Molecular Mechanisms of MAR Effects in Arabidopsis

拟南芥MAR效应的分子机制

• 批准号: 1020650
• 负责人: George Allen
• 金额: $ 62.7万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-10-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1020650&HistoricalAwards=false
• 关键词: Molecular; Mechanisms; MAR; Effects; Arabidopsis

This project will lead to an increased understanding of how genomic processes are coordinated. The investigators are studying a DNA sequence called a Matrix Attachment Region (MAR), which can bind to a dynamic structure within a cell's nucleus called the nuclear matrix (or scaffold). MARs are thought to be important for DNA replication, nuclear organization and gene expression. This project, which uses the model plant Arabidopsis thaliana, will address a major question affecting the use of transgenic plants in agriculture. Frequently the expression of a transgenic trait is extremely variable from one transformant to the next, reflecting complete or partial silencing of transgene expression. Studies in both plants and animals have shown that adding MARs to transgenes can increase the average level of expression, probably by reducing the frequency of silencing. However, increased expression does not always occur, and even MAR- containing transgenes are sometimes silenced. This present project seeks to understand how MARs function, and why their effects are not always the same. In particular, the investigators will ask if MAR-flanked transgenes are transcriptionally silenced (not transcribed into RNA), or if the transgenes are actively transcribed, but the RNA is degraded as rapidly as it is produced. Importantly, they will use technology allowing them to compare transgenes with and without MARs at the known genomic positions, and to relate differences in MAR effects to differences in genomic features of each location, such as proximity to native genes or the presence of transposons, repetitive DNA, or modified versions of the histone proteins that package DNA into the chromosome. Direct measurements of transcription of each transgene will also be used to test the hypothesis that MARs act primarily at the transcriptional level. Broader Impacts. This research will contribute to ongoing efforts to link the biochemistry of gene expression with organizational structures in the nucleus, and to a better understanding of the mechanisms controlling gene silencing and gene expression. A better understanding of these issues, including the molecular mechanisms of MAR effects, will support more sophisticated applications in eukaryotic genetic engineering, including new technologies to reduce transgene silencing. Examples of potential applications include the use of MARs to stabilize disease resistance, as well as pathway engineering for crop improvement, bioenergy applications, or production of pharmaceuticals and chemical feedstocks in non-food species. The project will also provide training and mentoring for a graduate student and a postdoctoral scholar. In addition, funding from this project will allow continuation and expansion of a successful collaboration with two Granville County Middle School teachers and the North Carolina Museum of Life and Science. The project has the potential to reach at least 10 middle schools in the Granville and Durham Counties, with as many as 1,000 middle school students directly benefiting from distribution and implementation of 'Science in a Suitcase' instructional kits (10 teachers, 100 students per middle school engaged in math and science classes for three years). Specific objectives include: engaging 10 middle school math and science teachers each year in a two day instructional workshop; providing follow-up support through the Museum of Life and Science staff; and providing instructional kits at no-cost to teachers who participate in the workshop.

该项目将使人们对基因组过程的协调方式有了更多的了解。研究人员正在研究称为基质附着区域（MAR）的DNA序列，该序列可以与细胞核中的动态结构结合，称为核基质（或支架）。火星被认为对于DNA复制，核组织和基因表达很重要。该项目使用模型植物拟南芥，将解决影响转基因植物在农业中使用的一个主要问题。通常，转基因性状的表达从一个转化物到下一种变化物是极大的，反映了转基因表达的完整或部分沉默。在动植物中的研究表明，将火星添加到转基因可以增加平均表达水平，这可能是通过降低沉默的频率。但是，表达增加并不总是发生，甚至含有转基因的结婚基因有时会被沉默。当前的项目试图了解火星的功能，以及为什么它们的效果并不总是相同的。特别是，研究人员将询问是否在转录下（未转录为RNA），或者是否会积极转录转录，但RNA的产生迅速降解。重要的是，他们将使用技术，使他们能够比较有和没有MARS的转基因，并在已知的基因组位置上进行比较，并将MAR效应的差异与每个位置的基因组特征的差异相关联，例如与本地基因的接近度或转座子的存在或重复的DNA，重复的DNA，或者是将DNA的修饰版本的DNA或修饰的组蛋白蛋白包装到Chromosome中。每个转基因转录的直接测量也将用于测试MARS主要在转录水平作用的假设。更广泛的影响。这项研究将有助于持续的努力将基因表达的生物化学与核中的组织结构联系起来，并更好地理解控制基因沉默和基因表达的机制。更好地理解这些问题，包括MAR效应的分子机制，将支持在真核基因工程中更复杂的应用，包括减少转基因沉默的新技术。潜在应用的例子包括使用火星来稳定疾病抗性，以及用于作物改善，生物能源应用的途径工程，或在非食物种类中生产药物和化学原料。该项目还将为研究生和博士后学者提供培训和指导。此外，该项目的资金将允许与两位格兰维尔县中学教师和北卡罗来纳州生活与科学博物馆的成功合作继续和扩展。该项目有可能在格兰维尔和达勒姆县至少达到10所中学，其中多达1,000名中学学生直接受益于“手提箱中的科学”教学套件的分布和实施（10名老师，每位中学100名学生参加数学和科学课程三年）。具体目标包括：每年在为期两天的教学研讨会上与10名中学数学和科学老师互动；通过生活和科学博物馆提供后续支持；并为参加研讨会的老师提供无成本的教学套件。