Plastid transformation in Arabidopsis thaliana

设计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能量转化为农作物生物量的过程。在大多数植物中，叶绿体工程的效率非常低。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在模式植物拟南芥中高效设计叶绿体的方法。实验设计将直接适用于进化相关的十字花科作物、重要蔬菜西兰花和食用油的重要来源油菜。在这些作物中设计叶绿体的能力可能会导致显著的产量提高。该项目还将产生教育影响，为包括妇女和代表性不足的少数群体在内的本科生提供研究培训。罗格斯大学将通过罗格斯大学数学、科学和工程专业女性道格拉斯项目和罗格斯大学本科生阿雷斯蒂研究中心招收本科生。代表不足的少数族裔、低收入和第一代大学生也将从纽约法明代尔法明代尔州立学院的大学科技入学计划或世界助学金计划中招募。巴西圣保罗大学(USP)的一名本科生将获得夏季叶绿体生物技术研究经验，作为罗格斯大学/USP联合博士项目招生计划的一部分。叶绿体基因组的转化在烟草中是司空见惯的，但在模式植物拟南芥中，转化的频率要低100倍，这阻碍了它在研究叶绿体生物学问题方面的广泛应用。最近的数据显示，在带有ACC2基因缺陷的拟南芥中，叶绿体的转化效率提高了100倍。该突变系对壮观霉素高度敏感，壮观霉素是一种用于叶绿体转化的选择性物质。由于ACC2 T-DNA基因敲除系在这次加入中是可用的，因此在哥伦比亚的背景下获得了提高叶绿体转化频率的原理证据。然而，哥伦比亚大学的加入对进行叶绿体转化的培养细胞再生植株是顽固的。本研究的目的是为植物界提供拟南芥叶绿体转化研究工具。这将通过以下途径实现：(1)在可再生的RLD和Ws材料中获得ACC2基因敲除系；(2)从转体细胞中开发植株再生方案，以获得可育的转体植株；(3)获得哥伦比亚生态型的可再生形式；(4)构建适合于实现这些目标的叶绿体转化载体。这一结果将加速对叶绿体功能的研究，从而使人们能够更好地了解管理这一重要细胞器与核之间相互作用并对基本叶绿体功能做出贡献的‘规则’。该奖项由分子和细胞生物科学部的遗传机制计划和综合组织系统部的植物基因组研究计划共同资助；这两个计划都在生物科学局。

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.