CMS restorer genes and proteins of Brassica napus (canola)

细胞质雄性不育（Cytoplasmic male sterile， CMS）是高等植物中普遍存在的一种性状，它是由线粒体基因组中新型开放阅读框（open reading frame, ORFs）的表达引起的。在大多数情况下，被称为生殖恢复（Rf）的核基因已经被确定可以抑制CMS表型。这种核恢复基因对一种特殊形式的CMS具有特异性，并通过下调与CMS相关的新型线粒体ORF的表达而起作用。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“基因组冲突”或进化“军备竞赛”：母系遗传，CMS-赋予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， pol和nap的新特征，这两种形式原属于油菜植物油菜，这表明这些系统是最近进化的，因此可以深入了解CMS -核恢复系统进化的分子机制。特别是这些系统的恢复基因，Rfp（用于pol）和Rfn，映射到相同的染色体位点。******要进一步了解核恢复基因如何随着CMS相关线粒体基因的出现而进化，将需要对Rfn/Rfp位点进行分子表征，并确定负责pol和nap CMS形式恢复的单个基因。这项发现资助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。我的小组是第一个分离出核恢复基因的小组之一，在我们的例子中，Rfo基因恢复了萝卜的ogu CMS。我们发现Rfo编码p型五肽重复（PPR）蛋白，这一特征随后在其他恢复系中被发现。这类蛋白质含有典型的35个氨基酸重复序列的串联拷贝，并指定转录物修饰位点，如核酸酶处理。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）的一个区域。更高分辨率的基因定位研究表明，Rfn基因定位在同一区域。Rfn基因型BAC包含4个相关的rf样PPR编码基因。这三种最有可能的候选基因目前正被引入甘蓝型油菜中，以评估它们赋予rfn特异性转录物修饰和抑制nap形式CMS的能力。在接下来的两年，也就是这项发现基金的预期期限内，我们将通过基因转化研究完成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，确定Rfn特异性结合的rna，并确定其在目标orf222/nad5c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. **