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.

线粒体功能对植物新陈代谢和雄性生殖是必不可少的，但对线粒体基因表达的调控机制，特别是细胞核的作用，人们知之甚少。由于线粒体包含的基因很少，其绝大多数蛋白质都是核编码的，包括基因表达的第一步--转录启动所需的所有因子。该项目探讨了核基因产物在线粒体转录中的作用，以及线粒体基因表达在营养和生殖阶段的作用。这项工作选择的生物是玉米，这是一种重要的作物物种，可以获得合适的遗传和分子资源。该项目是通过体外转录来定义启动子调控元件的。随后，鉴定了编码RNA聚合酶核心亚单位的基因，并发现其产物RpoTm与T3和T7等噬菌体的产物有关。由于RpoTm本身不能识别线粒体启动子，因此对可能的转录特异性因子进行了搜索，并获得了四个候选因子。这些基因包括编码DNA结合蛋白的三个基因，以及细菌Sigma-70的同源基因。一种可能性是，这些蛋白质中的每一种都赋予聚合酶特定的特异性，如果是这样的话，这将允许在发育和配子发生期间微调线粒体的功能。这种对灵活性的需求可以解释为什么植物线粒体转录比真菌和动物中的同源系统更复杂。为了确定线粒体核糖核酸聚合酶的形式(S)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化方法进行实验。反向遗传学将需要对rpoTm的突变子、转座子插入、功能丧失等位基因和一个或多个可能的转录因子进行表型和生化表征。如果技术允许，将使用反义方法来更快地获得结果。突变植株已经获得了RpoTm，并将首先进行分析。除了对转录产物的直接测量外，可能还有对突变的特定发育反应。例如，rpoTm功能缺失等位基因可能会降低花粉活力和胚胎发育频率，但对雌配子体的活力没有明显影响。因此，将对分离植株的花粉形态和竞争能力以及胚胎结构进行检查。为了分析对营养组织的影响，将构建马赛克植物，这些植物将在其他野生类型的背景下产生突变部分。第二个平行的目标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。这将有助于更好地理解启动子识别的机制，并有助于解释突变的表型。同时使用遗传和生化技术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的研究，让他们及早接触植物生物学，这可能会鼓励他们走上这条职业道路，或者至少显著提高他们的科学素养。