CMS restorer genes and proteins of Brassica napus (canola)

细胞质雄性不育（CMS）是高等植物中广泛存在的一种性状，它是由线粒体基因组中新的开放阅读框（ORF）的表达引起的。在大多数情况下，核基因称为恢复的生育力（Rf）已被确定，可以抑制CMS表型。这种核恢复基因对特定形式的CMS具有特异性，并通过下调新的CMS相关线粒体ORF的表达来发挥功能。CMS -恢复基因系统提供了一种研究核基因组和线粒体基因组之间相互作用的方法，并且具有进化意义，因为它们代表了一种类型的“基因组冲突”或进化“军备竞赛”：母系遗传，赋予CMS的mtDNA可以在种群中传播，但随着花粉变得有限，选择压力建立了可以抑制CMS的恢复基因的进化。我们已经发现了两种形式的CMS的新特征，pol和nap，原生于油菜植物甘蓝型油菜，这表明这些系统最近进化，因此可以提供对CMS -核恢复系统进化的分子机制的深入了解。特别地，这些系统的恢复基因Rfp（用于pol）和Rfn定位于相同的染色体基因座。 进一步理解核恢复基因如何响应CMS相关线粒体基因的出现而进化，将需要Rfn/Rfp基因座的分子表征和负责恢复pol和nap CMS形式的单个基因的鉴定。这项发现补助金的主要目的是完成我们正在进行的旨在实现这一目标的研究。我的团队是最早分离出核恢复基因的团队之一，在我们的案例中，Rfo基因恢复了萝卜的Ogu CMS。我们发现，Rfo编码的P型五肽重复（PPR）蛋白，随后发现其他恢复的功能。这些蛋白质含有典型的35个氨基酸重复序列的串联拷贝，并指定转录修饰如核酸酶加工的位点。我们现在已经发现Rfp基因定位于由Rfn基因型产生的细菌人工染色体（BAC）的区域。更高分辨率的遗传作图研究表明，Rfn基因映射到相同的区域。Rfn基因型BAC包含4个相关的Rf-like PPR编码基因。三个最有可能的候选人目前正在引入B。napus植物，以评估它们赋予Rfn特异性转录修饰和抑制CMS的nap形式的能力。在接下来的两年里，也就是这项发现补助金的预期期限内，我们将通过遗传转化研究完成Rfn和Rfp基因的鉴定。我们也希望通过与法国凡尔赛INRA的科学家合作，鉴定出Rfn特异性结合的RNA，并鉴定出其在靶orf 222/nad 5c转录本上的结合位点。

Cytoplasmic male sterility (CMS) is a widespread trait among higher plants that results from the expression of novel open reading frames (ORFs) in the mitochondrial genome. In most cases, nuclear genes termed restorers-of-fertility (Rf ) have been identified that can suppress the CMS phenotype. Such nuclear restorer genes are specific for a particular form of CMS and function by down-regulating the expression of the novel, CMS-associated mitochondrial ORF. CMS – restorer gene systems offer a means of studying interactions between the nuclear and mitochondrial genomes and are of evolutionary interest because they represent a type of “genomic conflict” or evolutionary “arms race”: maternally inherited, CMS-conferring mtDNAs can spread in a population but as pollen becomes limiting, selective pressure builds for the evolution of a restorer gene that can suppress the CMS. We have discovered novel features of the two forms of CMS, pol and nap, native to the canola plant Brassica napus that suggest these systems evolved recently and hence can provide insight into the molecular mechanisms through which CMS – nuclear restorer systems evolve. In particular restorer genes for these systems, Rfp (for pol) and Rfn, map to the same chromosomal locus. Further understanding how nuclear restorer genes can evolve in response to the appearance of a CMS-associated mitochondrial gene will require a molecular characterization of the Rfn/Rfp locus and the identification of the individual genes that are responsible for restoration of the pol and nap CMS forms. The primary objective of this discovery grant is to complete our ongoing studies aimed at achieving this goal. My group was one of the first to isolate a nuclear restorer, in our case the Rfo gene which restores the ogu CMS of radish. We showed that Rfo encodes a P-type pentatricopeptide repeat (PPR) protein, a feature subsequently found for other restorers. Such proteins contain tandem copies of a canonical 35 amino acid repeat and specify sites of transcript modification such as nuclease processing. We have now found that the Rfp gene localizes to a region of a bacterial artificial chromosome (BAC) generated from the Rfn genotype. Higher resolution genetic mapping studies indicate that the Rfn gene maps to the same region. The Rfn genotype BAC contains 4 related Rf-like PPR encoding genes. The three most likely candidates are currently being introduced into B. napus plants in order to assess their capacity for conferring Rfn-specific transcript modifications and suppressing the nap form of CMS. Over the next two years, the expected duration of this Discovery Grant, we will complete the identification of the Rfn and Rfp genes through genetic transformation studies. We hope as well, through collaboration with scientists at INRA, Versailles, France to identify the RNAs that are specifically bound by Rfn and to identify its binding site on the target orf222/nad5c transcript.