存在显著抑制了大肠杆菌的生长，这表明该蛋白对大肠杆菌宿主细胞有毒性。为了进一步证实 orf288 在雄性不育中的功能，该基因在拟南芥 APETALA3 启动子的控制下与线粒体靶向前序列融合，并导入拟南芥。带有 orf288 的转基因植物中，几乎有 80%的植物无法发育出花药。还发现，orf288 的独立表达导致了转基因植物的雄性不育，即使没有转运前序列。此外，orf288 和绿色荧光蛋白（GFP）作为融合蛋白在拟南芥原生质体中的瞬时表达表明，ORF288 即使没有外部线粒体靶向肽也能够锚定在线粒体上。这些观察结果为 orf288 负责 Brassica juncea 中的 hau CMS 雄性不育提供了重要证据。
