Dissection of cis-regulatory Regions in Plants Using Recombination, Allelic Expression, and Site-directed Mutagenesis

知识价值。理解生物体发育的核心是驱动基因表达变异的潜在遗传机制。人们普遍认为，基因调控区域的序列变异在表达差异和生物多样性中起着重要作用，但对这些复杂启动子区域的结构和功能知之甚少。该项目通过将调控区域的功能模块分解为其控制单元，朝着解决这种表达变异的起源迈出了一步。来自广泛真核生物的证据表明，在单个物种中，等位基因表达的变异是一种常见现象，并且在不同的组织和不同的发育阶段以非常高的频率发生。利用拟南芥和玉米这两个资源丰富的模式植物类群，结合自然发生的减数分裂重组，将利用等位基因表达的变化来绘制不同植物组织中几种基因表达差异的调控成分。这些数据有望揭示两种不同植物谱系中等位基因表达变异的调控成分，并为更全面地了解植物启动子的结构和功能做出重大贡献。此外，在拟南芥相同基因的上游区域，使用原位定向诱变技术有望发现其他不影响等位基因表达差异的基因表达调控成分，但它们确实具有顺式调控控制的功能。这些数据，结合项目中等位基因表达部分的数据，将提供拟南芥和玉米区域植物启动子结构和功能的详细表征。该项目的完成将有助于更广泛地了解植物启动子的进化，特别是关于通过减数分裂重组进行模块交换的作用，并将为阐明植物基因表达的顺式调控机制提供一种有前途的方法。该项目不仅有望在分子和进化遗传学领域取得知识进步，而且还有望开发一些可能的实用工业技术，旨在加强和推进农业实践。更广泛的影响。这项研究适用于多种活动，包括外联、纳入代表性不足的群体成员以及对各级学生的科学培训。在西弗吉尼亚州，一小部分高中生上大学（约16%），其中20%是第一代大学生。这些项目将通过实验室实践的科学培训，为这些代表性不足的学生提供多种教育机会，从而导致具有里程碑意义的发现，并最终实现创新的现实世界农业应用，造福整个社会。其他未被充分代表的群体将通过麦克奈尔奖学金（McNair Scholarship）和美国国家科学基金会本科生研究教育（REU）计划获得机会。麦克奈尔奖学金旨在为来自弱势背景的学生提供支持。此外，西弗吉尼亚大学还通过科学与工程女性项目（WiSE）和暑期本科生研究体验项目（SURE）提供了许多教育推广机会。学生将有机会在年度国际会议上向更广泛的科学界传播他们的工作成果。此外，这项研究是WVU计算生物学计划的一部分，该计划是生物系，数学系和统计学系之间的跨部门努力，旨在为新兴的生物信息学领域创建本科和研究生课程。所有生成的脚本和数据将用于开发该倡议下的新课程。

Intellectual Merit. Central to the understanding of organismal development are the underlying genetic mechanisms that drive variation in gene expression. It is generally agreed that sequence variation in the regulatory regions of genes plays a major role in expression divergence and organismal diversity, yet little is know regarding the structure and function of these complex promoter regions. This project takes a step toward resolving the genesis of this expression variation by dissecting the functional modules of regulatory regions into their controlling units. Evidence from a wide range of eukaryotic organisms indicates that, within individual species, variation in allelic expression is a common phenomenon and occurs at a significantly high frequency in various tissues and during different developmental stages. Using two resource-rich model plant taxa, Arabidopsis thaliana and Zea mays (maize), variation in allelic expression, combined with naturally occurring meiotic recombination, will be used to map the regulatory components of these expression differences for several genes in various plant tissues. The data are expected to reveal the regulatory components of allelic expression variation in two distinct plant lineages and contribute significantly to a more complete understanding of the structure and function of plant promoters. Additionally, other regulatory components of gene expression that do not contribute to allelic expression differences, but do function as cis-regulatory controls, are expected to be discovered using in situ site-directed mutagenesis in upstream regions of the same genes in Arabidopsis. These data, combined with those from the allelic expression portion of the project, will provide a detailed characterization of plant promoter structure and function in the Arabidopsis and maize regions of interest. The completion of this project will contribute to a broader understanding of promoter evolution in plants, specifically with respect to the role of module swapping via meitoic recombination, and will also provide a promising methodology for elucidating mechanisms for the cis-regulatory control of plant gene expression. This project promises not only intellectual advancement in the fields of molecular and evolutionary genetics, but also the development of a number of possible practical industrial technologies designed to enhance and advance agricultural practices.Broader Impacts. This research lends itself to multiple activities involving outreach, the inclusion of members of underrepresented groups, and scientific training of students at all levels. In the state of West Virginia, a small fraction of high school students attend college (~16%), and 20% of those incoming undergraduates are first-generation college students. These projects will provide multiple educational opportunities for these underrepresented students via scientific training in laboratory practices that lead to landmark discoveries, and ultimately to the implementation of innovative real world agricultural applications that benefit society as a whole. Other underrepresented groups will be provided opportunities at WVU via the McNair Scholarship, designed to provide support for students from disadvantaged backgrounds, and the NSF Research Education for Undergraduates (REU) program. Also, a number of educational outreach opportunities exist at WVU via the program for Women in Science and Engineering (WiSE), and the Summer Undergraduate Research Experience (SURE) program. Students will be provided the opportunity to disseminate the results of their work among the broader scientific community at annual international meetings. In addition, this research is designed as part of the WVU Computational Biology Initiative, an interdepartmental effort among the Departments of Biology, Mathematics and Statistics to create undergraduate and graduate programs in the emerging field of bioinformatics. All scripts and data generated will be used to develop new courses under the Initiative.