Plastid transformation in Arabidopsis thaliana

改造叶绿体基因组是提高光合作用效率的关键，光合作用是将太阳光转化为作物生物量的过程。在大多数植物中，叶绿体工程效率很低。然而，最近的一项突破揭示了这一过程的遗传瓶颈，该项目将利用新的信息来开发模式植物拟南芥叶绿体的有效工程方法。该实验设计将直接适用于进化相关的十字花科作物、重要蔬菜西兰花和重要食用油来源油菜。在这些作物中改造叶绿体的能力可能会显著提高产量。该项目还将对教育产生影响，为本科生，包括妇女和代表性不足的少数民族提供研究培训。本科生将通过罗格斯大学数学、科学和工程女性道格拉斯项目和罗格斯大学阿雷斯蒂本科生研究中心从罗格斯大学招募。未被充分代表的少数族裔、低收入和第一代大学生也将从纽约州法明代尔州法明代尔州立学院的大学科学技术入学计划或世界第一资助计划中招募。罗格斯大学将向巴西圣保罗大学（USP）的一名本科生提供叶绿体生物技术的暑期研究经验，这是罗格斯大学/USP联合博士项目招收学生计划的一部分。质体基因组的转化在烟草中很常见，但在模式植物拟南芥（Arabidopsis thaliana）中的频率要低100倍，这阻碍了其在质体生物学研究中的广泛应用。最近的数据显示，携带缺陷ACC2基因的拟南芥质体转化效率提高了100倍。这个突变系对大观霉素过敏，大观霉素是用于质体转化的选择剂。在哥伦比亚背景下获得了增加质体转化频率的原理证明，因为ACC2 T-DNA敲除系在该加入中可用。然而，哥伦比亚号植株难以从进行质体转化的培养细胞中再生。本研究旨在为植物界提供拟南芥质体转化的研究工具。这将通过(1)在可再生的RLD和Ws中获得ACC2敲除系来实现；(2)利用转质体细胞开发植物再生方案，获得可育的转质体植株；(3)获得哥伦比亚生态型的可再生形态；(4)构建适合实现这些目标的质体转化载体。该结果将加速对质体功能的研究，从而更好地理解控制这一重要细胞器与细胞核之间相互作用的“规则”，并有助于基本的质体功能。该奖项由分子和细胞生物科学部的遗传机制项目和综合有机体系统部的植物基因组研究项目共同资助；这两个项目都在生物科学理事会。

Engineering the chloroplast genome is key to improving the efficiency of photosynthesis, the process that converts sunlight energy into biomass in crops. In most plants, chloroplast engineering is very inefficient. However, a recent breakthrough has revealed the genetic bottleneck for this process, and this project will leverage the new information to develop methods for efficient engineering of chloroplasts in the model plant, Arabidopsis thaliana. The experimental design will be directly applicable to crops in the evolutionary related Brassicaceae family, broccoli, an important vegetable, and oilseed rape, an important source of edible oil. The ability to engineer chloroplasts in these crops could lead to significant production improvements. The project will also have educational impact by providing research training for undergraduates, including women and underrepresented minorities. Undergraduates will be recruited from Rutgers University through The Douglass Project for Rutgers Women in Math, Science and Engineering, and The Rutgers Aresty Research Center for Undergraduates. Under-represented minorities, low-income, and first-generation college students will also be recruited from the Collegiate Science and Technology Entry Program or the First in the World Grant programs at Farmingdale State College, Farmingdale, NY. A summer research experience in chloroplast biotechnology will be offered to an undergraduate student from the University of Sao Paulo (USP), Brazil as part of a plan to recruit students for the joint Rutgers/USP PhD program. Transformation of the plastid genome is routine in tobacco, but 100-fold less frequent in the model plant, Arabidopsis thaliana, preventing its widespread use for studying questions in plastid biology. Recent data have revealed that plastid transformation efficiency is 100-fold enhanced in Arabidopsis plants with a defective ACC2 gene. This mutant line is hypersensitive to spectinomycin, the selective agent used for plastid transformation. Proof of principle for increased plastid transformation frequency was obtained in the Columbia background, because ACC2 T-DNA knockout lines are available in this accession. However, the Columbia accession is recalcitrant to plant regeneration from cultured cells where plastid transformation is carried out. The goal of this research is to provide Arabidopsis plastid transformation research tools for the plant community. This will be achieved through (1) obtaining ACC2 knockout lines in the regenerable RLD and Ws accessions; (2) developing plant regeneration protocols from transplastomic cells to obtain fertile transplastomic plants; (3) obtaining regenerable forms of the Columbia ecotype; and (4) constructing plastid transformation vectors that are suitable to achieve these goals. The results will accelerate studies of plastid function, thereby allowing better understanding of the 'rules' that govern interactions between this important organelle and the nucleus and contribute to essential plastid functions.This award is co-funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences and by the Plant Genome Research Program in the Division of Integrative Organismal Systems; both programs are in the Directorate for Biological Sciences.