Molecular Mechanisms of MAR Effects in Arabidopsis

该项目将导致增加对基因组过程如何协调的理解。研究人员正在研究一种称为基质附着区（MAR）的DNA序列，它可以与细胞核内称为核基质（或支架）的动态结构结合。MAR被认为对DNA复制、核组织和基因表达很重要。该项目使用模式植物拟南芥，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基因组到下一个基因组是极其可变的，反映了转基因表达的完全或部分沉默。对植物和动物的研究表明，在转基因中加入MAR可以增加平均表达水平，可能是通过减少沉默的频率。然而，增加的表达并不总是发生，甚至含MAR的转基因有时也被沉默。本项目旨在了解MAR如何发挥作用，以及为什么它们的效果并不总是相同。特别是，研究人员将询问MAR侧翼的转基因是否在转录上沉默（不转录成RNA），或者转基因是否被活跃地转录，但RNA的降解速度与其产生的速度一样快。重要的是，他们将使用允许他们在已知基因组位置比较有和没有MAR的转基因的技术，并将MAR效应的差异与每个位置的基因组特征的差异联系起来，例如与天然基因的接近程度或转座子的存在，重复DNA或将DNA包装到染色体中的组蛋白的修饰版本。每个转基因的转录的直接测量也将用于测试MAR主要在转录水平起作用的假设。更广泛的影响。这项研究将有助于将基因表达的生物化学与细胞核中的组织结构联系起来，并更好地了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MAR稳定抗病性，以及用于作物改良的途径工程，生物能源应用或非食品物种的药物和化学原料生产。该项目还将为一名研究生和一名博士后学者提供培训和指导。此外，该项目的资金将允许继续和扩大与两名格兰维尔县中学教师和北卡罗来纳州生命和科学博物馆的成功合作。该项目有可能覆盖格兰维尔和达勒姆县的至少10所中学，多达1 000名中学生直接受益于“手提箱中的科学”教学包的分发和实施（每所中学10名教师，100名学生参加数学和科学课程，为期三年）。具体目标包括：每年让10名中学数学和科学教师参加为期两天的教学研讨会;通过生命和科学博物馆的工作人员提供后续支持;并向参加研讨会的教师免费提供教学工具包。

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.