Characterization of novel subcellular structures in Arabidopsis thaliana

拟南芥新型亚细胞结构的表征

• 批准号: 8148126
• 负责人: Ernest Y Kwok
• 金额: $ 14.5万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8148126
• 关键词: Address; Amino Acid Sequence; Amino Acids; Arabidopsis; Bacteria; Binding; Biochemical Pathway; Biotechnology; Carbon; Cell Culture Techniques; Cell model; Cells; Centrifugation; Chemicals; Chloroplasts; Code; Collection; Communities; Complementary DNA; Cytosol; Economics; Endoplasmic Reticulum; Engineering; Environment; Enzymes; Fertilizers; Fluorescence; Fluorescence Microscopy; Fluorescence-Activated Cell Sorting; Food; Fossil Fuels; Future; Genetic Engineering; Goals; Golgi Apparatus; Green Fluorescent Proteins; Health; Human; Industrial Waste; Insertional Mutagenesis; Interphase Cell; Label; Light; Location; Macromolecular Complexes; Mammalian Cell; Membrane; Microscopy; Modification; Mouse-ear Cress; Nutrient; Organelles; Pathway interactions; Peptide Hydrolases; Peptide Mapping; Peptide Sequence Determination; Pesticides; Pharmaceutical Preparations; Plant Model; Plant Physiology; Plant Proteins; Plants; Play; Production; Protein Engineering; Proteins; Proteomics; Role; Scientist; Screening procedure; Sequence Analysis; Signal Transduction; Site-Directed Mutagenesis; Solutions; Sorting - Cell Movement; Source; Spottings; Staging; Staining method; Stains; Structure; Subcellular structure; System; Tissues; Transgenes; Transgenic Organisms; Transgenic Plants; Trypsin; Two-Dimensional Gel Electrophoresis; Vacuole; Visual; Weight; base; cell type; drug production; improved; inhibitor/antagonist; interest; novel; peroxisome; planetary Atmosphere; plant genetics; protein expression; research study; tandem mass spectrometry; therapeutic protein; tool

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.

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。可以设计植物以生产治疗蛋白作为药物。农作物可以经过遗传修饰，以生产更营养且需要更少的化肥和农药的食物。还可以设计植物以清理环境或更换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。在药物生产领域，这种限制尤其具有挑战性，在药物生产的领域中，植物产生的蛋白质量会影响药物的经济可行性。 解决植物中蛋白质生产低的问题的一种方法是在植物细胞中找到新的地方来存储工程蛋白质。该提案的目的是发现和表征植物细胞中新隔室的转基因蛋白的积累。目前，将蛋白靶向细胞质和膜结合细胞器，例如内质网，仅导致每克植物组织的蛋白质浓度低。与其他策略（例如细菌和哺乳动物细胞培养物中的蛋白质表达）相比，这降低了植物生物技术的获利能力。如果成功，该项目将确定新的细胞器或亚细胞结构，以提高植物遗传工程的效率。该项目还将确定如何将蛋白质靶向这些新位置。此外，该项目可以将新的生化途径确定为未来植物基因工程的目标。 该项目将开始在模型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，在绿色荧光蛋白（GFP）和随机植物蛋白之间表达不同的融合（1）。这些随机蛋白是将GFP发送到不同细胞内室的靶向信号。在某些情况下，已经发现GFP在植物生物学家从未观察到的细胞区域积累。该项目将旨在表征这些新的细胞器以及如何将蛋白质靶向它们。 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GFP在其中照亮了植物细胞中的新结构。然后将转基因从候选线中克隆出来，以确定哪些蛋白质序列将GFP靶向其特定位置。此序列信息将用于确定将蛋白靶向新细胞器的最小要求。最后，为了充分表征细胞器，将通过肽指纹进行纯化并分析蛋白质含量。 公共卫生相关性：生物技术医生是基因工程工厂生产药物，增加营养含量，使用更少的化学品，清理工业废物并生产碳中性燃料的养分，更便宜。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找到新的隔间，以促进更有效地利用植物来改善人类健康。