Characterization of novel subcellular structures in Arabidopsis thaliana

描述（由申请人提供）：植物生物技术在许多方面具有改善人类健康的潜力。植物可以被改造成产生治疗性蛋白质作为药物。作物可以经过基因改造，生产出更有营养的食物，并且需要更少的化肥和杀虫剂。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。但是植物生物技术面临的一个关键问题是无法在植物细胞中产生大量的转基因蛋白。这一限制在药物生产领域尤其具有挑战性，因为植物产生的蛋白质数量会影响药物的经济可行性。解决植物蛋白产量低问题的一种方法是在植物细胞中寻找新的地方来储存工程蛋白。本提案的目标是发现和表征植物细胞中积累转基因蛋白的新区室。目前，将蛋白质靶向到细胞质和膜结合细胞器（如内质网）只会导致每克植物组织中蛋白质的浓度很低。与细菌和哺乳动物细胞培养的蛋白质表达等其他策略相比，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到这些新位置。此外，该项目可能为未来植物基因工程发现新的生化途径。该项目将从寻找模式植物拟南芥中的新细胞器开始。已经产生了108个独特的转基因拟南芥，它们表达绿色荧光蛋白（GFP）与随机植物蛋白之间的不同融合(1)。这些随机蛋白作为靶向信号，将绿色荧光蛋白发送到不同的细胞内区室。在某些情况下，已经发现绿色荧光蛋白在植物生物学家以前从未观察到的细胞区域积累。这个项目将致力于表征这些新的细胞器，以及蛋白质如何靶向它们。该项目的初始筛选阶段将使用荧光显微镜来识别候选植物，其中绿色荧光蛋白照亮了植物细胞中的新结构。然后将转基因从候选细胞系中克隆出来，以确定哪些蛋白质序列将GFP靶向到其特定位置。该序列信息将用于确定将蛋白质靶向到新细胞器的最低要求。最后，为了充分表征细胞器，它们将被纯化并通过肽指纹分析蛋白质含量。

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.