Genetic and Biochemical Analysis of Plant Mitochondrial Transcription

植物线粒体转录的遗传和生化分析

• 批准号: 0090658
• 负责人: David Stern
• 金额: $ 32.5万
• 依托单位: Boyce Thompson Institute Plant Research
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-15 至 2005-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0090658&HistoricalAwards=false
• 关键词: Genetic; Biochemical; Analysis; Plant; Mitochondrial

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.

线粒体功能对于植物代谢和男性生育力至关重要，但对调节线粒体基因表达的机制尤其是核的作用，但知之甚少。 由于线粒体几乎没有基因，因此其绝大多数蛋白质是编码的核，包括基因表达第一个投入步骤所需的所有因素，即转录起始。 该项目介绍了核基因产物在线粒体转录中的作用，并通过扩展是线粒体基因表达在营养和生殖阶段中的作用。为这项工作选择的生物是玉米，这是一个重要的农作物物种，可为其提供适当的遗传和分子资源。该项目是使用体外转录来定义启动子调节元件的项目。随后，鉴定出编码RNA聚合酶核心亚基的基因，发现rPOTM与T3和T7等噬菌体相关。由于单独的RPOTM无法识别线粒体启动子，因此对可能的转录特异性因子进行了搜索，并获得了四个候选者。这些包括三个编码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对聚合酶具有特殊的特异性，如果是，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将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，并确定每种方法在植物功能中的作用，将进行实验，以使用反向遗传和生化方法。反向遗传学将需要突变器转座子插入RPOTM的功能丧失等位基因和一个或多个可能的转录因子的表型和生化表征。如果技术允许，将采用反义方法来更快地获得结果。已经获得了RPOTM的突变植物，将首先分析。除了直接测量转录产物外，可能还对突变有特定的发育反应。例如，RPOTM的功能丧失等位基因可能会降低花粉活力和胚胎发育的频率，但对女性配子体的生存能力没有明显的影响。 因此，将在隔离植物以及胚胎结构中检查花粉形态和竞争力。为了分析植物组织的影响，将构建镶嵌植物，该植物将在原本野生型的背景下发展突变扇区。第二和平行的目标是使用上述蛋白质在细菌或昆虫细胞中表达后，重建RNA聚合酶。 这将使人们对启动子识别机理有更深入的了解，并有助于解释突变型。遗传和生化技术的伴随使用将为参与该项目的人提供广泛的培训。 此外，本科生将参与遗传方面，使他们尽早接触植物生物学，这些植物生物学可能会鼓励他们走上这一职业道路，或者至少会大大提高其科学素养。