Characterization of novel subcellular structures in Arabidopsis thaliana

拟南芥新型亚细胞结构的表征

基本信息

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
项目类别：
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

项目摘要

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）：植物生物技术具有在许多方面改善人类健康的潜力。植物可以被改造为生产治疗性蛋白质作为药物。农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。植物还可以被设计来净化环境或替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产生大量转基因蛋白。这种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量影响药物的经济可行性。解决植物蛋白质产量低问题的一种方法是在植物细胞中寻找新的位置来储存工程蛋白质。该提案的目标是发现并表征植物细胞中用于积累转基因蛋白的新区室。目前，将蛋白质靶向细胞质和膜结合细胞器（如内质网）仅导致每克植物组织中的蛋白质浓度较低。与细菌和哺乳动物细胞培养物中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，该项目将鉴定出新的细胞器或亚细胞结构，从而提高植物基因工程的效率。该项目还将确定如何将蛋白质定位到这些新位置。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。该项目将首先在模式植物拟南芥中寻找新的细胞器。已生产出 108 种独特的转基因拟南芥，它们表达绿色荧光蛋白 (GFP) 和随机植物蛋白之间的不同融合 (1)。这些随机蛋白质充当靶向信号，将 GFP 发送到不同的细胞内区室。在某些情况下，人们发现 GFP 在植物生物学家以前从未观察到的细胞区域中积累。该项目旨在表征这些新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 GFP 照亮植物细胞新结构的候选植物。然后，转基因将从候选品系中克隆出来，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，将对其进行纯化并通过肽指纹分析分析蛋白质含量。公共健康相关性：生物技术学家正在对植物进行基因工程，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、清理工业废物以及生产碳中性燃料。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内发现新的隔室，从而促进更有效地利用植物来改善人类健康。