Genetic and Biochemical Analysis of Plant Mitochondrial Transcription

线粒体的功能是植物代谢和雄性育性所必需的，然而对线粒体基因表达的调控机制，特别是细胞核的作用知之甚少。 由于线粒体含有很少的基因，其绝大多数蛋白质是核编码的，包括基因表达的第一个承诺步骤，转录起始所需的所有因子。 该项目涉及核基因产物在线粒体转录中的作用，并通过扩展，在营养和生殖阶段线粒体基因表达的作用。玉米是一种重要的农作物，有着丰富的遗传和分子资源，本研究采用体外转录技术来确定启动子调控元件。随后，鉴定了编码RNA聚合酶的核心亚基的基因，并且发现产物RpoTm与T3和T7等的RNA聚合酶的核心亚基相关。由于RpoTm单独不能识别线粒体启动子，因此进行了可能的转录特异性因子的搜索，并获得了四个候选者。这些基因包括三个编码DNA结合蛋白的基因，以及细菌sigma-70的直系同源物。 一种可能性是，这些蛋白质中的每一种都为聚合酶提供了特定的特异性，如果是这样的话，这将允许在发育和配子发生过程中对线粒体功能进行微调。这种对灵活性的需求可以解释为什么植物线粒体转录会比真菌和动物中的同源系统更复杂。 为了确定线粒体RNA聚合酶的形式和组成，以及它们在植物功能中的作用，将使用反向遗传和生物化学方法进行实验。反向遗传学将需要突变体转座子插入rpoTm和一种或多种可能的转录因子的功能丧失等位基因的表型和生物化学表征。如果技术允许，将使用反义方法更快地获得结果。已经获得了RpoTm的突变体植物，并将首先进行分析。除了直接测量转录产物外，可能存在对突变的特定发育反应。例如，rpoTm的功能丧失等位基因可能降低花粉活力和胚胎发育的频率，但对雌配子体的活力没有明显的影响。 因此，花粉形态和竞争力将在分离植物，以及胚结构进行检查。为了分析营养组织中的效应，将构建嵌合体植物，其将在其他野生型背景中发展突变体区段。第二个和平行的目的是在细菌或昆虫细胞中表达后，使用上述蛋白质重建RNA聚合酶。 这将导致对启动子识别机制的更好理解，并有助于解释多态性表型。同时使用遗传和生物化学技术将为参与该项目的人员提供广泛的培训。 此外，本科生将参与遗传学方面的研究，让他们尽早接触植物生物学，这可能会鼓励他们走上这条职业道路，或者至少大大提高他们的科学素养。

Mitochondrial function is essential for plant metabolism and male fertility, yet little is known of the mechanisms regulating mitochondrial gene expression, in particular the role of the nucleus. Because mitochondria contain few genes, the vast majority of its proteins are nucleus encoded, including all factors required for the first committed step in gene expression, transcription initiation. This project addresses the role of nuclear gene products in mitochondrial transcription and by extension, the role of mitochondrial gene expression during vegetative and reproductive stages. The organism chosen for this work is maize, an important crop species for which appropriate genetic and molecular resources are available.The project was initiated using in vitro transcription to define promoter regulatory elements. Subsequently, a gene encoding the core subunit of the RNA polymerase was identified and the product, RpoTm, was found to be related to those of phages such as T3 and T7. Because RpoTm alone cannot recognize mitochondrial promoters, a search for possible transcription specificity factors was carried out, and four candidates were obtained. These include three genes that encode DNA binding proteins, and an ortholog of bacterial sigma-70. One possibility is that each of these proteins lends a particular specificity to the polymerase and if so, this would allow a fine-tuning of mitochondrial function during development and gametogenesis. This need for flexibility could explain why plant mitochondrial transcription would be more complex that the cognate systems in fungi and animals. To determine the form(s) and composition of mitochondrial RNA polymerase, and the role of each of these in plant function, experiments will be conducted that use reverse genetic and biochemical approaches. Reverse genetics will entail the phenotypic and biochemical characterization of Mutator transposon insertion loss-of-function alleles of rpoTm and one or more of the possible transcription factors. If technology permits, an antisense approach will be used to obtain results more rapidly. Mutant plants have already been obtained for RpoTm and will be analyzed first. In addition to direct measurements of transcription products, there may be specific developmental responses to the mutations. For example, the loss-of-function allele of rpoTm may reduce pollen vigor and the frequency of embryo development, but has no apparent effect on the viability of female gametophytes. Therefore, pollen morphology and competitiveness will be examined in segregating plants, as well as embryo structure. To analyze effects in vegetative tissues, mosaic plants will be constructed that will develop mutant sectors in an otherwise wild-type background. A second and parallel objective is to reconstitute the RNA polymerase using the proteins described above, following expression in bacterial or insect cells. This will lead to a greater understanding of the mechanism of promoter recognition, and aid in interpreting the mutantphenotypes. The concomitant use of genetic and biochemical techniques will provide broad training to those involved in the project. In addition, undergraduate students will be involved in the genetic aspects, giving them early exposure to plant biology that may encourage them in this career path, or at least dramatically improve their scientific literacy.