SBIR/STTR Phase I: A High Throughput Gene Discovery System in Arabidopsis using Geminivirus gene expression vectors

这项小型企业技术转移(STTR)第一阶段项目建议开发一种基于甘蓝曲叶病毒的双生病毒衍生基因表达系统，以在瞬变、高通量模型中测试农艺重要基因的效果。虽然基因组学革命是了解基因功能的重要一步，但最重要的挑战是将生物信息学数据与实际功能反应相关联。范式遗传公司目前正在使用植物模式物种--拟南芥，专门为解决序列和功能数据之间的瓶颈而开发的管道中。通过基因工程获得高或低表达单个基因的拟南芥植株，在发育、生化和分子平台上进行分析。虽然这种管道在许多方面都取得了成功，但仍然存在两个缺点：(1)产生稳定的拟南芥转化体所需的时间，以及(2)一次只能测试一个基因的能力。为了解决这些问题，这项提议启动了使用双生病毒载体的额外平台的开发。首先，将进行拟南芥生态型和诱变种群的遗传筛选。这一筛选旨在分离支持病毒复制和传播的无症状拟南芥品系。其次，基于病毒水平较低可以减少植物症状发展的假设，病毒载体将被修改为在较低水平复制。这项研究的结果将被用于启动高通量分析，以确定农学上重要基因的功能。此外，这些结果将导致对病毒/寄主相互作用的重要农学理解。该项目在作物改良中的商业应用。

This Small Business Technology Transfer (STTR) Phase I Project proposes to develop a geminivirus-derived gene expression system, based on cabbage leaf curl virus, to test the effect of agronomically important genes in a transient, high throughput model. While the genomics revolution is a major step towardunderstanding gene function, the foremost challenge is correlating bioinformatics data with actualfunctional responses. Paradigm Genetics, Inc., currently uses the plant model species, Arbidopsis thaliana in a pipeline developed specifically to address the bottleneck between sequence and functional data. Arabidopsis plants genetically engineered to over or underexpress single genes are analyzed in developmental, biochemical and molecular platforms. While this pipeline has been successful in many ways, two disadvantages remain : (1) the amount of time required to generate stable Arabidopsis transformants, and (2) the ability to test only one gene at a time. To address these issues, this proposal initiates the development of an additional platform using geminivirus vectors. First, a genetic screen of Arabidopsis ecotypes and a mutagenized population will be carried out. This screen is designed to isolate symptomless Arabidopsis lines supporting viral replication and spread. Second, based on the hypothesis that lower viral levels can decrease symptom development in the plant, the viral vector will be modified to replicate at lower levels. Results from this research will be used to start a high throughput assay to determine functions of agronomically important genes. Additionally, these results will lead to important agronomic understandings of virus/host interactions.The commercial applications of this project are in crop plant improvement.