CAREER: Tailoring hormone responses in plants via synthetic signal integration devices

职业：通过合成信号集成设备定制植物中的激素反应

• 批准号: 1750006
• 负责人: Anna Stepanova
• 金额: $ 127.59万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-10-01 至 2024-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1750006&HistoricalAwards=false
• 关键词: CAREER; Tailoring; hormone; responses; plants

The plant hormone, auxin is an important plant-made growth regulator that governs multiple aspects of plant growth and physiology and acts at every stage in the life of the plant. The goal of this project is to generate new molecular tools to precisely control where and when auxin is made as a way to manipulate plant growth, particularly in the root. Taking advantage of the powerful gene editing and targeting technology (CRISPR), this project generates programmable genetic devices (biological logic gates), that make it possible to turn genes of interest (such as auxin biosynthesis genes) on and off at will. The basic principles and tools developed in this project have the potential for broad applications in agriculture and beyond. The protocols and materials generated in this study serve as the basis for a new Synthetic Biology course for upper level undergraduate and beginning graduate students at NC State University. In addition, the investigator has adapted some of these materials for workshops that provide training and resources for high school teachers. The investigator also establishes a molecular biology tool "lending library" that enables local teachers to execute molecular experiments in their classrooms. Furthermore, continuation of the previously established bilingual (English and Spanish) hands-on Plants4Kids demonstrations with live plants and seeds at the NC Museum of Natural Sciences and local elementary schools brings the excitement of experimental sciences to the youngest audiences. These activities expose young people to scientific methods and demystify agricultural and plant genetic techniques.Biology research is often restricted by the choice of promoters available to drive the expression of genes of interest in complex patterns and at desired levels. To overcome this limitation, a series of easily programmable CRISPR-based synthetic genetic circuits are developed to integrate multiple inputs from previously described synthetic and natural promoters. These new tools take advantage of crRNA:tracrRNA gRNA pairs and combine inputs from two or more different drivers/promoters to delimit where a dCas9-based synthetic transcription factors activate or represses target gene expression. The proposed approach rests on two or more pseudo-orthogonal gRNA pairs and is compatible with the construction of all basic logic gates to produce multiple derived patterns of targeted gene expression. In theory, this technology can produce hundreds of orthogonal genetic components that in combination perform complex logic operations. The new strategy is not only scalable, but also enables tuning of gene expression levels, thus providing an unprecedented degree of flexibility in targeted gene expression. To demonstrate the utility of the new approach, the investigator regulates the tissue specific expression of local auxin production in roots in an auxin-deficient mutant and interrogates the effects on root architecture; thus addressing a long-standing question on the role of local auxin production in maintaining the stem cell niche or plant roots.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.

植物激素，生长素是一种重要的植物生长调节剂，它控制着植物生长和生理的多个方面，并在植物生命的每个阶段起作用。该项目的目的是生成新的分子工具，以精确控制生长素的何时以及何时制造植物生长，尤其是在根部。利用强大的基因编辑和靶向技术（CRISPR），该项目生成了可编程的遗传设备（生物逻辑门），这使得可以随意转或关闭感兴趣的基因（例如生长素生物合成基因）。该项目中开发的基本原理和工具具有在农业及其他地区进行广泛应用的潜力。本研究中生成的方案和材料是北卡罗来纳州立大学的上层本科生和初学者的新合成生物学课程的基础。此外，调查人员还将其中一些材料调整为为高中教师提供培训和资源的研讨会。研究人员还建立了一种分子生物学工具“贷款库”，该工具使当地教师能够在课堂上执行分子实验。此外，在北卡罗来纳州自然科学博物馆和当地小学博物馆与现场植物和种子一起进行了以前建立的双语（英语和西班牙语）动手植物的延续，并为最年轻的观众带来了实验科学的兴奋。这些活动使年轻人采取了科学方法，并揭开了农业和植物遗传技术的神秘面纱。生物学研究通常受到选择的启动子的选择来限制，从而在复杂的模式和所需水平上推动感兴趣的基因表达。为了克服这一限制，开发了一系列易于编程的基于CRISPR的合成遗传回路，以整合来自先前描述的合成和天然启动子的多个输入。这些新工具利用了CRRNA：tracrrna grna对，并将来自两个或更多不同驱动因素/启动子的输入结合到界限，以划界基于DCAS9的合成转录因子激活或抑制靶基因表达。所提出的方法取决于两个或多个伪正交的GRNA对，并且与所有基本逻辑门的构造兼容，以产生靶向基因表达的多种派生模式。从理论上讲，该技术可以生产数百种结合进行复杂逻辑操作的正交遗传成分。新策略不仅是可扩展的，而且还可以调整基因表达水平，从而在靶向基因表达中提供了前所未有的灵活性。为了证明新方法的实用性，研究者调节了生长素缺陷型突变体中根中生长素产生的组织特异性表达，并询问对根系结构的影响。因此，关于局部生长素生产在维持干细胞生态位或植物根源中的作用的长期问题。该奖项反映了NSF的法定任务，并使用基金会的知识分子优点和更广泛的影响评估标准，被认为值得通过评估来提供支持。

项目成果

Deciphering the molecular basis of tissue-specific gene expression in plants: Can synthetic biology help?

• DOI: 10.1016/j.pbi.2022.102241
• 发表时间: 2022-08
• 期刊: CURRENT OPINION IN PLANT BIOLOGY
• 影响因子: 9.5
• 作者: Yaschenko, Anna E.;Fenech, Mario;Mazzoni-Putman, Serina;Alonso, Jose M.;Stepanova, Anna N.
• 通讯作者: Stepanova, Anna N.

Plant Biology Research: What Is Next?

• DOI: 10.3389/fpls.2021.749104
• 发表时间: 2021
• 期刊: Frontiers in plant science
• 影响因子: 5.6
• 作者: Stepanova AN

From Ethylene-Auxin Interactions to Auxin Biosynthesis and Signal Integration

从乙烯-生长素相互作用到生长素生物合成和信号整合

• DOI: 10.1105/tpc.19.00339
• 发表时间: 2019
• 期刊: The Plant Cell
• 作者: Stepanova, Anna N.;Alonso, Jose M.
• 通讯作者: Alonso, Jose M.

Auxin Interactions with Other Hormones in Plant Development

• DOI: 10.1101/cshperspect.a039990
• 发表时间: 2021-10-01
• 期刊: COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY
• 影响因子: 7.2
• 作者: Mazzoni-Putman, Serina M.;Brumos, Javier;Stepanova, Anna N.
• 通讯作者: Stepanova, Anna N.

Editorial overview: Toward deciphering the molecular basis of plant phenotypic plasticity

编辑概述：破译植物表型可塑性的分子基础

• DOI: 10.1016/j.pbi.2021.102107
• 发表时间: 2021
• 期刊: Current Opinion in Plant Biology
• 影响因子: 9.5
• 作者: Qiao, Hong;Stepanova, Anna N.
• 通讯作者: Stepanova, Anna N.