TRANSFORM-PGR: Manipulating Agrobacterium-mediated transformation and T-DNA integration for plant synthetic biology and genome engineering

TRANSFORM-PGR：操纵农杆菌介导的转化和 T-DNA 整合，用于植物合成生物学和基因组工程

• 批准号: 1725122
• 负责人: Stanton Gelvin
• 金额: $ 146.71万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1725122&HistoricalAwards=false
• 关键词: TRANSFORM; PGR; Manipulating; Agrobacterium; mediated; TRANSFORM; PGR; Manipulating; Agrobacterium; mediated

Non-technical paragraph:The introduction of DNA into plants (plant transformation to generate genetically modified or transgenic plants) has become a core technology for basic plant research and the agricultural biotechnology industry. Agrobacterium-mediated plant genetic transformation is the most commonly used method to generate transgenic plants. The bacterium Agrobacterium can transfer large DNA-molecules (T-DNA) capable of encoding numerous genes sufficient to, e.g., encode a novel biosynthetic pathway. Thus, Agrobacterium-mediated transformation is a key tool for synthetic biology. Although the Agrobacterium host range is broad, many agronomic important species or specific cultivars remain recalcitrant to transformation. In addition, T-DNA integrates randomly into the plant genome. Consequently, T-DNA may disrupt genes important for plant development and productivity. Random T-DNA integration often occurs in genomic regions that silence encoded transgenes, leading to unpredictable and unstable transgene expression. It is therefore important to develop novel technologies to increase transformation efficiency of a broader range of crop species and agronomic important varieties, and to insert T-DNA into defined locations of any plant genome. This project will develop novel technologies to broaden the plant host range of Agrobacterium, and to direct the integration of T-DNA to specific plant chromosomal regions pre-selected by the scientist. In addition, this project will develop novel technologies to deliver genes to plants efficiently without subsequent integration into the plant genome. This latter technology is important for delivering plant genome engineering reagents without maintaining these reagents after they have accomplished their tasks. Technical paragraph:This project proposes tool development for the plant research community to help enable functional genomics for a broad spectrum of species. Because Agrobacterium-mediated transformation is the preferred DNA transfer approach, one goal of this project is to increase the efficiency of transformation by debilitating plant defense responses to Agrobacterium. This will be accomplished by engineering Agrobacterium strains that can secrete Type III effectors and suppress plant defense responses to enhance transformation. Because controlled T-DNA integration and predictable transgene expression is important for synthetic biology, a second goal is to build a system to facilitate effective and precise DNA integration into plant genomes. This system will be developed using CRISPR/Cas9 to generate breaks in specific tomato genomic sequences that will "trap" T-DNA. These integration sites will be chosen to maximize the probability of stable transgene expression through numerous plant generations and under field conditions. The expression of targeted and randomly integrated transgenes will be assessed. Agrobacterium is also used to deliver genome engineering reagents to plants. However, integration of these reagents is undesirable, and they usually are segregated out of the engineered plants. This is difficult for vegetative-propagated species. The third goal of this project is to develop Agrobacterium strains that can efficiently deliver but not integrate T-DNA encoding genome editing reagents, or will secrete these reagents through a Type III secretion system. This will be accomplished by altering Agrobacterium VirD2, the protein that leads T-DNA from the bacterium into the plant and which is important for T-DNA integration.

非技术段落：将DNA引入植物（产生转基因或转基因植物的植物转化）已成为基础植物研究和农业生物技术行业的核心技术。农杆菌介导的植物遗传转化是产生转基因植物的最常用方法。细菌农杆菌可以传递能够编码足以编码新型生物合成途径的大量基因的大型DNA分子（T-DNA）。因此，农杆菌介导的转化是合成生物学的关键工具。尽管农杆菌宿主范围很广，但许多农艺重要物种或特定品种仍然是转化的顽固性。另外，T-DNA随机整合到植物基因组中。因此，T-DNA可能会破坏对植物发育和生产力重要的基因。随机T-DNA的整合通常发生在沉默的转基因的基因组区域，导致不稳定和不稳定的转基因表达。因此，重要的是要开发新的技术，以提高农作物物种和农艺重要品种的转化效率，并将T-DNA插入任何植物基因组的定义位置。该项目将开发新的技术，以扩大植物宿主的农业宿主范围，并指导T-DNA与科学家预先选择的特定植物染色体区域的整合。此外，该项目将开发出新的技术，以有效地将基因传递给植物，而不会随后整合到植物基因组中。后一种技术对于在完成任务后不维护这些试剂的情况下提供植物基因组工程试剂很重要。技术段落：该项目提出了植物研究社区的工具开发，以帮助为广泛的物种启用功能基因组学。由于农杆菌介导的转化是首选的DNA转移方法，因此该项目的一个目标是通过使植物防御对农杆菌的防御反应来提高转化的效率。这将通过工程农业菌株来实现，该农业菌株可以分泌III型效应子并抑制植物防御反应以增强转化。由于受控的T-DNA整合和可预测的转基因表达对于合成生物学很重要，因此第二个目标是建立一个系统，以促进有效而精确的DNA整合到植物基因组中。该系统将使用CRISPR/CAS9开发，以在特定的番茄基因组序列中产生断裂，以“捕获” T-DNA。这些整合位点将被选择以最大程度地提高稳定的转基因表达的概率，并在田间条件下通过众多植物世代和田间条件。将评估靶向和随机整合的转基因的表达。农杆菌还用于向植物提供基因组工程试剂。但是，这些试剂的整合是不可取的，通常将它们隔离在工程植物中。对于营养传播的物种来说，这很难。该项目的第三个目标是开发可以有效传递但不能整合编码基因组编辑试剂的T-DNA的农业菌株，或者将通过III型分泌系统分泌这些试剂。这将通过改变Vird2的农杆菌（从细菌进入植物的T-DNA的蛋白质）来实现，这对于T-DNA整合很重要。