CMS restorer genes and proteins of Brassica napus (canola)

甘蓝型油菜 (canola) 的 CMS 恢复基因和蛋白质

• 批准号: RGPIN-2015-05193
• 负责人: Brown, Gregory
• 金额: $ 2.19万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613407
• 关键词: CMS; restorer; genes; proteins; Brassica

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.

细胞质雄性不育(CMS)是高等植物普遍存在的一种性状，是线粒体基因组表达新的开放阅读框架(ORF)的结果。在大多数情况下，被称为育性恢复者(RF)的核基因已经被发现可以抑制CMS的表型。这种核恢复基因是特定形式的CMS所特有的，并通过下调与CMS相关的新型线粒体ORF的表达来发挥功能。CMS-恢复系基因系统为研究核和线粒体基因组之间的相互作用提供了一种手段，具有进化意义，因为它们代表着一种“基因组冲突”或进化的“军备竞赛”：母系遗传的、赋予CMS的mtDNA可以在种群中传播，但随着花粉变得有限，可以抑制CMS的恢复基因的进化产生选择压力。我们已经发现了油菜特有的两种形式的CMS，Pol和NAP的新特征，这表明这些系统是最近进化的，因此可以为CMS-核恢复系统进化的分子机制提供洞察力。特别是，这些系统的恢复基因rfp(用于poll)和rfn映射到相同的染色体位置。 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，需要对Rfn/RFP基因座进行分子表征，并确定负责Pol型和NAP CMS形式恢复的单个基因。这项发现拨款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。我的团队是第一批分离出核恢复系的人之一，在我们的案例中，RFO基因恢复了萝卜的ogu CMS。我们发现RFO编码一个P型五肽重复(PPR)蛋白，这是后来为其他修复者发现的一个特征。这些蛋白质含有35个氨基酸重复序列的串联拷贝，并指定转录修饰的位置，如核酸酶处理。我们现在已经发现RFP基因定位于由Rfn基因产生的细菌人工染色体(BAC)的一个区域。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BAC包含4个相关的Rf样PPR编码基因。目前正在将这三个最有可能的候选基因引入甘蓝型油菜植株，以评估它们赋予Rfn特异转录修饰和抑制CMS的NAP形式的能力。在未来两年，也就是这笔探索资助金的预计期限内，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特异结合的RNAs，并确定其在目标orf222/nad5c转录本上的结合位点。