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

本小型企业技术转移（STTR）一期项目拟开发一种基于白菜卷叶病毒的双病毒衍生基因表达系统，在瞬时高通量模型中测试农学上重要基因的作用。虽然基因组学革命是了解基因功能的重要一步，但最大的挑战是将生物信息学数据与实际功能反应相关联。Paradigm Genetics, Inc.目前在一个专门开发的管道中使用植物模式物种，Arbidopsis thaliana，以解决序列和功能数据之间的瓶颈。拟南芥植物基因工程过表达或过表达单一基因在发育，生化和分子平台分析。虽然这条管道在很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需要大量的时间，(2)一次只能测试一个基因。为了解决这些问题，本提案启动了使用双病毒载体的额外平台的开发。首先，将进行拟南芥生态型的遗传筛选和诱变群体。该筛选旨在分离支持病毒复制和传播的无症状拟南芥系。其次，基于较低的病毒水平可以减少植物症状发展的假设，病毒载体将被修改以在较低水平上复制。这项研究的结果将用于启动高通量测定，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/宿主相互作用的重要农艺学理解。该项目的商业应用是在作物植株改良方面。

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.