TRTech-PGR: Manipulating plant karyotypes by synthetic centromere formation

TRTech-PGR：通过合成着丝粒形成操纵植物核型

• 批准号: 2040218
• 负责人: R Kelly Dawe
• 金额: $ 232.54万
• 依托单位: University of Georgia Research Foundation Inc
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2040218&HistoricalAwards=false
• 关键词: TRTech; PGR; Manipulating; plant; karyotypes

An important goal of plant synthetic biology is to design artificial chromosomes with custom genes and pathways that can improve plant performance in areas such as drought tolerance. The overall aim of this project is to develop strategies for the introduction and inheritance of custom designed artificial plant chromosomes. This work will be communicated to the public through two “Plants by Design” research symposia, research demonstrations at the Bell Museum at the University of Minnesota, and interactive instructional events at local farmers markets in Athens Georgia. Years of research have provided a comprehensive understanding of what centromeres are made of and what proteins bind to them. This project will test the investigators' knowledge by creating synthetic centromeres with defined components. In prior work the investigators inserted long arrays of LexO binding sites on maize chromosome 4 and showed that a fusion protein containing a LexA DNA binding domain and CENH3 (LexA-CENH3) binds to LexO arrays and recruits other kinetochore proteins. In this project, they will determine whether LexA-CENH3-activated synthetic centromeres are capable of driving independent chromosome segregation. They will use several methods to break chromosome 4 and liberate the end fragments to become independent neochromosomes, which they will test for mitotic and meiotic stability. They will also create new LexO arrays and determine the minimum size necessary to activate functional centromeres. They will further extend the work to Nicotiana benthamiana, where methods exist to transform protoplasts with large molecules. They will build synthetic chromosome vectors based on the LexA-CENH3 system and test their properties in culture and regenerated plants. If successful, the results will expand knowledge of plant centromeres and provide first-generation synthetic chromosome vectors for plant genome engineering.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

植物合成生物学的一个重要目标是设计具有定制基因和途径的人工染色体，可以提高植物在耐旱性等方面的性能。该项目的总体目标是制定定制设计的人工植物染色体的引入和遗传策略。这项工作将通过两次“设计植物”研究研讨会、明尼苏达大学贝尔博物馆的研究演示以及佐治亚州雅典当地农贸市场的互动教学活动向公众传播。 多年的研究已经全面了解了着丝粒的组成以及与它们结合的蛋白质。该项目将通过创建具有确定成分的合成着丝粒来测试研究人员的知识。在之前的工作中，研究人员在玉米 4 号染色体上插入了长的 LexO 结合位点阵列，并表明含有 LexA DNA 结合域和 CENH3 (LexA-CENH3) 的融合蛋白与 LexO 阵列结合并招募其他着丝粒蛋白。在这个项目中，他们将确定 LexA-CENH3 激活的合成着丝粒是否能够驱动独立的染色体分离。他们将使用多种方法来断裂 4 号染色体并释放末端片段，使其成为独立的新染色体，并测试其有丝分裂和减数分裂稳定性。他们还将创建新的 LexO 阵列并确定激活功能性着丝粒所需的最小尺寸。他们将进一步将工作扩展到本塞姆氏烟草，那里存在用大分子转化原生质体的方法。他们将基于 LexA-CENH3 系统构建合成染色体载体，并测试其在培养和再生植物中的特性。如果成功，研究结果将扩展植物着丝粒的知识，并为植物基因组工程提供第一代合成染色体载体。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Synthetic maize centromeres transmit chromosomes across generations

• DOI: 10.1038/s41477-023-01370-8
• 发表时间: 2022-09
• 期刊: Nature Plants
• 影响因子: 18
• 作者: R. Dawe;J. Gent;Yibing Zeng;Han Zhang;Fang-fang Fu;Kyle W. Swentowsky;D. W. Kim;Na Wang;Jianing Liu;Rebecca D. Piri

An extensible vector toolkit and parts library for advanced engineering of plant genomes

• DOI: 10.1002/tpg2.20312
• 发表时间: 2023-03-09
• 期刊: PLANT GENOME
• 影响因子: 4.2
• 作者: Chamness, James C.;Kumar, Jitesh;Voytas, Daniel F.
• 通讯作者: Voytas, Daniel F.