TRTech-PGR: Manipulating plant karyotypes by synthetic centromere formation

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造染色体，以提高植物在抗旱性等领域的表现。该项目的总体目标是制定引入和遗传定制设计的人造植物染色体的策略。这项工作将通过两个“植物设计”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着丝粒是由什么组成的，以及什么蛋白质与着丝粒结合。这个项目将通过创建具有特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LexO结合位点的长阵列，并表明包含LexA DNA结合域和CENH3(LexA-CENH3)的融合蛋白与LexO阵列结合，并招募其他动粒蛋白。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。他们将使用几种方法来打破4号染色体，并释放末端片段，成为独立的新染色体，他们将测试有丝分裂和减数分裂的稳定性。他们还将创建新的LexO阵列，并确定激活功能着丝粒所需的最小尺寸。他们将进一步将这项工作扩展到本特米纳烟草，那里存在用大分子转化原生质体的方法。他们将构建基于LexA-CENH3系统的合成染色体载体，并在培养和再生植物中测试它们的特性。如果成功，这一结果将扩大植物着丝粒的知识，并为植物基因组工程提供第一代合成染色体载体。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.