Characterization of novel subcellular structures in Arabidopsis thaliana

描述（由申请人提供）：植物生物技术有可能在许多方面改善人类健康。植物可以被改造成生产作为药物的治疗性蛋白质。农作物可以通过基因改造生产出更有营养的食物，并减少化肥和杀虫剂的使用。植物也可以被改造成清洁环境或取代威胁大气的化石燃料。但植物生物技术面临的一个关键问题是无法在植物细胞中生产大量转基因蛋白质。这种限制在药物生产领域特别具有挑战性，其中植物产生的蛋白质的量影响药物的经济可行性。 解决植物中蛋白质产量低的问题的一种方法是在植物细胞中找到新的地方来储存工程蛋白质。该提案的目标是发现和表征植物细胞中用于积累转基因蛋白质的新隔室。目前，将蛋白质靶向细胞质和膜结合的细胞器如内质网导致每克植物组织中蛋白质的浓度仅低。与其他策略（如在细菌和哺乳动物细胞培养中表达蛋白质）相比，这降低了植物生物技术的盈利能力。如果成功，该项目将确定新的细胞器或亚细胞结构，这将提高植物基因工程的效率。该项目还将确定如何将蛋白质靶向这些新位置。此外，该项目可能会确定新的生化途径作为未来植物基因工程的目标。 这个项目将开始在模式植物拟南芥中寻找新的细胞器。已经产生了一组108个独特的转基因拟南芥，其表达绿色荧光蛋白（GFP）和随机植物蛋白之间的不同融合（1）。这些随机蛋白质作为靶向信号将GFP发送到不同的细胞内区室。在某些情况下，绿色荧光蛋白被发现积累在植物生物学家以前从未观察到的细胞区域。该项目旨在表征这些新的细胞器以及蛋白质如何靶向它们。 该项目的初步筛选阶段将使用荧光显微镜来识别候选植物，其中GFP照亮了植物细胞中的新结构。然后将转基因从候选株系中克隆出来，以确定哪些蛋白质序列将GFP靶向其特定位置。该序列信息将用于确定将蛋白质靶向新细胞器的最低要求。最后，为了充分表征细胞器，将通过肽指纹图谱对它们进行纯化和分析蛋白质含量。 公共卫生相关性：生物技术专家正在对植物进行基因工程改造，以生产药物，增加营养成分，使用更少的化学品以更便宜的价格种植，清理工业废物，并生产碳中性燃料。植物科学家面临的最大挑战之一是无法在植物细胞中产生高水平的蛋白质。该项目将在植物细胞中发现新的隔间，以促进更有效地利用植物来改善人类健康。

DESCRIPTION (provided by applicant): Plant biotechnology has the potential to improve human health on a number of fronts. Plants can be engineered to produce therapeutic proteins as drugs. Crops can be genetically modified to produce food that is more nutritious and to require less chemical fertilizers and pesticides. Plants can also be engineered to clean up the environment or replace the fossil fuels that threaten the atmosphere. But one critical problem faced by plant biotechnology is the inability to produce large amounts of transgenic proteins in plant cells. This limitation is particularly challenging in the realm of drug production where the amount of protein produced by a plant influences the economic viability of the drug. One way to address the problem of low protein production in plants is to find new places in the plant cell to store engineered proteins. The goal of this proposal is to discover and characterize new compartments in the plant cell for accumulation of transgenic proteins. Currently, targeting proteins to the cytosol and membrane bound organelles like the endoplasmic reticulum results in only low concentrations of the protein per gram of plant tissue. This reduces the profitability of plant biotechnology when compared to other strategies like protein expression in bacteria and mammalian cell cultures. If successful, this project will identify new organelles or subcellular structures that will improve the efficiency of plant genetic engineering. The project will also determine how to target proteins to these new locations. In addition, this project may identify new biochemical pathways as targets for future plant genetic engineering. This project will begin by looking for new organelles in the model plant Arabidopsis thaliana. A set of 108 unique transgenic Arabidopsis have been produced that express different fusions between the green fluorescent protein (GFP) and random plant proteins (1). These random proteins serve as targeting signals to send the GFP to different intracellular compartments. In some cases, the GFP has been found to accumulate in regions of the cell never before observed by plant biologists. This project will aim to characterize these new organelles and how proteins can be targeted to them. The initial screening stage of this project will use fluoresce microscopy to identify candidate plants in which GFP lights up new structures in the plant cell. The transgenes will then be cloned out of the candidate lines to determine what protein sequences are targeting the GFP to their particular locations. This sequence information will be used to determine the minimal requirements for targeting proteins to the new organelle. Finally, to fully characterize the organelles, they will be purified and analyzed for protein content by peptide fingerprinting. PUBLIC HEALTH RELEVANCE: Biotechnologists are genetically engineering plants to produce drugs, to increase nutrient content, to be grown cheaper using fewer chemicals, to clean up industrial wastes, and to produce carbon-neutral fuels. One of the biggest challenges faced by plant scientists is the inability to produce high levels of proteins in plant cells. This project will find new compartments within plant cells that will promote more efficient use of plants to improve human health.