Supplement request for 1F32GM116361-01

 描述(申请人提供)：所有真核生物都有或曾经有线粒体。这些细胞器是约20亿年前真核生物进化早期与细菌共生的遗迹。因此，线粒体保留了自己的基因组(MtDNA)。虽然它只编码核DNA(NucDNA)编码的一小部分基因，但它的产物必须与核DNA编码的对应物密切相互作用，才能产生维持真核细胞功能所需的能量。线粒体DNA的突变可能导致这种相互作用的中断，给生物体健康带来可怕的后果。在动物中，线粒体DNA突变比核DNA积累得更快，一种关于衰老的一般理论认为，体细胞线粒体DNA突变的积累会导致衰老和与年龄相关的疾病的发生。然而，并不是所有真核生物的线粒体DNA突变率都升高。为了了解潜在的mtDNA突变如何影响生物体的健康和适应，拟议的研究将检查植物属Silene中的有丝分裂核错配，当一个种群/物种的mtDNA在远亲的核背景下表达时就会发生这种情况。Silene包含了进化缓慢和快速进化的线粒体DNA物种，使其成为这项研究的理想模型。这项拟议的研究解决的首要问题是确定有丝分裂-核相互作用如何影响生物生理学、生态学和进化。具有不同进化关联程度的Silene物种和种群将进行杂交，以产生具有一系列预测的有丝分裂核错配严重程度的后代。将对这些杂交后代进行标准生长表型、代谢率和繁殖力的检测。氧化磷酸化(OXPHOS)的熟练程度将通过评估OXPHOS复合体II和IV的活性在对照组和不匹配的个体中进行评估。复合体IV由线粒体DNA和核DNA编码的亚基组成，在错配的个体中活性降低，而复合体II用作阴性对照，因为它只由核DNA编码的亚基组成。此外，将通过RNA-Seq来评估对照组和不匹配个体的子集之间的差异基因表达，以测试错配如何影响机体功能的几种替代假设。最后，将在不同的干旱制度下评估对照和不匹配的个体，以确定特定的有丝分裂核背景是否可能适应特定的环境。这项工作将通过利用一个非常合适的模型来扩展我们对有丝分裂-核基因组相互作用的理解，该模型补充了以前的研究。它的结果将与人类健康中正在进行的问题相关，包括提出衰老理论，并评论正在进行的关于线粒体替代疗法(又名“三亲”体外受精)长期后果的辩论。

 DESCRIPTION (provided by applicant): All eukaryotes have or once had mitochondria. These organelles are the remains of an ancient symbiosis with a bacterium early in eukaryotic evolution some 2 billion years ago. As such, mitochondria retain their own genome (mtDNA). Although it only encodes a small fraction of the genes encoded by nuclear DNA (nucDNA), its products must interact closely with their nucDNA-encoded counterparts in order to generate the energy needed for maintaining eukaryotic cellular functions. Mutations in mtDNA can lead to a breakdown in this interaction, with dire consequences for organismal health. Mutations in mtDNA accumulate rapidly compared to nucDNA in animals, and one general theory of ageing posits that the accumulation of somatic mtDNA mutations leads to ageing and the onset of age-related diseases. However, not all eukaryotes have elevated mtDNA mutation rates. To understand how underlying mtDNA mutations influence organismal health and fitness, the proposed research will examine mito-nuclear mismatch in the plant genus Silene, which occurs when mtDNA from one population/species is expressed against the nuclear background of a distant relative. Silene contains species with both slowly and rapidly evolving mtDNA, making it an ideal model for this research. The overarching question addressed by the proposed research is to determine how mito-nuclear interactions influence organismal physiology, ecology, and evolution. Silene species and populations with varying degrees of evolutionary relatedness will be crossed in order to produce progeny with a range of predicted mito-nuclear mismatch severities. Standard growth phenotypes, metabolic rates, and fecundities will be assayed in these hybrids. Oxidative phosphorylation (OXPHOS) proficiency will be assessed in both control and mismatched individuals by assessing OXPHOS complex II and IV activity. Complex IV consists of both mtDNA and nucDNA-ecoded subunits and should show reduced activity in mismatched individuals, while complex II serves as a negative control, since it is composed solely of nucDNA-encoded subunits. Furthermore, differential gene expression will be assessed between a subset of control and mismatched individuals via RNA-Seq to test between several alternative hypotheses for how mismatch affects organismal function. Finally, control and mismatched individuals will be assessed under variable drought regimes to determine if specific mito-nuclear backgrounds may be adapted to particular environments. This work will extend our understanding of mito-nuclear genomic interactions by utilizing a well- suited model that complements previous studies. Its results will be relevant to ongoing issues in human health including advancing theories of ageing and commenting on ongoing debates regarding the long-term consequences of mitochondrial replacement therapy (aka, "three-parent" in-vitro fertilization).