The genome sequence of Arabidopsis suggests that it contains approximately 30,700 protein-coding genes. A major goal of the 2010 program is to understand how each of these genes function during plant growth, development, and responses to biotic and abiotic cues. Global studies to analyze gene and protein function have largely focused on gene expression, gene disruption, protein interactions and protein localization. To elucidate biochemical activities of the proteome at the global scale, this project will continue to develop a protein chip for Arabidopsis. In the pilot project funded by NSF 2010 program, high throughput techniques for cloning and expression of 1000 Arabidopsis ORFs were optimized to produce high quality active proteins for the generation of an Arabidopsis protein chip. In this project, a collection of expression clones for 4,000 predicted Arabidopsis ORFs will be generated for tandem affinity purification (TAP) tag fusions. These proteins will be expressed in the plant-based transient expression system to produce and purify proteins. The proteins will be printed on various printing surfaces to produce protein microarrays, which will then be used to optimize protocols for analysis of protein activities. The reagents generated will be made available to the entire scientific community and the information will be available through our website (www.gersteinlab.org/proj/atpchip/).
Broader Impacts: This project will provide a valuable resource for a variety of applications aimed at the high-throughput study of protein function in Arabidopsis. The project will generate a suite of protocols specific to plant proteomes and generate a resourceful set of plant expression clones and other reagents that are expected to significantly enhance the analysis of protein function in Arabidopsis. Furthermore, the methods developed and information gained from this study can also be directly applied to the analysis of other agriculturally and horticultural plants. In addition to generating a community resource, the project provides the unique opportunity to elevate the awareness and importance of genomics and proteomics to a wide range of individuals including visiting scholars from around the world, educators from small colleges, public secondary school teachers in and around the New Haven area and under represented students at the undergraduate and graduate level. Specifically, participation in Yale University STARS program will provide opportunities for under represented students to conduct cutting edge research in proteomics lab.
